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Márcia Teresa da Silva Rodrigues
Setembro de 2011 U
M
in ho |2
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DEVELOPMENT OF TISSUE ENGINEERED
STRATEGIES COMBINING STEM CELLS
AND SCAFFOLDS AIMED TO REGENERATE
BONE AND OSTEOCHONDRAL INTERFACES
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ci a Te re sa d a Si lv a R
od rig
ue s Universidade do Minho
Escola de Engenharia
Tese de Doutoramento em Engenharia de Tecidos,
Medicina Regenerativa e Células Estaminais
Márcia Teresa da Silva Rodrigues
Setembro de 2011
DEVELOPMENT OF TISSUE ENGINEERED
STRATEGIES COMBINING STEM CELLS
AND SCAFFOLDS AIMED TO REGENERATE
BONE AND OSTEOCHONDRAL INTERFACES
Universidade do Minho
Escola de Engenharia
Trabalho efectuado sob a orientação do
Professor Rui L. Reis
e da
Doutora Manuela E. Gomes
É AUTORIZADA A REPRODUÇÃO PARCIAL DESTA TESE APENAS PARA
EFEITOS DE INVESTIGAÇÃO, MEDIANTE DECLARAÇÃO ESCRITA DO
INTERESSADO, QUE A TAL SE COMPROMETE
Márcia Teresa da Silva Rodrigues
- iii Acknowledgments
During my PhD, several people contributed to the accomplishment of this important step in my
life, and to whom I wish to express my gratefulness.
First and foremost, I would like to thank Prof. Rui Reis for the opportunity of performing my
PhD in 3B’s Research Group. Prof. Rui has been an inspiration as a mentor and as a leader,
from whom I learnt that high goals are accessible with the right dose of motivation, resilience,
effort and determination. Thank you for believing in my abilities and for the chance to
overcome many scientific challenges in the last 6 years. This journey has been a unique and
enriching experience both personally and professionally, for all defying challenges and
conquered achievements. I also appreciate the opportunity of participating in parallel projects
and scientific meetings that sometimes seemed an overwhelming assignment. Prof. Rui pulled
me out from the comfort zone, which made me question and test my limits. This exercise
made me stronger and open-minded to forthcoming scientific challenges.
I also wish to express my gratitude to Prof. Manuela Gomes, or should I say Manela, my cosupervisor in this project. Thank you for everything… basically! For your guidance, support, and
dedication to your students, and to research… Your amazing strength and determination in
pursuing all of your goals is inspirational, and encouraged me moving forward every step of
the way. Throughout this journey, I became a better and more confident scientist, mostly
because you believed in me and you brought up in me abilities that I didn’t think I have.
Thank you for sharing with me that cosy felling of companionship, when mentoring and
friendship come together. Because of that I can only remember good things, even in the most
stressful moments, as you were always there for me (even when you were overwhelmed in
work or at home with your newborn children).
I really enjoy working with you and having you as a mentor and a very good friend…
I also would like to acknowledge Prof. James Yoo and Dr Sang Jin Lee for permitting me to do
part of my PhD plan at WFIRM, and for all the support given in USA. Working at WFIRM gave
me the opportunity to learn and grow scientifically. It was a positive experience I will always
remember.
- iv I gratefully acknowledge the funding institutions that materialized my PhD project. First of all
my appreciation goes to the Portuguese Foundation for Science and Technology (FCT) for
awarding me a PhD grant. I am also grateful to the projects: Hippocrates and Find&Bind as well
as the network of excellence Expertissues for all the support in achieving my PhD plan. Finally,
I also recall Calouste Gulbenkian Foundation in this acknowledgment for supporting my
attendance to International Conferences.
To all researchers from the Biomaterials group, and technicians that participated in my project
at WFIRM, with a special thanks to Dr Bu Kyu Lee for the guidance in the surgeries, and also for
all scientific details in the bone project, and to Tom Shiner, who assisted me with my work.
A special thanks also to Pedro Baptista who helped me most with my integration process at
Winston-Salem, and with many scientific questions and doubts.
To my dear friends Liliya and Olga, thank you for your friendship and for a closer feeling of
home…
I could never forget, of course, my dear friends and colleagues at 3Bs Research Group that
helped me to improve not only my scientific knowledge but also my social and human skills:
- Belinha, it has been great to work with you, I loved the brainstorming we had for our
projects. I will never forget your daily support when I was homesick in the USA. The distance
made our friendship grow stronger.
- To my colleagues that participated in the projects compiled to make this Thesis possible:
Albino Martins, Vitor Araújo, Nathalie Gröen, and Sofia Caridade.
- To other colleagues and friends that did not actively participate in this Thesis but took also
part in my scientific journey: Sílvia Gomes, Helena Lima, Simone Silva, Maria Susano, Alexandra
Gonçalves, Ana Dias, and João Requicha. Thank you for your support and friendship…
I also wish to express my gratitude to Dra Isabel Dias and Dr Carlos Viegas, who performed the
orthopaedic surgeries in the goat model, and for all assistance and dedication.
Thank you Fernando Muñoz and Monica, for assisting me with your expertise in the processing
of calcified bone samples, and with the histological and histomorphometric analysis.
Very special thanks to my close friends: Elena Popa and Pedro Carvalho, who shared with me
most of the cheerful moments as well as the most stressful situations of this voyage. Every
- v time I decided to test my working abilities (quite frequently I must say) and embrace one task
after another combining a few projects simultaneously, you were there and assisted me with
little things that made a huge difference; “Little drops of water make the mighty ocean!”
Thank you so much! My PhD would not be the same without you, guys!
I cannot forget my dear cousin and friend Gi, who was invaluable in her help with the image
formatting throughout this Thesis, as well as with all the formatting cues of the submitted
papers.
I am deeply thankful to my family for all the support, love, dedication and comprehension,
especially my dear parents and my grandmother. Thank you for listening and for
understanding my choices and decisions, and encouraging me to pursue my goals.
Finally, I have to thank to my beloved husband, Jorge, for his endless support and patience, for
his understanding and love all these years, and ever since we are together. Every time I
complained about how difficult and stressful science was, you would listen and reply with a
smile. Despite the distance in some periods of my PhD, you were always there for me. I learnt
from you that the glass is never half empty rather than half full. This thought was present in
many complex situations and helped me focus on what was really important to accomplish my
goals.
- vi - vii Abstract
Bone is a specialized tissue characterized by its rigidity and hardness, yet light weighed to
fulfill diverse functions as mineral storage, organ protection or body support and locomotion.
Despite its extraordinary healing ability, bone response may be unsuccessful to repair severe
damage caused by injury or degenerative diseases. Furthermore, when bone is affected, other
tissues and interfaces might be quite distressed as well. Cartilage and bone interface of the
joints (osteochondral interfaces) is particularly affected by traumatic injuries and aging
diseases. The challenge lies in balancing the structural, functional and biological needs of bone
and cartilage in a stable milieu. As currently used therapies do not provide the ideal treatment,
the development of biological substitutes through tissue engineering (TE) approaches may
provide the ultimate solutions to restore, maintain, or improve bone and osteochondral tissue
function.
Scaffolds play an imperative role in most TE strategies, where they are expected to guide
cellular distribution and colonization, similarly to the natural occurring communications
between cells and tissue, and to provide mechanical support during tissue regeneration.
Nevertheless, in large damaged areas, scaffolding alone might be insufficient to promote a
satisfactory healing response. Culturing stem cells onto the scaffolds has demonstrated to
promote the regeneration of damaged tissues. Stem cells (SCs) can be found in almost every
tissue, evidencing their role in repairing injuries. Bone marrow stem cells (BMSCs) are the most
studied, and promising candidates for autologous TE approaches minimizing disease
transmission risks but shown to be donor age-affected and had limited self-renewal capability.
These limitations directed research into other stem cells sources, such as amniotic fluid (AF),
that have shown to be an almost unlimited SCs source with high proliferative and osteogenic
potential. Along with the almost endless ability to expand without telomere shortening,
amniotic fluid stem cells (AFSCs) share with embryonic stem cells some markers and a high self
renewal capacity.
In this Thesis several potential approaches were considered aiming at bone and
osteochondral TE, focusing on distinct scaffold design and composition, previously or newly
developed, and distinct stem cells sources. Different animal models were used to evaluate the
proposed strategies with scaffolds and/or cell-scaffold constructs.
As a first approach, a multilayered scaffold was developed composed of tricalcium
phosphate (TCP) granules entrapped in a polycaprolactone (PCL) nanofiber mesh, inspired
from the natural organic-inorganic nanostructure of bone. A synergistic effect of PCL-TCP
- viii scaffolds and mechanical stimulation was observed in the osteogenic differentiation of BMSCs
cultured onto these scaffolds, resulting in the production of a mineralized ECM, even in basal
medium. Composite multilayered scaffolds showed an interesting behavior under dynamic
conditions using cell culturing media without osteogenic supplements.
Another approach consisted in the combination of wet-spinning technology and a calcium
silicate solution to produce SPCL (starch and polycaprolactone blend) wet-spun fiber meshes
with functionalized silanol groups (SPCL-Si). The purpose was to developed new bioactive
materials linking the properties of classical bioactive ceramics, and the processability and
degradability of an organic polymer. SPCL-Si scaffolds own intrinsic properties to sustain in
vitro osteogenic features, and thus holding a great potential for bone engineering approaches.
Additionally, this Thesis aimed at designing a new construct for the repair of osteochondral
(OC) interfaces, proposing a novel bilayered scaffold, combining the well described agarose
gels for cartilage and the promising SPCL scaffolds for bone, encapsulated/seeded with
amniotic stem cells (AFSCs). An OC engineered system was successfully developed, where both
osteo- or chondrogenic differentiated AFSCs maintained long term viability and phenotypic
expression, even in basal medium after assembling of the bilayered construct.
Another major original objective of this Thesis was to explore the potential of amniotic
fluid stem cells (AFSCs), as compared to bone marrow stem cells (BMSCs), for bone TE
applications. Besides their source, the environmental conditions are known to influence cell
response, and thus, both AFSCs and BMSCs were seeded/cultured in either 2D or 3D (using
SPCL scaffolds) conditions. AFSCs and BMSCs expressed different bone-related markers at
different time points. This study demonstrated that the selection of a particular stem cell type
may not be a simple and direct process and relies on the target TE strategy.
Finally, non-critical sized defects were induced in goat femurs so as to understand the role
of the scaffold material -SPCL- and the influence of culturing autologous mesenchymal cells
with/without pre-culture in osteogenic medium. Neobone formation and cellular distribution
was increased in cell seeded SPCL scaffolds (pre-differentiation condition), showing the
relevance of implanted cells in the bone regeneration process, and suggesting the importance
of the stage of osteogenic differentiation of seeded cells.
In a similar approach, femoral critical sized defects were induced in nude rats and SPCL
scaffolds were implanted with or without AFSCs under different stages of osteogenic
differentiation. The bridging effect between the bone segments was more prominent in
scaffolds with osteogenic committed cells, and large blood vessels were observed, especially in
SPCL scaffolds seeded with undifferentiated cells or osteogenic-like cells. Both in vivo studies
- ix showed the potential of SPCL scaffolds as a tissue 3D support for the regeneration of bone and
underlined the importance of stem cells and stem cells stage of differentiation for achieving
enhanced bone tissue regeneration.
In summary, the described scaffold design and composition show a great potential to be
tailored to specific applications in bone tissue regeneration strategies. Nevertheless, SPCL
meshes obtained from melt spun fibers are clearly one step ahead as scaffold structures,
showing to provide the necessary support for bone and osteochondral TE strategies using
different sources of stem cells. Most importantly, the work described in this Thesis clearly
demonstrated that bone tissue engineering requires the presence of stem cells, and that their
pre-differentiation into the osteoblastic phenotype facilitates bone regeneration.
- x - xi Resumo
O osso é um tecido especializado, caracterizado pela sua rigidez e dureza embora a sua
estrutura leve permita a realização de diversas funções, incluíndo a reserva de minerais,
protecção de órgãos ou suporte do organismo e locomoção. Apesar da sua capacidade
regenerativa extraordinária, a resposta natural do osso revela-se, por vezes, limitada na
reparação de danos severos resultantes de traumas graves ou doenças degenerativas. Além
disso, quando o osso se deteora, outros tecidos e interfaces poderão estar igualmente
comprometidos. A interface cartilagem-osso presente nas articulações (interfaces
osteocondrais) é particularmente afectada por traumas e doenças associadas ao
envelhecimento. Uma vez que as terapias convencionais não garantem o tratamento ideal
destes tecidos, o desenvolvimento de substitutos biológicos através da engenharia de tecidos
propõe-se como uma alternativa para restaurar, manter ou melhorar a funcionalidade do osso
e do sistema osteocondral.
Na maioria das estratégias em engenharia de tecidos, as estruturas tridimensionais de
suporte ou scaffolds desempenham um papel fundamental, conduzindo a distribuição e
colonização celulares, de modo semelhante à comunicação que ocorre naturalmente entre as
células e o tecido, proporcionando também um suporte mecânico durante o processo de
regeneração. No entanto, em áreas danificadas de grandes dimensões, as estruturas
tridimensionais de suporte poderão ser insuficientes para promover uma resposta terapêutica
satisfatória. A cultura de células estaminais nestas estruturas tem demonstrado estimular a
regeneração de tecidos lesados; células estas que podem ser encontradas em quase todos os
tecidos, evidenciando o seu papel na reparação de lesões. Actualmente, as células estaminais
da medula óssea (bone marrow stem cells, BMSCs) têm sido as mais estudadas, revelando-se
candidatas promissoras para aplicações autólogas em engenharia de tecidos, reduzindo o risco
de transmissão de doenças, embora o seu potencial possa ser comprometido pela idade do
dador e pela limitada capacidade de auto-renovação. Estas limitações conduziram a
investigação científica na procura de novas fontes de células estaminais, como o fluído
amniótico que demonstrou ser uma fonte quase inesgotável de células estaminais com grande
potencial de proliferação e de diferenciação osteogénica. Além disso, as células estaminais do
fluído amniótico (amniotic fluid stem cells, AFSCs) partilham com as células estaminais
embrionárias marcadores genéticos e uma elevada capacidade de auto-renovação.
Nesta Tese foram estudadas diferentes estratégias de engenharia de tecidos, considerando
o seu potencial para aplicação em osso e em interfaces osteocondrais. Estas estratégias
- xii baseiam-se na utilização de estruturas distintas de suporte tridimensionais (scaffolds),
desenvolvidos prévia ou especificamente para este projecto, e em células estaminais de
diferentes fontes. Também foram analisados vários modelos animais para avaliação in vivo das
estratégias desenvolvidas, nomeadamente as estruturas de suporte tridimensionais na
presença/ausência de células estaminais.
Numa primeira abordagem, desenvolveu-se uma estrutura de suporte tridimensional
constituída por grânulos de fosfatos de tricálcio envolvidos numa matriz de nano fibras de
policaprolactona (PCL). Esta estrutura foi inspirada na nanoestrutura orgânica-inorgânica do
osso. Observou-se um efeito sinergístico das estruturas de suporte tridimensional de PCLfosfatos e da estimulação mecânica no processo de diferenciação osteogénica das BMSCs
cultivadas nestes suportes, resultando na produção de uma matriz extracelular mineralizada,
mesmo em meio de cultura basal. As estruturas compósitas de suporte tridimensional de
multi-camada demonstraram ser relevantes em condições dinâmicas utilizando meios de
cultura celular sem suplementos osteogénicos.
Numa segunda estratégia, combinou-se a tecnologia de extrusão líquida (wet-spining) e
uma solução de silicato de cálcio para produzir uma matriz de suporte tridimensional de uma
mistura polimérica de amido e policaprolactona (starch-polycaprolactone blend, SPCL) com
grupos funcionais de silanol (SPCL-Si). O objectivo consistiu no desenvolvimento de novos
materiais bioactivos, reunindo as propriedades dos materiais cerâmicos clássicos e a
processabilidade e degradabilidade de um polímero orgânico. As matrizes desenvolvidas
apresentam propriedades intrínsecas capazes de sustentar as propriedades osteogénicas das
células in vitro, e, consequentemente, com potencial para engenharia de tecidos ósseos.
Nesta Tese foi também investigado o desenvolvimento de estruturas de suporte
tridimensionais híbridas com células estaminais para a regeneração de interfaces
osteocondrais, através da criação de uma estrutura de suporte tridimensional de bi-camada,
combinando os géis de agarose, já estudados para a cartilagem, e as estruturas de suporte
tridimensionais promissoras de SPCL para osso, cultivados ou encapsulados, respectivamente
com AFSCs. Foi desenvolvido um sistema osteocondral, no qual as células estaminais do fluído
amniótico se diferenciaram nas linhagens condro- e osteogénicas, mantendo a viabilidade e a
expressão fenotípica ao longo do tempo, após a montagem do sistema de bicamada e em meio
de cultura basal.
O potencial das células estaminais do fluído amniótico (AFSCs) foi também explorado e
comparado com as células estaminais da medula óssea (BMSCs) para aplicações em
engenharia de tecido ósseo. A origem e o meio envolvente são condicionantes da resposta
- xiii celular e, assim sendo, as AFSCs e as BMSCs foram cultivadas em ambientes de duas e três
dimensões (utilizando as estruturas de suporte tridimensionais de SPCL). Os dois tipos
celulares revelaram diferentes perfis de expressão de vários marcadores associados à
diferenciação osteogénica, analisados ao longo do tempo em cultura, demonstrando que a
selecção de um tipo de células estaminais não é uma escolha simples e directa, e que se
encontra dependente da estratégia específica que se pretende.
Em seguida, estudos de defeitos ósseos não-críticos foram realizados em fémures de cabras
com o propósito de analisar o papel das estruturas de suporte tridimensionais de SPCL e da
influência do cultivo de células autólogas da medula óssea, com ou sem pré-cultura em meio
de diferenciação osteogénico. A formação de novo tecido ósseo e a distribuição celular foram
mais pronunciadas nos locais implantados com os suportes tridimensionais híbridos com
células pré-diferenciadas, evidenciando a importância das células implantadas e sugerindo
também a relevância do estadio de diferenciação celular no processo regenerativo ósseo.
Posteriormente, foram induzidos defeitos críticos em fémures de ratos atímicos, onde se
implantaram também estruturas de suporte tridimensionais de SPCL, na presença ou ausência
de AFSCs, em diversos estadios de diferenciação osteogénica. A aproximação dos dois
segmentos ósseos no defeito criado foi mais pronunciado na presença das estruturas de
suporte tridimensionais híbridas com AFSCs comprometidas com o fenótipo osteogénico,
enquanto que os maiores vasos sanguíneos foram observados nos defeitos preenchidos com
estruturas de suporte tridimensionais hibridadas com AFSCs no estadio indiferenciado ou com
AFSCs no estadio considerado osteogénico. Ambos os estudos in vivo demonstraram o
potencial das estruturas tridimensionais de SPCL para suporte e regeneração de tecido ósseo,
salientando uma vez mais a importância das células estaminais e do estadio de diferenciação
celular numa melhor regeneração tecidular.
Resumidamente, o design e composição das estruturas de suporte tridimensionais,
descritos anteriormente, demonstraram potencial para aplicação em estratégias de
regeneração óssea. No entanto, as matrizes de SPCL obtidas por melt spun, estão um passo à
frente como estruturas de suporte uma vez que demonstraram garantir o suporte
tridimensional de estratégias para osso e interface osteocondral, utilizando células estaminais
de diferentes origens. Em conclusão, o trabalho descrito nesta Tese demonstra claramente
que a engenharia de tecidos do osso requer a presença de células estaminais e que a sua prédiferenciação no fenótipo osteogénico facilita a regeneração óssea.
_____________________________________________________________________________
O resumo em português desta Tese não se encontra redigido segundo o novo acordo ortográfico.
- xiv - xv Table of Contents
Acknowledgments ................................................................................................................... iii
Abstract .................................................................................................................................. vii
Resumo.................................................................................................................................... xi
Table of Contents.........................................................................................................................xv
Short Curriculum Vitae .......................................................................................................... xxiii
This Thesis is based on the following publications .................................................................. xxv
List of Figures ....................................................................................................................... xxvii
List of Tables ..................................................................................................................... xxxiii
List of Abbreviations and Symbols ........................................................................................ xxxv
SECTION I
GENERAL INTRODUCTION ......................................................................................................... 1
Chapter I
CURRENT STRATEGIES FOR OSTEOCHONDRAL REGENERATION: FROM STEM CELLS TO PRE-
CLINICAL APPROACHES ............................................................................................................. 3
I.1.Abstract ........................................................................................................................... 5
I.2.Introduction ..................................................................................................................... 6
I.2.1. Osteochondral defects (OCD) ............................................................................. 7
I.2.2. Weight bearing influence in biomechanics of the joint ....................................... 8
I.2.3. Current treatments in clinical field ..................................................................... 8
I.2.4. TE strategies to improve available treatments .................................................... 9
I.2.4.1. Cells to promote healing ................................................................................ 9
I.2.4.2. Biomaterials: human designs to mimic natural extracellular material ........... 11
I.2.4.3. Assisted Devices: bioreactor systems ........................................................... 13
I.2.5. In vivo models for osteochondral tissue engineering ........................................ 14
I.3.Conclusions .................................................................................................................... 15
I.4.References ..................................................................................................................... 15
SECTION II
MATERIALS AND METHODS .................................................................................................... 21
Chapter II
MATERIALS & METHODS ......................................................................................................... 23
- xvi II.1.Abstract ........................................................................................................................ 25
II.2.Materials ....................................................................................................................... 25
II.2.1. Polycaprolactone ............................................................................................. 25
II.2.2. Starch-polycaprolactone blend......................................................................... 26
II.2.3. Tri-calcium phosphates .................................................................................... 27
II.2.4. Agarose............................................................................................................ 27
II.3.Scaffold fabrication ....................................................................................................... 28
II.3.1. Melt fiber extrusion - Fiber bonding ................................................................. 29
II.3.2. Electrospinning ................................................................................................ 29
II.3.3. Tricalcium Phosphates production by solid state reaction ................................ 30
II.3.4. Wet-spinning ................................................................................................... 30
II.3.4.1. Designing of an in situ functionalized surface ........................................... 30
II.4.Scaffold characterization ............................................................................................... 31
II.4.1. In vitro bioactivity of SPCL-Si wet-spun fiber mesh scaffolds............................. 31
II.4.2. Scanning electron microscopy .......................................................................... 32
II.4.3. Dynamic Mechanical Analysis ........................................................................... 32
II.4.4. Micro-computed tomography .......................................................................... 32
II.4.5. Thin-film X-ray diffraction ................................................................................ 33
II.4.6. Fourier transform infrared spectroscopy with attenuated total reflectance ...... 34
II.4.7. Inductively coupled plasma optical emission spectrometer .............................. 34
II.5.Scaffold sterilization prior to cell culturing studies ......................................................... 35
II.6.Biological assays ............................................................................................................ 35
II.6.1. Harvesting, isolation and culture of stem cells .................................................. 35
II.6.1.1. Harvesting and isolation of goat bone marrow stromal cells ..................... 35
II.6.1.2. Harvesting and isolation of human amniotic fluid stem cells ..................... 37
II.6.2. Cell culturing and expansion ............................................................................ 37
II.6.2.1. Goat bone marrow stromal cells ............................................................... 38
II.6.2.2. Human amniotic fluid stem cells ............................................................... 38
II.6.2.3. Human mesenchymal stem cells from bone marrow ................................ 38
II.6.3. Osteogenic differentiation ............................................................................... 39
II.6.4. Osteochondral differentiation .......................................................................... 39
II.6.5. Seeding on 2D cultures .................................................................................... 40
II.6.6. Seeding cells onto 3D matrices ......................................................................... 40
- xvii II.6.6.1. gBMSCs, hAFSCs or hBMSCs onto SPCL scaffolds ...................................... 40
II.6.6.2. gBMSCs onto SPCL-Si scaffolds ................................................................. 41
II.6.6.3. Encapsulating hAFSCs into agarose gels .................................................... 41
II.6.7. Characterization of cell-scaffold constructs ...................................................... 41
II.6.7.1. Cell viability methods – MTS and Calcein AM ............................................ 42
II.6.7.2. Cellular proliferation assay (DNA quantification) ...................................... 43
II.6.7.3. Histology .................................................................................................. 44
II.6.7.4. Osteogenic markers.................................................................................. 44
II.6.7.4.1. Alkaline Phosphatase Activity (ALP) ....................................................... 44
II.6.7.4.2. Alizarin Red Staining .............................................................................. 46
II.6.7.4.3. Fourier Transformed Infrared Spectroscopy with Attenuated Total
Reflectance ............................................................................................................. 46
II.6.7.4.4. Immunocytochemistry: Collagen I and Osteocalcin ................................ 47
II.6.7.4.5. Immunofluorescence: Collagen I and RunX-2 ......................................... 47
II.6.7.5. Chondrogenic markers ............................................................................. 48
II.6.7.5.1. Safranin O staining ................................................................................ 48
II.6.7.5.2. Immunofluorescence: aggrecan and collagen II ..................................... 48
II.6.7.6. Evaluation of cell morphology and distribution ......................................... 49
II.6.7.6.1. Confocal laser scanning microscopy (CLSM) ........................................... 49
II.6.7.6.2. SEM....................................................................................................... 49
II.6.7.6.3. Energy dispersive X-ray analysis or EDS analysis .................................... 50
II.7.Animal models studies .................................................................................................. 50
II.7.1. Goat model – non critical defects ..................................................................... 50
II.7.1.1. Implantation surgery ................................................................................ 51
II.7.1.2. Imprinting new bone with fluorescent dyes .............................................. 52
II.7.1.3. Explant retrieval and characterization ...................................................... 52
II.7.1.3.1. Histological characterization.................................................................. 52
II.7.2. Nude rat model - non-union defects................................................................. 53
II.7.2.1. Implantation ............................................................................................ 54
II.7.2.2. Explant retrieval and characterization ...................................................... 55
II.7.2.2.1. Micro-computed tomography (bone formation assessment) ................. 55
II.7.2.2.2. X-rays .................................................................................................... 55
II.7.2.2.3. Histological Characterization ................................................................. 56
II.7.2.2.4. Histomorphometric analysis .................................................................. 56
- xviii II.8. Statistical analysis......................................................................................................... 57
II.9. References ................................................................................................................... 57
SECTION III
SCAFFOLD DESIGN AND CHARACTERIZATION .......................................................................... 63
Chapter III
SYNERGISTIC EFFECT OF SCAFFOLD COMPOSITION AND DYNAMIC CULTURING ENVIRONMENT
IN MULTI-LAYERED SYSTEMS FOR BONE TISSUE ENGINEERING ............................................... 65
III.1. Abstract....................................................................................................................... 67
III.2. Introduction ................................................................................................................ 68
III.3. Materials and Methods ............................................................................................... 69
III.3.1. Development of the nanofibrous multilayered composite scaffolds ................. 69
III.3.2. Characterization of the composite scaffolds ..................................................... 70
III.3.2.1. Thin-film X-ray diffraction (TF-XRD) .......................................................... 70
III.3.2.2. Fourier Transformed Infrared Spectroscopy with attenuated total
reflectance (FTIR-ATR) ................................................................................................. 70
III.3.2.3. Scanning electron microscopy (SEM) ........................................................ 70
III.3.3. In vitro culture of bone marrow mesenchymal stromal cells onto nanofibrous
scaffolds 70
III.3.3.1. Cell proliferation and osteogenic differentiation in multi-stacked
nanofibrous scaffolds .................................................................................................. 71
III.3.3.1.1. DNA assay ............................................................................................ 71
III.3.3.1.2. ALP assay ............................................................................................. 72
III.3.3.1.3. Alizarin Red staining ............................................................................. 72
III.3.3.1.4. Immunocytochemistry ......................................................................... 72
III.3.4. Statistical Analysis ............................................................................................ 72
III.4. Results and Discussion ................................................................................................. 73
III.5. Conclusions ................................................................................................................. 78
III.6. References .................................................................................................................. 79
Chapter IV
FUNCTIONAL BIODEGRADABLE SCAFFOLDS FOR BONE TISSUE ENGINEERING: BIOACTIVITY
PROFILE AND OSTEOGENIC DIFFERENTIATION OF MARROW MESENCHYMAL STROMAL CELLS 83
IV.1. Abstract ...................................................................................................................... 85
IV.2. Introduction ................................................................................................................ 86
IV.3. Materials and Methods ............................................................................................... 87
IV.3.1. Materials ......................................................................................................... 87
- xix IV.3.2. Wet-spun fiber mesh scaffolds processing ....................................................... 88
IV.3.3. Evaluation of the in vitro bioactivity of wet-spun fiber mesh scaffolds.............. 88
IV.3.3.1. Scanning electron microscopy (SEM) ........................................................ 88
IV.3.3.2. Thin-film X-ray diffraction (TF-XRD) .......................................................... 89
IV.3.3.3. Micro-computed tomography (µ-CT) analysis: .......................................... 89
IV.3.3.4. Dynamic mechanical analysis (DMA):........................................................ 89
IV.3.3.5. Induced-coupled plasma emission spectroscopy (ICP) .............................. 90
IV.3.4. Cell culture study ............................................................................................. 90
IV.3.4.1. Harvesting and seeding gBMSCs onto wet-spun fiber mesh scaffolds ....... 90
IV.3.4.2. Cell viability assay ..................................................................................... 91
IV.3.4.3. Cell proliferation assay ............................................................................. 91
IV.3.4.4. ALP assay ................................................................................................. 91
IV.3.4.5. SEM.......................................................................................................... 91
IV.3.5. Statistical Analysis ............................................................................................ 92
IV.4. Results and Discussion ................................................................................................ 92
IV.4.1. Bioactivity assessment ..................................................................................... 92
IV.4.1.1. DMA analysis ............................................................................................ 96
IV.4.2. Biological assays............................................................................................... 97
IV.5. Conclusions ............................................................................................................... 100
IV.6. References ................................................................................................................ 100
Chapter V
BILAYERED CONSTRUCTS AIMED AT OSTEOCHONDRAL STRATEGIES: THE INFLUENCE OF MEDIA
SUPPLEMENTS IN THE OSTEO- AND CHONDRO-GENIC DIFFERENTIATION OF AMNIOTIC FLUID-
DERIVED STEM CELLS ............................................................................................................ 105
V.1. Abstract ..................................................................................................................... 105
V.2. Introduction ............................................................................................................... 108
V.3. Materials and Methods .............................................................................................. 110
V.3.1. Viability assay with Calcein AM ...................................................................... 111
V.3.2. SEM (scanning electronic microscopy)............................................................ 112
V.3.3. Histological characterization .......................................................................... 112
V.4. Results and Discussion ............................................................................................... 113
V.4.1. AFSCs-agarose system .................................................................................... 113
V.4.2. Agarose-SPCL bilayered system ...................................................................... 116
V.4.2.1. AFSCs-SPCL layer .................................................................................... 116
- xx V.5. Conclusions ................................................................................................................ 123
V.6. References ................................................................................................................. 124
SECTION IV
DOES STEM CELL ORIGIN INFLUENCE STEM CELL RESPONSE ? ............................................... 127
Chapter VI
AMNIOTIC FLUID STEM CELLS VERSUS BONE MARROW MESENCHYMAL STEM CELLS AS A
SOURCE FOR BONE TISSUE ENGINEERING ............................................................................. 129
VI.1. Abstract .................................................................................................................... 131
VI.2. Introduction .............................................................................................................. 132
VI.3. Materials and Methods ............................................................................................. 134
VI.3.1. Cell culture..................................................................................................... 134
VI.3.2. Calcein AM assay ........................................................................................... 134
VI.3.3. Alkaline Phosphatase (ALP) staining ............................................................... 135
VI.3.4. Alizarin Red Staining (AR) ............................................................................... 135
VI.3.5. Immunofluorescence ..................................................................................... 135
VI.3.6. Scanning electronic microscopy (SEM) ........................................................... 136
VI.4. Results and Discussion .............................................................................................. 136
VI.4.1. Osteogenic differentiation of AFSCs and BMSCs in a 2D culture environment. 136
VI.4.2. Osteogenic differentiation of hAFSCs and hBMSCs seeded on SPCL scaffolds (3D
environment) ................................................................................................................ 138
VI.5. Discussion ................................................................................................................. 141
VI.6. Conclusions ............................................................................................................... 143
VI.7. References ................................................................................................................ 144
SECTION V
IN VIVO STUDIES ................................................................................................................... 147
Chapter VII
TISSUE ENGINEERED CONSTRUCTS BASED ON SPCL SCAFFOLDS CULTURED WITH GOAT
MARROW CELLS: FUNCTIONALITY IN FEMORAL DEFECTS ...................................................... 149
VII.1. Abstract ................................................................................................................... 151
VII.2. Introduction ............................................................................................................. 152
VII.3. Materials and Methods ............................................................................................ 154
VII.3.1. Production of SPCL scaffolds .......................................................................... 154
VII.3.2. gBMSCs harvesting ......................................................................................... 154
VII.3.3. In vitro cell seeding and culture ...................................................................... 155
- xxi VII.3.4. In vitro characterization of cells-scaffold constructs ....................................... 155
VII.3.5. Animals Study ................................................................................................ 156
VII.3.5.1. Implantation procedures ........................................................................ 157
VII.3.5.2. Harvesting samples after implantation ................................................... 158
VII.3.6. Statistical Analysis .......................................................................................... 159
VII.4. Results and Discussion ............................................................................................. 159
VII.4.1. In vitro characterization of autologous gBMSCs-SPCL constructs .................... 159
VII.4.2. In vivo studies ................................................................................................ 161
VII.4.2.1. Histologic and Fluorescence analysis ...................................................... 161
VII.5. Conclusions .............................................................................................................. 164
VII.6. References ............................................................................................................... 165
Chapter VIII
THE EFFECT OF THE DIFFERENTIATION STAGE OF AMNIOTIC FLUID STEM CELLS SEEDED ONTO
BIODEGRADABLE SCAFFOLDS IN THE REGENERATION OF NON-UNION DEFECTS ................... 169
VIII.1. Abstract.................................................................................................................. 171
VIII.2. Introduction ............................................................................................................ 172
VIII.3. Materials and Methods ........................................................................................... 174
VIII.3.1. In vitro Study .............................................................................................. 174
VIII.3.1.1. Alizarin Red (AR) and Alkaline phosphatase (ALP) staining ...................... 174
VIII.3.1.2. SEM (scanning electronic microscopy) .................................................... 175
VIII.3.1.3. FTIR-ATR (Fourier transform attenuated total reflectance infrared
spectroscopy) ............................................................................................................ 175
VIII.3.1.4. Immunofluorescence.............................................................................. 176
VIII.3.2. In vivo Study ............................................................................................... 176
VIII.3.2.1. µ-CT scanning ......................................................................................... 177
VIII.3.2.2. Tissue processing ................................................................................... 177
VIII.3.2.3. Histomorphometrical Analysis ................................................................ 178
VIII.3.3. Statistical Analysis ...................................................................................... 178
VIII.4. Results and Discussion............................................................................................. 178
VIII.4.1. In vitro study .............................................................................................. 178
VIII.4.2. In vivo study ............................................................................................... 182
VIII.4.2.1. Neobone formation assessment ............................................................. 183
VIII.4.2.2. Histology characterization ...................................................................... 185
VIII.4.2.2.1. Histomorphometric Analysis ............................................................ 186
- xxii VIII.5. Conclusions ............................................................................................................. 190
VIII.6. References .............................................................................................................. 191
SECTION VI
GENERAL CONCLUSIONS ....................................................................................................... 195
Chapter IX
FINAL REMARKS AND FUTURE STUDIES ................................................................................. 197
IX.1 .General Conclusions .................................................................................................. 199
IX.2. Final Remarks and Future Studies .............................................................................. 204
- xxiii Short Curriculum Vitae
Márcia Teresa da Silva Rodrigues was born in Porto (Portugal) in 1981. She was graduated
in Applied Biology at the School of Sciences in University of Minho (Portugal) in June 2004.
She started her PhD in 2005 at the 3Bs Research Group-University of Minho (under the
supervision of Professor Manuela E. Gomes and Professor Rui L. Reis) in cooperation with the
Department of Regenerative Medicine at the Wake Forrest Institute of Regenerative Medicine
(Winston-Salem, NC, USA) under the local supervision of Dr Sang Jin Lee and Professor James
Yoo.
She was also involved in the preparation of European (FP7) and National (Portuguese
Foundation for Science and Technology) research projects proposals. She was co-responsible
for the internal cell culture training at the 3B’s Research Group and the Headquarters of the
European Institute of Excellence on Tissue Engineering and Regenerative Medicine. She is also
a Credited Technician on Animal Experimentation, accreditation Issued by Direcção Geral de
Veterinária, Ministério da Agricultura, do Desenvolvimento Rural e das Pescas on February 8th
2007, after performing the Laboratory Animal Science Course by FELASA in 2006.
During the course of her PhD, she was part of the organization committee of the TERMISEU, held in Porto, Portugal (2008). She was invited to be a scientific abstract reviewer in the
annual conference of Society for Biomaterials in 2008 (Atlanta, USA) and 2009 (San Antonio,
USA), and a reviewer for the Journal of Tissue Engineering and Regenerative Medicine.
As a result from her research, she has participated in the most relevant conferences and
workshops in the Biomaterials and Regenerative Medicine field, with a total of 13 oral
communications and 15 poster presentations. She has been awarded a Fundação Calouste
Gulbenkian Travel award for attendance at Society of Biomaterials Meeting 2006 (Chicago,
USA) and the award for the “Best 50 abstracts awards” submitted to the Tissue Engineering
and Regenerative Medicine International Society - European Chapter Conference (TERMIS-EU)
in 2008.
Presently, Márcia Teresa da Silva Rodrigues is author of 9 published papers in international
refereed journals, 1 paper in press, 5 papers submitted for publication, 1 invited review paper
and 1 book chapter.
- xxiv - xxv This Thesis is based on the following publications
Papers in international refereed journals:
Rodrigues MT, Gomes ME and Reis RL, Current strategies in osteochondral regeneration: from
stem cells to pre-clinical approaches, Current Opinion in Biotechnology, Tissue, Cell and
Pathway Engineering issue 22: 1-8 - invited review paper (2011).
Rodrigues MT, Martins A, Dias IR, Viegas CAA, Neves NM, Gomes ME and Reis RL, Synergistic
effect of scaffold composition and dynamic culturing environment in multi-layered systems for
bone tissue engineering, accepted for publication in Journal of Tissue Engineering and
Regenerative Medicine (2011).
Rodrigues MT, Groen N, Leonor I, Carvalho PP, Dias, Caridade S, Mano JF, van Blitterswijk CA,
Gomes ME and Reis RL, Functional biodegradable scaffolds for Bone Tissue Engineering degradation behaviour, bioactivity prolife and osteogenic differentiation of marrow
mesenchymal cells, submitted
Rodrigues MT, Lee SJ, Gomes ME, Reis RL, Atala A, Yoo J, Bilayered constructs aimed at
osteochondral strategies: the influence of media supplements in the osteo and chondrogenic
differentiation of amniotic fluid-derived stem cells, submitted
Rodrigues MT, Lee SJ, Gomes ME, Reis RL, Atala A, Yoo J, Amniotic fluid stem cells versus bone
marrow mesenchymal stem cells for bone tissue engineering, submitted
Rodrigues MT, Gomes ME, Viegas CAA, Azevedo JT, Dias IR, Gúzon FM and Reis RL, Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects, Journal of Tissue Engineering and Regenerative Medicine 2011; 5: 41-49.
Rodrigues MT, Lee BK, Shiner T, Lee SJ, Gomes ME, Reis RL, Atala A, Yoo J, The effect of the
differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds in the
regeneration of non-union defect, submitted
Book Chapters:
Gomes ME, Oliveira JT , Rodrigues MT, Santos MI , Tuzlakoglu K, Viegas C A A, Dias IR and Reis
RL, Sarch-polycaprolactone based scaffolds in bone and cartilage tissue engineering
approaches, In Natural-based polymers for biomedical applications, eds. RL Reis, J Mano, N
Neves, H Azevedo , AP Marques, ME Gomes, Woodhead Publishing, Cambridge, 337-356
(2008).
- xxvi - xxvii List of Figures
Chapter I
Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
Figure I.2.1 – Clinical and Tissue Engineering strategies to promote the regeneration of
osteochondral defects. ............................................................................................................. 6
Chapter II
Materials and Methods
Figure II.2.1 – Ring opening polymerization of ε-caprolactone to polycaprolactone. ................ 25
Figure II.2.2 – Chemical structure of starch components: amylose (A) and amylopectin
(B). .......................................................................................................................................... 26
Figure II.2.3 – Structure of agarose molecule .......................................................................... 28
Figure II.6.1 - Harvesting marrow from the goat iliac crest. ..................................................... 36
Figure II.6.2 - 2D culture of gBMSCs after 10 days in basal medium. ........................................ 38
Figure II.6.3 – Calcein AM cleavage by viable cells. .................................................................. 43
Figure II.7.1 - Non-critical size defects drilled in the posterior femur of a goat model. ............. 52
Chapter III
Synergistic effect of scaffold composition and dynamic culturing environment in multilayered systems for bone tissue engineering
Figure III.4.1 - FTIR-ATR analysis of multi-layered scaffolds. Membranes of PCL nanofiber
mesh (PCL) were used as controls. .......................................................................................... 73
Figure III.4.2 - XRD analysis of multi-layered PCL-TCP scaffolds. Membranes of PCL
nanofiber mesh (PCL) were used as controls. .......................................................................... 74
Figure III.4.3 – SEM analysis of multilayer scaffolds; the upper SEM micrographs refer to
the PCL-TCP multilayer scaffolds while the lower pictures correspond to the control of
PCL nanofiber mesh (left to right: 100 x, 300 x and 1000 x magnifications). ............................. 74
Figure III.4.4 – gBMSCs proliferation given by DNA quantification was assessed onto PCLTCP scaffolds and PCL membranes (control) after 7, 14 or 21 days in basal or osteogenic
media in either static (st) or dynamic (dyn) conditions. Symbols *, **, and *** denote
study groups with statistically significant differences (p<0.05), as using One Way ANOVA
method. .................................................................................................................................. 75
- xxviii Figure III.4.5 – gBMSCs osteogenic differentiation given by ALP/DNA ratio was assessed
onto PCL-TCP scaffolds and PCL membranes (control) after 7, 14 or 21 days in basal or
osteogenic media in either static (st) or dynamic (dyn) conditions. Symbols *, and **
denote st groups with statistically significant differences (p<0.05), as using One Way
ANOVA method. ..................................................................................................................... 76
Figure III.4.6 – Osteogenic phenotype characterization of gBMSCs seeded onto PCL-TCP
scaffolds after 21 days in culture with basal or osteogenic media, either in static or
dynamic conditions. Immunocytochemistry (ICC) was performed for Collagen I and
Osteocalcin, as well as Alizarin Red staining. Insets represent PCL meshes (control) under
the same conditions. ............................................................................................................... 77
Chapter IV
Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and
osteogenic differentiation of marrow mesenchymal stromal cells
Figure IV.4.1 - TF-XRD patterns of SPCL fiber meshes produced by wet-spinning, where a
calcium silicate solution was used as a coagulation bath (SPCL-Si), and subsequently
soaked in SBF for 7 days. SPCL was used as control. ................................................................ 93
Figure IV.4.2 - 2D and 3D micro-CT and SEM images of SPCL-Si scaffolds before and after
1, 3 or 7 days in SBF. Control scaffolds of SPCL (without silanol groups) (CTR) after 7 days
in SBF. Blue corresponds to the apatite deposition.................................................................. 94
Figure IV.4.3 - Changes in calcium (Ca), phosphorus (P) and silicon (Si) concentration in
the SBF solution after different immersion periods of the SPCL or SPCL-Si scaffolds.
Symbols o and + denote study groups with statistically significant differences (o=p<0.001
and +=p<0.05, respectively), as using Two Way ANOVA method. ............................................ 94
Figure IV.4.4 - Variation of the storage modulus as a function of frequency between 0.1
and 15 Hz after equilibration at 37 ºC with the samples immersed in PBS solution. ................. 96
Figure IV.4.5 - Results obtained from the MTS test performed on gBMSCs seeded onto
SPCL-Si scaffolds (SPCL-Si) and SPCL-Si scaffolds pre-coated with an apatite layer (SPCLSi-7SBF), and cultured in osteogenic medium for 7 or 14 days. Symbol * denote study
groups with statistically significant differences (p<0.05), as using Two Way ANOVA
method. .................................................................................................................................. 97
Figure IV.4.6 - Results from ALP assays performed on seeded onto SPCL-Si scaffolds
(SPCL-Si) and SPCL-Si pre-coated with an apatite layer layer (SPCL-Si-7SBF)and, after
culture in osteogenic medium for 7 or 14 days. ....................................................................... 98
Figure IV.4.7 - SEM pictures showing the gBMSCs morphology when seeded onto SPCL-Si
(SPCL-Si) scaffolds and SPCL-Si scaffolds pre-coated with apatite (SPCL-Si-7SBF), followed
by culture in osteogenic medium for 7 or 14 days. Inset micrographs refer to scaffolds,
SPCL-Si (C2) and SPCL-Si pre-coated (C4) after 7 days in osteogenic medium. .......................... 98
- xxix Figure IV.4.8 - Assessment of calcified matrix production by gBMSCs seeded onto SPCL-Si
scaffolds (SPCL-Si) after 14 days in osteogenic medium. .......................................................... 99
Chapter V
Bilayered constructs aimed at osteochondral strategies: the influence of media supplements
in the osteo and chondrogenic differentiation of amniotic fluid-derived stem cells
Figure V.4.1 – Calcein AM stained samples of AFSCs encapsulated in agarose gels after 7,
14, and 21 days in chondrogenic and basal medium (control). .............................................. 113
Figure V.4.2 – Immunofluorescent analysis of collagen type II expression in AFSCs
encapsulated in agarose gels after 7, 14, and 21 days in chondrogenic and basal media
(control). Magnification, 200X. .............................................................................................. 114
Figure V.4.3 - Immunofluorescent analysis for aggrecan expression in AFSCs
encapsulated in agarose gels after 7, 14 and 21 days in chondrogenic or basal media
(control). Magnification, 200X. .............................................................................................. 115
Figure V.4.4 - SEM micrographs of the osteogenic layer (AFSCs seeded onto SPCL
scaffold) of the bilayered scaffolds after 7 or 14 days in OC culture media. IGF-1(+)
indicates culture medium supplemented with IGF-1 while IGF-1(-) refers to the same
culture medium without IGF-1. The basal culture medium consisted of the basic medium
currently used for AFSC expansion and maintenance, and was used as the control
medium in these studies. ...................................................................................................... 116
Figure V.4.5 - Immunofluorescence for RunX-2 expression in the osteogenic layer (AFSCs
seeded onto SPCL scaffolds) of the bilayered scaffolds after 14 days (14d) in the OC
culture media. IGF-1(+) indicates culture medium supplemented with IGF-1 while IGF-1() refers to the same culture medium without IGF-1. “Basal” refers to the basic medium
currently used for AFSC expansion and maintenance, and was used as a control in this
assay. .................................................................................................................................... 119
Figure V.4.6 - Viability of the chondrogenic layer (AFSCs in agarose gels) of the bilayered
scaffold after 7 (7d) or 14 days (14d) in the OC culture media. IGF-1(+) indicates culture
medium supplemented with IGF-1 while IGF-1 (-) refers to the same culture medium
without IGF-1. “Basal” represents the basic medium currently used for AFSC expansion
and maintenance, and was used as a control. ....................................................................... 120
Figure V.4.7 - Collagen type II expression (200x magnification) in the chondrogenic layer
(AFSCs in agarose gels) of the bilayered scaffold after 7 or 14 days in OC culture medium.
IGF-1(+) indicates culture medium supplemented with IGF-1 while IGF-1 (-) refers to the
same culture medium without IGF-1. Cultures maintained in basal medium were used as
controls. ............................................................................................................................... 121
Figure V.4.8 - Aggrecan immunofluorescence (200 x magnification) in the chondrogenic
layer (AFSCs in agarose gels) of the bilayered scaffold after 7 or 14 days in OC culture
- xxx medium. IGF-1(+) indicates culture medium supplemented with IGF-1 while IGF-1(-)
refers to the same culture medium without IGF-1. Culture in basal medium was used as
a control. .............................................................................................................................. 121
Figure V.4.9 - Safranin-O (cartilage-specific) staining of the chondrogenic layer (AFSCs in
agarose gels) of the bilayered scaffold after 14 days in OC culture medium. IGF-1(+)
indicates culture medium supplemented with IGF-1 while IGF-1 (-) refers to the same
culture medium without IGF-1. “Basal” represents the basic medium currently used for
AFSC expansion and maintenance, and was used as a control. Magnification, 200X. ............. 122
Chapter VI
Amniotic fluid stem cells versus bone marrow mesenchymal stem cells as a source for bone
tissue engineering
Figure VI.4.1– Viability assay (green) and Alizarin Red (red) staining of hAFSCs and hMSCs
cultured in 2D environment for 0, 7, 14 or 21 days in osteogenic medium. ........................... 136
Figure VI.4.2- ALP staining of hAFSCs and hMSCs cultured in 2D environment for 0, 7, 14
or 21 days in osteogenic medium. ......................................................................................... 137
Figure VI.4.3 – Calcium quantification (g/ml/well) of AFSCs and BMSCs in 2D cultures in
osteogenic medium for 0, 7, 14 or 21 days. ........................................................................... 138
Figure VI.4.4 – Viability assay of AFSCs and BMSCs seeded onto SPCL scaffolds and
cultured in osteogenic medium for 0, 7, 14 or 21 days. ......................................................... 138
Figure VI.4.5 - Scanning electron microscopy of AFSCs and BMSCs seeded onto SPCL
scaffolds in osteogenic culture for 0, 7, 14 or 21 days............................................................ 139
Figure VI.4.6 - Calcium quantification (g/ml/construct) of AFSCs- and BMSCs-SPCL
constructs in osteogenic medium for 0, 7, 14 or 21 days. ...................................................... 139
Figure VI.4.7 – Immunofluorescence of RunX2 expression in AFSCs- and BMSCs-SPCL
constructs in osteogenic medium for 0, 7, 14 or 21 days. ...................................................... 140
Figure VI.4.8 – Immunofluorescence of Collagen I expression in AFSCs- and BMSCs-SPCL
constructs in osteogenic medium for 0, 7, 14 or 21 days. ...................................................... 141
Chapter VII
Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells:
functionality in femoral defects
Figure VII.3.1 - Diagram of the implantation site: A) empty drill defects, B) defects filled
with SPCL (no cells), defects filled with cell-SPCL constructs after C) 1 day of culture and
D) 7 days of culture in osteogenic medium. Implants were placed in the same anatomical
site relative to both posterior femurs in each animal. ........................................................... 157
- xxxi Figure VII.4.1 - SEM micrographs of SPCL scaffolds seeded with gBMSCs and in vitro
cultured in osteogenic culture for 1 day (A) or 7 days (B)....................................................... 159
Figure VII.4.2 - In vitro double strand DNA concentration in SPCL scaffolds seeded with
gBMSCs cultured in osteogenic culture for 1 and 7 days. ....................................................... 160
Figure VII.4.3 - In vitro ALP activity in SPCL scaffolds seeded with gBMSCs cultured in
osteogenic culture for 1 or 7 days. ........................................................................................ 160
Figure VII.4.4 - Drill sections marked with Lévai-Laczkó staining. In A) Control 1 – empty
drill defects, B) Control 2 – defects filled with SPCL (no cells), C) defects filled with cellsSPCL constructs after C1) 1 day of culture and C2) 7 days of culture in osteogenic
medium. ............................................................................................................................... 162
Figure VII.4.5 - Drill sections marked with Xylenol Orange (red), Calcein Green (green)
and Tetracycline (not observed) fluorescence stainings. In A) Control 1 – empty drill
defects, B) Control 2 – defects filled with SPCL (no cells), C) defects filled with cell-SPCL. ...... 162
Figure VII.4.6 – New bone formation percentage in the different induced drills: A) empty
drill defects, B) defects filled with SPCL (no cells), defects filled with cell-SPCL constructs
after C) 1 day of culture and D) 7 days of culture in osteogenic medium. .............................. 163
Figure VII.4.7 - New bone roundness measured for the different induced drills: A) empty
drill defects, B) defects filled with SPCL (no cells), defects filled with cell-SPCL constructs
after C) 1 day of culture and D) 7 days of culture in osteogenic medium. .............................. 164
Chapter VIII
The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable
scaffolds in the regeneration of non-union defects
Figure VIII.4.1 - SEM micrographs of hAFSCs-SPCL constructs after 0, 7, 14 or 21 days in
osteogenic medium. ............................................................................................................. 179
Figure VIII.4.2 – Characterization of AFSCs-SPCL constructs for ALP staining after 0, 7, 14
or 21 days in osteogenic culture. ........................................................................................... 179
Figure VIII.4.3 - Characterization of AFSCs-SPCL constructs for collagen I by
immunofluorescence, after 0, 7, 14 or 21 days in osteogenic culture. ................................... 179
Figure VIII.4.4 – Characterization of AFSCs-SPCL constructs by SEM and EDS analysis.
SEM magnified images of calcium phosphate nodules at the surface and inside (inset) of
these constructs after 21 days in osteogenic medium. The presence of calcium and
phosphorus atoms was detected by EDS analysis after 0, 7, 14 and 21 days in osteogenic
culture. SPCL spectrum represents a SPCL scaffolds without seeded cells.............................. 180
Figure VIII.4.5 - Calcium content measurement in AFSCs-SPCL constructs after 0, 7, 14 or
21 days in osteogenic culture. ............................................................................................... 181
- xxxii Figure VIII.4.6 - Characterization of AFSCs-SPCL constructs by FT-IR analysis. The
presence of calcium and phosphorus groups was detected after 0, 7, 14 or 21 days in
osteogenic culture. SPCL spectrum represents a SPCL scaffolds without seeded cells. ........... 182
Figure VIII.4.7 - Picture of dissected femurs after an end point. Femur on the left
represents a femur post-implantation and on the right, the left rear femur, control. ............ 182
Figure VIII.4.8 - m-CT images obtained from all defect conditions after 4 and 16 weeks of
implantation. ........................................................................................................................ 183
Figure VIII.4.9 – Volumetric measurements of the defect section obtained from mCT
analysis using Mimics software, representing the bone neoformation area. .......................... 184
Figure VIII.4.10 – Detailed image of native bone and SCPL scaffold interface in vivo after
4 weeks showing the native bone remodeling process aiming at defect regeneration
(both pictures are 200x magnified). ...................................................................................... 185
Figure VIII.4.11 – Histometric analysis for collagen I expression (%) in the studied
conditions. Values are represented by mean  standard error of mean. Symbol * denote
study groups with statistically significant differences (p<0.05), as using Two Way ANOVA
method. ................................................................................................................................ 186
Figure VIII.4.12 – Histometric analysis for osteocalcin expression (%) in the studied
conditions. Values are represented by mean  standard error of mean. Symbol * denote
study groups with statistically significant differences (p<0.05), as using Two Way ANOVA
method. ................................................................................................................................ 187
Figure VIII.4.13 – Histometric analysis for VEGF expression (%) in the studied conditions.
Values are represented by mean  standard error of mean. .................................................. 188
Figure VIII.4.14 – Detection of blood vessel formation in H&E stained sections for all
conditions studied after 4 or 16 weeks of implantation. Pictures were 200x magnified. ........ 189
- xxxiii List of Tables
Chapter I
Current strategies for osteochondral regeneration: from stem cells to pre-clinical
approaches
Table I.2.1 - Overview of the scaffold-cells constructs that have been studied for
osteochondral tissue applications in pre-clinical models in the past 5 years. 13
Chapter V
Bilayered constructs aimed at osteochondral strategies: the influence of media supplements
in the osteo and chondrogenic differentiation of amniotic fluid-derived stem cells
Table V.4.1 - Results obtained from EDS analysis concerning atomic percentage (At %) of
several ions present in AFSCs-SPCL layer after 7 (7d) and 14 (14d) days in osteochondral
culture medium (broader analysis). 116
Table V.4.2 - Results obtained from EDS analysis concerning atomic percentage (At %) of
several ions present in AFSCs-SPCL layer after 7 (7d) and 14 (14d) days in osteochondral
culture medium. EDS analysis was performed in mineralization aggregate areas of the
constructs. 118
- xxxiv - xxxv List of Abbreviations and Symbols
2
2D: bidimensional
3
3D: tridimensional
-A-
α-amylase: alpha-amylase
α-MEM: alpha-Modification Eagle Medium
A/B: antibiotic/antimicotic
AFSCs: amniotic fluid stem cells
ALP: alkaline phosphatase
AR: alizarin red
ARS: alizarin red staining
-B-
ß-TCP: beta tricalcium phosphate
BM: bone marrow
-C-
cm2: square centimeter
Ca or Ca2+: Calcium
Calcein AM: Calcein acetoxymethyl ester
CaP: calcium phosphates
CD: cluster differentiation
Cl-: chloride ion
CaCl2: calcium chloride
CPDA-1: citrate-phosphate-dextroseadenine 1
-D-
DAPI: 4',6-diamidino-2-phenylindole
DMA: Dynamic Mechanical Analysis
DMEM: Dulbecco Modified Eagle Medium
DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
dsDNA: double-stranded DNA
-E-
ECM: extracellular matrix
EDS: energy dispersion spectroscopy
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme linked immunosorbent
assay
ES-FBS: embryonic stem cell screened FBS
Ex/Em: Excitation/Emission
-F-
FBS: foetal bovine serum
FDA: Food and Drug Administration
FTIR-ATR: Fourier transformed infra-red
spectroscopy with attenuated total
reflenctance
-G-
g: gram
gBMSCs: goat bone marrow cells
GFP: green fluorescent protein
-H-
h: hour
hAFSCs: human amniotic fluid stem cells
hBMSCs: human mesenchymal stem cells
HA: hydroxyapatite
HAc: hyaluronic acid
HCl: hydrochloric acid
HCO3
-: bicarbonate ion
HPO4
2-: orthophosphate ion
-I-
ICP: Induced-coupled plasma emission
spectroscopy
IGF-1: insuling growth factor 1
IM: intramuscular
ITS: insulin transferin selenium
-K-
kPa: kilo pascal
kV: kilo volt
kVp: peak kilovoltage
K+: potassium
KCl: potassium chloride
K2PO4
.3H2O
-M-
µA: micro Ampere
µg: microgram
µm: micrometer
mA: mili Ampere
m-CT or µ-CT: micro Computed
Tomography
- xxxvi mL: milliliter
mg: miligram
mm: milimiter
ms: milisecond
mM: milimolar
µmol/ml-1: micro mol per mL
M: molar
Mg or Mg2+: magnesium
MgCl2
.6H2O: Magnesium Chloride
Hexahydrate
MSCs: mesenchymal stem cells
MTS: (3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)
-N-
nm: nanometer
Na+: sodium
NaOH: sodium hydroxide
NaCl: sodium cloride
NaHCO3: sodium carbonate
NaSO4: sodium sulfate
-O-
OC: osteochondral
OCa: osteocalcin
OPF: oligo-polyethylene-glycol fumarate
-P-
P: phosphorus
Pa: passage
PBS: phosphate buffer saline
PCL: polycaprolactone
PGA: polyglycolic acid
PLGA: polylactic-co-glycolic acid
PLAGA: poly(lactic acid-co-glycolic acid
P/S: penicillin /streptamicine
-R-
RNA: ribonucleic acid
rpm: rotation per minute
-S-
SEM: scanning electronic microscopy
Si: silicium
Si-OH: silanol groups
SBF: simulated body fluid
SPCL: blend of corn starch with
polycaprolactone
SPCL-Si: blend of corn starch with
polycaprolactone with funcionalized silanol
groups (with a silicate precipitation
solution)
SO4
2-: sulphate
-T-
TCPs: tricalcium phosphates
TE: tissue engineering
TF-XRD: Thin-film X-ray diffraction
TGF-ß1: transforming growth factor beta 1
(CH2OH)3CNH3: tris-hydroxymethylaminomethane
-U-
UI or U.I.: International Units
UK: United Kingdom
US: United States
USA: United States of America
-X-
XRD: X-ray diffraction
-W-
WFIRM: Wake Forest Institute and
Regenerative Medicine
wt/v: weight/volume
1
SECTION I
GENERAL INTRODUCTION
2
3
Chapter I
CURRENT STRATEGIES FOR OSTEOCHONDRAL REGENERATION:
FROM STEM CELLS TO PRE-CLINICAL APPROACHES
This chapter is based on the following publication:
Rodrigues MT, Gomes ME, Reis RL, Current strategies for osteochondral regeneration:
from stem cells to pre-clinical approaches. Current Opinion in Biotechnology 22: 1-8 invited review paper (2011).
4
- 5 -
Chapter I
CURRENT STRATEGIES FOR OSTEOCHONDRAL REGENERATION:
FROM STEM CELLS TO PRE-CLINICAL APPROACHES
I.1. Abstract
Damaged cartilage tissue has no functional replacement alternatives and current therapies
for bone injury treatment are far from being the ideal solutions emphasizing an urgent need
for alternative therapeutic approaches for osteochondral regeneration.
The tissue engineering field provides new possibilities for therapeutics and regeneration in
rheumatology and orthopaedics, holding the potential for improving the quality of life of
millions of patients by exploring new strategies towards the development of biological
substitutes to maintain, repair and improve osteochondral tissue function. Numerous studies
have focused on the development of distinct tissue engineering strategies that could result in
promising solutions for this delicate interface. In order to outperform currently used methods,
novel tissue engineering approaches propose, for example, the design of multi-layered
scaffolds, the use of stem cells, bioreactors or the combination of clinical techniques.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 6 -
I.2. Introduction
Osteochondral (OC) interfaces are part of the joint, being a specialized and integrated
structure consisting of multiple connective tissue elements, including muscles, tendons,
ligaments, synovium, cartilage, and bone, organized to permit stability and movement of the
human skeleton.
OC injuries can lead to joint malfunction and ultimately to the development of degenerative
diseases such osteoarthritis. With an increasing aging population, OA represents a significant
socio-economical burden worldwide. Although several procedures are available on the clinical
market, an ideal solution has yet to be found in order to fulfill all necessary requirements for a
long term successful regenerative approach.
This paper is aimed at reviewing distinct strategies aiming at a successful OC regeneration,
involving cells, scaffolds, bioreactors or a combination of these elements. The rationale for
currently used techniques as well as some promising studies in animal models will be also
discussed in this review in order to highlight the state-of-the-art in OC over the past few years
(Figure I.2.1).
Figure I.2.1 – Clinical and Tissue Engineering strategies to promote the regeneration of
osteochondral defects.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 7 -
One of the most challenging goals in bone and cartilage tissue engineering (TE) is the
creation of an engineered OC interface to repair damaged areas. Similarly to the natural
milieu, an engineered interface should distribute everyday mechanical stresses with lowfriction load bearing, while interacting with different structural and biological needs in a stable
environment. This is particularly more demanding and unique if one considers the distinctive
requirements of bone and cartilage tissues as well as the several OC systems found in the
human body, dependent on their location and functionality.
Several materials, shapes, stiffness and chemical compositions were described for bone[18] and cartilage scaffolds[8-21], considering the relevance of scaffold architecture to sustain
the mechanical stresses of the joint as well as to guide the cells into the desired phenotype,
and promoting a complete integration of the OC system in order to restore tissue functionality.
The selection of cells plays also an important element in this delicate interface headed for
engineered grafts. Several potential cells sources were successfully described for bone[22-26],
and cartilage[17, 22-27], which are likely to be useful for OC strategies[8].
The subsequent step towards the clinical application is the up-scale and custom made
production of the OC implants to fit perfectly to the injured area and to provide the biological
and structural needs required to restore tissue function. In order to automate and make the
system cost-effective, several bioreactor models[28, 29] were designed and have been
showing promising results.
I.2.1. Osteochondral defects (OCD)
Most OC lesions or defects (OCD) and OC injury-associated diseases lead to loss of integrity
or stability at the articular surface with resultant decrease range of motion of the involved
joint, and, ultimately, premature osteoarthritis[30]. Although OCDs occur as a result of
repetitive trauma within the joint, several factors, such as ischemia, genetics, abnormal
vasculature, and metabolic disorders are associated to body processes leading to loss of
cartilage[31] or to relevant changes in the architecture or composition of the bone[32].
Furthermore, joint healing is strongly dependent on age, as age is the strongest known risk
factor for development of OA[33] and depth of injury is also age associated[34]. Aged cartilage
also induces changes in chondrocyte function and material properties, and responds
differently to cytokines and growth factors[33].
The location of a particular defect[35] does influence repair response of the cartilage as
well as the mechanical alignment of the joint[36]. OC lesions are identified most frequently in
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 8 -
the femoral condyles[37], capitellum of the elbow[38], dome of the talus[30], and the dorsal
surface of the patella[37].
I.2.2. Weight bearing influence in biomechanics of the joint
Homeostasis of articular cartilage depends on mechanical loads generated during daily
activity. Some joint areas are particularly more affected by weight pressure than others, which
may progress to a more degenerative and diffuse joint involvement that translates to the
patient by causing pain, swelling, clicking, and instability. Ultimately, inappropriate joint loads
are associated to focal stress and result in focal degeneration of cartilage, as occurs in OA, and
increase the stress on subchondral bone. Changes in pressure and shear stress induced by joint
movement may induce changes in matrix protein expression and in the release of nitric oxide
associated to joint pathogenesis[39].
The stress may also vary throughout the cartilage on a joint surface, because loading is not
completely uniform, leading to gradients in stress and pressure[40]. This effect is evident in
most patients, where the surface of the joints does not conform perfectly under loading and
may result in an increased risk for OA progression[33].
I.2.3. Current treatments in clinical field
Currently available treatments depend upon the size of the OC defect and the condition of
the overlying cartilage. Using reparative surgery, cartilage treatments include arthroscopic
debridement, abrasion arthroplasty and microfracture. These procedures stimulate the body
to heal the injury, mainly resulting in the formation of fibrocartilage[41]. Fibrocartilage is a scar
tissue presenting diminished resilience, reduced stiffness, and poor wear characteristics when
compared to hyaline cartilage. Thus, fibrocartilage is unlikely to withstand physiological
loading and cannot guarantee to function successfully in long term. Nevertheless, other
options are available with restorative surgery, namely, autografts recurring to mosaicoplasty
procedures, allografts[42, 43] and biologic replacement using cultured autologous
chondrocytes[44, 45]. The biggest challenge with autografts is to achieve a final round shape
that mimics the surface of the articular joints. Allografts procedure is similar to autografts[46]
and mostly used after other surgeries have failed. It is not recommended for patients with OA,
and the limited supply of donor tissue is a major problem of this practice.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 9 -
Autologous chondrocyte transplantation/implantation[44] has also been described to help
restoring the structural make up of the articular cartilage. The intermediate and long-term
functional and clinical results are promising, especially regarding the durability of the repair in
human patients follow up[45].
More recently[47], tissues from the covering of bone and cartilage are implanted into the
lesion through periosteal and perichondral grafting to promote the repair and functionality of
cartilage.
Despite the availability of procedures, all current treatment options inflict further tissue
destruction before any therapeutic effect can be achieved.
I.2.4. TE strategies to improve available treatments
I.2.4.1. Cells to promote healing
Despite current knowledge in OC field, the selection of a cell source to promote efficient OC
regeneration is a major issue that must to be considered. Ideally, a cell source should enable
insignificant donor morbidity or tissue scarcity, resurface joints with cartilage, have no
limitations in the amounts available and be easy to maintain/expand in vitro, be readily
available, have no issues of immunogenicity or diseases transmission risks and be of low cost.
Tissue insufficient supply and morbidity, and host immune responses and disease
transmission risks limit condrocyte and osteoblast as ideal cells in OC strategies. Among adult
stem cells, bone marrow mesenchymal stem cells (BMSCs)[15, 16, 19, 20, 22 ,48-51] and
adipose-derived stem cells (ASCs)[9, 12, 26, 48, 50, 52] are the most investigated.
Nevertheless, some studies described a higher chondro-[50] and osteogenic[48] potential of
BMSCs when compared to ASCs. The effectiveness of autologous BMSCs transplantation for
the repair of full-thickness articular cartilage defects was assessed in patellae lesions of two
human patients[53]. A similar approach was also considered to repair full thickness femoral
condyle defect in an athlete, who had reattained his previous activity level and experienced
neither pain nor other complications[27].
Other cell sources, including synovial tissue and periosteum-derived stem cells have also
showed potential for osteo- and chondro-genic differentiation[24, 54]. Cells from synovial
membrane are harvested with minimal complications at the donor site due to a high selfregenerative capability[24] of synovial tissue. The periosteum is a specialized fibrous tissue
composed of fibroblast, osteoblast, and progenitor cells that may also be a possible cell source
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 10 -
for OC TE based on its accessibility, rapid proliferation and differentiation potential[54].
Furthermore, after skeletal surgery procedures, periosteum is often used as a covering layer
over tissue to stimulate local regeneration. Despite the potential, periosteum-derived cells
should be more investigated for cellular therapies[55].
Umbilical cord stem cells (UCSCs) together with amniotic fluid derived stem cells (AFSCs)
were also introduced to cartilage and bone TE [8, 23, 25] presenting interesting characteristics,
since they are easier to obtain and represent an almost unlimited stem cell sources. Some risks
were associated to human AFSCs harvesting but, as pregnant women are older than ever
before, amniocentesis is likely to become a routine procedure in future years. More recently,
cells from human fetal membranes and placenta, with similar features to human UCMSCs and
AFSCs, have also been successfully differentiated into osteogenic and chondrogenic
lineages[56]. Although embryonic stem cells (ES)[57] hoped for a promising future in
regenerative medicine, its use is still ethically controversial and has major ethical
considerations associated. Notwithstanding that human ES cells express molecules which could
cause immune rejection[57] and present a high genomic instability[57], ES cells transplantation
in a collagen gel have shown to induce the formation of cartilage tissue[17] under mechanical
condition in rats aiming at OC regeneration.
More recently, iPS technology, where iPS cells are generated by reprogramming of somatic
cells through the exogenous expression of transcription factors, holds great promise for
regenerative medicine in autologous cell replacement therapies and in genetic defects by
restoring cellular function[58]. Nevertheless and due to iPS recent development, cell
characterization and in vivo functionality are to be addressed in bone and cartilage fields.
Stem cells obtained from different sources are likely to enable the most successful
outcomes in OC regenerative approaches. Besides intrinsic characteristics of stem cells from a
particular source, other factors should be monitored aiming at a successful strategy; such as
final application, patient age, defect location and damage size. Cell culture media to induce
chondrogenesis and osteogenesis of undifferentiated cells or maintain and proliferate primary
chondrocytes and osteoblasts in an ex vivo atmosphere is commercially available. However, a
common osteochondrocytic medium to co-culture or simultaneously differentiate bone and
cartilage cells was not fully established yet, although some attempts have been described [8,
15, 59]. This approach can be advantageous to simplify cell culturing procedures and,
simultaneously, reduce the time and production costs of an engineered graft towards a clinical
scenario.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 11 -
I.2.4.2. Biomaterials: human designs to mimic natural extracellular material
The implantation of cells in the afflicted area could be a direct approach in OC strategies,
but the request for a support material to promote regeneration, especially in large sized
defects, is to be critically considered. This idea is inspired in nature itself as, in the body, the
majority of cells subsist in a 3D world, anchored onto a network of extracellular matrix (ECM),
which scaffolding design proposes to recreate.
Scaffold characteristics will greatly influence cells and should mimic the complex and
demanding environment to which cells are exposed to. Besides the tissue structural support
and stimulation, either chemically or mechanically, the optimal scaffold should assist tissue
functionality promoting the easy diffusion of nutrients, growth factors and cellular waste
products[60]. Additionally, the ideal scaffold should be biocompatible and its biodegradability
adjustable to the time required for tissue regeneration[60].
In the last few years, thousands of scaffolds have been proposed for reparative strategies
made from different materials and production methodologies, with varying properties and
composition. An OC scaffold should combine the better of the two worlds in a functional and
integrated system. Lots of effort has been undertaken to achieve this goal and the most
common approach is an independent cartilage or bone strategy, likely because chondrocytes
and bone cells present different function-related characteristics including metabolic and
structural features, yet communicating and interacting, in a unique culturing system.
Natural based polymers such as agarose[15, 61], starch[9], chitosan[9, 13, 14, 62], silk[14],
gellan gum[12], hyaluronic acid[16], collagen[17, 63] or blends of these materials[9, 14, 18,
21], and synthetic materials such as polylactic acid (PLA)[8], polycaprolactone (PCL)[20] and
oligo-polyethylene-glycol fumarate[49] have been proposed for cartilage applications. Most of
these materials are processed into hydrogel and gel based matrices, which hold particular
relevance for cartilage strategies because of their high water content, tissue-like elastic
properties and the ability to encapsulate cells[64]. Also, gel structures partially tolerate shock
absorption and deformation mimicking articular cartilage characteristics.
However, cartilage repair in OC interfaces should be accompanied by an adequate
restoration of the underlying subchondral bone, enhancing the in locus integration of the OC
system.
The minerals and the collagen fibers in the matrix are responsible for bone hardness and
resistance. Nevertheless, the constant remodeling makes bone very plastic and capable of
internal structural changes according to the stresses it is subjected to. Thus, bone regeneration
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 12 -
requires scaffolds with high mechanical and osteoconductive properties, and structurally
strong enough to sustain weight bearing loads and avoid cartilage calcification, which leads to
tissue malfunction and death. Scaffolds should also be biodegradable to keep up with the
natural bone remodeling process. Despite the brittle behaviour and low tensile strength,
inappropriate for significant torsion areas such as long bones, hydroxyapatite (HA) and
tricalcium phosphate (TCP) are the most studied ceramics due to their osteoconductive and
high mechanical properties, and are already used in some clinical aplications[4,6]. Other
materials, including silk[2, 7] , PCL and PCL blends[1, 3, 5], and PLA[8], have also been
effectively tested as delivery systems[2] or artificial ECM[8], mimicking and recreating in some
extend the structural organization of bone[1, 3, 7].
Some OC approaches successfully evaluated the in vivo application of scaffolds made of
collagen fibrils with HA nanoparticles without implanted cells[65], which can be of particular
importance if one considers the practical and commercial standpoint, as the engineered
product could be a ready-to-use graft for surgery procedures. Furthermore, this approach
would avoid tissue morbidity and scarcity of autologous cell sources or even immune reactions
from allogenic sources and problems related to cell culturing methodologies (e.g., animal
origin supplements).
Other strategies focus on the cellular interactions of implanted cells in the tissue
surroundings, considering the reduced metabolism of cartilage. Chondrocytes in adult
individuals do not divide or establish cell-to-cell contacts but are responsible to produce
cartilage dense ECM[34], thus maintaining cartilage integrity.
Especially in elder patients, implanted cells could meliorate the native ECM properties, and
improve the functionality of damaged tissue by stimulating fresh ECM production. In bony
defects, the integration of cells in the implant may stimulate bone marrow cells and establish a
metabolic balance favoring the neobone formation. Furthermore, in critical sized defects, cells
are likely to participate in a molecular communication level bridging the native tissues to the
implant towards a successful OC regeneration.
Different approaches to design an OC scaffold including hydrogels[49], combination of two
distinct layers[21, 29, 62, 63] or a gradient scaffold[65], usually an association of a gel or a
foam and a ceramic, have been developed as alternatives to this problem (Table I.2.1).
These complex scaffolds favor the integration into the native tissue after implantation and
guide the cells, into the desired phenotype, according to the prearranged environment created
from scaffold physical and chemical properties.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 13 -
Table I.2.1 - Overview of the scaffold-cells constructs that have been studied for osteochondral
tissue applications in pre-clinical models in the past 5 years.
Scaffolds Cells ref.
OPF with gelatin microparticle hydrogel Cell-free / marrow mesenchymal stem cells [49]
PCL/TCP-PCL scaffold Cell-free / marrow mesenchymal stromal cells [20]
Hyaluronic acid gel sponges Autologous mesenchymal stromal cells [16]
Hyaluronate-type I collagen-fibrin
scaffold
Cell-free /autologous chondrocytes [18]
Hyaluronic acid-atelocollagen/ß-TCP
bilayered scaffold
Cell free / chondrocytes [21]
Collagen/HA gradient scaffold Cell free / autologous chondrocytes [65,66]
Poly(lactide-co-glycolide)/nano-HA
scaffold
Cell free / marrow mesenchymal stem cells [51]
Polylactic acid (PLA)- coated polyglycolic
acid (PGA) scaffold
Cell free / Autologous marrow mesenchymal
stem cells
[19]
Collagen /-TCP bilayered scaffold Cell free [63]
More recently, emerging approaches include the incorporation of bone and/or cartilage
growth factors in scaffolds[49, 63] to stimulate native tissue formation and differentiation in
vivo. The inclusion of growth factors can ultimately recruit host cells into the damaged site,
initiating a healing pathway, which could be promising for the treatment of OCDs.
I.2.4.3. Assisted Devices: bioreactor systems
The limited diffusion in static culture environments may constrain tissue ingrowth in
engineered scaffolds. Bioreactors are usually designed to control the transport of nutrients
and oxygen to cells in constructs promoting cellular expansion, and in some cases, enabling
mechanical stimulation of cultured cells, thus enhancing cell differentiation and ECM
formation.
The challenge is, once again, finding a compromise considering the different intrinsic
properties of cartilage and bone tissues. In a bioreactor system, dynamic compression should
be applied for cartilage ECM stimulation while, for bone, medium perfusion is required to
control mass transport and provide shear-stress to stimulate neobone formation. To overcome
this issue, studies have focused on the development of double chamber bioreactors with
physical separation; described to fulfil the needs of tissue-specific mechanical forces for OC
stimulation[28, 29].
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 14 -
The next step, barely explored, would be the automation of bioreactors controlled by
computer software. The customization of engineered grafts through the development of
anatomically moulded surfaces[61] have showed potential results headed to translational OC
interfaces. As follows bioreactors would be a reliable system of automation and
standardization of cell and scaffold methodologies reducing the time and production costs of
functional custom-designed grafts.
I.2.5. In vivo models for osteochondral tissue engineering
Animal studies still represent an essential tool to understand the biologic behavior of
healing and tissue regeneration in vivo, though differences in the anatomy and metabolism of
animal models must be considered in an experimental setup with human correlations.
Different animal models have been used in OC studies [16, 18-21, 49, 51, 63, 65]. Rats
present distinctive characteristics, such as athymic nude or transgenic animals, not easily
available in larger animal models. This model has been used to test the efficacy of a
poly(lactide-co-glycolide)/nano-HA scaffold seeded with undifferentiated mesenchymal stem
cells in OC defects[51]. After 12 weeks, defects treated with these constructs showed smooth
and hyaline cartilage with abundant glycosaminoglycan and collagen type II deposition.
Rabbit also demonstrated to be a successful model for OC [16, 20, 21, 49], especially in
femoral regions with the successful application of hyaluronate-atelocollagen/beta-TCPhydroxyapatite scaffolds in the patellar grove[21], which promoted, in some extent, OC
regeneration without the formation of fibrocartilage.
Sheep is also a popular animal model due to their weight-bearing limbs and with metabolic
and bone remodelling rates similar to that of humans as well as the sequence of events in
bone graft incorporation and healing capacities. An OC interface was evaluated in sheep by the
implantation of a composite scaffold of collagen and HA with or without autologous
chondrocytes into a condyle critical defect[65]. Both conditions showed to support neobone
formation and hyaline-like cartilage regeneration. With a similar implant, collagen/TCP, OC
regeneration was evaluated in the trochlear groove of minipigs[63]. Although cells were
absence in this strategy, the incorporation in situ of growth factors in the construct leads to
fibrocartilage formation and partial reconstruction of the subchondral bone integrity in a short
term follow up.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 15 -
A pilot clinical trial with 13 patients using the collagen/HA cell free tri-layer scaffold[66]
mentioned above, indicated promising results with tissue recovery in some extent after a sixmonth follow up.
I.3. Conclusions
The currently available treatments based on “damage to heal approaches”, have a limited
success. With an increasing aging population, tissue engineering strategies provide important
cues and hope for the treatment of OC degeneration. Ultimately, the tissue engineered
implant should be able to stimulate and replace old tissue and native lethargic cells in order to
accomplish both regeneration and restoring function for a successful clinical achievement.
The challenge stands for the replication of the natural functional architecture and the
translation of promising strategies towards patients needs. Success lies on the delicate balance
of cartilage and bone characteristics combined in an engineered graft, and its integration in
vivo. The implant must participate in the regenerative process, considering the specific
properties of each OC interface, which can only be achieved through the design of scaffold
materials accommodating the specific characteristics of bone and cartilage tissues, and
providing stem cells with the necessary cues to satisfy both tissue cellular needs. The
application of cells in critical defects or elder patient injuries is likely to be beneficial in
stimulating native cells into the regenerative process. The use of bioreactors can improve the
functionality of such constructs, accelerate the production, create custom-made systems, and
reduce time costs for obtaining implants for OC applications.
I.4. References
1. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5: 41-49.
2. Bessa PC, Balmayor ER, Hartinger J, Zanoni G, Dopler D, Meinl A, Banerjee A, Casal M, Redl
H, Reis RL, et al.: Silk fibroin microparticles as carriers for delivery of human recombinant bone
morphogenetic protein-2: in vitro and in vivo bioactivity. Tissue Eng Part C Methods 2010, 16:
937-945.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 16 -
3. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2: 243-252.
4. Ogose A, Hotta T, Kawashima H, Kondo N, Gu W, Kamura T, Endo N: Comparison of
hydroxyapatite and beta tricalcium phosphate as bone substitutes after excision of bone
tumors. J Biomed Mater Res B Appl Biomater 2005, 72: 94-101.
5. Leonor I, Rodrigues MT, Gomes ME, Reis RL: In Situ Functionalization of Wet-Spun Fibre
meshes for Bone Tissue Engineering: One Step Approach. J Tissue Eng Regen Med 2011, 5:
104-111.
6. Muehrcke DD, Shimp WM, Aponte-Lopez R: Calcium phosphate cements improve bone
density when used in osteoporotic sternums. Ann Thorac Surg 2009, 88: 1658-1661.
7. Kim HJ, Kim UJ, Kim HS, Li C, Wada M, Leisk GG, Kaplan DL: Bone tissue engineering with
premineralized silk scaffolds. Bone 2008, 42: 1226-1234.
8. Wang L, Zhao L, Detamore MS: Human umbilical cord mesenchymal stromal cells in a
sandwich approach for osteochondral tissue engineering. J Tissue Eng Regen Med 2011. doi:
10.1002/term.370
9. Sa-Lima H, Caridade SG, Mano JF, Reis RL: Stimuli-responsive chitosan-starch injectable
hydrogels combined with encapsulated adipose-derived stromal cells for articular cartilage
regeneration. Soft Matter 2010, 6: 5184-5195.
10. Oliveira JT, Reis RL: Polysaccharide-based materials for cartilage tissue engineering
applications. J Tissue Eng Regen Med 2011, 5(6): 421-236.
11. Maher SA, Mauck RL, Rackwitz L, Tuan RS: A nanofibrous cell-seeded hydrogel promotes
integration in a cartilage gap model. J Tissue Eng Regen Med 2009, 4: 25-29.
12. Oliveira JT, Gardel LS, Rada T, Martins L, Gomes ME, Reis RL: Injectable gellan gum
hydrogels with autologous cells for the treatment of rabbit articular cartilage defects. J Orthop
Res 2010, 28: 1193-1199.
13. Malafaya PB, Oliveira JT, Reis RL: The effect of insulin-loaded chitosan particle-aggregated
scaffolds in chondrogenic differentiation. Tissue Eng Part A 2010, 16: 735-747.
14. Silva SS, Motta A, Rodrigues MT, Pinheiro AF, Gomes ME, Mano JF, Reis RL, Migliaresi C:
Novel genipin-cross-linked chitosan/silk fibroin sponges for cartilage engineering strategies.
Biomacromolecules 2008, 9: 2764-2774.
15. Grayson WL, Bhumiratana S, Grace Chao PH, Hung CT, Vunjak-Novakovic G: Spatial
regulation of human mesenchymal stem cell differentiation in engineered osteochondral
constructs: effects of pre-differentiation, soluble factors and medium perfusion. Osteoarthritis
Cartilage 2010, 18: 714-723.
16. Kayakabe M, Tsutsumi S, Watanabe H, Kato Y, Takagishi K: Transplantation of autologous
rabbit BM-derived mesenchymal stromal cells embedded in hyaluronic acid gel sponge into
osteochondral defects of the knee. Cytotherapy 2006, 8: 343-353.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 17 -
17. Nakajima M, Wakitani S, Harada Y, Tanigami A, Tomita N: In vivo mechanical condition
plays an important role for appearance of cartilage tissue in ES cell transplanted joint. J Orthop
Res 2008, 26: 10-17.
18. Filova E, Rampichova M, Handl M, Lytvynets A, Halouzka R, Usvald D, Hlucilova J, Prochazka
R, Dezortova M, Rolencova E, et al.: Composite hyaluronate-type I collagen-fibrin scaffold in
the therapy of osteochondral defects in miniature pigs. Physiol Res 2007, 56 Suppl 1: S5-S16.
19. Zhou G, Liu W, Cui L, Wang X, Liu T, Cao Y: Repair of porcine articular osteochondral defects
in non-weightbearing areas with autologous bone marrow stromal cells. Tissue Eng 2006, 12:
3209-3221.
20. Shao X, Goh JC, Hutmacher DW, Lee EH, Zigang G: Repair of large articular osteochondral
defects using hybrid scaffolds and bone marrow-derived mesenchymal stem cells in a rabbit
model. Tissue Eng 2006, 12: 1539-1551.
21. Ahn JH, Lee TH, Oh JS, Kim SY, Kim HJ, Park IK, Choi BS, Im GI: Novel hyaluronateatelocollagen/beta-TCP-hydroxyapatite biphasic scaffold for the repair of osteochondral
defects in rabbits. Tissue Eng Part A 2009, 15: 2595-2604.
22. Caplan AI: Adult mesenchymal stem cells for tissue engineering versus regenerative
medicine. J Cell Physiol 2007, 213: 341-347.
23. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC,
Snyder EY, Yoo JJ, et al.: Isolation of amniotic stem cell lines with potential for therapy. Nat
Biotechnol 2007, 25: 100-106.
24. Fan J, Varshney RR, Ren L, Cai D, Wang DA: Synovium-derived mesenchymal stem cells: a
new cell source for musculoskeletal regeneration. Tissue Eng Part B Rev 2009, 15: 75-86.
25. Arien-Zakay H, Lazarovici P, Nagler A: Tissue regeneration potential in human umbilical
cord blood. Best Pract Res Clin Haematol 2010, 23: 291-303.
26. Rada T, Reis RL, Gomes ME: Adipose tissue-derived stem cells and their application in bone
and cartilage tissue engineering. Tissue Eng Part B Rev 2009, 15: 113-125.
27. Kuroda R, Ishida K, Matsumoto T, Akisue T, Fujioka H, Mizuno K, Ohgushi H, Wakitani S,
Kurosaka M: Treatment of a full-thickness articular cartilage defect in the femoral condyle of
an athlete with autologous bone-marrow stromal cells. Osteoarthritis Cartilage 2007, 15: 226231.
28. Malafaya PB, Reis RL: Bilayered chitosan-based scaffolds for osteochondral tissue
engineering: influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity
studies in a specific double-chamber bioreactor. Acta Biomater 2009, 5: 644-660.
29. Chang CH, Lin FH, Lin CC, Chou CH, Liu HC: Cartilage tissue engineering on the surface of a
novel gelatin-calcium-phosphate biphasic scaffold in a double-chamber bioreactor. J Biomed
Mater Res B Appl Biomater 2004, 71: 313-321.
30. Naran KN, Zoga AC: Osteochondral lesions about the ankle. Radiol Clin North Am 2008, 46:
995-1002
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 18 -
31. Hunter DJ: Risk stratification for knee osteoarthritis progression: a narrative review.
Osteoarthritis Cartilage 2009, 17: 1402-1407.
32. Li B, Aspden RM: Composition and mechanical properties of cancellous bone from the
femoral head of patients with osteoporosis or osteoarthritis. J Bone Miner Res 1997, 12: 641651.
33. Sharma L, Kapoor D, Issa S: Epidemiology of osteoarthritis: an update. Curr Opin Rheumatol
2006, 18: 147-156.
34. Bhosale AM, Richardson JB: Articular cartilage: structure, injuries and review of
management. Br Med Bull 2008, 87: 77-95.
35. Kuettner KE, Cole AA: Cartilage degeneration in different human joints. Osteoarthritis
Cartilage 2005, 13: 93-103.
36. Moyer RF, Birmingham TB, Chesworth BM, Kean CO, Giffin JR: Alignment, body mass and
their interaction on dynamic knee joint load in patients with knee osteoarthritis. Osteoarthritis
and Cartilage 2010, 18: 888-893.
37. Peters TA, McLean ID: Osteochondritis dissecans of the patellofemoral joint. Am J Sports
Med 2000, 28: 63-67.
38. Baker CL, 3rd, Romeo AA, Baker CL, Jr.: Osteochondritis Dissecans of the Capitellum. Am J
Sports Med 2010, 38(9): 1917-1928.
39. Smith RL, Carter DR, Schurman DJ: Pressure and shear differentially alter human articular
chondrocyte metabolism: a review. Clin Orthop Relat Res 2004: S89-95.
40. Elder BD, Athanasiou KA: Hydrostatic pressure in articular cartilage tissue engineering:
from chondrocytes to tissue regeneration. Tissue Eng Part B Rev 2009, 15: 43-53.
41. Beris AE, Lykissas MG, Papageorgiou CD, Georgoulis AD: Advances in articular cartilage
repair. Injury 2005, 36 Suppl 4: S14-23.
42. Lattermann C, Romine SE: Osteochondral allografts: state of the art. Clin Sports Med 2009,
28: 285-301.
43. Gross AE, Shasha N, Aubin P: Long-term followup of the use of fresh osteochondral
allografts for posttraumatic knee defects. Clin Orthop Relat Res 2005: 79-87.
44. Gikas PD, Bayliss L, Bentley G, Briggs TW: An overview of autologous chondrocyte
implantation. J Bone Joint Surg Br 2009, 91: 997-1006.
45. Peterson L, Brittberg M, Kiviranta I, Akerlund EL, Lindahl A: Autologous chondrocyte
transplantation. Biomechanics and long-term durability. Am J Sports Med 2002, 30: 2-12.
46. Hangody L, Vasarhelyi G, Hangody LR, Sukosd Z, Tibay G, Bartha L, Bodo G: Autologous
osteochondral grafting--technique and long-term results. Injury 2008, 39 Suppl 1: S32-39.
47. Ulutas K, Menderes A, Karaca C, Ozkal S: Repair of cartilage defects with periosteal grafts.
Br J Plast Surg 2005, 58: 65-72.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 19 -
48. Niemeyer P, Fechner K, Milz S, Richter W, Suedkamp NP, Mehlhorn AT, Pearce S, Kasten P:
Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone
regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich
plasma. Biomaterials 2010, 31: 3572-3579.
49. Guo X, Park H, Young S, Kretlow JD, van den Beucken JJ, Baggett LS, Tabata Y, Kasper FK,
Mikos AG, Jansen JA: Repair of osteochondral defects with biodegradable hydrogel composites
encapsulating marrow mesenchymal stem cells in a rabbit model. Acta Biomater 2010, 6: 3947.
50. Im GI, Shin YW, Lee KB: Do adipose tissue-derived mesenchymal stem cells have the same
osteogenic and chondrogenic potential as bone marrow-derived cells? Osteoarthritis Cartilage
2005, 13: 845-853.
51. Xue D, Zheng Q, Zong C, Li Q, Li H, Qian S, Zhang B, Yu L, Pan Z: Osteochondral repair using
porous poly(lactide-co-glycolide)/nano-hydroxyapatite hybrid scaffolds with undifferentiated
mesenchymal stem cells in a rat model. J Biomed Mater Res A 2010, 94: 259-270.
52. Mizuno H: Adipose-derived stem cells for tissue repair and regeneration: ten years of
research and a literature review. J Nippon Med Sch 2009, 76: 56-66.
53. Wakitani S, Mitsuoka T, Nakamura N, Toritsuka Y, Nakamura Y, Horibe S: Autologous bone
marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in
human patellae: two case reports. Cell Transplant 2004, 13: 595-600.
54. Ringe J, Leinhase I, Stich S, Loch A, Neumann K, Haisch A, Haupl T, Manz R, Kaps C, Sittinger
M: Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue
engineering. J Tissue Eng Regen Med 2008, 2: 136-146.
55. Jansen EJ, Emans PJ, Guldemond NA, van Rhijn LW, Welting TJ, Bulstra SK, Kuijer R: Human
periosteum-derived cells from elderly patients as a source for cartilage tissue engineering? J
Tissue Eng Regen Med 2008, 2: 331-339.
56. Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A, Wengler GS, Parolini O:
Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue
Eng Regen Med 2007, 1: 296-305.
57. Stojkovic M, Lako M, Strachan T, Murdoch A: Derivation, growth and applications of human
embryonic stem cells. Reproduction 2004, 128: 259-267.
58. Kiskinis E, Eggan K: Progress toward the clinical application of patient-specific pluripotent
stem cells. J Clin Invest 2010, 120: 51-59.
59. Li J, Mareddy S, Tan DM, Crawford R, Long X, Miao X, Xiao Y: A minimal common
osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation
of bone marrow stromal cells in the construction of osteochondral graft. Tissue Eng Part A
2009, 15: 2481-2490.
60. Hutmacher DW, Schantz JT, Lam CX, Tan KC, Lim TC: State of the art and future directions
of scaffold-based bone engineering from a biomaterials perspective. J Tissue Eng Regen Med
2007, 1: 245-260.
Chapter I – Current strategies for osteochondral regeneration: from stem cells to pre-clinical approaches
- 20 -
61. Hung CT, Lima EG, Mauck RL, Takai E, LeRoux MA, Lu HH, Stark RG, Guo XE, Ateshian GA:
Anatomically shaped osteochondral constructs for articular cartilage repair. J Biomech 2003,
36: 1853-1864.
62. Oliveira JM, Rodrigues MT, Silva SS, Malafaya PB, Gomes ME, Viegas CA, Dias IR, Azevedo
JT, Mano JF, Reis RL: Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral
tissue-engineering applications: Scaffold design and its performance when seeded with goat
bone marrow stromal cells. Biomaterials 2006, 27: 6123-6137.
63. Gotterbarm T, Richter W, Jung M, Berardi Vilei S, Mainil-Varlet P, Yamashita T, Breusch SJ:
An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium
phosphate in deep osteochondral defects. Biomaterials 2006, 27: 3387-3395.
64. Nicodemus GD, Bryant SJ: Cell encapsulation in biodegradable hydrogels for tissue
engineering applications. Tissue Eng Part B Rev 2008, 14: 149-165.
65. Kon E, Delcogliano M, Filardo G, Fini M, Giavaresi G, Francioli S, Martin I, Pressato D,
Arcangeli E, Quarto R, et al.: Orderly osteochondral regeneration in a sheep model using a
novel nano-composite multilayered biomaterial. J Orthop Res 2010, 28: 116-124.
66. Kon E, Delcogliano M, Filardo G, Pressato D, Busacca M, Grigolo B, Desando G, Marcacci M:
A novel nano-composite multi-layered biomaterial for treatment of osteochondral lesions:
technique note and an early stability pilot clinical trial. Injury 2010, 41: 693-701.
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SECTION II
MATERIALS AND METHODS
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- 23 -
Chapter II
MATERIALS & METHODS
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Chapter II
MATERIALS AND METHODS
II.1. Abstract
This chapter describes all experimental methods and techniques as well as biomaterials and
cell protocols related to the scientific studies performed under the scope of this PhD Thesis.
Despite the fact that methodologies to be addressed in this chapter are succinctly
described in each of the following experimental chapters, herein are provided fully detailed
procedures complemented with the rationale for the selection of the materials and cells.
II.2. Materials
II.2.1. Polycaprolactone
Polycaprolactone (PCL) can be synthetized by a ring opening polymerization of a cyclic
lactone monomer such as ε-caprolactone (Figure II.2.1), resulting in a semicrystalline polymer
with a melting point of 59-64 ºC and a glass-transition temperature of -60 ºC[1].
Figure II.2.1 – Ring opening polymerization of ε-caprolactone to polycaprolactone.
Synthetic polymers, such as PCL, are typically more versatile in tailoring a wide range of
properties and structure features. Also, they represent a reliable source of raw materials,
degraded by hydrolysis of its ester linkages in physiological conditions[2, 3], and avoid
immunogenicity problems[4, 5]. Although not produced from renewable raw materials, PCL is
Chapter II – Materials and Methods
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fully biodegradable, with a slow degradation rate[1]. PCL also forms compatible blends and
copolymers with a wide range of polymers producing materials with unique elastomer
properties[4]. For all of these reasons, PCL is used to produce Food and Drug Administration
(FDA) approved medical devices, and widely studied for several tissue engineering (TE)
approaches and for other biomedical applications[6-10] including long term implantable
devices.
The PCL used in this Thesis is commercially available as TONE™ polymer purchased from
Union Chemicals and Plastics Divisions (Bound Brook, New Jersey, USA).
II.2.2. Starch-polycaprolactone blend
Natural polymers have been presented as an interesting option to the currently used metal
and synthetic materials, due to a higher biodegradability rate, non-cytotoxicity, and good
mechanical properties. These polymers are synthesized involving enzyme-catalyzed, chain
growth polymerization reactions of activated monomers, which are typically formed within
cells by complex metabolic processes.
Starch is a biopolymer produced by green plants as an energy store. It is quite abundant in
nature, and an almost unlimited source and low cost associated raw material. Starch consists
of multiple glucose units linked by glycosidic bonds, resulting in a combination of two
polymeric carbohydrates (polysaccharides) amylose (A), a linear and helical structure, and the
branched amylopectin (B) (Figure II.2.2).
A B
Figure II.2.2 – Chemical structure of starch components: amylose (A) and amylopectin (B).
Scaffolds, based on blends of starch polymer with several different synthetic polymers,
have been developed by our group using several methodologies[11-14], including by meltbased[15-17] and wet-spun[17-19] routes. Some of these scaffolds have previously shown
Chapter II – Materials and Methods
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great potential for TE strategies aiming at bone[11, 14-16, 18, 20, 21] and cartilage[22, 23]
applications.
Specifically, scaffolds based on blends of starch and polycaprolactone (SPCL) have been
successfully used in a great number of studies showing to support the adhesion and
proliferation of endothelial cells[21], chondrocytes[22] or osteoblasts, as well as the
proliferation and differentiation of mesenchymal stem cells[16, 20, 23] obtained from different
sources into osteoblasts[12] or chondrocytes[23]. Furthermore, in vivo studies not only
indicate a good integration of the starch based materials in the host[17] but also that these
biomaterials have a weak potential to stimulate an inflammatory reaction[24].
The 30:70 (wt %) SPCL blend used for the experimental studies described in the following
chapters was obtained from Novamont (Novara, Italy).
II.2.3. Tri-calcium phosphates
Ceramic materials have shown important scaffolding properties aiming at bone TE
strategies[22, 25, 26]. These achievements are supported by fact that hydroxyapatite (HA) is
the essential crystalline part of the calcified components of the skeleton, and is the principal
mineral in bone, enamel, dentin, and cementum[26].
Ceramic materials evidence a high modulus yet brittle behavior[27] and their processability
is limited. However, these materials offer advantages over most polymers regarding their
stimulating biological properties, such as osteoconductivity, and cell bioactivity[28]. Among
ceramics, HA and tri-calcium phosphates (TCPs) are the most commonly used in biomedical
research, being HA the preferred ceramic for bone strategies[9, 29, 30]. However, the
osteoconductive and bioreabsorvable nature of beta-TCPs are associated to an earlier
incorporation into surrounding bone in vivo may be advantageous to HA, which remains
unremodelled despite long periods upon implantation[31].
II.2.4. Agarose
Agarose is a natural based polysaccharide obtained from agar (Figure II.2.3) used for a
variety of life science applications, including immune-isolation purposes.
Agarose is heated to 90 ºC to form a polymeric solution by solving agarose powder with
water or phosphate saline buffer. When the temperature is lowered to close to room
temperature (fluctuations may take place considering the several commercially available
Chapter II – Materials and Methods
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agarose products) gelation occurs. Notwithstanding the wide application of agarose, gels
produced from agarose have been used as cell encapsulation systems for cartilage TE. Despite
the slow degradation profile, the soft and flexible structure provided by these gels recreates a
better 3D environment, suitable for chondrocyte maintenance[32] and mesenchymal stem cell
differentiation into the chondrogenic lineage[32].
Figure II.2.3 – Structure of agarose molecule
In this Thesis, agarose gels (2 %, Invitrogen) were used as encapsulating systems for hAFSCs,
as described in chapter V, by dissolving a 2 % agarose solution in ultra-pure water and cooling
it down in a flat plastic surface before cutting the hydrogel discs.
II.3. Scaffold fabrication
The design of a scaffold ultimately determines the functionality of the grown tissue.
Scaffold design comprehends the combination of the selected materials and methods,
considering a set of structure-properties specification for a target application. Several
techniques were considered in the experimental chapters of this Thesis, namely fiber bonding
of melt spun fibers, electro-spinning, and wet-spinning. The purpose of using these different
methodologies was to try to develop a range of scaffold designs/properties and determine the
ones that are likely to direct cell behavior in the most appropriate manner, considering the
aimed application.
Scaffolds produced by fiber bonding of melt spun fibers presented a microporous structure
with high porosity and interconnectivity so that cells can colonize the entire scaffold while
communicating with each other; these scaffolds were studied in chapter V, VI, VII, and VIII.
Conversely, electrospun scaffolds, studied under chapter III, resulted in a nanofiber mesh,
resembling the biological scale of extracellular matrix (ECM), where calcium phosphates were
included to improve the osteogenic potential of the mesh. Wet-spinning is another interesting
Chapter II – Materials and Methods
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methodology to develop scaffolds with high porosity and interconnectivity allowing to,
simultaneously, introduce chemical modifications, such as functionalizing scaffold surface for
stimulating the communication between scaffold and cells. Scaffolds obtained by wet spinning
were studied in chapter IV.
II.3.1. Melt fiber extrusion - Fiber bonding
SPCL (a blend of starch with polycaprolactone, 30/70 % wt) fiber mesh scaffolds were
produced by a fiber bonding process consisting of cutting and sintering the fibers, which were
previously obtained by an extrusion/melt spinning process. Briefly, a selected amount of fibers
was placed in a glass mould and heated in an oven at 150 ºC. Immediately after removing the
moulds from the oven, the fibers are slightly compressed by a Teflon cylinder (which runs
within the mould) and then cooled at –20 ºC. Then, samples were cut into cylinder discs,
according to the experimental needs following sterilization by ethylene oxide. Scaffolds
previously produced by this method showed interesting results for bone and cartilage TE
applications[16, 20, 21, 23], and their mechanical and degradation profiles have also been
described elsewhere[15].
II.3.2. Electrospinning
The use of nanotechnology to tailor orthopaedic scaffolds arises from the need to mimic
the ECM structure and complexity at a biological scale, since bone matrix is mainly composed
of an intricate nanofiber structure of nonstoichometric HA integrated in collagen fibers.
Electrospinning is an interesting approach for biomaterial processing enabling to control
morphology, porosity and composition of the polymer with nanoscale properties[8, 33].
Composite scaffolds were developed in chapter III, combining electrospinning with β-TCP
powder as indicated in the following section. PCL fiber meshes were produced using a
polymeric solution of 17 % (wt/v) of PCL in a mixture of chloroform (Aldrich) and N,NDimethylformamide (Aldrich) at a 7:3 ratio. This solution was electrospun at 9-10 kV, with a
flow rate of 1.0 mL/h collecting a random fiber mesh (20 cm away from the collector) on a flat
aluminum foil.
The electrospinning experiments were conducted at room temperature, as well as the
drying of the electrospun nanofiber meshes. PCL single layered membranes were used as
negative controls of the multi-layered PCL-β-TCP study.
Chapter II – Materials and Methods
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II.3.3. Tricalcium Phosphates production by solid state reaction
β-TCP can be produced through a solid state reaction[34], an interesting synthesis route
given its simplicity and yield. In our studies, the ceramic material, -TCP, was obtained from a
solid state reaction between stoichiometric amounts of calcium phosphate dibasic anhydrous
(Fluka) and calcium carbonate (Sigma) followed by a 24 h sinterization at 800 ºC. The powders
were hand-sieved with stainless steel sieves (mesh 225-106 μm).
After obtaining β-TCP powder, a polymeric solution of 17 % (wt/v) PCL was electrospun to
produce the polymeric nanofiber mesh layer and the TCPs were subsequently spattered over.
Composite scaffolds were developed by assembling a total of three stacked layers of
electrospun PCL-β-TCPs at a ratio of 0.5 g β-TCP/130 cm2 PCL meshes.
II.3.4. Wet-spinning
Wet-spinning technology requires polymer dissolution prior to scaffold formation and,
consequently, depends on polymer solubility and on solvent volatility. The development of
SPCL wet spun scaffolds was described previously[18]. Briefly, to obtain a polymer solution
with proper viscosity, SPCL was dissolved in chloroform (Sigma-Aldrich) at a concentration of
30 % (wt/v) followed by injection (internal diameter of 0.8 mm) to induce the formation of
fiber structures by precipitation in a methanol (Sigma-Aldrich) coagulation bath. A
programmable syringe pump (KD Scientific, World Precision Instruments) with a controlled
pumping rate of 2.5 ml/hour was used to run fibres formation. The fiber mesh structure was
formed by random movements of the syringe in the coagulation bath.
Scaffolds were air-dried at room temperature overnight to remove remaining solvent
residues.
II.3.4.1. Designing of an in situ functionalized surface
Silicon ion plays a significant role in bone and cartilage development of higher
organisms[35]. Furthermore, the release of silicon and calcium was reported to up-regulate
and activate genes in osteoprogenitor cells[36]. Thus, in chapter IV, we propose to evaluate
the relevance of the incorporation of silanol groups (Si-OH) in wet spun fiber mesh scaffolds of
SPCL.
Chapter II – Materials and Methods
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SPCL scaffolds with functionalized Si-OH groups were produced in a similar way to SPCL
scaffolds described in the previous section. The main difference is the coagulation bath;
instead of methanol, a calcium silicate solution was used with a molar ratio of
Si(OC2H5)4:H2O:C2H5OH:HCl:CaCl2, of 1.0 : 4.0 : 4.0 : 0.014 : 0.20 [18, 37] to assure the
osteoconductivity of the polymeric scaffolds. Afterwards, fiber meshes were air-dried at 60 ºC
for 24 h, and designated as SPCL-Si.
II.4. Scaffold characterization
Scaffold characterization is part of the process of designing a strategy for biomedical
applications. Physic-chemical properties provide important information about the biomaterial
and the manufacture of a 3D matrix while predicting some biological outcomes.
In the following sections, technical information is provided concerning analytic procedures
selected for scaffold evaluation, either chemical or physical techniques, under the scope of this
Thesis.
II.4.1. In vitro bioactivity of SPCL-Si wet-spun fiber mesh scaffolds
The concept of bioactivity is defined as one of the characteristics of an implant material
which allows it to form a bond with living tissues[38, 39]. The assessment of apatite formation
on the surface of a material in simulated body fluid (SBF) is useful for predicting the in vivo
bioactivity of the material not only qualitatively but also quantitatively. SBF solution presents
ion concentration of Na+ 142.0, K+ 5.0, Ca2+ 2.5, Mg2+ 1.5, Cl- 147.8, HCO3
- 4.2, HPO4
2- 1.0, SO4
2-
0.5 mM, similar to blood plasma’s.
Bioactivity of SPCL-Si wet-spun fiber meshes was determined by soaking the scaffolds in 10
mL of SBF at 36.5 ºC for up to 7 days. The SBF solution was prepared by dissolving reagentgrade chemicals of NaCl, NaHCO3, KCl, K2PO4
.3H2O, MgCl2
.6H2O, CaCl2 and NaSO4 into distilled
water, at pH 7.40 buffered with tris-hydroxymethyl-aminomethane ((CH2OH)3CNH3) and 1 M
hydrochloric acid at 36.5 ºC. After soaking, the substrates were removed from SBF, rinsed in
distilled water and air-dried.
The bioactivity assay was conducted in order to assess in vitro the bioactive potential of
wet-spun fiber meshes functionalized with silanol groups for bone TE applications.
Chapter II – Materials and Methods
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II.4.2. Scanning electron microscopy
The scanning electron microscopy (SEM) was used as complementary method to evaluate
the morphology of the scaffolds, including pore shape, size and distribution as well as the
interconnectivity between these pores. The morphology of multilayered scaffolds of PCL-TCP
from chapter III was evaluated by SEM to determine the distribution and integration of TCP
granules in the PCL fiber meshes. Additionally, in chapter IV, film morphology as well as the
presence of different elements was analyzed before and after immersing the scaffolds in
simulated body fluid.
Prior to SEM analysis, the scaffolds were sputter coated with gold (Fisons Instruments,
model SC502, England) for 2 minutes at 15 mA. Samples were assessed in a SEM (Leica
Cambridge, model S360) equipment and micrographs recorded at 15 kV ranging from 100 to
1000x magnification.
II.4.3. Dynamic Mechanical Analysis
Cells and tissues exhibit a viscoelastic behavior[40], which an implantable scaffold or
construct should match at physiological conditions[41] in order to participate in biophysical
and biological responses.
Dynamic mechanical analysis (DMA) measures the vistoelastic properties of materials as a
response to stress, temperature and frequency, and determines their time-dependent
mechanical performance.
Dynamic mechanical analysis of SPCL-Si scaffolds from chapter IV was performed using a
TRITEC8000B DMA from Triton Technology, equipped with a tensile mode. Before the analysis,
scaffolds were immersed in PBS until equilibrium was reached. After equilibration at 37 ºC,
DMA spectra were obtained with a frequency scan between 0.1 and 25 Hz. The experiments
were performed under constant strain amplitude (30 µm) and a static pre-load of 0.7 N was
applied to keep the sample tight. During DMA analysis, scaffolds were immersed in a liquid
bath in a Teflon® reservoir. Triplicates were used for each condition.
II.4.4. Micro-computed tomography
Micro-computed tomography (m-CT, µ-CT or micro-CT) has been successfully used in
different branches of science for the study of porous or cavity-containing structures. Micro-CT
Chapter II – Materials and Methods
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is a non-destructive model that uses x-rays to create multiple micrometer cross-sections of a
3D sample that later can be used to recreate a virtual 3D model. This technique is extremely
useful for scaffold characterization in view of the fact that besides the morphological aspect of
the scaffold, other properties such as porosity, pore interconnectivity or thickness can be
quantitatively determined.
Micro-CT is also an interesting tool for those studies where a calcified layer is an expected
result, either produced by the cells or as a precipitation coating as for bioactivity assays.
Calcified areas are easily detected in polymeric matrices by micro-CT due to the significant
differences found in the density of polymer scaffolds and calcium phosphates present in the
matrix[18]. Additionally, in small dimensioned scaffolds the distribution of the ECM over the
scaffold can be observed and measured with appropriate software.
Micro-CT, using a Skyscan 1072 scanner (Skyscan, Kontich, Belgium), was performed to
characterize SPCL-Si scaffolds after the bioactivity studies from chapter IV so as to observe the
distribution of the apatite layer on the scaffolds after simulated body fluid immersion. X-ray
scans were performed in triplicate using a resolution of 5.86 µm and integration time of 1.9
sec. The X-ray source was set at 40 keV / 248 μA. Approximately 400 projections were acquired
over a rotation range of 180 º with a rotation step of 0.45 º. Data sets were reconstructed
using standardized cone-beam reconstruction software (NRecon v1.4.3, SkyScan). Data sets of
200 slices were segmented into binary images with a dynamic threshold of 50 to 255 (grey
values) to identify the organic and inorganic phase. This data was used for morphometric
analysis (CTAnalyser, v 1.5.1.5, SkyScan) and to build 3D models (ANT 3D creator, v2.4,
SkyScan).
II.4.5. Thin-film X-ray diffraction
X-Ray Diffraction Analysis (XRD) is a versatile, non destructive tool for investigating the
crystallographic structure and sub-nanometric morphology of natural and synthetic materials.
Thin-film X-ray diffraction (TF-XRD, Philips X'Pert MPD, The Netherlands) was used to
identify any crystalline phase present on the scaffolds from chapter III and IV.
In the case of PCL-TCP nanofiber meshes, XRD provides information about the type of
calcium phosphate obtained by solid state reaction, whose granules were integrated in the
multilayered structure. Results were compared to membranes of PCL, the negative control of
this experiment.
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In chapter IV, XRD was performed to characterize the crystalline/amorphous nature of the
apatite films formed on the polymeric wet-spun fiber mesh after soaking in the calcium silicate
solution or after immersion in simulated body fluid (results were compared to non immersed
controls).
The data collection was performed by 2 scan method with 1 º as incident beam angle
using CuK X-ray line and a scan speed of 0.05 º/min in 2.
II.4.6. Fourier transform infrared spectroscopy with attenuated total reflectance
Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) is a
non-destructive, fast and highly sensitive tool for material analysis, providing an infrared
spectrum of absorption, emission, photoconductivity or Raman of a solid, liquid or gas. Data
obtained from the infrared spectrum represents the molecular absorption and transmission of
a specimen, creating a unique molecular fingerprint of the sample, thus permitting its
identification, quality and the amount of components in a mixture.
FTIR–ATR was performed using an IRPrestige-21 (Shimadzu, Japan) with an attenuated total
reflectance in PCL-TCP fiber mesh samples from chapter III, to determine if calcium phosphate
granules were integrated in the PCL-TCP fiber mesh.
All spectra were recorded using at least 64 scans and 2 cm−1 resolution, in the spectral
range 4000–600 cm−1.
II.4.7. Inductively coupled plasma optical emission spectrometer
Inductively coupled plasma optical emission spectrometer (ICP-OES) uses the inductively
coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at
wavelengths characteristic of a particular element. The intensity of this emission is indicative
of the concentration of the element within the sample.
Elemental concentrations of calcium, phosphorus and silicium were measured by
inductively coupled plasma atomic emission spectrometry (JY2000-2, Jobin Yvon, Horiba,
Japan). Samples were firstly collected and 0.22 µm filtered and kept at -80 ºC until usage. This
technique was used to analyse the simulated body fluid solution before and after immersing
the wet-spun fiber meshes with or without the silanol groups (bioactivity tests), in chapter IV.
Triplicate samples were considered for ICP analysis.
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II.5. Scaffold sterilization prior to cell culturing studies
Prior to cell culture experiments, samples were cut into discs, whose dimensions were
defined accordingly to the experiments to be performed, and ethylene oxide sterilized at
Pronefro (Maia, Portugal) or at the WFIRM (Winston-Salem, North Carolina, USA). Typical
conditions included a cycle temperature of 37 ºC, a moisture level of 50 %, a cycle time of 14
hours, and a chamber pressure of 50 kPa.
The multi-layered PCL-β-TCP scaffolds (developed on chapter III) were cut into 5 mm discs
followed by two 30 minute-cycle of UV radiation, instead of ethylene oxide sterilization.
II.6. Biological assays
II.6.1. Harvesting, isolation and culture of stem cells
The interest in the potential of adult stem cells has been increasing due to the limited
availability of tissue specific progenitor cells.
Bone marrow represents a source of mesenchymal adult stem cells[42-44] due to the
availability of established culture techniques to isolate and expand cell populations from
human donors. Additionally, expansion of bone marrow stem cells overcomes the tissue
morbidity and donor tissue scarcity, minimizing the problems associated with other types of
primary cells. In autologous strategies, the implantation of cells from the individual also
reduces disease transmission risks.
Conversely, amniotic fluid is arising as an interesting source of stem cells. Amniotic fluid
cells (AFSCs) are used for prenatal diagnosis of fetal abnormalities caused by genetic
mutations. Along with the almost endless ability to expand without telomere shortening,
AFSCs share with embryonic stem cells some markers and a high self renewal capacity without
associated ethical concerns[45].
II.6.1.1. Harvesting and isolation of goat bone marrow stromal cells
Goat bone marrow stromal cells (gBMSCs) were used in the studies described in chapter III,
IV, and VII. This animal model was selected for these studies, firstly due to their analogy to
human bone marrow stem cells (hBMSCs), and also because goats are a high level vertebrate
Chapter II – Materials and Methods
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with metabolic and bone remodelling rates similar to that of humans. The sequence of events
in bone graft incorporation and healing capacities[46] are also comparable to humans, which
explain how goats became increasingly popular as an animal model in the orthopaedic field.
Goats have been widely used in studies of biocompatibility, and bone formation and
regeneration[47-49]. Furthermore, the use of gBMSCs allows performing autologous
experiments, as goat bone marrow aspirates can be obtained by a biopsy procedure, and the
isolated cells can be cultured using serum from the same animal where ultimately, the same
cells or cell-scaffold constructs will be implanted.
The methodology optimized for harvesting goat bone marrow (BM) was based on the
techniques used to perform human BM biopsies from the iliac bone. Thus, through
percutaneous puncture, bone marrow was aspirated from the iliac wings of goats using a
biopsy needle, without erythrocyte contamination (Figure II.6.1).
Figure II.6.1 - Harvesting marrow from the goat iliac crest.
The experimental protocol for BM aspirate was performed according to the national
guidelines and after approval of the National Ethical Committee for Laboratory Animals from
Direcção Geral da Agricultura. Prior to general anesthesia, goat iliac regions were shaved and
disinfected. Animals were submitted to a pre-anesthetic medication with acepromazine
maleate (5 mg EV, Calmivet, Vetóquinol) and placed under general anesthesia by induction
with thiopenthal sodium (20-25 mg/Kg EV, Pentothal sodium, Abbott Labs), maintained by
inhalation of a mixture of 1.5 % isoflurane (IsoFlo, Abbott Labs, USA) and oxygen for a
maximum of 30 minutes.
A bone marrow aspiration needle (Inter.V, Medical Device Technologies, Inc., Denmark) and
a 10 mL syringe, already containing 1 mL heparin (5.000 U.I., Heparin sodium, B. Braun
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Medical, Inc.) to avoid marrow coagulation, was used to collect 10 mL of bone marrow from
the iliac crest of adult goats (Figure II.6.1), transferred into a sterile tube and resuspended in
30 mL of RPMI-1640 culture medium (Sigma-Aldrich), containing 1 % penicillin/streptomycin
(Gibco) plus 1 mL of heparin (5.000 U.I.).
Afterwards, goat marrow stromal cells (gBMSCs) were centrifuged for 10 minutes at 1200
rpm and a dense cellular pellet was collected and cultured in a basic culture medium – DMEM
(Dulbecco’s Modified Eagle’s Medium) (Sigma-Aldrich) supplemented with 10 % FBS (Gibco)
and 1 % A/B (Invitrogen). Four days after the harvesting procedure, cells were rinsed in sterile
PBS (Sigma) and medium was exchanged. Following gBMSCs expansion up to 90 % of
confluence, cells were cryopreserved at 1 passage (Pa) in a solution of FBS with 10 % DMSO
until needed for experimental purposes.
The advantage of this methodology is based on the acquisition of a higher quality of bone
marrow samples with a simple technique that, after optimization, can be systematically used in
subsequent experiments. The achieved optimization of this technique constitutes the first
report in the scientific literature of harvesting bone marrow aspirate from percutaneous
puncture in the goat model.
II.6.1.2. Harvesting and isolation of human amniotic fluid stem cells
Human amniotic stem cells (hAFSCs) were used in the studies described in chapters V, VI,
and VIII. AFSCs were obtained from human amniotic fluid specimens collected from
amniocentesis procedures, using backup cells that would otherwise be discarded. AFSCs were
immunoselected with magnetic microspheres isolating the c-Kit–positive population[45], as Ckit or CD 117 has been described to be the receptor for stem cell factor. The protocol has been
described in detail elsewhere[45]. AFS cells were grown in α-MEM medium (Gibco, Invitrogen)
containing 15 % embryonic stem cell screened FBS (ES-FBS, HyClone, USA), 1 % glutamine and
1 % penicillin/streptomycin (Gibco), supplemented with 18 % Chang B (Irvine Scientific), and 2
% Chang C (Irvine Scientific) at 37 ºC with 5 % CO2 atmosphere.
II.6.2. Cell culturing and expansion
Samples obtained from body tissues such as bone marrow or amniotic fluid usually have a
cell number that is not sufficient for TE applications. Thus, stem cells should be isolated,
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selected, and expanded towards the achievement of cell numbers enough to perform in vitro
studies, and eventually a cell number relevant for clinical applications.
II.6.2.1. Goat bone marrow stromal cells
Cryopreserved cells to be used in experimental studies were thawed by placing the frozen
cell vial in a 37 ºC water bath. When the vial was almost defrosted, gBMSCs were removed
from the bath and pipetted into cell culture flasks, and let to attach for 24 hours prior medium
exchange. In general, cells were expanded up to 90 % confluence and sub-cultured to 2 Pa
(Figure II.6.2) in basic medium (DMEM, 10 % FBS and 1 % A/B) before seeding/encapsulation
procedures.
Figure II.6.2 - 2D culture of gBMSCs after 10 days in basal medium.
In autologous approaches (chapter VII), gBMSCs were expanded in 10 % autologous serum
instead of FBS. Autologous serum was isolated from goat peripheral blood every 3 weeks and
centrifuged at 3,000 rpm for 10 minutes. Serum was stored at -20 ºC until usage.
II.6.2.2. Human amniotic fluid stem cells
Human AFSCs were isolated as previously described[45] and cultured in basic amniotic fluid
cell (BAFC) medium composed of α-MEM (HyClone) with 18 % Chang B (Irvine Scientific) and 1
% Chang C (Irvine Scientific) media as well as 2 % L-glutamine (HyClone) and 15 % ES-FBS.
II.6.2.3. Human mesenchymal stem cells from bone marrow
Human mesenchymal stem cells from bone marrow (hBMSCs) were purchased from Lonza®
and expanded as indicated in manufacturer datasheet. Basal culture medium was composed of
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α-MEM (HyClone) supplemented with 10 % ES-FBS (HyClone) and 1 % penicillin/streptavidin
solution.
II.6.3. Osteogenic differentiation
The osteogenic differentiation of mesenchymal stem cells is dependent on mechanical and
biochemical stimuli from their micro-environment. Dexamethasone, ascorbic acid and ßglycerol phosphate are known to induce osteogenic differentiation, and thus commonly used
as osteogenic supplements provided in cell culture medium. Dexamethasone is a synthetic
glucocorticoid shown to induce osteogenic differentiation of osteoprogenitors cells from fetal
calvaria-derived cells[50] and adult bone marrow stromal-derived cells[51, 52]. In human body,
ascorbic acid is an essential nutrient required for collagen synthesis, thus playing an essential
role in the structure and function of skeletal tissues. When ß-glycerol phosphate, a potential
source of phosphate ions[52], is also present, a zone of hydroxyapatite-containing mineral is
formed within collagen fibrils[53].
Thus, in order to induce the osteogenic phenotype, gBMSCs were cultured in minimal
essential medium Eagle’s α-modification (α-MEM; Sigma-Aldrich), A/B (1 %), FBS (10%) and
supplemented with osteogenic factors, namely dexamethasone (10−8 M; Sigma-Aldrich),
ascorbic acid (50 μg/ml; Sigma) and β-glycerophosphate (10 mM; Sigma) for up to 3 weeks.
Minor variations in medium composition are indicated in experimental studies performed
at WFIRM, USA (chapter V, VI and VIII). In this case, osteogenic medium prepared for hBMSCs
and hAFSCs was composed of DMEM (HyClone) supplemented with 10 % FBS (HyClone), Lascorbic acid (50 µM; Sigma), dexamethasone (100 nM; Sigma), and glycerol 2-phosphate
disodium salt hydrate (10 mM; Sigma).
II.6.4. Osteochondral differentiation
The composition of the co-culture media used to maintain the osteogenic and
chondrogenic phenotype of differentiated AFSCs (as described in chapter V) was prepared
considering the inclusion of both osteo- and chondrogenic factors usually used in osteo- or
chondrogenic culture media.
Several media from the literature were analyzed, and the most frequent supplements
combined to obtain a co-culture medium composed of DMEM-LG (HyClone), 2 % FBS
(HyClone), sodium pyruvate (1x, Sigma), ITS (1x, Sigma), 5 mM glycerol 2-phosphate (Sigma),
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50 µg/ml ascorbic acid (Sigma), 10 mM dexamethasone (Sigma), 40 g/ml L-proline (Sigma),
either in the presence or absence of 100 ng/ml IGF-1 (Invitrogen).
Since TGF- was removed from the media, another factor was added and its presence
tested in the medium: IGF-1. IGF-1 or insulin growth factor-1 has been described to participate
in both bone and cartilage development, which could maintain the induced phenotype of
AFSCs. After 7 or 14 days in the co-culture media, constructs were removed and characterized
for osteo- and chondrogenic specific markers. BAFC medium[45] was used as control of this
experiment.
II.6.5. Seeding on 2D cultures
In order to compare the potential of hBMSCs and hAFSCs for bone tissue engineering
applications (chapter VI), both cells were seeded onto culture treated 6-well plates (Costar) at
passage 5 and passage 24, respectively, at a concentration of 30,000 cells/well. Both type of
cells were cultured for 3 days with basal medium, and then, exchanged to osteogenic medium,
composed of DMEM (HyClone Laboratories Inc.) supplemented with 10 % FBS (HyClone), 100
nM dexamethasone (Sigma), 50 µM L-ascorbic acid (Sigma) and 10 mM glycerol 2-phosphate
disodium salt hydrate (Sigma), for up to 3 weeks (0, 7, 14 or 21 days). Before the osteogenic
characterization, cells were briefly rinsed in PBS (HyClone Laboratories Inc.), and fixed in 10%
neutral buffered formalin (Surgipath Medical Ind.).
II.6.6. Seeding cells onto 3D matrices
II.6.6.1. gBMSCs, hAFSCs or hBMSCs onto SPCL scaffolds
In chapter VII, a gBMSCs suspension of 2,500,000 cells/mL was prepared and seeded onto
the SPCL porous scaffolds in a drop-wise manner, at a cellular density of 500,000 cells per SPCL
scaffold (6 mm diameter x 2 mm height discs). Seeding chambers were used to improve cell
seeding efficiency by avoiding cellular dispersion. Afterwards, constructs were cultured in nontreated 12-well plates (Costar, Becton Dickinson), to reduce cellular adhesion to the plates.
Osteogenic medium described in previous sections was selected for 1 or 7 days of cell culturing
prior to implantation. In chapter VII, autologous sera (10 %) were used. Autologous culture
medium was changed twice a week.
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In a similar way to gBMSCs, hBMSCs (only in chapter VI) and hAFSCs were seeded onto
SPCL scaffolds (5 mm x 4 mm, 7 mm x 4 mm, and 6 mm x 3.8 mm cylinders in chapter V, VI and
VIII, respectively) at a concentration of 8.6 x105, 1.2 x106, and 1.2 x106 cells/scaffold,
respectively, and cultured in basal medium for 3 days before adding osteogenic media for up
to 3 weeks.
In chapter VI, hBMSCs and hAFSCs were seeded on culture treated 6-well plates (Costar) at
a concentration of 30,000 cells per well in order to compare their behaviour with cells cultured
in 3D SPCL scaffolds. Cells seeded on the 6-well plates were cultured for 3 days in basal
medium, and then, exchanged to osteogenic medium, for up to 3 weeks (0, 7, 14 or 21 days).
II.6.6.2. gBMSCs onto SPCL-Si scaffolds
In chapter IV, gBMSCs were seeded onto SPCL-Si scaffolds (5 mm diameter discs) before
and 7 days after scaffold immersion in a SBF solution, at a concentration of 100,000 cells per
scaffold. After the seeding, constructs were cultured in osteogenic medium for 7 or 14 days.
II.6.6.3. Encapsulating hAFSCs into agarose gels
In chapter V, a 2 % agarose solution was used as encapsulating gel. Firstly, the agarose
solution was prepared and kept at 37 ºC while hAFSCs were tripsinized and counted. Then,
hAFSCs were resuspended at a concentration of 2 x106 cells/mL of agarose. Cell-agarose
system was left to gelify for a couple of minutes at room temperature inside a biological hood.
Afterwards, 4 mm diameter discs were cut off from the cell encapsulated gel and placed in non
treated 24 multi-well plates (Costar) with basal medium.
After 2 days, chondrogenic medium was prepared with DMEM (HyClone) supplemented
with antibiotic/antimicotic solution, 50 µg/ml ascorbic acid (Sigma), 1 mM dexamethasone
(Sigma), 40 g/ml L-proline (Sigma), 100 g/ml sodium pyruvate (Sigma), 1 % ITS (100x, Sigma)
and 10 ng/ml TGF-ß1 (Sigma), and added to the encapsulated system. Medium was exchanged
once a week for up to 4 weeks in chondrogenic culture.
II.6.7. Characterization of cell-scaffold constructs
Cell seeded matrices were characterized in terms of cellular viability and proliferation as
well as of osteo- or chondro-genic differentiation markers to assess cellular phenotypes, after
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several pre-determined periods of culture. Cell viability was assessed by MTS or Calcein AM
methodologies while proliferation was determined by a double strand DNA quantification kit.
Osteogenic differentiation was evaluated based on the activity of alkaline phosphatase
(ALP) and the expression of RunX-2, a transcription factor involved in bone development.
Furthermore, morphological features and the production of a mineralized matrix in bone
strategies were also considered by SEM and EDS analysis, Alizarin Red staining as well as the
expression of collagen I, the major organic component of bone ECM, and osteocalcin, and
FTIR-ATR to assess chemical groups that may be present in the bony calcified matrix.
Chondrogenic differentiation was assessed through histological stains as Safranin O as well
as immunochemistry for collagen II, and aggrecan, major molecules present on the cartilage
ECM.
Techniques selected for cell-scaffold characterization are described in detail within the
following sections of this chapter.
II.6.7.1. Cell viability methods – MTS and Calcein AM
The MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) assay is a colorimetric method for determining the number of viable cells in
proliferation or cytotoxicity assays, and was used in chapter IV. The MTS compound is
bioreduced by cells, when reductase enzymes are active, into a colored formazan product that
is soluble in culture medium. The quantity of formazan product measured at 490 nm
absorbance, is directly proportional to the number of living viable cells in culture.
Calcein AM (Calcein acetoxymethyl ester) (Figure II.6.3) is a cell-permeant and nonfluorescent compound widely used for determining cell viability. In live cells the nonfluorescent calcein, AM is hydrolyzed by intracellular esterases into the strongly green
fluorescent anion calcein. The fluorescent calcein is well-retained in the cytoplasm in live cells,
and the fluorescence intensity is proportional to the amount of live cells. Calcein is a
photostable reagent not influenced by intracellular pH. Its bright green fluorescence can be
monitored at Ex/Em = 494 nm /520 nm.
This method was used in chapters V, VI, and VIII. After cell culturing according to
predefined end points, hAFSC- and hBMSC-SPCL constructs were rinsed in PBS (HyClone) and
incubated in culture medium with 3 µM Calcein AM (Molecular Probes, Invitrogen) for 30
minutes at 37 ºC in a 5 % CO2 environment. Afterwards, constructs were rinsed again in PBS
and fixed in 10 % buffered formalin (Surgipath Medical Industries, Inc.) overnight at 4 ºC,
Chapter II – Materials and Methods
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before observed under a confocal microscope. DAPI (Molecular Probes, Invitrogen), a
fluorescent marker that binds to nucleic acids, DNA and RNA, and can enter live or dead cells,
was used in chapter VI.
Figure II.6.3 – Calcein AM cleavage by viable cells.
The main difference in these methods is that MTS is a quantitative method, while Calcein
AM is qualitative. Small absorbance variations can be detected among samples with MTS
assay. Although counting cells with Calcein AM method is possible, it is very time consuming,
and with an intense fluorescence signal, counting individual cells can be a difficult task to
perform. Nevertheless, Calcein AM procedure presents additional advantages as it enables, for
instance observing the distribution and colonization of viable cells population on a scaffold or
plate.
II.6.7.2. Cellular proliferation assay (DNA quantification)
The cell proliferation assay described in this Thesis (chapter III, IV and VII) is an ultrasensitive fluorescent nucleic acid stain for quantitating double-stranded DNA (dsDNA) by
measuring the fluorescence produced when PicoGreen dye (Molecular Probes, P-7589) is
excited by UV light and bounds to double-stranded DNA (dsDNA) present in the tested sample.
In this assay, the constructs were harvested from the culture medium, rinsed in PBS,
followed by cell lysis in ultra pure water. Samples were kept at -80 ºC until testing. Afterwards,
samples were thawed and briefly sonicated for 15 minutes to rupture any remaining cell
Chapter II – Materials and Methods
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membrane. Cell lysate is incubated with a dye that fluoresces when bounds to DNA. The
intensity of the fluorescence from the DNA–dye complex is then measured with a standard
fluorometer. The number of cells in the sample is determined by comparing the fluorescence
intensity of the sample to a previously generated standard curve (concentrations ranging from
0.0 to 1.5 µg/mL). The standard curve relates the number of cells in a sample to the intensity
of the fluorescence from the DNA–dye complex. The fluorescence is read using a microplate
ELISA reader (Synergie HT, BioTek) at an excitation of 485/20 nm and an emission of 528/20
nm.
II.6.7.3. Histology
Histological analysis was performed in in vitro cultured AFSC-agarose systems from chapter
V to assess the chondrogenic differentiation through staining and immunostaining procedures.
Samples for histological analysis were firstly rinsed in PBS and fixed in a 4 % buffered
formalin solution overnight. Afterwards, samples were included in histological cassettes and
dehydrated in a series of ethanol concentration followed by xylene solution to remove water
from tissues in tissue processor equipment (Microm STP120, MICROM International GmbH,
Germany). AFSC-agarose constructs were wrapped in foam before placed inside the cassettes.
The foam encloses delicate and very small samples that could be lost or damaged during the
procedure.
Samples were then embedded in paraffin wax (Microm EC 350-2, ThermoScientific, Spain),
and included in blocks, and stored at room temperature. Blocks were then cut into 10 m thick
sections using a microtome, and then depparaffined. Since most stains and antibodies are
aqueous solutions, deparaffinization (removing paraffin wax and re-hydration of samples) is a
common step prior staining or immune-detection described below. After the histological
protocol is completed, sections must be mounted before observed under a microscope.
II.6.7.4. Osteogenic markers
II.6.7.4.1. Alkaline Phosphatase Activity (ALP)
Alkaline Phosphatases (ALP) are a group of enzymes found in several organs but the
isoenzyme ALP-2 is primarily found in the bone, with a particular influence in bone
Chapter II – Materials and Methods
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metabolism. As the name implies, this enzyme works best at an alkaline pH (a pH of 10), acting
by splitting off phosphorus (an acidic mineral) creating an alkaline pH.
Bone ALP is present in vesicles secreted by osteoblasts to induce bone mineralization, and
has been associated to the in vitro ECM development and maturation[54]. Consequently, ALP
activity has been considered a marker of the osteogenic differentiation process of stem cells.
ALP can be detected by different methods based on enzymatic reaction followed by
colorimetric analysis or by fast red violet dye. In this Thesis, ALP was evaluated according to
the procedures mentioned above; fast red violet dye in constructs with BMSCs- and AFSCs
seeded onto SPCL scaffolds from chapter VI and VIII, and a colorimetric assay using an ELISA
microplate reader in samples with gBMSCs- seeded onto TCP-PCL nanofibers or seeded onto
SPCL- and SPCL-Si- scaffolds from chapter III, VII, and IV, respectively.
The principle of the ALP staining, considered in chapter VI and VIII, relies on immersing the
samples in a Napthol AS-MX phosphate alkaline solution; a histochemical substrate used for
the detection of ALP activity, commonly used in conjuction with Fast Violet B salt (Sigma). After
each end point, cells were fixed in 10 % buffered formalin solution overnight at 4 ºC, and then
rinsed and kept in PBS until incubating the cells in a staining solution of 0.25 % Napthol AS-MX
phosphate alkaline solution (Sigma-Aldrich) and Fast Violet B salt (Sigma) for 30 minutes. To
remove the excess of non-specific staining, samples were rinsed in PBS and observed under an
inverted microscope (Leica, DMI4000B). Sample images were acquired using a camera QImaging (Retiga-2000RV) or, in the case of full sized constructs, observed directly and images
acquired using an Olympus SP-570UZ digital camera.
Beside the staining approach, ALP activity can be quantitatively evaluated using a
colorimetric development assay, which was used in chapter III, IV, and VII. Despite the fact that
this method is more sensitive to small ALP activity variations, it is an indirect evaluation
method as only ALP molecules released into the sample solution can be measured. Thus,
constructs were harvested from the culture medium, rinsed in PBS and placed in a microtube
containing 1 mL of ultra-pure water and kept frozen at -80 ºC until testing. Afterwards,
samples were thawed and briefly sonicated for 15 minutes in order to rupture any cell that
might have kept the membrane integrity after the osmostic treatment followed by a
freezing/thawer cycle. Then, a substrate solution consisting of 0.2 % (wt/v) p-nytrophenyl
phosphate (Sigma) in a substrate buffer with 1 M diethanolamine HCl (Merck), at pH 9.8 was
added to each sample, at a proportion of 60 l of solution to 20 l of sample.
The plate containing the samples was incubated in the dark for 45 minutes at 37 ºC,
followed by the addition of a stop solution (2 M NaOH (Panreac) plus 0.2 mM EDTA (Sigma)) to
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end the color development reaction. Standards were prepared with p-nytrophenol (10
µmol.ml-1) (Sigma) in order to achieve final concentrations ranging between 0.0 - 0.3 µmol.ml1. Samples and standards were analyzed in triplicates. The colorimetric assay measures the
conversion of the colorless substrate p-nitrophenol phosphate into the yellow soluble product
p-Nitrophenol by the enzyme ALP. P-nitrophenyl phosphate absorbs at 405 nm, and is the
preferred substrate for high sensitivity detection of ALP in ELISA assays. The absorbance was
read in a microplate ELISA reader (Synergie HT, BioTek) at 405 nm and concentration values
determined by intrapolation using a standard curve established by the standards.
II.6.7.4.2. Alizarin Red Staining
Alizarin red or 1,2-dihydroxyanthraquinone is an organic compound used for decades to
identify calcium-rich deposits[55] in tissue sections or cells in culture. The reaction is not
strictly specific for calcium, since magnesium, manganese, barium, strontium, and iron may
interfere, but these elements usually do not occur in sufficient concentration to interfere with
the staining. Calcium forms an Alizarin Red S-calcium complex in a chelation process, and the
end product is birefringent. Alizarin red staining is particularly versatile since the method can
be both qualitative and semi-quantitative as the dye can be extracted, dragging with it the
calcium ions, and readily assayed. Then, using a calibration curve and appropriate standards,
calcium concentration values can be achieved.
Alizarin Red was directly evaluated as a staining in constructs from chapter III and VI;
gBMSCs seeded onto PCL-TCP nanofibers and hAFSCs- and hBMSCs- seeded onto 6-well plates,
respectively. An alizarin red solution (0.2 % to 2 %, depending on specimens, Sigma-Aldrich)
was prepared, pH adjusted to 4.1-4.3 and samples stained by immersion for 2 minutes.
The alizarin red stain found in BMSC- and AFSC- samples from chapter VI, and in the BMSCand AFSC-SPCL constructs from chapter VI and VIII, was solubilized in cetylpyridinium chloride
(Sigma) at pH 7.0 for 15 minutes under milt agitation and calcium-bounded to alizarin red
quantified at 562 nm, using a plate reader (SpectraMax MS, Molecular Devices).
II.6.7.4.3. Fourier Transformed Infrared Spectroscopy with Attenuated Total Reflectance
FTIR spectroscopy has been successfully used to analyze the changes in minerals and
components of bone tissue since vibrational spectroscopy in the mid-infrared region can
provide molecular structure information about mineralized tissue[56].
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The composition of the human AFSC-SPCL constructs considered in chapter VIII was
analyzed by FTIR-ATR in order to detect molecular groups associated to ECM mineralization.
Before the analysis, samples were dehydrated in a series of ethanol concentrations and airdried at room temperature. FTIR-ATR was performed in the spectral range of 1800-600 cm-1
using the Spectrum 400 FT-IR/FT-NIR spectrometer (Perkin Elmer). Results were compared to
cell-free scaffolds in order to exclude the contributions from the scaffold composition.
II.6.7.4.4. Immunocytochemistry: Collagen I and Osteocalcin
Immunocytochemistry for Collagen I and Osteocalcin was performed on gBMSCs- PCL-TCP
samples from chapter III. Collagen I is the most abundant collagen of the human body and the
major protein present in bone ECM, responsible for strengthen and support of bone tissue. On
the other hand, osteocalcin (OCa) is a non-collagenous protein, produced by osteoblasts, that
participates in the matrix mineralization of bone[57] as OCa binds to hydroxyapatite in a
calcium-dependent manner[58].
Immunocytochemistry was performed according to R.T.U. Vectastain Universal Elite ABC kit
(PK 7200) from VectorLabs, using R.T.U Normal Horse Serum to avoid unspecific reactions, a
biotinilated secondary antibody (R.T.U. Biotinylated Universal Antibody, one-hour incubation),
and a Peroxidase Substract Kit (Vector SK-4100). 3,3’-Diaminobenzidine (DAB) was the
colorimetric substrate used in the assays.
Primary antibodies used included Collagen I (MAB339, Chemicon) and Osteocalcin
(Ab13418, Abcam), which were prepared using a 1/100 dilution. With the exception of primary
antibodies, which were incubated overnight at 4ºC, all the other steps of this procedure were
performed at room temperature.
II.6.7.4.5. Immunofluorescence: Collagen I and RunX-2
Immunofluorescence for Collagen I was performed on BMSC- and AFSC-SPCL constructs
from chapter VI and VIII, while RunX-2 expression was assessed in BMSC- and AFSC-SPCL
constructs from chapter V and VI. Cbfa1/RunX-2 is a key transcription factor associated with
osteoblast differentiation, essential for osteoblastic differentiation and skeletal
morphogenesis[59].
Collagen I from Southern Biotech (1310-01) and RunX-2 (ab23981) purchased to Abcam
were diluted to 1:100 and 1:20 ratio, respectively, and incubated overnight at 4 ºC.
Chapter II – Materials and Methods
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AlexaFluor 488 and 594 (Molecular Probes, Invitrogen; 1:200 dilution, one-hour incubation)
were used as fluorescent secondary antibodies.
Instead of animal serum, blocking step was performed using protein block serum free
(Dako). After the procedure, constructs were then observed under a confocal microscope
(Axiovert 100 M, Zeiss) equipped with argon/He-Ne laser sources.
II.6.7.5. Chondrogenic markers
II.6.7.5.1. Safranin O staining
Safranin, Safranin O or basic red 2 is a cationic dye frequently used as a counterstain, and
for the detection of mucin, cartilage[60] and mast cells.
Following deparaffinization of AFSC-agarose sections from chapter V, slides were stained
with Weigert’s iron hematoxylin working solution (Merck) for 7 minutes. Samples were rinsed
in tap water for 10 minutes, and subsequently rinsed quickly in 1 % acetic acid solution for
about 10 to 15 second and stained in 0.1 % safranin O solution (Fluka) for 5 minutes.
II.6.7.5.2. Immunofluorescence: aggrecan and collagen II
Immunofluorescence for aggrecan and collagen II was performed on AFSCs encapsulated in
agarose hydrogels from chapter V.
Aggrecan and collagen II, two of the main cartilagineous proteins, were evaluated to assess
the chondrogenic differentiation under the scope of this Thesis. Aggrecan is a chondroitin
sulfate proteoglycan that along with collagen II forms a major structural component of
cartilage. About 90 % of articular cartilage is made of Collagen II, a protein responsible to
entrap proteoglycan aggregates and provide the characteristic tensile strength to the tissue.
Samples were prepared as described previously in section II.6.7.6. Primary antibodies
Collagen II (MAB1330, Chemicon) and Aggrecan (MCA1452 Serotec) were prepared with a
1/100 dilution. AlexaFluor 488 (Invitrogen; 1:200 dilution, one-hour incubation) was used as
the fluorescent secondary antibody.
Chapter II – Materials and Methods
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II.6.7.6. Evaluation of cell morphology and distribution
Cell morphology and distribution throughout a 3D matrix was evaluated in cell-scaffold
constructs with confocal microscopy and scanning electron microscopy.
II.6.7.6.1. Confocal laser scanning microscopy (CLSM)
Confocal laser scanning microscopy (CLSM) is an optical imaging technique used to increase
contrast and optical resolution, overcoming the limitations of conventional widefield
microscopy. Simultaneously, CLSM facilitates the generation of high resolution 3D images from
a relatively thick sample[61]. In some cases, the 3D reconstruction allows a better
understanding of the distribution, organization or morphology of cells, or molecules of
interest.
Confocal microscopy was a very useful technique to assess cellular viability by Calcein AM in
human AFSCs and BMSCs seeded onto SPCL scaffolds and AFSCs encapsulated in agarose gels,
described in chapter V and VI. CLSM was also used for the detection of specific bone markers,
such as RunX-2 or collagen I in SPCL constructs seeded with AFSCs or BMSCs (chapter V, VI and
VIII).
Samples were prepared accordingly to Calcein AM and immunofluorescence protocols
above mentioned, before analyzed under a confocal microscope (Axiovert 100 M, Zeiss)
equipped with argon/He-Ne laser sources.
II.6.7.6.2. SEM
Cell seeded constructs[18,62] were also examined by SEM to assess cellular morphology,
distribution and relative proliferation between samples from different end points. In some
cases, it is also possible to assess ECM mineralization by energy dispersion spectroscopy (EDS)
coupled to SEM equipment.
All cell-scaffold constructs described in experimental chapters (chapters III, IV, V, VI, VII and
VIII), namely gBMSC-PCL-TCP, gBMSC-SPCL and gBMSC-SPCL-Si samples, as well as hBMSC- and
AFSC- seeded onto SPCL scaffolds, were observed by scanning electron microscopy (Leica
Cambridge, model S360 or Hitacho S-2600N, Hitachi Science Systems, Ltd).
Constructs were fixed in formalin, and the samples from chapter III, IV and VII were
dehydrated in a series of ethanol concentrations followed by air-dried. Conversely, constructs
Chapter II – Materials and Methods
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from chapter V, VI and VIII were critical point dried (EMS850X, Electron Microscopy Sciences)
after fixed in a formalin solution and dehydrated to 100 % ethanol.
All samples were gold sputtered (Fisons Instruments, model SC502 or Hummer 6.2
sputtering system, Anatech Ltd) for 2 minutes at 15 mA. Samples were further assessed by
SEM and micrographs recorded at 15 kV ranging from 100x to 5000 x magnification.
II.6.7.6.3. Energy dispersive X-ray analysis or EDS analysis
Energy dispersive X-ray analysis or EDS is a technique used to identify the elemental
composition of a sample, including bone tissue[63]. In terms of biological samples in this
Thesis, EDS analysis proved to be very useful for the detection of a calcified/mineralized ECM
(with calcium and phosphorus elements) produced by osteoblastic cells, when seeded in nonceramic scaffolds.
Since samples for SEM can be used for EDS analysis, constructs preparation is the same as
mentioned in the previous section. EDS and EDAX (Pegasus X4M) were used to determine the
presence/absence of calcium and phosphorus in the ECM matrix produced by hAFSCs and
hBMSCs cultured onto SPCL scaffolds (chapter V and VIII, respectively).
II.7. Animal models studies
II.7.1. Goat model – non critical defects
The selection of goat as an animal model was justified in a previous section (II.6.1.1.). The
primary goal of this study was to analyse the role of the scaffold material and of BMSCs in a TE
strategy aimed at bone regeneration. Thus, SPCL scaffolds, seeded or not seeded with gBMSCs
under different stages of osteogenic differentiation were implanted in non-critical sized
femoral defects.
Although many studies are conducted ectopically, orthotopic implants provide more
relevant data since implants are located in the tissue/area of their target application and thus,
any eventual site specific reaction such as cell recruitment or growth factor release either
associated to the tissue healing or to an inflammatory process is expected to be similar to the
ones that are likely to occur in a real clinical situation.
Chapter II – Materials and Methods
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Four skeletally adult female goats weighing 30-45 kg were used in the study described in
chapter VII. The housing care and experimental protocol were performed according to the
national guidelines, after approval by the National Ethical Committee for Laboratory Animals
(2007-07-27, document number 018939) and conducted in accordance with international
standards on animal welfare as defined by the European Communities Council Directive of 2
November 1986.
II.7.1.1. Implantation surgery
After general anaesthesia, each goat was positioned in lateral recumbency, prepared and
draped in a sterile manner to perform a surgical access to the lateral diaphysis of femur.
A skin incision was then performed from the greater throcanter and continued distally to
the lateral femoral condyle. The subcutaneous tissue, tensor fascia lata and lateral fascia of the
vastus muscle were incised. Biceps femoris muscles were retracted posteriorly, and the vastus
muscle was retracted anteriorly, after being detached from the linea aspera and femora shaft,
like the periosteum.
Non-critical size defects ( 6 mm and 3 mm depth) were drilled in the lateral diaphysis of
both posterior femurs of the 4 adult goats with a bone drill (Synthes, Switzerland). The drillhole technique selected was based on the one described by Hallfeldt et al.[64].
Eight drills were created bilaterally in the posterior femurs, with a separation distance
between drills of 3 cm, in two non parallel sections to avoid fracture tension or neobone
formation among drills. Two drills were left empty and 2 filled with scaffolds without cells, the
control for this experiment. The remaining drills were filled in with constructs osteogenically
cultured for 1 day (2 defects) or 7 days (2 defects).
Implants were gently pressed fit (Figure II.7.1). The muscle was replaced over the bone, and
the fascia lata and skin were closed with resorbable and non-resorbable sutures, respectively.
During the first post-operative week, animals were medicated with flunixin meglumin (1
mg/kg IM, Finadyne P.A., Schering-Plough II) for 2 days and amoxicillin (15 mg/kg IM,
Clamoxyl L.A., Pfizer) for 7 days. After implantation, animals were kept in a room, where
goats could move freely and full weight-bear on the posterior limbs during the complete postoperative period.
Chapter II – Materials and Methods
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Figure II.7.1 - Non-critical size defects drilled in the posterior femur of a goat model.
II.7.1.2. Imprinting new bone with fluorescent dyes
Fluorochrome sequential labelling of mineralizing tissues is commonly used in different
fields of clinical and basic research[65]. Fluorochrome use in animal models makes it possible
to determine the onset time and location of osteogenesis, which are the fundamental
parameters in bone tissue engineering studies.
Intravital fluorescence markers, namely, xylenol orange (90 mg/kg, Aldrich), calcein green
(10 mg/kg, Sigma) and tetracycline (25 mg/kg, Sigma) were injected subcutaneously after 2, 4
and 6 weeks of follow up, respectively, for monitoring bone formation and mineralization
along with the implantation period.
II.7.1.3. Explant retrieval and characterization
Six weeks after surgery, and 24 h after tetracycline injection, animal euthanasia was
performed using an overdose of pentobarbital sodium (Eutasil, Sanofi). The femurs were
explanted and cut into single-defect segments, which were fixed in a 4 % formaldehyde
solution (pH 7.2) (Sigma) until further usage.
II.7.1.3.1. Histological characterization
Single-defect segments were processed and embedded in glycol methacrylate solution
(Technovit 7200® VLC–Heraus Kulzer GmbH) for histologic assessment of calcified bone.
Chapter II – Materials and Methods
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Sections of 30 µm were prepared according to Donath et al. technique[66] using an ExaktCutting® System (Aparatebau GMBH, Germany).
Only the mid section of each bone segment was observed at the fluorescence microscope
(Olympus BX51) for fluorochrome detection, and then selected for histomorphometric
analysis.
Additional sections were stained with Lévai-Laczkó[67] to differentiate between native
bone and the new bone formation using a stereo microscope (Olympus SZX9).
Quantitative measurement for bone neoformation was carried out after selecting relevant
drill surrounding areas and quantified using Microimage 4.0 software.
II.7.2. Nude rat model - non-union defects
Non-unions or critical sized defects were induced in a nude rat model using human AFSCs.
The purpose of selecting the rat instead of the goat, one step backwards in the philogenetic
scale, relies on the fact that the rat is another popular subject in the TE field, and in particular
in orthopaedics research. Regardless of the size, rat’s popularity is mainly due to its low cost,
easy handling, housing, operating, and follow up than goats. The rat plays an important role in
studies of biocompatibility, including subcutaneous or intramuscular implantation of
biomaterials[17,24] to bone defect repair[68,69]. The availability of rats with distinctive
characteristics, such as athymic nude[69] or transgenic animals not easily available in larger
animal models, amplifies the study possibilities in xenograft research and, consequently the
implantation of human cells in a non-human host.
Despite the fact that AFS cells are unlikely to cause any minimal immune reaction, due to
their described non-immunogenic features, AFSCs were used in critical-sized defects in nude
rats. Several main reasons can justify this selection since in the human fetus, precursor cells,
which will lately evolve into immune cells, can be found as early as 4-8 weeks of gestation, and
we wanted to prevent any possible interference with a human cell source. Furthermore, AFSCs
present exciting properties that can be used as an alternative source to autologous
approaches, as a highly proliferative and universally available source of stem cells.
Geriatric animals were used to better mimic the regenerative process in bone, which is
particularly affected in elder patients. Thus, male athymic nude rats (36-40 week old) weighing
420-560 g (n=60) were purchased from Charles River and used in the study described in
chapter VIII. Since this study was performed in collaboration with WIRM, all procedures were
Chapter II – Materials and Methods
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performed in accordance with Wake Forest University Animal Care and Use Committee (ACUC)
approved protocols (protocol number; A07-063/A10-032).
During the entire study, adequate measures were taken to minimize any pain or discomfort
to the animals.
II.7.2.1. Implantation
Animals were randomly assigned into 5 groups (n=6 / group) as follows: 1) no-repair group
(empty defects, no implant), 2) scaffold-only group, and 3) scaffold with undifferentiated
hAFSC cell group, 4) scaffold with hAFSC osteogenically committed group, and 5) scaffold with
hAFSCs differentiated into osteoblast-like cells group.
Anesthesia was induced with 3 % isoflurane (USP, Novaplus) prior to surgical procedures.
The dorsolateral side of the right leg was shaved and sterilized with routine aseptic agent.
Under aseptic conditions, a 20 mm long incision was made on the skin over the femur of the
right hind limb. The skin and the gluteus muscle were dissected to approach the femur. A
periosteal incision was made on the periosteum of the femur, and the periosteum and the
attached muscles were elevated to expose the femur. Retracting adjacent tissues, a 3.0 mm
thick and 2.2 cm long custom-made bone plate was placed along the intact femur and was
fixed with 4 stainless steel screws using a micro drill system (BS72-4950, Harvard apparatus,
USA) to stabilize the femur after scaffold implantation.
A 5 mm bone segment in between screwed areas at both ends was removed from the
femur using a bone cutting bur. The created defects were thoroughly irrigated with sterile
saline to avoid residual bone particles at the site. Subsequently, the scaffolds and cell-scaffold
constructs from the different study groups, according to our experimental design, were
implanted to the bony defects and fixed in a press-fit manner. After inserting the
scaffolds/constructs, the muscle and the skin were closed layer by layer using 5-0 Vicryl sutures
(Ethicon, USA). The animals were maintained postoperatively according to the guideline of the
ACUC.
Radiographic 2D images of the femoral defects were obtained from each animal every 4
weeks, using C-arm equipment (Siremobil Compact L, Siemens) to monitor plate stability and
select animals for micro-CT analysis. Animals were kept under general anaesthesia during both
(x-rays and micro-CT) procedures in order to minimize stress and assure animal welfare.
Chapter II – Materials and Methods
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II.7.2.2. Explant retrieval and characterization
Rats were euthanized 4 or 16 weeks after implantation accordingly to ACUC guidelines.
Right femurs were retrieved, rinsed in PBS, placed in 10 % buffered formalin for 96 h and then
kept in 60 % ethanol until histological processing.
II.7.2.2.1. Micro-computed tomography (bone formation assessment)
Bone ingrowth studies have been investigated involving micro-CT analysis for assessment of
structural characteristics and mineralization of a tissue in continuous regeneration[70],
creating new possibilities for biomedical applications.
Rats were anaesthetized during the real time live imaging of the femoral defects, obtained
using a μ-CT equipment, Siemens MicroCAT II (Siemens Preclinical Solutions, Knoxville, TN),
where a MicroCAT II version 1.9d software was used to acquire raw data.
Scans were performed with an x-ray voltage of 70 kVp / 500 μA, and an exposure time of
1600 ms (BIN Factor of 2, 360° rotation, 360 steps, 36 micron isotropic voxel dimension).
RVA version 4.2.9 and COBRA EXXIM version 4.9.52 were used to reconstruct the raw data
into raw slice images, and then Amira version 3.1 was used for conversion to DICOM images.
Analysis was done after transfer of images to TeraRecon AquariusNET Server (TeraRecon, Inc.,
San Mateo, CA) using TeraRecon software AquariusNET version 1.8.1.6. Another software
packages used for image analysis and volumetric measurements included Mimics version 13
(Materialise, Leuven, Belgium).
II.7.2.2.2. X-rays
X-ray is a form of electromagnetic radiation that can penetrate solid objects, which has
proved useful for medical purposes, including the detection of pathological conditions of the
skeletal system as well as some disease process in soft tissues.
In this Thesis (chapter VIII), radiographic 2D images of the femoral defects were obtained
from each animal every 4 weeks up to a maximum of 16 weeks, using C-arm equipment
(Siremobil Compact L, Siemens) to monitor plate stability and select animals for micro-CT
analysis.
Chapter II – Materials and Methods
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II.7.2.2.3. Histological Characterization
Explants were decalcified using an acid solution of Immunocal® (Decal Chemical Corp), and
tissue processed in a graded series of ethanol, xylene and embedded in paraffin blocks.
Sections of 10 m were obtained using a microtome, and stained with H&E. For this purpose,
slides were stained with Mayer’s hematoxylin for 4 minutes, followed by a rinse in water to
remove excess of stain and 3 immersions in ammonia water. Then, samples were washed in
tap water for 3 minutes and stained with eosin for another 3 minutes.
Hematoxylin, a base dye, stains nuclei blue due to an affinity to nucleic acids in the cell
nucleus; eosin, an acidic dye, stains the cytoplasm pink.
After staining, samples were rinsed again in water and dehydrated before mounting the
slides with non-aqueous medium, and observed under a microscope (Imager Z1m, Zeiss)
equipped with an AxioCam MRc5 camera (Zeiss).
Immunocytochemistry was performed for bone related markers: collagen I and osteocalcin,
as well as an angiogenic marker, VEGF, in explant sections from chapter VIII.
Primary antibodies anti- collagen I (1310-01, Southern Biotech; 1:20 dilution), osteocalcin
(V-19, Santa Cruz Biotechnology, 1:100) and VEGF (147, Santa Cruz Biotechnology, 1:100) were
diluted in antibody diluent with background reducing components from Dako (Denmark).
Biotinylated secondary antibodies were obtained from Vector Lab (Burlingame, CA, USA) as
well as R.T.U. HRP/Straptavidin (SA-5704). Immunochemistry visualization was assessed by
NovaRED substrate kit (SK-4800, Vector Lab).
Specimen slides were observed under a microscope (Imager Z1m, Zeiss, Germany)
equipped with a digital camera (AxioCam MRc5).
II.7.2.2.4. Histomorphometric analysis
Histomorphometrical analysis was carried out to quantify stained areas in samples
retrieved from in vivo experiments of chapter VIII, after immunostaining for bone related
markers, namely osteocalcin and collagen I as well as vascular markers by VEGF expression. For
this purpose, an interest area was selected, and kept constant for all slides, where protein
expression was detected and analyzed using the Cell D software (analysis image processing)
and MicroImage software from Olympus Optical Co.
Chapter II – Materials and Methods
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II.8. Statistical analysis
Several statistical methods were used in this Thesis, accordingly to the experimental assay
requirements.
In chapter III, the statistical analysis was carried out using One Way ANOVA test and Tukey's
Multiple Comparison Test (p<0.05), and results presented as mean  standard deviation.
In chapter IV, results are also presented as means  standard deviation. Statistical
differences between sample groups (p<0.001 or p<0.05) were assessed by two Way ANOVA.
Statistical analysis was also carried out in chapter VII by mean  standard error of mean
using T-Test and 2-way ANOVA for in vitro and for in vivo measurements, respectively. At least
4 samples were considered in the in vitro assays (DNA, ALP, SEM) while 16 samples were
considered in vivo for each condition; A) empty drill, B) drill with SPCL scaffold, drills with SPCL
seeded with gBMSCs for either C) 1 or D) 7 days in osteogenic medium).
Results from chapter VIII, are presented as means  standard deviation in in vitro assays,
while results from histomorphometric analysis are presented as means  standard error of
mean. Two Way ANOVA test was applied to assess statistical significant differences between
sample groups followed by Bonferroni’s Multiple Comparison test (*=p<0.05).
References
1.Middleton JC, Tipton AJ: Synthetic biodegradable polymers as orthopedic devices.
Biomaterials 2000, 21(23): 2335-46.
2. Pitt CG, Gratzl MM, Kimmel GL, Surles J, Schindler A: Aliphatic polyesters II. The degradation
of poly (DL-lactide), poly (epsilon-caprolactone), and their copolymers in vivo. Biomaterials
1981, 2(4): 215-20.
3. Tsuji H, Ikada Y: Blends of Aliphatic Polyesters. II. Hydrolysis of Solution-Cast Blends from
Poly(L-lactide) and Poly (e-caprolactone) in Phosphate-Buffered Solution. Journal of Applied
Polymer Science 1998, 67: 495-415.
4. Tsuji H, Ikada Y: Blends of Aliphatic Polyesters. I. Physical Properties and Morphologies of
Solution-Cast Blends frorn Poly(DL-lactide) and Poly(e-caprolactone). Journal of Applied
Polymer Science 1996, 60: 2367-2375.
5. Bezwada RS, Jamiolkowski DD, Lee IY, Agarwal V, Persivale J, Trenkabenthin S, Erneta M,
Suryadevara J, Yang A, Liu S: Monocryl(R) Suture, a New Ultra-Pliable Absorbable
Monofilament Suture. Biomaterials 1995, 16(15): 1141-1148.
Chapter II – Materials and Methods
- 58 -
6. Shin M, Yoshimoto H, Vacanti JP: In vivo bone tissue engineering using mesenchymal stem
cells on a novel electrospun nanofibrous scaffold. Tissue Eng 2004, 10(1-2): 33-41.
7. Srouji S, Kizhner T, Suss-Tobi E, Livne E, Zussman E: 3-D Nanofibrous electrospun
multilayered construct is an alternative ECM mimicking scaffold. J Mater Sci Mater Med 2008,
19(3): 1249-55.
8. Martins A, Araujo JV, Reis RL, Neves NM: Electrospun nanostructured scaffolds for tissue
engineering applications. Nanomedicine (Lond) 2007, 2(6): 929-42.
9. Gupta D, Venugopal J, Mitra S, Giri Dev VR, Ramakrishna S: Nanostructured biocomposite
substrates by electrospinning and electrospraying for the mineralization of osteoblasts.
Biomaterials 2009, 30(11): 2085-94.
10. Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS: Multilineage differentiation of human
mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 2005,
26(25): 5158-66.
11. Salgado AJ, Coutinho OP, Reis RL: Novel starch-based scaffolds for bone tissue engineering:
cytotoxicity, cell culture, and protein expression. Tissue Eng 2004, 10(3-4): 465-74.
12. Salgado AJ, Coutinho OP, Reis RL, Davies JE: In vivo response to starch-based scaffolds
designed for bone tissue engineering applications. J Biomed Mater Res A 2007, 80(4): 983-9.
13. Elvira C, Mano JF, San Roman J, Reis RL: Starch-based biodegradable hydrogels with
potential biomedical applications as drug delivery systems. Biomaterials 2002, 23(9): 1955-66.
14. Balmayor ER, Tuzlakoglu K, Marques AP, Azevedo HS, Reis RL: A novel enzymaticallymediated drug delivery carrier for bone tissue engineering applications: combining
biodegradable starch-based microparticles and differentiation agents. J Mater Sci Mater Med
2008, 19(4): 1617-23.
15. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2(5): 243-52.
16. Gomes ME, Bossano CM, Johnston CM, Reis RL, Mikos AG: In vitro localization of bone
growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and
cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12(1): 177-88.
17. Santos TC, Marques AP, Horing B, Martins AR, Tuzlakoglu K, Castro AG, van Griensven M,
Reis RL: In vivo short-term and long-term host reaction to starch-based scaffolds. Acta
Biomater 2010, 6(11): 4314-26.
18. Leonor I, Rodrigues MT, Gomes ME, Reis RL: In Situ Functionalization of Wet-Spun Fibre
meshes for Bone Tissue Engineering: One Step Approach. J Tissue Eng Regen Med 2011, 5:
104-111.
19. Tuzlakoglu K, Pashkuleva I, Rodrigues MT, Gomes ME, van Lenthe GH, Muller R, Reis RL: A
new route to produce starch-based fiber mesh scaffolds by wet spinning and subsequent
Chapter II – Materials and Methods
- 59 -
surface modification as a way to improve cell attachment and proliferation. J Biomed Mater
Res A 2010, 92(1): 369-77.
20. Gomes ME, Holtorf HL, Reis RL, Mikos AG: Influence of the porosity of starch-based fiber
mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal
cells cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12(4): 801-9.
21. Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, Kirkpatrick CJ: Response of micro- and
macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.
Biomaterials 2007, 28(2): 240-8.
22. Oliveira JT, Crawford A, Mundy JM, Moreira AR, Gomes ME, Hatton PV, Reis RL: A cartilage
tissue engineering approach combining starch-polycaprolactone fibre mesh scaffolds with
bovine articular chondrocytes. J Mater Sci Mater Med 2007, 18(2): 295-302.
23. Gonçalves A, Costa P, Rodrigues MT, Dias IR, Reis RL, Gomes ME: Effect of flow perfusion
conditions in the chondrogenic differentiation of bone marrow stromal cells cultured onto
starch based biodegradable scaffolds Acta Biomaterialia 2011, 7: 1644-1652
24. Marques AP, Reis RL, Hunt JA: An in vivo study of the host response to starch-based
polymers and composites subcutaneously implanted in rats. Macromol Biosci 2005, 5(8): 775785.
25. Tisdel CL, Goldberg VM, Parr JA, Bensusan JS, Staikoff LS, Stevenson S: The influence of a
hydroxyapatite and tricalcium-phosphate coating on bone growth into titanium fiber-metal
implants. J Bone Joint Surg Am 1994, 76(2): 159-171.
26. Schmitz JP, Hollinger JO, Milam SB: Reconstruction of bone using calcium phosphate bone
cements: a critical review. J Oral Maxillofac Surg 1999, 57(9): 1122-1126.
27. Collins AM, Skaer NJV, Gheysens T, Knight D, Bertram C, Roach HI, Oreffo ROC, Von-Aulock
S, Baris T, Skinner J, Mann S: Bone-like resorbable silk-based scaffolds for load-bearing
osteoregenerative applications. Advanced Materials 2009, 21: 75-78.
28. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR: Biodegradable and bioactive porous
polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006, 27(18):
3413-3431.
29. Ngiam M, Liao S, Patil AJ, Cheng Z, Yang F, Gubler MJ, Ramakrishna S, Chan CK: Fabrication
of mineralized polymeric nanofibrous composites for bone graft materials. Tissue Eng Part A
2009, 15(3): 535-546.
30. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL: Electrospun silk-BMP-2 scaffolds for bone tissue
engineering. Biomaterials 2006, 27(16): 3115-3124.
31. Ogose A, Hotta T, Kawashima H, Kondo N, Gu W, Kamura T, Endo N: Comparison of
hydroxyapatite and beta tricalcium phosphate as bone substitutes after excision of bone
tumors. J Biomed Mater Res B Appl Biomater 2005, 72(1): 94-101.
32. Mauck RL, Yuan X, Tuan RS: Chondrogenic differentiation and functional maturation of
bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage 2006,
14(2): 179-189.
Chapter II – Materials and Methods
- 60 -
33. Pham QP, Sharma U, Mikos AG: Electrospinning of polymeric nanofibers for tissue
engineering applications: a review. Tissue Eng 2006, 12(5): 1197-1211.
34. Fernandez E, Gil FJ, Ginebra MP, Driessens FC, Planell JA, Best SM: Calcium phosphate bone
cements for clinical applications. Part II: precipitate formation during setting reactions. J Mater
Sci Mater Med 1999, 10(3): 177-183.
35. Pietak AM, Reid JW, Stott MJ, Sayer M: Silicon substitution in the calcium phosphate
bioceramics. Biomaterials 2007, 28(28): 4023-4032.
36. Hench LL: Genetic design of bioactive glass. Journal of the European Ceramic Society 2009,
29(7): 1257-1265.
37 Oyane A, Kawashita M, Nakanishi K, Kokubo T, Minoda M, Miyamoto T, Nakamura T:
Bonelike apatite formation on ethylene-vinyl alcohol copolymer modified with silane coupling
agent and calcium silicate solutions. Biomaterials 2003, 24(10): 1729-1735.
38 Kokubo T, Takadama H: How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials 2006, 27(15): 2907-2915.
39. Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T: Solutions able to reproduce in vivo
surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res 1990, 24(6):
721-34.
40. Darling EM, Topel M, Zauscher S, Vail TP, Guilak F: Viscoelastic properties of human
mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes. J
Biomech 2008, 41(2): 454-464.
41 Mano JF, Reis RL, Cunha AM. Dynamic mechanical analysis in polymers for biomedical
applications. In: Reis RL, Cohn D, eds. Polymer based systems on tissue engineering,
replacement and Regeneration. Dordrecht, The Netherlands: Kluwer Academic Publishers,
2002: 139.
42. Mauney JR, Volloch V, Kaplan DL: Role of adult mesenchymal stem cells in bone tissue
engineering applications: current status and future prospects. Tissue Eng 2005, 11(5-6): 787802.
43. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem
cells. Science 1999, 284(5411): 143-7.
44. Tuan RS, Boland G, Tuli R: Adult mesenchymal stem cells and cell-based tissue engineering.
Arthritis Res Ther 2003, 5(1): 32-45.
45. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC,
Snyder EY, Yoo JJ, Furth ME, Soker S, Atala A: Isolation of amniotic stem cell lines with
potential for therapy. Nat Biotechnol 2007, 25(1): 100-106.
46. Pearce AI, Richards RG, Milz S, Schneider E, Pearce SG: Animal models for implant
biomaterial research in bone: a review. Eur Cell Mater 2007, 13: 1-10.
Chapter II – Materials and Methods
- 61 -
47. Kruyt MC, Dhert WJ, Yuan H, Wilson CE, van Blitterswijk CA, Verbout AJ, de Bruijn JD: Bone
tissue engineering in a critical size defect compared to ectopic implantations in the goat. J
Orthop Res 2004, 22(3): 544-51.
48. Li X, Feng Q, Liu X, Dong W, Cui F: Collagen-based implants reinforced by chitin fibres in a
goat shank bone defect model. Biomaterials 2006, 27(9): 1917-1923.
49. Zhu L, Liu W, Cui L, Cao Y: Tissue-engineered bone repair of goat-femur defects with
osteogenically induced bone marrow stromal cells. Tissue Eng 2006, 12(3): 423-433.
50. Bellows CG, Heersche JN, Aubin JE: Determination of the capacity for proliferation and
differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Dev
Biol 1990, 140(1): 132-138.
51. Aubin JE: Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for
heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem 1999, 72(3): 396410.
52. Maniatopoulos C, Sodek J, Melcher AH: Bone formation in vitro by stromal cells obtained
from bone marrow of young adult rats. Cell Tissue Res 1988, 254(2): 317-330.
53. Franceschi RT, Iyer BS, Cui YQ: Effects of Ascorbic-Acid on Collagen Matrix Formation and
Osteoblast Differentiation in Murine Mc3T3-E1 Cells. Journal of Bone and Mineral Research
1994, 9(6): 843-854.
54. Lian JB, Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation
of bone cell development and tissue formation. Crit Rev Oral Biol Med 1992, 3(3): 269-305.
55. Puchtler H, Meloan SN, Terry MS: On the history and mechanism of alizarin and alizarin red
S stains for calcium. J Histochem Cytochem 1969, 17(2): 110-124.
56. Boskey A, Pleshko Camacho N: FT-IR imaging of native and tissue-engineered bone and
cartilage. Biomaterials 2007, 28(15): 2465-2478.
57. Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, Collart D, Zambetti G, Stein G:
Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression. Proc
Natl Acad Sci U S A 1989, 86(4): 1143-1147.
58. Lian JB, Stein GS, Stewart C, Puchacz E, Mackowiak S, Aronow M, Vondeck M, Shalhoub V:
Osteocalcin - Characterization and Regulated Expression of the Rat Gene. Connective Tissue
Research 1989, 21(1-4): 391-399.
59. Byers BA, Garcia AJ: Exogenous Runx2 expression enhances in vitro osteoblastic
differentiation and mineralization in primary bone marrow stromal cells. Tissue Eng 2004,
10(11-12): 1623-1632.
60. Rosenberg L: Chemical basis for the histological use of safranin O in the study of articular
cartilage. J Bone Joint Surg Am 1971, 53(1): 69-82.
61. Jones CW, Smolinski D, Keogh A, Kirk TB, Zheng MH: Confocal laser scanning microscopy in
orthopaedic research. Prog Histochem Cytochem 2005, 40(1): 1-71.
Chapter II – Materials and Methods
- 62 -
62. Oliveira JM, Rodrigues MT, Silva SS, Malafaya PB, Gomes ME, Viegas CA, Dias IR, Azevedo
JT, Mano JF, Reis RL: Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral
tissue-engineering applications: Scaffold design and its performance when seeded with goat
bone marrow stromal cells. Biomaterials 2006, 27(36): 6123-37.
63. Mehta R, Chowdhury P, Ali N: Scanning electron microscope studies of bone
samples:influence of simulated microgravity. Nuclear instruments and methods in physics
research B 2007, 261: 908-912.
64. Hallfeldt KK, Stutzle H, Puhlmann M, Kessler S, Schweiberer L: Sterilization of partially
demineralized bone matrix: the effects of different sterilization techniques on osteogenetic
properties. J Surg Res 1995, 59(5): 614-620.
65. van Gaalen SM, Kruyt MC, Geuze RE, de Bruijn JD, Alblas J, Dhert WJ: Use of fluorochrome
labels in in vivo bone tissue engineering research. Tissue Eng Part B Rev 2010, 16(2): 209-217.
66. Donath K. Preparation of histologic sections by the cutting-grinding technique for hard
tissue and other material not suitable to be sectioned by routine methods. In: Exakt-KulzerPublication, ed. Equipment and methodical performance, 1995.
67. Jeno L, Geza L: A simple differential staining method for semi-thin sections of ossifying
cartilage and bone tissues embedded in epoxy resin. Mikroskopie 1975, 31(1-2): 1-4.
68. Vogelin E, Jones NF, Huang JI, Brekke JH, Lieberman JR: Healing of a critical-sized defect in
the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone
morphogenetic protein. J Bone Joint Surg Am 2005, 87(6): 1323-1331.
69. Jager M, Degistirici O, Knipper A, Fischer J, Sager M, Krauspe R: Bone healing and migration
of cord blood-derived stem cells into a critical size femoral defect after xenotransplantation. J
Bone Miner Res 2007, 22(8): 1224-1233.
70. Jones AC, Arns CH, Sheppard AP, Hutmacher DW, Milthorpe BK, Knackstedt MA:
Assessment of bone ingrowth into porous biomaterials using MICRO-CT. Biomaterials 2007,
28(15): 2491-2504.
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SECTION III
SCAFFOLD DESIGN AND CHARACTERIZATION
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Chapter III
SYNERGISTIC EFFECT OF SCAFFOLD COMPOSITION
AND DYNAMIC CULTURING ENVIRONMENT IN MULTI-LAYERED SYSTEMS
FOR BONE TISSUE ENGINEERING
This chapter is based on the following publication:
Rodrigues MT, Martins A, Dias IR, Viegas CAA, Neves NM, Gomes ME, and Reis RL,
Synergistic effect of scaffold composition and dynamic culturing environment in multilayered systems for bone tissue engineering, accepted for publication in Journal of
Tissue Engineering and Regenerative Medicine
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Chapter III
SYNERGISTIC EFFECT OF SCAFFOLD COMPOSITION AND DYNAMIC CULTURING ENVIRONMENT
IN MULTI-LAYERED SYSTEMS FOR BONE TISSUE ENGINEERING
III.
III.1. Abstract
Bone extracellular matrix (ECM) is composed of mineralized collagen fibrils which support
biological apatite nucleation that participates in bone outstanding properties. Understanding
and mimicking morphological and physiological parameters of bone at a biological scale is a
major challenge in tissue engineering scaffolding. Emergent new (nano)technologies offer the
possibility to improve scaffold design that may be critical to obtain highly functional tissue
substitutes for bone applications.
This study aims to develop novel biodegradable composite scaffolds made of tricalcium
phosphate (TCPs) and electrospun nanofibers of poly(ε-caprolactone) (PCL), in order to
combine the osteoconductivity of TCPs with the biocompatibility and elasticity of PCL, thereby
mimicking bone structure and composition. We hypothesized that scaffolds with such
structure/composition would stimulate the proliferation and differentiation of bone marrow
mesenchymal stromal cells (BMSCs) towards the osteogenic phenotype.
Composite scaffolds were developed by electrospining using consecutive stacked layers of
PCL and TCPs, and then characterized by Fourier Transform Infrared spectroscopy, X-Ray
diffraction and scanning electronic microscopy. To assess cellular behavior, goat bone marrow
mesenchymal stromal cells (gBMSCs) were seeded onto composite scaffolds and cultured in
either static or dynamic conditions, using both basal and osteogenic differentiation medium
during 7, 14 or 21 days. Cellular proliferation was quantified and osteogenic differentiation
was confirmed by alkaline phosphatase activity, alizarin red staining and
immunocytochemistry for osteocalcin and type I collagen. The results suggest that these PCLTCP scaffolds provide a 3D support for gBMSCs proliferation and osteogenic differentiation,
including the production of ECM matrix. In fact, the presence of TCPs is shown to positively
stimulate the osteogenic process, especially under dynamic conditions, where the
environment induced by PCL-TCP scaffolds is sufficient to promote osteogenic differentiation
even in basal medium conditions. The enhancement of the osteogenic potential in dynamic
conditions evidences the synergistic effect of scaffolds composition and dynamic stimulation in
gBMSCs osteogenic differentiation.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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III.2. Introduction
Bone fracture defects, bone loss, infection or bone tumoral resections are serious problems
requiring for bone surgery, and the implantation of auto- or allo-grafts has long been used as a
standard procedure for bone reconstruction. Nevertheless, graft strategies are associated with
significant disadvantages, namely tissue morbidity at the harvesting site, donor scarcity and
immune response and disease transmission risks. To overcome these limitations, the challenge
stands for designing novel tissue engineered products.
The selection of a biomaterial plays a key role in the development of an engineered product
aimed at tissue regeneration and repair, as materials can tailor the biophysical and
biochemical milieu at a molecular level, directing cellular behavior and function.
Synthetic polymers, such as poly ε-caprolactone (PCL) are typically more versatile in
tailoring a wide range of properties and structure features. Also, they represent a reliable
source of raw materials, with adequate mechanical properties, and avoid immunogenicity
problems[1, 2]. Nevertheless, polymers lack properties to stimulate biological functions, such
as osteoconductivity, and cell bioactivity[3]. Concerning these properties, ceramic materials
offer advantages over polymers, but their high modulus yet brittle behavior[4] limit their
processability into 3D scaffolds[5, 6].
Designing scaffolds using nanotechnology tools aims at mimicking the extracellular matrix
(ECM) structure and complexicity at a biological scale, as bone natural matrix is mainly
composed of an intricate nanofiber structure of nonstoichometric hydroxyapatite integrated in
collagen fibers. At a submicron level, this organic-inorganic combination confers the intrinsic,
unique biomechanical and functional properties of bone 3D architecture.
The products of electrospining technology are micro- and nano-structured scaffolds made
of an ultra-fine and continuous fiber network with variable pore-size distribution, high microporosity and high surface-to-volume ratio, morphologically similar to natural ECM[7]. Several
materials, including synthetic- and natural-origin polymers[8-16] and proteins[9, 10, 12, 17,
18], have been successfully electrospun into nanofiber scaffolds, which interacted positively
with intercellular communications by sustaining cell adhesion, proliferation and differentiation
towards the osteogenic phenotype both in vitro[9, 11, 14, 16, 18] and in vivo[9, 11, 15].
Considering the properties and flexibility of electrospun nanofiber meshes, the
incorporation of biologically active factors, like matrix proteins[19] or minerals as calcium
phosphates[8, 11-13], either in the spun fibers or as coatings, makes them attractive to
improve scaffold designs for bony functional substitutes. Moreover an integrated composite
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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scaffold of a polymer with a ceramic material offers a chemical environment that would
resemble the organic and inorganic components of bone native matrix[20].
In this study, we formulated a novel multilayer composite structure combining the
osteoinductive properties of beta-tricalcium phosphate powder with a degradable polymer.
PCL was selected due to its degradability and cellular compatibility, and because this material
has been widely studied for biomedical applications[8, 9, 13-15]. Several studies suggested
hydroxyapatite as the ceramic material preferred for bone strategies[8, 12, 19]. Nevertheless,
the osteoconductive and bioreabsorvable nature of beta-tricalcium phosphate together with
an earlier incorporation into surrounding bone in vivo may be advantageous to hydroxyapatite,
which remains unremodeled despite long periods upon implantation[21].
The design of these 3D fibrous scaffolds is expected to enhance the synergistic effects of
ceramic (osteoconductivity and bioactivity) and biopolymer features (elasticity and shape
control). The possibility to combine and stack layers in such simple and thin structures can
improve their tridimensionality, which has been described to be critical in structure, function
and morphology of body tissues.
The limited diffusion in standard static culture environments is another important issue to
considering when designing bone regeneration strategies, since this may constrain cell
proliferation and uniform distribution in tissue engineered constructs[10, 22]. Therefore, we
also propose to evaluate the effect of dynamic culturing in the osteogenic differentiation
process of goat bone marrow mesenchymal stromal cells (gBMSCs), cultured in the multi-layer
scaffolds, either in the presence of basic or osteogenic differentiation media.
III.3. Materials and Methods
III.3.1. Development of the nanofibrous multilayered composite scaffolds
The ceramic material, beta-tricalcium phosphate (-TCP), was obtained from a solid state
reaction between stoichiometric amounts of calcium phosphate dibasic anhydrous (Fluka) and
calcium carbonate (Sigma) followed by a 24 h sinterization at 800 ºC. The powders were handsieved with stainless steel sieves (mesh 225 - 106 μm). A polymeric solution of 17 % (w/v)
polycaprolactone (PCL) in a mixture of chloroform (Aldrich) and N,N-dymethylformamide
(Aldrich) at a 7:3 ratio was processed by electrospinning technique. This solution was
electrospun at 9-10 kV, with a flow rate of 1.0 mL/h collecting a random fiber mesh (20 cm
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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away from the collector) on a flat aluminium foil. Composite scaffolds were then developed by
assembling three stacked layers of electrospun PCL fibers and TCPs at a ratio of 0.5 g TCP per
PCL mesh. 0.25 g of TCP powder was dispersed between each two consecutive PCL layers. The
resulting PCL-TCP scaffolds, as well as TCPs powder and PCL meshes alone, were characterized
by FTIR, XRD and SEM, as described below.
III.3.2. Characterization of the composite scaffolds
III.3.2.1. Thin-film X-ray diffraction (TF-XRD)
TF-XRD patterns were recorded on a Philips X’Pert MPD (Philips, The Netherlands)
diffractometer using CuKα radiation to analyze the surface composition of the specimens.
III.3.2.2. Fourier Transformed Infrared Spectroscopy with attenuated total reflectance
(FTIR-ATR)
FTIR-ATR was performed using IRPrestige-21 (Shimadzu, Japan) equipment. Spectra were
taken with a resolution of 2 cm-1 and averaged over 64 scans, covering the wave number range
of 4400-400 cm-1.
III.3.2.3. Scanning electron microscopy (SEM)
SEM analysis was performed to characterize the morphology of the developed structures
using Hitachi S-2600N (Hitachi Science Systems, Ltd) equipment. Prior to any SEM
observations, sample surfaces were gold sputtered.
III.3.3. In vitro culture of bone marrow mesenchymal stromal cells onto nanofibrous
scaffolds
To assess cellular behavior, scaffolds were cut into 5 mm diameter discs, and then sterilized
by means of two 30 minute-cycle of UV irradiation.
PCL nanofiber meshes without TCPs were considered as controls of the experiment.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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Goat bone marrow mesenchymal stromal cells (gBMSCs) were harvested from iliac crests of
adult goats and expanded in basal medium composed of DMEM (Dulbecco Modified Eagle
Medium, Sigma-Aldrich), supplemented with 10 % FBS (Invitrogen) and 1 % antibioticantimicotic solution (A/A) (Gibco), and then seeded onto the PCL-TCP composites at a
concentration of 5.0 x104 cells/mesh or scaffold. After the seeding, cell-scaffold constructs
were maintained in non treated 48 multi-well plates (Costar) in basal medium for 24 h and
then cultured under static or dynamic conditions in either basal or osteogenic differentiation
medium during 7, 14 or 21 days.
Dynamic conditions were provided by an orbital shaker (Digisystem) under constant mild
agitation (60 rpm), working permanently until the end of the experiment and kept in the same
CO2 incubator where constructs under static conditions were also cultured.
Osteogenic medium was prepared with -MEM (Minimal Essential Medium Eagle alpha
modification, Sigma-Aldrich), 10 % FBS and 1 % A/A and osteogenic supplements namely
ascorbic acid (50 g/ml) (Sigma-Aldrich), dexamethasone (10-8 M) (Sigma-Aldrich) and glycerophosphate (10 mM) (Sigma-Aldrich). In all conditions, cell culture medium was changed
twice a week.
III.3.3.1. Cell proliferation and osteogenic differentiation in multi-stacked nanofibrous
scaffolds
Cell proliferation was assessed by a DNA quantification assay while the osteogenic
differentiation was assessed by ALP quantification, alizarin red staining and
immunocytochemistry (ICC) for osteocalcin (OCa) and type I collagen.
III.3.3.1.1. DNA assay
Proliferation potential of gBMSCs seeded onto the developed scaffolds was considered by
double strand DNA quantification (dsDNA). For this purpose, a fluorimetric dsDNA
quantification kit (PicoGreen, Molecular Probes) was used.
The fluorescence was read using a microplate ELISA reader (BioTek) at an excitation of
485/20 nm and an emission of 528/20 nm.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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III.3.3.1.2. ALP assay
ALP activity was measured in this study, as an osteogenic cell marker. A substrate solution
consisting of 0.2 % (wt/v) p-nytrophenyl phosphate (Sigma) in a substrate buffer with 1 M
diethanolamine HCl (Merck), at pH 9.8 was added to each sample. The plate was incubated in
the dark for 45 minutes at 37 ºC, followed by the addition of a stop solution (2 M NaOH
(Panreac) plus 0.2 mM EDTA (Sigma)). Samples and standards (the latter prepared with pnytrophenol (10 µmol.ml-1) (Sigma)) were analyzed in triplicates. The absorbance was read
using a microplate ELISA reader (BioTek) at 405 nm.
III.3.3.1.3. Alizarin Red staining
Cell-scaffold constructs were removed from culture and fixed in 4 % formalin (Sigma)
solution overnight at 4 ºC. A 2 % Alizarin Red solution (Sigma) was prepared (pH adjusted to
4.1-4.3) to stain calcium ions that might be present resultant from mineral ECM produced by
cells. Constructs were stained with alizarin red solution for about 2 minutes, washed with PBS
until removal of excess of stain and let dry.
III.3.3.1.4. Immunocytochemistry
After overnight fixation with 4 % formalin, samples were kept in PBS until
immunocytochemistry (ICC) analysis. ICC was performed according to R.T.U. Vectastain
Universal Elite ABC kit (PK 7200) kit from VectorLabs, using R.T.U Normal Horse Serum to avoid
unspecific reactions, a biotinilated secondary antibody (R.T.U. Biotinylated Universal Antibody)
and a Peroxidase Substract Kit (Vector SK-4100). Primary antibodies Collagen I (MAB339,
Chemicon) and Osteocalcin (Ab13418, Abcam) were prepared using a 1/100 dilution.
III.3.4. Statistical Analysis
Statistical analysis was carried out using One Way ANOVA test and Tukey's Multiple
Comparison Test (p<0.05). All results are presented as mean  standard deviation.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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In the present study controls of the experiment were considered using PCL nanofiber
meshes without TCPs.
III.4. Results and Discussion
Goat model is a popular animal model for orthopedics, whose stem cells can be applied to
autologous approaches in bone TE. Previous studies have demonstrated the potential of
gBMSCs to differentiate into the osteogenic lineage[23-25]. Furthermore, PCL nanofiber
meshes, used in this study as positive controls for determining the applicability and
improvement of multi-layered PCL-TCP scaffolds in bone-related strategies, have shown to
promote cellular adhesion, proliferation and osteogenic differentiation of mesenchymal stem
cells[26].
Multi-layered composite scaffolds were achieved through the dispersion of TCP onto
electrospun PCL fibers. The calcium phosphate obtained from the chemical reaction presented
a rhombohedrical structure, typical of -TCP, and the vibrational bands associated to the
phosphate groups, as demonstrated by FTIR (Figure III.4.1) and XRD (Figure III.4.2) data.
Figure III.4.1 - FTIR-ATR analysis of multi-layered scaffolds. Membranes of PCL nanofiber mesh
(PCL) were used as controls.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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Figure III.4.2 - XRD analysis of multi-layered PCL-TCP scaffolds. Membranes of PCL nanofiber
mesh (PCL) were used as controls.
SEM observation indicated a dispersion of TCPs granules within the electrospun PCL
nanofibers structure, in the produced composite scaffolds (Figure III.4.3).
Figure III.4.3 – SEM analysis of multilayer scaffolds; the upper SEM micrographs refer to the
PCL-TCP multilayer scaffolds while the lower pictures correspond to the control of PCL
nanofiber mesh (left to right: 100 x, 300 x and 1000 x magnifications).
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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A random distribution was observed not only with TCPs granules but also in nanofibers of
the mesh-like stratified structure. Adittionally, TCPs mineral particles were integrated within
the structure of multi-layered nanofiber scaffolds.
In our study, gBMSCs attached, and proliferated into the PCL-TCP scaffolds, both in static
and dynamic conditions, as well as in basal and osteogenic culture conditions. Similar results
were achieved in control PCL meshes.
In terms of proliferation, gBMSCs showed two different behaviors (p>0.05); cell
proliferation in PCL meshes increased with time in culture, while in PCL-TCP constructs, cells
increased up to 14 days in basal or osteogenic culture conditions and then, the cell number
decreased at week 3 (Figure III.4.4). This decreasing tendency of proliferation in PCL-TCP
scaffolds may be a result of the osteogenic differentiation process, undertaken by the gBMSCs.
Some studies indicate an inverse relationship between proliferation and differentiation of
osteoprogenitor cells during bone formation[27].
Figure III.4.4 – gBMSCs proliferation given by DNA quantification was assessed onto PCL-TCP
scaffolds and PCL membranes (control) after 7, 14 or 21 days in basal or osteogenic media in
either static (st) or dynamic (dyn) conditions. Symbols *, **, and *** denote study groups with
statistically significant differences (p<0.05), as using One Way ANOVA method.
ALP activity levels (Figure III.4.5) were not detected for samples cultured for 7 or 14 days,
probably due to low basal concentrations of ALP enzyme in the samples.
The ALP/DNA ratio in PCL meshes cultured under dynamic conditions using osteogenic
media is very similar to that found for PCL-TCP constructs cultured under static conditions in
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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the same medium, after 3 weeks in culture. Nevertheless, ALP is highly expressed in PCL-TCP
constructs cultured under dynamic culture conditions in osteogenic medium as compared to
all the remaining study groups (p<0.05). ALP activity data indicated a positive and synergistic
effect of TCPs and mechanical stimulation on the formation of ECM by osteogenically
differentiated gBMSCs.
Figure III.4.5 – gBMSCs osteogenic differentiation given by ALP/DNA ratio was assessed onto
PCL-TCP scaffolds and PCL membranes (control) after 7, 14 or 21 days in basal or osteogenic
media in either static (st) or dynamic (dyn) conditions. Symbols *, and ** denote st groups
with statistically significant differences (p<0.05), as using One Way ANOVA method.
Furthermore, to confirm gBMSCs differentiation onto PCL-TCP scaffolds, osteogenic
phenotype characterization was assessed by immunocytochemistry analysis for type I collagen
and osteocalcin (OCa), and alizarin red staining for calcium detection, after 21 days in culture
(Figure III.4.6).
Collagen I is the major protein present in bone ECM. All PCL-TCP scaffolds seeded with
gBMSCs were positively stained for Collagen I, although a more intense immunostain was
observed in constructs cultured under dynamic conditions (Figure III.4.6). In gBMSC-PCL
meshes (control), collagen I was more intense in basal medium culture. These results showed
that a collagen I-rich ECM is being produced by gBMSCs either in PCL-TCP scaffolds or PCL
meshes.
In addition to a collagen matrix, a calcified milieu is an important part of the native bone
tissue. Osteocalcin (OCa) is a non-collagenous protein, produced by osteoblasts that
participates in the matrix mineralization of bone[28]. OCa was found to be present in gBMSCPCL-TCP constructs under dynamic environment, while in static cultures, OCa was only
detected in the presence of constructs cultured in osteogenic medium. Furthermore, in
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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gBMSC-PCL meshes (control), OCa was only detected under static conditions in osteogenic
differentiation medium (Figure III.4.6).
Figure III.4.6 – Osteogenic phenotype characterization of gBMSCs seeded onto PCL-TCP
scaffolds after 21 days in culture with basal or osteogenic media, either in static or dynamic
conditions. Immunocytochemistry (ICC) was performed for Collagen I and Osteocalcin, as well
as Alizarin Red staining. Insets represent PCL meshes (control) under the same conditions.
Alizarin red staining was assessed to detect calcium ions, essential for the ECM
mineralization. After 21 days in culture, gBMSC-PCL-TCP constructs exhibited a stronger stain
when compared to gBMSC-PCL membrane controls (Figure III.4.6). The stain was more intense
under dynamic conditions, both in basal or osteogenic media, as well as in constructs cultured
in osteogenic differentiation medium in a static environment, indicating the production of
calcified ECM.
OCa and alizarin red staining followed a similar pattern in gBMSC-PCL-TCP constructs, which
confirms the formation of a mineralized ECM, as OC is thought to bind to hydroxyapatite in a
calcium-dependent manner[29], during the natural development of bone.
Even if in all culture conditions, gBMSC-PCL-TCP constructs expressed osteogenic markers
to some extent, the combination of these markers was stronger in dynamic conditions,
especially for Alizarin Red staining. It is important to highlight that mineralization nodules
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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(observed as darker red spots) are visible only in the dynamic culture conditions and in the
multilayer composite scaffolds, corroborating the previous results of ALP activity. The fact that
ALP, an enzyme associated to ECM development and maturation[27], is not expressed by cells
seeded onto PCL-TCP constructs in basal/dynamic environment is quite interesting, since these
cells are able to produce calcified ECM (Figure III.4.6). Salasznyk et al. reported the bone
isoform of ALP is found only in osteogenic supplemented mesenchymal stem cells[30], which
justifies the basal ALP activity levels in basal/dynamic environment, despite the mineralization
of ECM. In their study, cells in basal medium did not differentiate when ALP was not
expressed; but the presence of PCL-TCP constructs could stimulate gBMSCs to select a
different pathway in the absence of osteogenic medium that could be also encouraged by the
dynamic environment, as calcified ECM is reduced in basal/static conditions. Furthermore, in
the presence of PCL-TCP scaffolds, gBMSCs achieved osteogenic differentiation and ECM
mineralization even in the absence of osteogenic supplements after 3 weeks in culture.
Mechanical stimuli are required to maintain a healthy bone, and described to induce the
up-regulation of osteogenic marker genes and increase matrix mineralization of the ECM in the
presence of substrates coated with ECM proteins[31]. Furthermore, cyclic mechanical
stretching was also described to participate in an increased ALP activity and mineralized matrix
deposition[31].
In our study, the particular structure of PCL-TCP scaffolds mimicking bone natural ECM,
both in structure and composition, stimulated gBMSCs to produce ECM matrix (collagen I and
OCa). Under dynamic conditions, the environment induced by PCL-TCP scaffolds is sufficient to
promote osteogenic differentiation even in basal medium conditions.
In summary, PCL-TCP scaffolds participate in the osteogenic process of gBMSCs, even in
basal medium and static conditions. Nevertheless, the osteogenic potential is enhanced in
dynamic environments, evidencing the synergistic effect of TCPs and mild mechanical
stimulation in gBMSCs osteogenic phenotype.
III.5. Conclusions
The production of multi-layered PCL-TCP scaffolds was successfully achieved as well as the
in vitro assessment of their application aiming at bone tissue strategies. Moreover, considering
that bone ECM is essentially an organic-inorganic composite and nano-scaled organized, the
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
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developed multi-layered composite can be a promising system in the regenerative medicine
field.
The results obtained suggest that the combination of PCL with calcium phosphates
generated scaffolds with improved capacity to promote gBMSCs adhesion, proliferation, and
expression of osteogenic phenotype, when compared to the synthetic polymer alone.
Furthermore, the dynamic environment positively affected the gBMSCs behavior, and
enhanced gBMSCs phenotypic expression and mineralized matrix synthesis. Interestingly, the
synergistic effect of mild agitation and PCL-TCP scaffolds stimulated gBMSCs differentiation
and production of a calcified ECM, even in the absence of osteogenic supplements from the
culture medium.
The development of PCL nanofibers with ceramic materials, in a multi-layered structure, is
an innovative methodology with great potential for the development of more adequate
scaffolds for bone tissue engineering approaches.
III.6. References
1. Tsuji H, Ikada Y: Blends of aliphatic polyesters .1. Physical properties and morphologies of
solution-cast blends from poly(DL-lactide) and poly(epsilon-caprolactone). Journal of Applied
Polymer Science 1996, 60: 2367-2375.
2. Bezwada RS, Jamiolkowski DD, Lee IY, Agarwal V, Persivale J, Trenkabenthin S, Erneta M,
Suryadevara J, Yang A, Liu S: Monocryl(R) Suture, a New Ultra-Pliable Absorbable
Monofilament Suture. Biomaterials 1995, 16: 1141-1148.
3. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR: Biodegradable and bioactive porous
polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006, 27:
3413-3431.
4. Collins AM, Skaer NJV, Gheysens T, Knight D, Bertram C, Roach HI, Oreffo ROC, Von-Aulock S,
Baris T, Skinner J, et al.: Bone-like resorbable silk-based scaffolds for load-bearing
osteoregenerative applications. Advanced Materials 2009, 21: 75-78.
5. Yang S, Leong KF, Du Z, Chua CK: The design of scaffolds for use in tissue engineering. Part I.
Traditional factors. Tissue Eng 2001, 7: 679-689.
6. Chen GP, Ushida T, Tateishi T: Scaffold design for tissue engineering. Macromolecular
Bioscience 2002, 2: 67-77.
7. Martins A, Reis RL, Neves NM: Electrospinning: processing technique for tissue engineering
scaffolding. International Materials Reviews 2008, 53: 257-274.
C
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
- 80 -
8. Gupta D, Venugopal J, Mitra S, Giri Dev VR, Ramakrishna S: Nanostructured biocomposite
substrates by electrospinning and electrospraying for the mineralization of osteoblasts.
Biomaterials 2009, 30: 2085-2094.
9. Srouji S, Kizhner T, Suss-Tobi E, Livne E, Zussman E: 3-D Nanofibrous electrospun
multilayered construct is an alternative ECM mimicking scaffold. J Mater Sci Mater Med 2008,
19: 1249-1255.
10. Hosseinkhani H, Hosseinkhani M, Tian F, Kobayashi H, Tabata Y: Ectopic bone formation in
collagen sponge self-assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion
culture bioreactor. Biomaterials 2006, 27: 5089-5098.
11. Ko EK, Jeong SI, Rim NG, Lee YM, Shin H, Lee BK: In vitro osteogenic differentiation of
human mesenchymal stem cells and in vivo bone formation in composite nanofiber meshes.
Tissue Eng Part A 2008, 14: 2105-2119.
12. Ngiam M, Liao S, Patil AJ, Cheng Z, Yang F, Gubler MJ, Ramakrishna S, Chan CK: Fabrication
of mineralized polymeric nanofibrous composites for bone graft materials. Tissue Eng Part A
2009, 15: 535-546.
13. Araujo JV, Martins A, Leonor IB, Pinho ED, Reis RL, Neves NM: Surface controlled
biomimetic coating of polycaprolactone nanofiber meshes to be used as bone extracellular
matrix analogues. J Biomater Sci Polym Ed 2008, 19: 1261-1278.
14. Li WJ, Tuli R, Huang X, Laquerriere P, Tuan RS: Multilineage differentiation of human
mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 2005, 26:
5158-5166.
15. Shin M, Yoshimoto H, Vacanti JP: In vivo bone tissue engineering using mesenchymal stem
cells on a novel electrospun nanofibrous scaffold. Tissue Eng 2004, 10: 33-41.
16. Xin X, Hussain M, Mao JJ: Continuing differentiation of human mesenchymal stem cells and
induced chondrogenic and osteogenic lineages in electrospun PLGA nanofiber scaffold.
Biomaterials 2007, 28: 316-325.
17. Li M, Mondrinos MJ, Gandhi MR, Ko FK, Weiss AS, Lelkes PI: Electrospun protein fibers as
matrices for tissue engineering. Biomaterials 2005, 26: 5999-6008.
18. Sefcik LS, Neal RA, Kaszuba SN, Parker AM, Katz AJ, Ogle RC, Botchwey EA: Collagen
nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human
adipose stem cells. J Tissue Eng Regen Med 2008, 2: 210-220.
19. Li C, Vepari C, Jin HJ, Kim HJ, Kaplan DL: Electrospun silk-BMP-2 scaffolds for bone tissue
engineering. Biomaterials 2006, 27: 3115-3124.
20. Zhang YZ, Su B, Venugopal J, Ramakrishna S, Lim CT: Biomimetic and bioactive nanofibrous
scaffolds from electrospun composite nanofibers. Int J Nanomedicine 2007, 2: 623-638.
21. Ogose A, Hotta T, Kawashima H, Kondo N, Gu W, Kamura T, Endo N: Comparison of
hydroxyapatite and beta tricalcium phosphate as bone substitutes after excision of bone
tumors. J Biomed Mater Res B Appl Biomater 2005, 72: 94-101.
Chapter III – Synergistic effect of scaffold composition and dynamic culturing environment in multi-layered systems
for bone tissue engineering
- 81 -
22. Yu X, Botchwey EA, Levine EM, Pollack SR, Laurencin CT: Bioreactor-based bone tissue
engineering: the influence of dynamic flow on osteoblast phenotypic expression and matrix
mineralization. Proc Natl Acad Sci U S A 2004, 101: 11203-11208.
23. Oliveira JM, Rodrigues MT, Silva SS, Malafaya PB, Gomes ME, Viegas CA, Dias IR, Azevedo
JT, Mano JF, Reis RL: Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral
tissue-engineering applications: Scaffold design and its performance when seeded with goat
bone marrow stromal cells. Biomaterials 2006, 27: 6123-6137.
24. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5: 41-49.
25. Leonor I, Rodrigues MT, Gomes ME, Reis RL: In Situ Functionalization of Wet-Spun Fibre
meshes for Bone Tissue Engineering: One Step Approach. J Tissue Eng Regen Med 2011, 5:
104-111.
26. Binulal NS, Deepthy M, Selvamurugan N, Shalumon KT, Suja S, Mony U, Jayakumar R, Nair
SV: Role of Nanofibrous Poly(Caprolactone) Scaffolds in Human Mesenchymal Stem Cell
Attachment and Spreading for In Vitro Bone Tissue Engineering-Response to Osteogenic
Regulators. Tissue Eng Part A 2010, 16: 393-404.
27. Lian JB, Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation
of bone cell development and tissue formation. Crit Rev Oral Biol Med 1992, 3: 269-305.
28. Lian JB, Stein GS, Stewart C, Puchacz E, Mackowiak S, Aronow M, Vondeck M, Shalhoub V:
Osteocalcin - Characterization and Regulated Expression of the Rat Gene. Connective Tissue
Research 1989, 21: 391-399.
29. Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, Collart D, Zambetti G, Stein G:
Structure of the Rat Osteocalcin Gene and Regulation of Vitamin-D-Dependent Expression.
Proceedings of the National Academy of Sciences of the United States of America 1989, 86:
1143-1147.
30. Salasznyk RM, Klees RF, Westcott AM, Vandenberg S, Bennett K, Plopper GE: Focusing of
gene expression as the basis of stem cell differentiation. Stem Cells Dev 2005, 14: 608-620.
31. Huang CH, Chen MH, Young TH, Jeng JH, Chen YJ: Interactive effects of mechanical
stretching and extracellular matrix proteins on initiating osteogenic differentiation of human
mesenchymal stem cells. J Cell Biochem 2009, 108: 1263-1273.
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Chapter IV
FUNCTIONAL BIODEGRADABLE SCAFFOLDS FOR
BONE TISSUE ENGINEERING: BIOACTIVITY PROFILE AND OSTEOGENIC
DIFFERENTIATION OF MARROW MESENCHYMAL STROMAL CELLS
This chapter is based on the following publication:
Rodrigues MT, Gröen N, Leonor I, Carvalho PP, Caridade S, Mano JF, Dias IR, van Blitterswijk
CA, Gomes ME, and Reis RL, Functional biodegradable scaffolds for Bone Tissue Engineering:
bioactivity profile and osteogenic differentiation of marrow mesenchymal stromal cells,
submitted
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- 85 -
Chapter IV
FUNCTIONAL BIODEGRADABLE SCAFFOLDS FOR BONE TISSUE ENGINEERING:
BIOACTIVITY PROFILE AND OSTEOGENIC DIFFERENTIATION OF MARROW MESENCHYMAL
STROMAL CELLS
IV.1. Abstract
Bone is a complex tissue, whose functionality can be restricted by trauma or degenerative
diseases. Tissue engineering plays a key role in developing alternative strategies for bone
regeneration, where scaffolds are expected to guide cellular distribution and colonization,
similarly to the natural occurring communications between cells and tissue, and provide for
needed support during tissue regeneration.
Cell behavior has been suggested to be mostly based on the response to the surface
composition and topography of the support material. Surface design of biodegradable
polymers has gained much attention and specifically the coating of an apatite layer is
considered to be a promising approach in bone strategies, in order to stimulate the direct
bonding to living bony tissue. Incorporation of functional groups into the surface of polymeric
scaffolds has been also described to be capable of inducing an apatite layer formation, critical
for cellular and molecular communications to most of implanted scaffolds aiming functional
tissue regeneration. Therefore, the present study focuses on a fiber mesh scaffold produced
from a blend of starch with polycaprolactone (SPCL) using wet-spinning technology by a
method that enables the in situ incorporation of silanol groups (Si-OH).
These scaffolds (SPCL-Si) were designed into a well-defined 3D porous-architecture, highly
interconnected, whose bioactive behaviour was analyzed considering its possible role in vivo
during the regeneration/repair of bone tissue.
Results indicated that apatite nucleation is induced by SiOH groups incorporated on SPCL
scaffolds after incubation in simulated body fluid (SBF), confirming its bioactive properties.
Cell culture assays indicated that goat marrow mesenchymal stromal cells not only
proliferate and differentiate into the osteogenic phenotype onto SPCL-Si scaffolds, but seem to
prefer SPCL-Si scaffolds alone rather than SPCL-Si scaffolds coated with an apatite layer formed
by immersion in SBF. Therefore, SPCL-Si scaffolds present intrinsic properties to sustain in vitro
osteogenic features, with great potential aiming at bone engineered approaches.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.2. Introduction
Bone is one of the major tissues affected by trauma, age- and metabolic-related diseases
which translate to a decline in the quality of life of thousands of patients worldwide, thus
representing a major medical and a socio-economical problem. The challenge stands for
developing multifunctional biomaterials[1] to keep up with the process of tissue repair, and
restoring functionality by promoting polymer interactions with in vivo biological materials, and
substitute currently used autografts, the gold standard for bone replacement.
The degree of success of bone tissue engineering (TE) approaches is greatly dependent on
the intrinsic properties of materials used to obtain a biocompatible and biodegradable scaffold
with osteoinductive, osteoconductive and osteogenic properties in order to assist the
attachment, proliferation and differentiation of seeded cells toward the desired bone tissue[2,
3].
The use of starch blended with polycaprolactone to develop new scaffolds has been earlier
investigated for TE, including bone regenerative approaches, demonstrating promising
results[4-10]. However, all biodegradable polymers used within the TE field present a
drawback for bone applications as they do not disclose osteoinductive properties, i.e., they are
not able to induce bone formation by themselves[11].
It is well known that the direct bonding between an implant and bone may occur if a layer
of bone-like mineral forms on the surface of the implant. Therefore, the focus on the surface
design of biodegradable polymers has gained much attention as bioactivity can be induced on
non-bioactive surfaces. Functional groups, as for example, silanol (Si‐OH), can be incorporated
on the surface of polymeric scaffolds, inducing apatite nucleation in a simulated body fluid
(SBF) solution, resulting in the formation of an apatite layer[12-15]. Although calcium and
phosphorus are the main elements of inorganic bone, other ions, whose role has not been fully
studied yet, are key elements in the functionality of the bone metabolic system. Among them,
the silicium (Si), a trace element found in animals and human nutrition, has been associated
with calcium in the mineralization process, and is extremely important in active calcification
sites in young bones[16], as a regulatory factor in bone formation. Furthermore, silicon
compounds stimulate the DNA synthesis in osteoblast-like cells[17], the osteogenic
differentiation of mesenchymal stem cells[15, 18, 19], and the collagen I synthesis at a
physiological level[19].
A previous work[15] by our group, described the development of bioactive fiber meshes, in
a new reliable and economical methodology that enables the control of pore size, shape and
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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orientation, based on a simple wet-spinning technique, using a calcium silicate solution as
coagulation bath. Si-OH groups were successfully incorporated on the surface of the fibers,
thus allowing for obtaining a bioactive scaffold without the need for further coating or
chemical modifications.
The present study analyzed the effect of Si-OH groups on the structure and properties of
wet spun SPCL fiber mesh scaffolds, in terms of bioactivity profile and biological response of
bone marrow stromal cells (BMSCs), aiming at bone regeneration strategies. For this purpose,
the bioactivity of wet spun SPCL fiber mesh scaffolds was assessed in vitro by means of soaking
the samples in simulated body fluid (SBF), whose ion concentration is similar to those of
human plasma. Dynamical-mechanical analysis was performed in order to assess the
mechanical properties of SPCL and SPCL with silanol groups scaffolds under simulated
physiological conditions. Bone marrow stromal cells have been described to have a great
potential for bone regeneration strategies[20-23] as these cells can be easily guided into the
osteogenic lineage, with applicability for autologous approaches[8, 24, 25]. The presence of
seeded cells onto scaffolds should assist bridging the gap between structural support and in
locus cellular and molecular communication towards functional tissue regeneration. Since
apatite is naturally present in bone tissue, the apatite coating of the scaffold is expected to
play an important role in the integration of the implant with the native tissue[19]. Thus, in this
study it was also assessed the response of goat BMSCs towards the influence of the apatite
coating on the surface of the scaffolds as compared with the bioactive uncoated wet spun
starch‐PCL fiber mesh scaffolds with functionalized silanol groups, in terms of osteogenic
differentiation and extracellular matrix maturation.
IV.3. Materials and Methods
IV.3.1. Materials
A biodegradable thermoplastic blend of corn starch with polycaprolactone (30/70 wt %),
SPCL, previously described in other works by our group[4, 5, 8-10] has been selected for this
study. Chloroform (CHCl3) and methanol (CH3OH) were obtained from Sigma-Aldrich.
Tetraethoxysilane (TEOS: Si(OC2H5)4) and calcium chloride (CaCl2) were obtained from
Sigma-Aldrich. Ethyl alcohol (C2H5OH) was obtained from Panreac.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
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IV.3.2. Wet-spun fiber mesh scaffolds processing
The polymer, a blend of corn starch with polycaprolactone (SPCL) was dissolved in
chloroform at a concentration of 30 % (w/v). Then, the polymer solution was loaded into a
disposable syringe, with a 0.8 mm internal diameter needle. A programmable syringe pump
(KD Scientific, World Precision Instruments, UK) controlled the injection rate of the polymer
solution in order to form the fiber mesh in the coagulation bath.
Two different coagulation baths were used: (i) methanol, the control, and (ii) a calcium
silicate solution with a molar ratio Si(OC2H5)4 / H2O / C2H5OH / HCl / CaCl2 of 1.0 / 4.0 / 4.0 /
0.014 / 0.20 [15, 26], where the HCl was used as a catalyst. Control scaffolds in the methanol
bath (SPCL scaffolds), were dried overnight at room temperature while fiber mesh scaffolds
produced calcium silicate solution (SPCL-Si scaffold) were air dried at 60 ºC for 24 h.
All scaffolds were cut into 5 mm diameter discs and sterilized by ethylene oxide.
IV.3.3. Evaluation of the in vitro bioactivity of wet-spun fiber mesh scaffolds
The in vitro bioactivity tests of the SPCL and SPCL-Si wet-spun fiber meshes were carried
out by soaking samples in 10 mL of acellular simulated body fluid (SBF) at 36.5 ºC for up to 7
days. The SBF presents ions concentration similar to the one found in the human blood
plasma[13, 27].
After each period of immersion the test samples were removed from SBF, washed with
distilled water and dried at room temperature. The wet-spun fiber mesh scaffolds before and
after bioactivity tests were analyzed by several techniques, as described in the sub-sections
below.
IV.3.3.1. Scanning electron microscopy (SEM)
SEM with an attached energy dispersive electron probe X-ray analyser (EDS) (Leica
Cambridge S360, UK) was used to observe the morphology of the wet-spun mesh scaffolds
(SPCL and SPCL-Si) after the assays.
Previously to EDS and SEM analysis, sample surfaces were carbon coated (Fisons
Instruments, Evaporation PSU CA508, UK) and gold sputtered (Fisons Instruments, Sputter
Coater SC502, UK), respectively.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.3.3.2. Thin-film X-ray diffraction (TF-XRD)
TF-XRD was performed using a RINT2500 equipment (Rigaku Co., Japan) aiming to identify
crystalline phases present on the polymeric wet-spun fiber mesh, with or without silanol
groups, after immersion in SBF (results were compared to non immersed controls), and to
characterize the crystalline/amorphous nature of the calcium phosphate films.
The data was collected by 2 scan method with 1º as incident beam angle using CuK X-ray
line and a scan speed of 0.05 º/min in 2.
IV.3.3.3. Micro-computed tomography (µ-CT) analysis:
Micro-CT analysis was conducted to evaluate morphology, porosity, pore interconnectivity
as well as mean pore size, and mean fiber thickness after bioactivity assays, using a highresolution μ-CT, Skyscan 1072 scanner (Skyscan, Kontich, Belgium).
X-ray scans were performed in triplicate using a resolution of pixel size of 5.86 µm and
integration time of 1.9 sec. The X-ray source was set at 40 keV of energy and 248 μA of
current. Approximately 400 projections were acquired over a rotation range of 180º with a
rotation step of 0.45º. Data sets were reconstructed using standardized cone-beam
reconstruction software (NRecon v1.4.3, SkyScan).
Representative data sets of 200 slices were segmented into binary images with a dynamic
threshold of 50 to 255 (grey values) in order to identify the organic and inorganic phase. This
data was used for morphometric analysis (CTAnalyser, v 1.5.1.5, SkyScan) and to build 3D
models (ANT 3D creator, v2.4, SkyScan).
IV.3.3.4. Dynamic mechanical analysis (DMA):
Viscoelastic measurements were performed using a TRITEC8000B DMA from Triton
Technology (Belgium)[14], equipped with a tensile mode. SPCL and SPCL-Si scaffolds were cut
in 5 mm diameter discs to meet the distance between the clamps. The geometry of the
samples was measured, samples clamped in the DMA apparatus and then immersed in a PBS
solution. DMA spectra were obtained during a frequency scan between 0.1 and 25 Hz. The
experiments were performed under constant strain amplitude (30 µm). A static pre-load of 0.7
N was applied during the test to keep the sample tight. During the DMA analysis, scaffolds
were immersed in a liquid bath in a Teflon® reservoir. Three samples were used per condition.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.3.3.5. Induced-coupled plasma emission spectroscopy (ICP)
Elemental concentrations were measured in SBF, before and after immersing wet-spun
fiber meshes (with or without Si groups), using inductively coupled plasma atomic emission
spectrometry (ICP: JY2000-2, Jobin Yvon, Horiba, Japan).
Samples were collected at the end of each test period, filtered with a 0.22 µm filter and
kept at -80ºC until usage; for each condition and immersion time triplicate samples were
analyzed.
IV.3.4. Cell culture study
In a previous study[15], SPCL (control) and SPCL-Si scaffolds produced by wet spinning
were seeded with goat bone marrow stromal cells (gBMSCs) in osteogenic medium for up to
14 days. Preliminary data indicated that gBMSCs preferred SPCL-Si for cellular proliferation and
osteogenic differentiation, evidencing the importance of silanol groups in SPCL-Si scaffolds for
bone strategies. Thus, the present study focused on assessing the response of gBMSCs to SPCL
scaffolds functionalized with silanol groups as compared to SPCL-Si pre-coated with an apatite
layer, obtained by 7 days of immersion in SBF.
IV.3.4.1. Harvesting and seeding gBMSCs onto wet-spun fiber mesh scaffolds
Goat bone marrow mesenchymal stromal cells (gBMSCs) were harvested from the iliac
crests of adult goats and cultured in DMEM (Dulbecco Modified Eagle Medium, Sigma)
supplemented with 10 % foetal bovine serum (Gibco) and 1% antibiotic/antimicotic solution
(Gibco) and cryopreserved. Cells were thawed, expanded and sub-cultured twice (2 Pa) before
being seeded onto SPCL-Si scaffolds or SPCL-Si scaffolds after a 7 day incubation in SBF (SPCLSi-7SBF) at a concentration of 100,000 cells/scaffold. After seeding, cells were cultured in
alpha-MEM (Sigma) and in the presence of osteogenic supplements, namely 10-8 M
dexamethasone (Sigma), 50 g/ml ascorbic acid (Sigma), 10 mM -glycerophosphate (Sigma)
for 7 and 14 days.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.3.4.2. Cell viability assay
The MTS test (Promega, USA) was used to assess cell viability in SPCL-Si or SPCL-Si-7SBF
scaffolds seeded with gBMSCs, after 7 and 14 days of culture.
After each culturing end point, cells were rinsed in PBS and then incubated in a MTS
solution (1 fraction of MTS reagent + 5 fraction of basal culture medium without phenol red
(Sigma)) for 3 hours at 37 ºC in a 5% CO2 environment. Afterwards, absorbance was read at
490nm in a microplate ELISA reader equipment (BioTek).
IV.3.4.3. Cell proliferation assay
Proliferation of gBMSCs seeded onto constructs was also analyzed by double strand DNA
quantification (dsDNA). For this purpose, a fluorimetric dsDNA quantification kit (PicoGreen,
Molecular Probes) was used. The fluorescence was read using a microplate ELISA reader
(BioTek) at an excitation of 485/20 nm and an emission of 528/20 nm.
IV.3.4.4. ALP assay
ALP activity has been considered an early osteogenic marker to assess osteogenic
differentiation of gBMSCs seeded onto scaffolds under study. A substrate solution was added
to each sample consisting of 0.2 % (wt/v) p-nytrophenyl phosphate (Sigma, USA) in a substrate
buffer with 1 M diethanolamine HCl (Merck, Germany), at pH 9.8. Samples were then
incubated in the dark for 45 minutes at 37 ºC. After the incubation period, a stop solution (2 M
NaOH (Panreac, Spain) plus 0.2 mM EDTA (Sigma, USA)), was added to each well containing
the sample.
Standards were prepared with p-nytrophenol (10 µmol.ml-1) (Sigma, USA). Samples and
standards were prepared in triplicates. The absorbance was read using a microplate ELISA
reader (BioTek) at 405nm.
IV.3.4.5. SEM
gBMSCs morphology after culturing onto SPCL-Si and SPCL-Si-7SBF fiber meshes was
observed by SEM. Before sputter coat with gold, cell-scaffold constructs were rinsed in PBS,
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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fixed in a 2.5% solution of gluteraldehyde (Sigma) overnight, dehydrated in a series of ethanol
concentrations and air dried.
IV.3.5. Statistical Analysis
Results from ICP and DMA tests as well as biology assays are presented as means 
standard deviation.
Statistical analysis was carried out using two Way ANOVA test was also applied to check the
existence of statistical differences between sample groups (p<0.001, or p<0.05).
IV.4. Results and Discussion
IV.4.1. Bioactivity assessment
To understand the mechanism of apatite formation on bioactive materials, Kokubo et
al.[13, 28] proposed a protein-free and acellular simulated body fluid (SBF) with pH 7.40 and
ionic composition (Na+ 142.0, K+ 5.0, Ca2+ 2.5, Mg2+ 1.5, Cl- 147.8, HCO3
- 4.2, HPO4
2- 1.0, SO4
2-
0.5 mM) similar to the ones found in the human blood plasma. The bioactivity of artificial
materials is commonly evaluated by examining the formation of apatite on the surface of the
scaffold after immersion in SBF.
The figure IV.4.1 exhibits the TF-XRD patterns obtained for the SPCL fiber meshes produced
by wet-spinning, and SPCL fiber mesh functionalized with silanol groups (SPCL-Si), after soaking
in SBF for 7 days.
The TF-XRD patterns of the surface of the SPCL-Si sample, after a 7 day immersion in SBF,
exhibit several broad diffraction peaks, whose position and intensities can be assigned to an
apatite-like phase (ASTM JCPDS 9-432) (Figure IV.4.1). The peaks in 2 and their
correspondence to the diffraction planes of apatite are: 10.82º (1 0 0), 25.87º (0 0 2), and
31.75º (2 2 1). The apatite film formed presents low crystallinity, as the apatite peaks were
comparatively broader than the crystalline apatite.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
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Figure IV.4.1 - TF-XRD patterns of SPCL fiber meshes produced by wet-spinning, where a
calcium silicate solution was used as a coagulation bath (SPCL-Si), and after 7 days of
immersion in SBF (SPCL-Si 7d in SBF). SPCL was used as control. Apatite characteristic peaks are
represented in the spectrum with black dots ().
SPCL scaffolds, obtained by precipitation in methanol, could not induce the formation of an
apatite layer even after 7 days in SBF. Conversely, in SPCL-Si scaffolds, obtained by
precipitation in a calcium silicate bath, the formation of an apatite layer can be observed after
only 1 day of immersion in a SBF solution, despite the fact that, after that time, the scaffold
was not completely covered with the apatite layer, as observed by SEM (Figure IV.4.2).
Micro-CT analysis was used to follow up the formation and growth of an apatite layer (blue
color) as function of time (Figure IV.4.2). Scaffold porosity, around 49.77 %, is similar to the
values found before soaking the scaffolds in SBF, 56.84 %. As the immersion time in SBF
increases, the apatite layer becomes more dense and compact but still homogeneously
distributed, and thus without compromising the overall morphology and interconnectivity of
the 3D-fiber mesh scaffolds.
These results clearly indicate that the presence of functional silanol groups is responsible
for apatite formation in SPCL scaffolds after immersion in SBF solution (Figure IV.4.2).
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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Figure IV.4.2 - Morphological characterization by means of micro-CT and SEM micrographs of
SPCL-Si scaffolds before and after 1, 3 or 7 days of immersion in SBF. Control (CTR) scaffolds of
SPCL (without silanol groups) after 7 days in SBF. The blue color regions in micro-CT images
correspond to the apatite deposition.
The concentrations of calcium, phosphorus and silicon in the SBF solution, after immersion
of SPCL with and without Si-OH groups, measured by ICP analysis, are shown in Figure IV.4.3.
Figure IV.4.3 - Changes in calcium (Ca), phosphorus (P) and silicon (Si) concentration in the SBF
solution after different immersion periods of the SPCL or SPCL-Si scaffolds. Symbols o and +
denote study groups with statistically significant differences (o=p<0.001 and +=p<0.05,
respectively), as using Two Way ANOVA method.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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For up to 7 days, an increment in silicon concentration was observed for SPCL-Si scaffolds.
This increment is related to the release of silicon ions from the SPCL-Si fiber mesh scaffold,
leading to the formation of Si-OH groups, responsible for the apatite nucleation. These results
indicate that an organized arrangement of functional groups, Si-OH groups, on SPCL-Si fiber
mesh scaffold is the key point to render a bioactive behavior, as the classic ceramic materials,
such as Bioglass[29]. For example, when this bioactive ceramic is soaked in SBF, the first
reaction of this type of bioactive glass surface is ion exchange, in which Ca2+ and Na+ in the
glass exchange for H3O
+ in the solution, resulting in an increase in pH of the solution as well as
in the formation of a hydrated silica gel layer[29]. The formation of hydrated silica gel layer on
the surface of Bioglass, which is abundant in Si – OH groups, provides favourable sites for the
calcium phosphate nucleation[30, 31]. Furthermore, the water molecules in the SBF react with
the Si-O-Si bond to form additional Si-OH groups[32]. Then, these functional groups induce
apatite nucleation, and the released Ca2+ and Na+ ions accelerate apatite nucleation by
increasing of the ionic activity product (IAP) of apatite in the fluid. Also, Tanashi et al.[33]
reported that Si-OH groups were effective in apatite nucleation. Therefore, the mineralization
induced by the bioactive ceramics is due to the formation of specific surface functional groups
such as Si-OH, which serve as effective sites for heterogeneous nucleation of calcium
phosphate[34]. Additionally, an increase of IAP in the surrounding fluid could thereby promote
the calcium phosphate nucleation and growth on the surface of bioactive ceramics[34].
In the case of SPCL scaffolds, no traces of Si element were detected in the control. For SPCLSi, the differences in Si concentration are not statistically significant (p>0.05) along consecutive
soaking periods, since the amount of Si released is just enough to promote the formation of
the apatite layer, detected by TF-XRD and µ-CT.
A slightly decrease in the calcium concentration is observed for the first 24 hours, which
can be explained by the release of calcium ions available in the silica phase bond to the SPCL
(Figure IV.4.3). This release into the SBF can lead to an increase of the ionic activity product of
the surrounding fluid with respect to apatite. The Si-OH groups induced the apatite nucleation,
and the increased ionic activity product accelerates the nucleation rate of apatite. Once
apatite nuclei are formed, they can spontaneously grow into a uniform layer by consuming the
calcium and phosphate ions from the SBF, since SBF is already highly supersaturated with
respect to apatite[35].
As the soaking time in SBF increases, the calcium and phosphorus concentrations decreased
gradually, as the apatite layer is forming on the fiber mesh scaffolds while consuming the
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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calcium and phosphate ions in SBF solution. The decrease in Ca and P concentrations is more
evident in SPCL-Si scaffolds (p<0.001) after 3 and 7 days in the SBF solution.
In summary, the results obtained from the in vitro bioactivity tests clearly indicated that the
presence of Si-OH functional groups in wet spun SPCL fiber meshes is a prerequisite for the
apatite formation in simulated body fluid.
IV.4.1.1. DMA analysis
Figure IV.4.4 presents the dynamical mechanical behavior of the SPCL and SPCL-Si scaffolds
with the variation of the frequency, assessed under simulated physiological condition, i.e., in a
hydrated environment and at 37 ºC [36-38]. The storage modulus (E’) of all samples tends to
increase with increasing frequency as observed in Figure IV.4.4.
However, for the SPCL samples E’ increases from 5.54 MPa to 7.04 MPa while for the SPCLSi samples, E’ increases (p<0.001) from 11.38 MPa to 14.81 MPa indicating that the SPCL-Si
possess a higher stiffness than SPCL’s. This is possibly explained by the fact that the in situ
functionalization improves the mechanical properties of the scaffold.
Thus, these results demonstrate that the described methodology allows obtaining bioactive
polymeric scaffolds without prejudice to its mechanical properties.
Figure IV.4.4 - Variation of the storage modulus as a function of frequency between 0.1 and 15
Hz after equilibration at 37 ºC with the samples immersed in PBS solution.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.4.2. Biological assays
Results obtained from previous experiments showed that the presence of silanol groups in
SPCL scaffolds enhanced the proliferation and osteogenic differentiation of gBMSCs[15],
evidencing the importance of these groups for bone TE strategies. Since studies on SPCL vs
SPCL-Si wet spun scaffold were already conducted, in our study we did not include SPCL as
experimental control, and focus the evaluation on the effect of pre-coating SPCL-Si scaffolds
with an apatite layer.
In general, gBMSCs kept a good cell viability and proliferation rate when seeded onto both
types of scaffolds. Nevertheless, viability levels are higher in the presence of SPCL-Si scaffolds
alone, without the apatite coating, which tends to increase with the culturing time (p<0.01)
(Figure IV.4.5).
Figure IV.4.5 - Results obtained from the MTS test performed on gBMSCs seeded onto SPCL-Si
scaffolds (SPCL-Si) and SPCL-Si scaffolds pre-coated with an apatite layer (SPCL-Si-7SBF), and
cultured in osteogenic medium for 7 or 14 days. Symbol * denote study groups with
statistically significant differences (p<0.05), as using Two Way ANOVA method.
The ALP/dsDNA ratio represents the amount of activity of ALP produced by the cells seeded
on the scaffolds. In SPCL-Si previously coated with an apatite layer and seeded/cultured with
gBMSCs, the ALP/dsDNA values are lower and decreased from day 7 to day 14, while in SPCL-Si
constructs, ALP/DNA ratio seems to be stabilized and maintained through the culturing time
(Figure IV.4.6).
Since ALP is a glycoprotein associated with the formation and maturation of the
extracellular matrix[39], lower ALP levels indicate that the presence of an apatite layer on the
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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surface of SPCL-Si scaffolds may interfere with the development and maturation of the ECM
produced by gBMSCs in SPCL-Si scaffolds. Also, and considering the expression of this
osteogenic marker, cells seeded onto SPCL-Si and SPCL-Si pre-coated scaffolds are likely to be
at different stages of the osteogenic process.
Figure IV.4.6 - Results from ALP assays performed on seeded onto SPCL-Si scaffolds (SPCL-Si)
and SPCL-Si pre-coated with an apatite layer layer (SPCL-Si-7SBF) and, after culture in
osteogenic medium for 7 or 14 days.
In SEM micrographs (Figure IV.4.7), cells are shown to be more proliferative and
homogeneously distributed onto SPCL-Si scaffolds both after 7 and 14 days in osteogenic
culture. Furthermore, cells tend to proliferate by bridging between fibres, yet without closing
the pores.
Figure IV.4.7 - SEM pictures showing the gBMSCs morphology when seeded onto SPCL-Si
(SPCL-Si) scaffolds and SPCL-Si scaffolds pre-coated with apatite (SPCL-Si-7SBF), followed by
culture in osteogenic medium for 7 or 14 days. Inset micrographs refer to scaffolds, SPCL-Si
(C2) and SPCL-Si pre-coated (C4) after 7 days in osteogenic medium.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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Although calcium phosphate coating is considered to be excellent in promoting cell
adhesion and stimulating cells into the osteogenic phenotype, the viability, colonization of the
scaffolds, and osteogenic related markers indicate that the presence of silanol groups has a
stronger positive impact in cells response. This was confirmed by the results obtained from all
assays performed on samples for both for 7 and 14 days in culture.
Images obtained from the micro-CT analysis performed on constructs cultured for 14 days
in osteogenic medium, showed the formation of a mineralized matrix in SPCL-Si scaffolds
indicating that the presence of silicium improved the deposition of a calcified matrix produced
by gBMSCs (Figure IV.4.8).
Figure IV.4.8 - Assessment of calcified matrix production by gBMSCs seeded onto SPCL-Si
scaffolds (SPCL-Si) after 14 days in osteogenic medium by µCT analysis.
Cells seeded onto SPCL-Si scaffolds seem to prefer the superficial silicium molecules to
proliferate and to support the biomimetic growth of natural apatite for ECM synthesis. The
favorable results of SPCL-Si alone may indicate that the mechanism involving silicium is likely
to be more similar to the in vivo natural process than the SBF coating. Although silicium exists
in minimal amounts in bone, has an important role in bone metabolism and osteoblast
behaviour in terms of proliferation and differentiation[15-19].
These results also explain the ALP activity levels of gBMSCs seeded on SPCL-Si scaffolds as
osteogenesis in guided bone regeneration has been described to be preceded by a localized,
marked expression of ALP in an organized connective tissue environment[40].
The development of an apatite layer in SPCL-Si scaffold is atypical for biodegradable
polymeric scaffolds, which has been commonly associated to ceramic materials[41]. Moreover
the results suggest that SPCL-Si scaffolds interact more actively with cells without the presence
of a calcium phosphate coating, confirming the osteoconductive properties associated to
scaffolds containing silicium[15, 42] aiming at bone tissue strategies.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
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IV.5. Conclusions
In this study, the potential of functionalized wet-spun fibers for bone TE was assessed by
physical, chemical and biological characterization.
The precipitation of SPCL in the calcium silicate solution leads to an incorporation of Si-OH
groups in the structure of SPCL-Si scaffold and SPCL-Si scaffolds were shown to induce apatite
nucleation (but not SPCL scaffolds) when incubated with SBF, confirming its bioactive
properties. Dynamic mechanical analysis showed that SPCL-Si scaffold present higher stiffness
than SPCLs, thus improved mechanical properties which are likely to be beneficial for bone
related applications. Thus, the developed wet spun fiber meshes with incorporated Si-OH
functional groups combine the properties of classical bioactive ceramics, and the degradability
of an organic polymer.
The current work also confirms that not only Si–OH groups improve cellular functionality
towards the osteoblastic phenotype, evidencing osteogenic and osteoconductive properties,
but gBMSCs seems to prefer SPCL-Si scaffolds instead of SPCL-Si scaffolds coated with an
apatite layer. The results obtained in SPCL-Si-gBMSC constructs may be related to the
biological importance of silicium in bone metabolic processes, underlying the potential of
SPCL-Si scaffolds for bone regeneration strategies.
IV.6. References
1. Place ES, George JH, Williams CK, and Stevens MM: Synthetic polymer scaffolds for tissue
engineering. Chem Soc Rev 2009, 38(4): 1139-1151.
2. Shin H, Zygourakis K, Farach-Carson MC, Yaszemski MJ, and Mikos AG: Modulation of
differentiation and mineralization of marrow stromal cells cultured on biomimetic hydrogels
modified with Arg-Gly-Asp containing peptides. J Biomed Mater Res A 2004, 69(3): 535-543.
3. Liu C, Xia Z, and Czernuszka JT: Design and development of three-dimensional scaffolds for
tissue engineering. Chemical Engineering Research & Design 2007, 85(A7): 1051-1064.
4. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, and Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2(5): 243-252.
C
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
- 101 -
5. Santos MI, Tuzlakoglu K, Fuchs S, Gomes ME, Peters K, Unger RE, Piskin E, Reis RL, and
Kirkpatrick CJ: Endothelial cell colonization and angiogenic potential of combined nano- and
micro-fibrous scaffolds for bone tissue engineering. Biomaterials 2008, 29(32): 4306-4313.
6. Tuzlakoglu K, Bolgen N, Salgado AJ, Gomes ME, Piskin E, and Reis RL: Nano- and micro-fiber
combined scaffolds: a new architecture for bone tissue engineering. J Mater Sci Mater Med
2005, 16(12): 1099-1104.
7. Tuzlakoglu K, Pashkuleva I, Rodrigues MT, Gomes ME, van Lenthe GH, Muller R, and Reis
RL: A new route to produce starch-based fiber mesh scaffolds by wet spinning and subsequent
surface modification as a way to improve cell attachment and proliferation. J Biomed Mater
Res A 92(1): 369-377.
8. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, and Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5: 41-49
9. Gomes ME, Bossano CM, Johnston CM, Reis RL, and Mikos AG: In vitro localization of bone
growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and
cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12(1): 177-188.
10. Gomes ME, Holtorf HL, Reis RL, and Mikos AG: Influence of the porosity of starch-based
fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow
stromal cells cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12(4): 801-809.
11. Laurencin CT, Ambrosio AM, Borden MD, and Cooper JA, Jr.: Tissue engineering: orthopedic
applications. Annu Rev Biomed Eng 1999, 1: 19-46.
12. Rhee SH: Bone-like apatite-forming ability and mechanical properties of poly(epsiloncaprolactone)/silica hybrid as a function of poly(epsilon-caprolactone) content. Biomaterials
2004, 25(7-8): 1167-1175.
13. Kokubo T, Takadama H: How useful is SBF in predicting in vivo bone bioactivity?
Biomaterials 2006, 27(15): 2907-2915.
14. Ohtsuki C, Kamitakahara M, and Miyazaki T: Coating bone-like apatite onto organic
substrates using solutions mimicking body fluid. J Tissue Eng Regen Med 2007, 1(1): 33-38.
15. Leonor I, Rodrigues MT, Gomes ME, and Reis RL: In Situ Functionalization of Wet-Spun
Fibre meshes for Bone Tissue Engineering: One Step Approach. J Tissue Eng Regen Med 2011,
5: 104-111.
16. Carlisle EM: Silicon: a possible factor in bone calcification. Science 1970, 167(916): 279-280.
17. Keeting PE, Oursler MJ, Wiegand KE, Bonde SK, Spelsberg TC, and Riggs BL: Zeolite A
increases proliferation, differentiation, and transforming growth factor beta production in
normal adult human osteoblast-like cells in vitro. J Bone Miner Res 1992, 7(11): 1281-1289.
18. Obata AKasuga T: Stimulation of human mesenchymal stem cells and osteoblasts activities
in vitro on silicon-releasable scaffolds. J Biomed Mater Res A 2009, 91(1): 11-17.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
- 102 -
19. Reffitt DM, Ogston N, Jugdaohsingh R, Cheung HF, Evans BA, Thompson RP, Powell JJ, and
Hampson GN: Orthosilicic acid stimulates collagen type 1 synthesis and osteoblastic
differentiation in human osteoblast-like cells in vitro. Bone 2003, 32(2): 127-135.
20. Krampera M, Pizzolo G, Aprili G, and Franchini M: Mesenchymal stem cells for bone,
cartilage, tendon and skeletal muscle repair. Bone 2006, 39(4): 678-83.
21. Mauney JR, Volloch V, and Kaplan DL: Role of adult mesenchymal stem cells in bone tissue
engineering applications: current status and future prospects. Tissue Eng 2005, 11(5-6): 787802.
22. Tuan RS, Boland G, and Tuli R: Adult mesenchymal stem cells and cell-based tissue
engineering. Arthritis Res Ther 2003, 5(1): 32-45.
23. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, and Marshak DR: Multilineage potential of adult human mesenchymal
stem cells. Science 1999, 284(5411): 143-147.
24. Zhu L, Liu W, Cui L, and Cao Y: Tissue-engineered bone repair of goat-femur defects with
osteogenically induced bone marrow stromal cells. Tissue Eng 2006, 12(3): 423-433.
25. Kruyt MC, Dhert WJ, Yuan H, Wilson CE, van Blitterswijk CA, Verbout AJ, and de Bruijn JD:
Bone tissue engineering in a critical size defect compared to ectopic implantations in the goat.
J Orthop Res 2004, 22(3): 544-551.
26. Oyane A, Kawashita M, Nakanishi K, Kokubo T, Minoda M, Miyamoto T, and Nakamura T:
Bonelike apatite formation on ethylene-vinyl alcohol copolymer modified with silane coupling
agent and calcium silicate solutions. Biomaterials 2003, 24(10): 1729-1735.
27. Kokubo T: Design of bioactive bone substitutes based on biomineralization process.
Materials Science & Engineering C-Biomimetic and Supramolecular Systems 2005, 25(2): 97104.
28. Kokubo T, Kushitani H, Sakka S, Kitsugi T, and Yamamuro T: Solutions able to reproduce in
vivo surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res 1990,
24(6): 721-734.
29. Filgueiras MR, La Torre G, and Hench LL: Solution effects on the surface reactions of a
bioactive glass. J Biomed Mater Res 1993, 27(4): 445-453.
30. Li P, Ohtsuki C, Kokubo T, Nakanishi K, Soga N, Nakamura T, and Yamamuro T: Effects of
ions in aqueous media on hydroxyapatite induction by silica gel and its relevance to bioactivity
of bioactive glasses and glass-ceramics. J Appl Biomater 1993, 4(3): 221-229.
31. Wen HB, Moradian-Oldak J, Zhong JP, Greenspan DC, and Fincham AG: Effects of
amelogenin on the transforming surface microstructures of Bioglass in a calcifying solution. J
Biomed Mater Res 2000, 52(4): 762-773.
32. Kokubo T, Kim HM, and Kawashita M: Novel bioactive materials with different mechanical
properties. Biomaterials 2003, 24(13): 2161-2175.
Chapter IV – Functional biodegradable scaffolds for bone tissue engineering: bioactivity profile and osteogenic
differentiation of marrow mesenchymal stromal cells
- 103 -
33. Tanahashi M, Yao T, Kokubo T, Minoda M, Miyamoto T, Nakamura T, and Yamamuro T:
Apatite coated on organic polymers by biomimetic process: improvement in its adhesion to
substrate by NaOH treatment. J Appl Biomater 1994, 5(4): 339-347.
34. Kim HM: Ceramic bioactivity and related biomimetic strategy. Current Opinion in Solid
State & Materials Science 2003, 7(4-5): 289-99.
35. Kim HM: Bioactive ceramics: challenges and perspectives. Journal Ceramic Society of Japan
2001, 109: S49-S57.
36. Mano JF, Neves NM, Reis RL, Mechanical Characterization of Biomaterials. In
Biodegradable Systems in Tissue Engineering and Regenerative Medicine, Reis RL, Roman JS,
Editor. 2005, CRC Press.
37. Mano JF: Viscoelastic properties of chitosan with different hydration degrees as studied by
dynamic mechanical analysis. Macromol Biosci 2008, 8(1): 69-76.
38. Mano JF R, Reis RL, Cunha AM, Dynamic Mechanical Analysis in Polymers for Medical
Applications in Mechanical Characterization of Biomaterials. In Biodegradable Systems in
Tissue Engineering and Regenerative Medicine, Reis RL CD, Editor. 2002, Kluwer Academic
Publishers. p. 139-164.
39. Lian JB, Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation
of bone cell development and tissue formation. Crit Rev Oral Biol Med 1992, 3(3): 269-305.
40. Stucki U, Schmid J, Hammerle CF, and Lang NP: Temporal and local appearance of alkaline
phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical
study in humans. Clin Oral Implants Res 2001, 12(2): 121-127.
41. Vallet-Regi M: Ceramics for medical applications. Journal of the Chemical Society-Dalton
Transactions 2001, (2): 97-108.
42. Huang Y, Jin X, Zhang X, Sun H, Tu J, Tang T, Chang J, and Dai K: In vitro and in vivo
evaluation of akermanite bioceramics for bone regeneration. Biomaterials 2009, 30(28): 50415048.
- 104 -
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Chapter V
BILAYERED CONSTRUCTS AIMED AT
OSTEOCHONDRAL STRATEGIES: THE INFLUENCE OF MEDIA
SUPPLEMENTS IN THE OSTEO- AND CHONDRO-GENIC DIFFERENTIATION
OF AMNIOTIC FLUID-DERIVED STEM CELLS
This chapter is based on the following publication:
Rodrigues MT, Lee SJ, Gomes ME, Reis RL, Atala A, and Yoo J, Bilayered constructs
aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells, submitted
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- 105 -
Chapter V
BILAYERED CONSTRUCTS AIMED AT OSTEOCHONDRAL STRATEGIES:
THE INFLUENCE OF MEDIA SUPPLEMENTS IN THE OSTEO- AND CHONDROGENIC
DIFFERENTIATION OF AMNIOTIC FLUID-DERIVED STEM CELLS
V.
V.1. Abstract
The development of osteochondral (OC) tissue engineered interfaces would be a novel
treatment for traumatic injuries and aging associated diseases that affect body joints. This
study reports the development of a bilayered osteochondral construct based on biodegradable
and natural based polymers. Amniotic fluid-derived stem cells (AFSCs) were either
differentiated into cells of the osteogenic lineage after seeding onto starch-polycaprolactone
(SPCL) scaffolds or into cells of the chondrogenic lineage after encapsulation in agarose gels.
After these two constructs were assembled into a single construct, this bilayered system was
cultured for 1 or 2 weeks in OC-defined culture media containing bone and cartilage growth
factors (glycerol-2-phosphate, L-ascorbic acid, dexamethasone, and sodium pyruvate, ITS, Lproline, respectively). Additionally, the effect of the presence or absence of insulin-like growth
factor-1 (IGF-1) in the culture medium was assessed. Cell viability and expression of bone- and
cartilage-specific markers were analyzed in order to determine the influence of the culture
media on cell phenotype. The results indicated that, after osteogenic differentiation, AFSCs
that had been seeded onto SPCL scaffolds did not require OC medium to maintain their
phenotype. In fact, they produced a protein-rich, mineralized extracellular matrix (ECM) for up
to 2 weeks. However, AFSCs differentiated into chondrocyte-like cells appeared to require OC
medium, but not IGF-1, to synthesize an ECM and maintain the chondrogenic phenotype. Thus,
the results obtained show that IGF-1 was not essential for creating osteochondral constructs
with AFSCs, and the OC supplements used appear to be quite important to generate cartilage
in long-term tissue engineering approaches for osteochondral interfaces. In summary, this
study suggests that constructs generated from agarose-SPCL bilayered scaffolds containing
pre-differentiated AFSCs may be useful for potential applications in regeneration strategies for
damaged or diseased joints.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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V.2. Introduction
Osteochondral (OC) interfaces are one of the most susceptible areas in the human body to
traumatic injuries and aging associated diseases, such as, for instance, osteoarthritis[1-3].
Understanding and mimicking the complexity of the OC system is critical for designing a
successful tissue engineering approach that can restore the functionality of a joint. However,
bone and cartilage, which are the tissues that make up the OC interface, have different
molecular compositions and cellular organizations, and consequently, differences in structural
and mechanical properties between these tissues. Thus, current tissue engineering strategies
are hampered by the difficulties inherent in designing a seamless interface between these two
distinct tissues.
First, the ideal cell source for OC engineering strategies has not yet been found. The cell
source should be proliferative, yet it should possess the phenotypic plasticity to differentiate
into the various cell types that make up the OC interface. In addition, the cells should be able
to maintain the differentiated phenotype in the presence of other cell types and after removal
of the selective differentiation medium used in culture. The amniotic fluid has been recently
appointed as a source of stem cells[4, 5, 6]. Amniotic fluid-derived stem cells (AFSCs) have the
capacity to differentiate along both the chondrogenic[4, 5] and osteogenic[4] lineages.
Importantly, they also share certain phenotypic characteristics with both adult and embryonic
stem cells[6], and they have shown to be multipotential cells with no expansion limitations[4].
Also, the use of AFSCs does not raise the ethical concerns that are associated with the use of
embryonic stem cells for research and therapy[6, 7].
OC interfaces are exposed to a number of different in vivo stresses and strains that result
from the patient’s daily activities and movements. Therefore, any cells used for OC tissue
engineering must be supported by a scaffold that can withstand these stresses while
regeneration takes place. In addition to providing mechanical support, a scaffold for OC
regeneration should generate an efficient and integrated interface which enables different cell
types to communicate and interact while keeping these various cells and their functions in the
proper structural compartment.
Agarose gels have previously been used for cell culture and cartilage tissue engineering
strategies[8-10]. The soft, flexible structure of this natural based gel recreates a 3D
environment suitable for chondrocyte maintenance[11] as well as the differentiation of
mesenchymal stem cells (MSCs) into the chondrogenic lineage[11]. In contrast, SPCL scaffolds,
which are a blend of starch and polycaprolactone, have been previously used in tissue
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
- 109 -
engineering strategies designed for bone regeneration and replacement[12-15], as well as for
cartilage applications[16, 17]. SPCL scaffolds are biodegradable and biocompatible, and they
are based on naturally occurring materials. These scaffolds support the adhesion and
proliferation of several cell types, including endothelial[12] and stem cells[13]. Furthermore,
these scaffolds assist bone neoformation in vivo, especially when seeded previously with
cells[13].
In addition to physical support (scaffold) and a biological interface (cells), the biochemical
factors involved in stimulating cell communication, differentiation, and maintenance of
phenotype should also be considered when designing a tissue engineering approach.
Transforming growth factor-beta (TGF-) is frequently used as a standard factor in media
designed to induce chondrogenic differentiation. TGF- is part of the bone morphogenic
protein (BMP) superfamily, and is involved in chondrogenesis, including differentiation of
progenitor cells into chondrocytes[18]. Additionally, TGF- also induces proliferation and ECM
production in articular chondrocytes[19, 20].
In bone differentiation, dexamethasone, ascorbic acid and -glycerophosphate are
commonly used supplements for culture media designed to induce osteogenic differentiation
of various cell types. Dexamethasone, a synthetic glucocorticoid, has been shown to induce
osteogenic differentiation of osteoprogenitor cells from adult bone marrow stromal-derived
cells[21, 22]. Ascorbic acid plays an essential role in the structure and function of skeletal
tissues as it is required for human collagen synthesis. Moreover, when a potential source of
phosphate ions, such as -glycerophosphate[22], is also present, a zone of hydroxyapatitecontaining mineralized ECM is formed within the collagen fibrils[23], and this mineralization of
the matrix is essential for formation of bone tissue. However, although osteo- and
chondrogenic media are frequently described in the literature and are used to induce the
differentiation of cells into these lineages, an efficient osteochondrogenic medium, which
would be able to support both osteo- and chondrogenesis and the co-culture of both bone and
cartilage cells, has not been completely established, although some attempts have been
described[24].
In this study, we aimed to develop a novel approach for designing functional scaffolds that
would support an OC interface. A novel OC medium, designed to support differentiation and
maintenance of both bone and cartilage cell phenotypes, was also developed by combining
some of the growth factors and nutrients associated with osteo- and chondrogenic media.
Furthermore, insulin-like growth factor (IGF) was also evaluated as a potential factor for
supporting OC constructs in vitro, since it is involved in several developmental and
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
- 110 -
physiological functions[25] of bone and cartilage, including cartilage and bone development;
chondrocyte proliferation and ECM synthesis[26, 27], and osteoblast proliferation and bone
formation[28].
In order to test the OC media and our novel scaffold design, human AFSCs were either
cultured on SPCL scaffolds and differentiated into osteogenic cells or encapsulated in agarose
gel and differentiated into chondrogenic cells. These two cell seeded scaffolds were then
brought together in order to form a combination of scaffolds that would provide a highly
supportive scaffold for bone formation as well as a soft matrix for cartilage growth. The
resulting single constructs were cultured for 1 or 2 weeks in our novel OC media, either with or
without IGF-1. At each time point, the constructs were characterized regarding the expression
of bone and cartilage specific markers to evaluate the influence of the culture media on the
osteogenic or chondrogenic phenotype of the differentiated cells.
V.3. Materials and Methods
Human AFSCs were cultured in basic amniotic fluid cell (BAFC) medium. The BAFC medium
(500 mL) contained liquid α-MEM (HyClone Laboratories Inc., USA) with 18 % Chang B (Irvine
Scientific, USA) and 1 % Chang C (Irvine Scientific) media as well as 2 % L-glutamine (HyClone
Laboratories Inc.) and 15 % embryonic screened fetal bovine serum (ES-FBS, HyClone
Laboratories Inc.) as previously described[4]. Briefly, back-up human amniocentesis cultures
were harvested by trypsinization, and immunoselected with c-kit. AFS cells were then
subcultured at a dilution of 1:8 and not permitted to expand beyond 60 % of confluence. The
AFS cells passaged 2–3 times (passage 18-20) in BAFC medium. Then AFSCs were either
encapsulated in agarose discs (2 %, Invitrogen, USA) at a concentration of 2 x106 cells/mL for
the chondrogenic layer of the bilayered scaffold or seeded onto SPCL scaffolds at a
concentration of 8.6 x105 cells/scaffold for the osteogenic layer. SPCL fibers were obtained by
a fiber melt extrusion/fiber bonding process. Fiber-mesh scaffolds were prepared by cutting
and sintering SPCL fibers by a melt-spinning process as previously described[14]. SPCL
scaffolds were selected as 3D environment as these scaffolds have been extensively studied
and characterized for bone TE strategies[12-16]. Prior to the cell culture assays, all scaffolds
were cut into cylinders of 4 mm diameter and 5 mm length, and sterilized using ethylene
oxide.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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SPCL scaffolds seeded with hAFSCs and agarose gels with encapsulated hAFSCs were
cultured for 2 days in BAFC culture medium, and then the medium was exchanged for either
osteogenic or chondrogenic media, respectively. The chondrogenic medium consisted of
DMEM (HyClone Laboratories Inc.) supplemented with 1 % antibiotic/antimicotic solution, 50
µg/mL L-ascorbic acid (Sigma, St Louis, MO, USA), 1 mM dexamethasone (Sigma), 40 g/mL Lproline (Sigma), 100 g/mL sodium pyruvate (Sigma), 1 % ITS (100x, Sigma) and 10 ng/mL TGF1 (Sigma). The osteogenic medium was composed of DMEM (HyClone Laboratories Inc.)
supplemented with 10 % FBS (HyClone Laboratories Inc.) and the following osteogenic
supplements: 100 nM dexamethasone (Sigma), 50 µM L-ascorbic acid and 10 mM glycerol-2phosphate disodium salt hydrate (Sigma). Chondrogenic differentiation of AFSCs in the
agarose gels was assessed after 7, 14 and 21 days in the chondrogenic media, and then the
gels were characterized for cellular viability with a Calcein AM assay, safranin-O staining, and
immunofluorescence for aggrecan and collagen type II.
After both chondrogenic and osteogenic differentiation occurred in the two scaffolds, the
constructs were combined by adding a drop of agarose (2 %) between the cell-encapsulated
and the SPCL scaffolds to promote bonding of the two layers. Then, the bilayered constructs
were cultured for 2 additional weeks in co-culture medium under static conditions to evaluate
whether this medium could maintain differentiated AFSCs in both chondrogenic and
osteogenic phenotypes simultaneously. The co-culture media was composed of DMEM-LG
(HyClone Laboratories Inc.), 4 g/mL sodium pyruvate, ITS (1x), 5 mM glycerol-2-phosphate,
50 µg/mL L-ascorbic acid, 10 mM dexamethasone, and 40 g/mL L-proline. In some studies,
the growth factor IGF-1 (100 ng/mL, Invitrogen) was added to the co-culture medium (IGF-1
(+)).
After 7 and 14 days in the co-culture media, constructs were removed and characterized
for the osteo- or chondrogenic markers mentioned above. BAFC medium (i.e., the basal
conditions) was used as a control for this experiment.
V.3.1. Viability assay with Calcein AM
AFSCs encapsulated in the agarose gels were rinsed in PBS (HyClone Laboratories Inc.) and
then were incubated in PBS containing 3 µM Calcein AM (Molecular Probes, Invitrogen) for 30
min at 37 ºC in a 5 % CO2 environment. This incubation was followed by a quick rinse in PBS
and overnight fixation in 10 % buffered formalin (Surgipath Medical Ind., Inc, USA) at 4 ºC.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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AFSC viability was detected using a confocal microscope (Axiovert 100 M, Zeiss, Germany)
equipped with argon/He-Ne laser sources.
V.3.2. SEM (scanning electronic microscopy)
Bilayered constructs were rinsed in PBS, fixed in 10 % buffered formalin overnight, and
dehydrated in a series of ethanol concentrations and critical point dried (EMS850X, Electron
Microscopy Sciences, USA). The scaffolds were then sputtered with gold (Hummer 6.2
sputtering system, Anatech Ltd, USA). This procedure destroys the structure and morphology
of AFSC-agarose systems and therefore, SEM observation was only used for the AFSC-SPCL
constructs of the OC system (Hitachi S-2600N, Hitachi Science Systems, Ltd, Japan).
Additionally, in order to detect specific ions that eventually became present at the surface of
cell seeded SPCL constructs on the ECM, such as calcium (Ca) and phosphorus (P), an energy
dispersive spectroscopy (EDS) analysis was performed with EDAX (Pegasus X4M, EDS/EBSD,
EDAX B.V., Netherlands).
V.3.3. Histological characterization
For histological evaluation, the samples were rinsed in PBS, fixed in 10 % buffered formalin
overnight and processed using a tissue processor (Microm STP120, MICROM International
GmbH, Germany). Next, each sample was embedded in paraffin blocks (Microm EC 350-2,
ThermoScientific, Spain), and 10 m thick sections were cut and stained with safranin-O
(Fluka, Switzerland). Following deparaffinization of AFSC-agarose samples, slides were stained
with Weigert’s iron hematoxylin working solution (Sigma-Aldrich, Germany) for 7 minutes.
Samples were rinsed in tap water for 10 minutes, and subsequently rinsed quickly in 1 %
acetic acid (Fluka) solution for about 10 to 15 seconds and stained in 0.1 % safranin O solution
for 5 minutes.
Immunofluorescence analysis for collagen type II and aggrecan was also carried out.
Sections were cut from the paraffin blocks, deparaffinized and rehydrated with a graded series
of ethanol concentrations. Prior to incubation with primary antibodies for either mouse anticollagen II (MAB1330, Millipore, Spain) or mouse anti-human aggrecan (MCA1452, Serotec,
Germany), slides were blocked with bovine serum albumin (BSA; 3 % in PBS) (Sigma-Aldrich,
Germany) for 40 minutes. Samples were incubated with primary antibodies overnight in a 4 ºC
room. After washing, the samples were incubated with rabbit anti-mouse Alexa Fluor 488Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
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conjugated secondary antibody (A11059, Invitrogen, Spain) for 1 hour at room temperature.
Samples were observed under a microscope (Zeiss Imager Z1m, Germany) and images were
acquired using a digital camera (AxioCam MRm5).
V.4. Results and Discussion
V.4.1. AFSCs-agarose system
The AFSCs remained viable during the 21-day culture period regardless of the type of media
used (basal or chondrogenic), as shown in Figure V.4.1.
Figure V.4.1 – Calcein AM stained samples of AFSCs encapsulated in agarose gels after 7, 14,
and 21 days in chondrogenic and basal medium (control).
Nevertheless, the intensity of Calcein AM fluorescence found in samples cultured in basal
culture media appeared to be higher than that observed in samples cultured in chondrogenic
media.
Since basal medium is specifically formulated for cell maintenance and expansion, AFSCs
may have been able to proliferate at a higher rate when cultured in basal medium.
Conversely, chondrogenic medium directs AFSCs towards the chondrogenic differentiation
process and thus, stimulates the cells to stop proliferating and begin expressing ECM proteins.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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This result is expected, because during mammalian development, proliferation usually inhibits
differentiation, while differentiation is accompanied by cell cycle withdrawal[29]. The inverted
relationship between proliferation and differentiation has been previously reported in
osteoprogenitor cells[30].
Besides water, the cartilage matrix consists of macromolecules in which collagen type II
(around 80 % of the total tissue collagen) and proteoglycans (aggrecan) are the main structural
representatives[31], and these molecules are responsible for tissue formation. Therefore, the
differentiation of AFSCs into chondrocyte-like cells was assessed by examining the expression
of collagen type II and aggrecan by immunofluorescent analysis.
Collagen type II expression was detected as early as 7 days into culture of AFSCs
encapsulated in agarose gels, and by the end of week 3, the expression appeared to increase,
especially when chondrogenic medium was used for culture. This is particularly evident if the
amount of collagen type II in chondrogenic and basal medium cultured constructs is compared,
as expression of collagen type II in the latter tends to decline with time in culture (Figure
V.4.2).
Figure V.4.2 – Immunofluorescent analysis of collagen type II expression in AFSCs encapsulated
in agarose gels after 7, 14, and 21 days in chondrogenic and basal media (control).
Magnification, 200X.
Collagen type II expression is observed throughout the differentiation process of MSCs into
chondrocytes[32]. Collagen is initially expressed at the final stage of undifferentiated MSCs,
and then increases significantly as cells became chondroprogenitors, and again as fully
differentiated chondrocytes[32]. This continuous expression of collagen II during chondrogenic
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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differentiation may explain why collagen type II is expressed at all time points in this study.
Since AFSCs share several markers with MSCs[4, 33]; the chondrogenic differentiation of AFSCs
may also follow a similar time course in terms of collagen type II expression, and be
continuously expressed during our experiment.
However, it has been shown that the aggrecan is available in smaller amounts in cartilage
ECM than collagen type II[31], and we have also observed this in our study (Figure V.4.3).
Figure V.4.3 - Immunofluorescent analysis for aggrecan expression in AFSCs encapsulated in
agarose gels after 7, 14 and 21 days in chondrogenic or basal media (control). Magnification,
200X.
In basal medium, AFSCs encapsulated in agarose gels expressed a low level of aggrecan for
the 3 weeks of this experiment. Conversely, in chondrogenic culture medium, aggrecan
expression is more evident, reaching a peak at 2 weeks that is followed by a slight decrease at
3 weeks. This pattern of expression is supported by data showing that during skeletogenesis,
chondroprogenitor determination and chondrocyte differentiation are accompanied by
dynamic ECM remodeling[32]. Aggrecan expression in the ECM increases as chondrocytes
differentiate and become hypertrophic[32]. In addition, chondroprogenitor cells and
chondrocytes express high levels of collagen type II and increasing levels of aggrecan in the
ECM. Nevertheless, as chondrocytes move into a hypertrophic state, low expression levels of
collagen type II and high aggrecan levels are observed[32]. Our results show that in the
agarose gels, strong collagen type II expression and lower expression of aggrecan are present
with some fluctuations from week 2 to week 3. This suggests that by week 3, AFSCs may still be
in an early stage of chondrocyte differentiation.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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V.4.2. Agarose-SPCL bilayered system
V.4.2.1. AFSCs-SPCL layer
The AFSCs seeded onto the SPCL scaffold proliferated well and were able to differentiate to
osteogenic cell types in the different culture media. In the SEM micrographs of these
constructs (Figure V.4.4), the cells were distributed throughout the scaffold, and some
mineralization nodules could be seen at the surface of the SPCL fibers, suggesting the
formation of a calcified ECM. Larger mineralized aggregates were found when IGF-1 was
added to the OC culture medium after 1 week of culture in this medium. Although these
nodules did not increase in size with time, the mineralization observed in the SPCL constructs
was stable and was still evident after 2 weeks of culture in the OC medium.
Figure V.4.4 - SEM micrographs of the osteogenic layer (AFSCs seeded onto SPCL scaffold) of
the bilayered scaffolds after 7 or 14 days in OC culture media. IGF-1(+) indicates culture
medium supplemented with IGF-1 while IGF-1(-) refers to the same culture medium without
IGF-1. The basal culture medium consisted of the basic medium currently used for AFSC
expansion and maintenance, and was used as the control medium in these studies.
EDS analysis was also performed at the surface of the constructs after assembly of the OC
constructs. Two EDS analyses were considered; the first was broader and included several
areas of the construct that contained cells with and without mineralization nodules (Table
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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V.4.1), and the second analysis included only areas in which mineralization was present (Table
V.4.2).
Table V.4.1 - Results obtained from EDS analysis of the atomic percentage (At %) of several
ions present in AFSCs-SPCL layer after 7 and 14 days in the OC culture media.
At (%) Si P Ca Ca/P
7 d IGF-1 (+) 0.22 0.66 0.95 1.439
7 d IGF-1 (‒) 0.09 0.56 0.55 0.982
7 d Basal 0.20 1.52 1.61 1.059
14 d IGF-1 (+) 0.13 0.58 0.47 0.810
14 d IGF-1 (‒) 0.07 0.55 0.57 1.036
14 d Basal 0.08 0.46 0.46 1.000
Represented ions: Si-silicium, P-phosphorus, Ca-calcium. Ca/P stands for the ratio
Calcium/Phosphorus.
Silicon (Si), a trace element that has an essential role in bone formation and is thought to
be involved in the synthesis and/or stabilization of collagen[34] was found in all of the
constructs at different end points. Si levels tended to decrease with culture time in OC media,
although this decrease was larger in constructs cultured in basal medium.
The detection of calcium (Ca) and phosphorus ions (P) via EDS has been associated with the
production of mineralized matrices by cells. In this study, although Ca and P levels decreased
with culture time when the constructs were maintained in both basal and OC media without
IGF-1, the Ca/P ratio was close to 1 at all time points. However, when IGF-1 was added to the
media, the Ca/P ratio detected in the constructs cultured for 7 days was 1.439. This ratio is
more similar to tricalcium phosphate (TCP) ratio[35].
In general, the decrease observed in the percentage of atomic concentrations with the
culture end points may be related to the presence of a saturation point at which a balance of
mineralized matrices might be established. When osteoblasts become entrapped in bone ECM,
these cells eventually begin to die as part of the natural process of bone formation.
Furthermore, if the cell proliferation rate is reduced because the cells have become entrapped
in the mineralized matrices they produced, the synthesis of ECM will also decrease and
stabilize. Nevertheless, when the analysis was performed specifically on mineralized nodules,
some variations in the amounts of the studied ions were observed (Table V.4.2).
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
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Table V.4.2 - Results obtained from EDS analysis of the atomic percentage (At %) of several
ions present in AFSCs-SPCL layer after 7 and 14 days in the OC culture media. EDS analysis was
performed on areas of the constructs containing mineralized aggregates.
At (X) Si P Ca Ca/P
7 d IGF-1 (+) 0.41 4.44 4.94 1.113
7 d IGF-1 (‒) 0.28 3.06 3.34 1.092
7 d Basal 0.33 5.78 6.85 1.185
14 d IGF-1 (+) 0.14 2.51 2.53 1.008
14 d IGF-1 (‒) 0.10 2.88 3.07 1.066
14 d Basal 0.24 3.73 6.19 1.660
Represented ions: Si-silicium, P-phosphorus, Ca-calcium. Ca/P stands for the ratio
Calcium/Phosphorus.
When only mineralized areas were studied, Si values decreased with time. Although
calcium and phosphorus slightly decreased with culture time, the atomic concentrations of
both of these minerals were higher than the ones observed in the broader analysis shown in
Table V.4.1. This result is expected, since the EDS focused on areas of mineralization in the
second analysis. When the Ca/P was calculated, all culture conditions but one resulted in a
ratio around 1. The constructs cultured for 14 days in basal medium after the OC assembly
were found to have a Ca/P ratio of 1.66, which is nearly the same as the Ca/P ratio found in
naturally occurring bone hydroxyapatite, which is 1.67[35, 36]. During the process of bone
hardening during aging, the Ca:P ratio gradually increases from 1 to 1.67, the hydroxyapatite
ratio reported for healthy bone[37].
Although IGF-1 has been shown to be important for bone and cartilage tissue development,
we did not find that the presence of this growth factor had any effect on the maintenance of
the osteogenic phenotype of cells cultured in OC medium with IGF-1. Furthermore, the best
results in terms of mineralization were observed in basal medium conditions after 2 weeks in
culture, when the constructs demonstrated a Ca/P ratio similar to that seen in natural bone.
Runx-2 is an important transcription factor involved in osteogenic development and
exogenous expression, which has been shown to enhance osteoblast-specific gene expression
in rat bone marrow stromal cells as well as biological mineral deposition[38]. In our previous
study, hAFSCs were shown to express RunX-2 during osteogenic differentiation over a period
of up to 3 weeks in osteogenic medium. In the present study, we analyzed possible differences
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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in RunX-2 expression 2 weeks after the novel OC media was added to the constructs (Figure
V.4.5).
Figure V.4.5 - Immunofluorescence for RunX-2 expression in the osteogenic layer (AFSCs
seeded onto SPCL scaffolds) of the bilayered scaffolds after 14 days (14d) in the OC culture
media. IGF-1(+) indicates culture medium supplemented with IGF-1 while IGF-1(-) refers to the
same culture medium without IGF-1. “Basal” refers to the basic medium currently used for
AFSC expansion and maintenance, and was used as a control in this assay.
Runx-2 expression was higher when the cells were cultured in OC media than when they
were cultured in basal medium. In the basal medium, RunX-2 expression was almost
nonexistent, which indicates that in OC media, with or without IGF, the osteogenic process is
dynamic and evolving, and that mineralization may be taking place, since high levels of Runx2
expression have been shown to maintain the mineralization capacity of expanded marrow
cells[38].
Cell viability in the hAFSC-containing agarose gels was qualitatively analyzed using a Calcein
AM assay after the chondrogenic differentiation process.
Figure V.4.6 indicates that most cells remained viable during the culture period, and
viability was independent of the medium used for culture. Furthermore, cellular viability was
high both at the surface/border and in the center areas of the gels, indicating that the agarose
gels were permeable enough to allow the exchange of nutrients and gases between the
culture media and the encapsulated cells.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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Figure V.4.6 - Viability of the chondrogenic layer (AFSCs in agarose gels) of the bilayered
scaffold after 7 (7d) or 14 days (14d) in the OC culture media. IGF-1(+) indicates culture
medium supplemented with IGF-1 while IGF-1 (-) refers to the same culture medium without
IGF-1. “Basal” represents the basic medium currently used for AFSC expansion and
maintenance, and was used as a control.
Collagen type II expression within the gels was assessed by immunofluorescence. Figure
V.4.7 indicates that collagen type II was present in the constructs for up to 14 days after the
addition of OC media. However, expression of this chondrogenic marker was lower in the
assembled bilayered construct than in AFSC-containing agarose gels alone, and this was true
whether the constructs were cultured in basal or chondrogenic media.
A possible explanation is that the OC media is a rich cocktail that, together with the
presence of the AFSC-SPCL constructs, might interfere with the chondrogenic process of
encapsulated AFSCs.
Aggrecan levels were also measured in the bilayered constructs. They were found to be
similar to the levels observed in the AFSC-agarose gels alone. This suggests that aggrecan
expression is more stable than collagen type II expression in the presence of OC media (Figure
V.4.8).
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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Figure V.4.7 - Collagen type II expression (200x magnification) in the chondrogenic layer (AFSCs
in agarose gels) of the bilayered scaffold after 7 or 14 days in OC culture medium. IGF-1(+)
indicates culture medium supplemented with IGF-1 while IGF-1 (-) refers to the same culture
medium without IGF-1. Cultures maintained in basal medium were used as controls.
Figure V.4.8 - Aggrecan immunofluorescence (200 x magnification) in the chondrogenic layer
(AFSCs in agarose gels) of the bilayered scaffold after 7 or 14 days in OC culture medium. IGF1(+) indicates culture medium supplemented with IGF-1 while IGF-1(-) refers to the same
culture medium without IGF-1. Culture in basal medium was used as a control.
Safranin-O staining is used to identify areas of cartilage production in tissue sections. When
we performed this stain on sections taken from the bilayered OC constructs, the chondrogenic
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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layer (AFSC-agarose gel) stained positively in sections from all samples cultured in OC medium
without IGF-1 after 14 days of culture, as shown in Figure V.4.9.
Figure V.4.9 - Safranin-O (cartilage-specific) staining of the chondrogenic layer (AFSCs in
agarose gels) of the bilayered scaffold after 14 days in OC culture medium. IGF-1(+) indicates
culture medium supplemented with IGF-1 while IGF-1 (-) refers to the same culture medium
without IGF-1. “Basal” represents the basic medium currently used for AFSC expansion and
maintenance, and was used as a control. Magnification, 200X.
In basal medium as well as in medium containing IGF-1, the safranin-O staining intensity is
very low. This suggests that the combination of other factors besides IGF-1 in the
osteochondral medium maintains a higher level of chondrogenesis in the constructs, thus
increasing the safranin-O staining in the constructs cultured in the OC medium.
Despite the fact that collagen type II and aggrecan expression were lower in the OC
medium than in regular chondrogenic medium, these proteins were still present in the
chondrogenic layer of the bilayered constructs cultured in OC medium, indicating that in this
media, AFSCs continue to produce ECM.
However, because aggrecan levels remain low with time, and are independent of the
culture media, it is unlikely that AFSC-derived chondrocyte-like cells became hypertrophic in
these constructs. The decrease in protein production may have been a result of the reduced
proliferation rate observed in these samples, which is typical of differentiated chondrocytes.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
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V.5. Conclusions
The results obtained in this study demonstrated that AFSCs seeded onto SPCL scaffolds or
encapsulated in agarose gel systems remained viable even after these constructs were
cultured for additional time in the novel OC culture media described here. Furthermore, in the
osteogenic layer (SPCL), osteogenically differentiated AFSCs did not require the OC media to
maintain an osteogenic phenotype for up to 14 days, as they were able to continue producing
mineralized ECM even when cultured in basal medium. In agarose gels, encapsulated AFSCs
differentiated into chondrocyte-like cells that produced an ECM rich in collagen type II and
aggrecan. When cultured in OC media, the expression of these markers decreased, but
safranin-O staining clearly indicated that the cells continued to produce a cartilage-like ECM in
OC medium without IGF supplement.
In this bilayered system, IGF was not essential for the maintenance of chondrogenic or
osteogenic phenotypes in differentiated AFSCs as evidenced by marker expression studies.
However the other supplements in the OC medium were shown to be very important for
achieving long-term differentiation results.
This OC co-culture medium could be advantageous not only to simplify cell culture
procedures but also to allow physical interaction between osteogenic and chondrogenic
phenotypes while ensuring that the induced phenotype of chondrocytes and osteoblasts is
maintained. In addition, this medium would reduce the time and production costs of a tissue
engineered product and move it closer to a clinically applicable strategy for joint repair. Finally,
the development of a bilayered OC system for tissue engineering of bone/cartilage interfaces
could better mimic the integration of bone and cartilage in an OC defect by providing scaffolds
that address tissue specific needs.
In this study, a bilayered scaffold of this type was successfully created in which both
osteogenically and chondrogenically differentiated AFSCs maintained long term viability and
phenotypic expression in vitro. Thus, the integrated agarose-SPCL scaffold proved to be
functional in vitro and may lead to the development of new strategies for OC repair and
regeneration.
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and chondrogenic differentiation of amniotic fluid-derived stem cells
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V.6. References
1. Naran KN, Zoga AC: Osteochondral lesions about the ankle. Radiol Clin North Am 2008,
46(6): 995-1002
2. Madry H, van Dijk CN, and Mueller-Gerbl M: The basic science of the subchondral bone.
Knee Surg Sports Traumatol Arthrosc 2010, 18(4): 419-433.
3. Pape D, Filardo G, Kon E, van Dijk CN, and Madry H: Disease-specific clinical problems
associated with the subchondral bone. Knee Surg Sports Traumatol Arthrosc 2010, 18(4): 448462.
4. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC,
Snyder EY, Yoo JJ, Furth ME, Soker S, and Atala A: Isolation of amniotic stem cell lines with
potential for therapy. Nat Biotechnol 2007, 25(1): 100-6.
5. Kolambkar YM, Peister A, Soker S, Atala A, and Guldberg RE: Chondrogenic differentiation
of amniotic fluid-derived stem cells. J Mol Histol 2007, 38(5): 405-413.
6. Prusa AR, Marton E, Rosner M, Bernaschek G, and Hengstschlager M: Oct-4-expressing cells
in human amniotic fluid: a new source for stem cell research? Hum Reprod 2003, 18(7): 14891493.
7. Fauza D: Amniotic fluid and placental stem cells. Best Pract Res Clin Obstet Gynaecol 2004,
18(6): 877-891.
8. Diduch DR, Jordan LC, Mierisch CM, and Balian G: Marrow stromal cells embedded in
alginate for repair of osteochondral defects. Arthroscopy 2000, 16(6): 571-577.
9. Gu WY, Yao H, Huang CY, and Cheung HS: New insight into deformation-dependent
hydraulic permeability of gels and cartilage, and dynamic behavior of agarose gels in confined
compression. J Biomech 2003, 36(4): 593-598.
10. Buschmann MD, Gluzband YA, Grodzinsky AJ, Kimura JH, and Hunziker EB: Chondrocytes in
agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res 1992,
10(6): 745-758.
11. Mauck RL, Yuan X, and Tuan RS: Chondrogenic differentiation and functional maturation of
bovine mesenchymal stem cells in long-term agarose culture. Osteoarthritis Cartilage 2006,
14(2): 179-189.
12. Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, and Kirkpatrick CJ: Response of microand macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.
Biomaterials 2007, 28(2): 240-248.
13. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, and Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5(1): 41-49.
C
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
- 125 -
14. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, and Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2(5): 243-252.
15. Gomes ME, Holtorf HL, Reis RL, and Mikos AG: Influence of the porosity of starch-based
fiber mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow
stromal cells cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12(4): 801-809.
16. Gonçalves A, Costa P, Rodrigues MT, Dias IR, Reis RL, and Gomes ME: Effect of flow
perfusion conditions in the chondrogenic differentiation of bone marrow stromal cells cultured
onto starch based biodegradable scaffolds Acta Biomaterialia 2011, 7: 1644-1652.
17. Oliveira JT, Crawford A, Mundy JM, Moreira AR, Gomes ME, Hatton PV, and Reis RL: A
cartilage tissue engineering approach combining starch-polycaprolactone fibre mesh scaffolds
with bovine articular chondrocytes. J Mater Sci Mater Med 2007, 18(2): 295-302.
18. Leonard CM, Fuld HM, Frenz DA, Downie SA, Massague J, and Newman SA: Role of
transforming growth factor-beta in chondrogenic pattern formation in the embryonic limb:
stimulation of mesenchymal condensation and fibronectin gene expression by exogenenous
TGF-beta and evidence for endogenous TGF-beta-like activity. Dev Biol 1991, 145(1): 99-109.
19. Yoo JU, Barthel TS, Nishimura K, Solchaga L, Caplan AI, Goldberg VM, and Johnstone B: The
chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells. J Bone
Joint Surg Am 1998, 80(12): 1745-1757.
20. Grimaud E, Heymann D, and Redini F: Recent advances in TGF-beta effects on chondrocyte
metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth
Factor Rev 2002, 13(3): 241-257.
21. Aubin JE: Osteoprogenitor cell frequency in rat bone marrow stromal populations: role for
heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem 1999, 72(3): 396410.
22. Maniatopoulos C, Sodek J, and Melcher AH: Bone formation in vitro by stromal cells
obtained from bone marrow of young adult rats. Cell Tissue Res 1988, 254(2): 317-330.
23. Franceschi RT, Iyer BS, and Cui Y: Effects of ascorbic acid on collagen matrix formation and
osteoblast differentiation in murine MC3T3-E1 cells. J Bone Miner Res 1994, 9(6): 843-854.
24. Li J, Mareddy S, Tan DM, Crawford R, Long X, Miao X, and Xiao Y: A minimal common
osteochondrocytic differentiation medium for the osteogenic and chondrogenic differentiation
of bone marrow stromal cells in the construction of osteochondral graft. Tissue Eng Part A
2009, 15(9): 2481-2490.
25. Butler AA, Yakar S, Gewolb IH, Karas M, Okubo Y, and LeRoith D: Insulin-like growth factor-I
receptor signal transduction: at the interface between physiology and cell biology. Comp
Biochem Physiol B Biochem Mol Biol 1998, 121(1): 19-26.
Chapter V – Bilayered constructs aimed at osteochondral strategies: the influence of media supplements in the osteo
and chondrogenic differentiation of amniotic fluid-derived stem cells
- 126 -
26. Martel-Pelletier J, Boileau C, Pelletier JP, and Roughley PJ: Cartilage in normal and
osteoarthritis conditions. Best Pract Res Clin Rheumatol 2008, 22(2): 351-384.
27. Vinatier C, Bouffi C, Merceron C, Gordeladze J, Brondello JM, Jorgensen C, Weiss P,
Guicheux J, and Noel D: Cartilage tissue engineering: towards a biomaterial-assisted
mesenchymal stem cell therapy. Curr Stem Cell Res Ther 2009, 4(4): 318-329.
28. Meinel L, Zoidis E, Zapf J, Hassa P, Hottiger MO, Auer JA, Schneider R, Gander B, Luginbuehl
V, Bettschart-Wolfisberger R, Illi OE, Merkle HP, and von Rechenberg B: Localized insulin-like
growth factor I delivery to enhance new bone formation. Bone 2003, 33(4): 660-672.
29. Beier F, Leask TA, Haque S, Chow C, Taylor AC, Lee RJ, Pestell RG, Ballock RT, and LuValle P:
Cell cycle genes in chondrocyte proliferation and differentiation. Matrix Biology 1999, 18(2):
109-120.
30. Lian JB, Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation of
bone cell development and tissue formation. Crit Rev Oral Biol Med 1992, 3(3): 269-305.
31. Aigner T, Stove J: Collagens-major component of the physiological cartilage matrix, major
target of cartilage degeneration, major tool in cartilage repair. Adv Drug Deliv Rev 2003,
55(12): 1569-1593.
32. Tuan RS: Biology of developmental and regenerative skeletogenesis. Clin Orthop Relat Res
2004, (427 Suppl): S105-17.
33. Hipp J, Atala A: Sources of stem cells for regenerative medicine. Stem Cell Rev 2008, 4(1): 311.
34. Jugdaohsingh R: Silicon and bone health. J Nutr Health Aging 2007, 11(2): 99-110.
35. Vallet-Regi M: Ceramics for medical applications. Journal of the Chemical Society-Dalton
Transactions 2001, (2): 97-108.
36. Palmer LC, Newcomb CJ, Kaltz SR, Spoerke ED, and Stupp SI: Biomimetic systems for
hydroxyapatite mineralization inspired by bone and enamel. Chem Rev 2008, 108(11): 47544783.
37. Dorozhkin SV: Calcium Orthophosphates as Bioceramics: State of the Art. Journal of
Functional Biomaterials 2010, 1: 22-107.
38. Byers BA, Garcia AJ: Exogenous Runx2 expression enhances in vitro osteoblastic
differentiation and mineralization in primary bone marrow stromal cells. Tissue Eng 2004,
10(11-12): 1623-1632.
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SECTION IV
DOES STEM CELL ORIGIN INFLUENCE STEM CELL RESPONSE ?
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Chapter VI
AMNIOTIC FLUID STEM CELLS VERSUS BONE MARROW MESENCHYMAL
STEM CELLS AS A SOURCE FOR BONE TISSUE ENGINEERING
This chapter is based on the following publication:
Rodrigues MT, Lee SJ, Gomes ME, Reis RL, Atala A, and Yoo J, Amniotic fluid stem cells
versus bone marrow mesenchymal stem cells as a source for bone tissue engineering,
submitted
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Chapter VI
AMNIOTIC FLUID STEM CELLS VERSUS BONE MARROW MESENCHYMAL STEM CELLS
AS A SOURCE FOR BONE TISSUE ENGINEERING
VI.1. Abstract
The ideal or universal cell source for tissue engineering (TE) is yet to be found. The widely
studied bone marrow mesenchymal stem cells (BMSCs) have been evidencing important
osteogenic characteristics, therefore becoming the gold standard for orthopaedic regeneration
strategies. However, novel stem cell sources, for instance, amniotic fluid stem cells (AFSCs) are
arising showing important and unique features that might lead to novel successful approach
towards bone regeneration.
This study aims to originally compare the osteogenic potential of BMSCs and AFSCS towards
bone TE strategies, under distinct culturing environments. The purpose is to analyse the originrelated response of stem cells to different external environments during the osteogenic
differentiation process. Thus, the osteogenic differentiation was evaluated both on 2D and 3D,
using a culture treated plate, and by means of seeding/culturing the cells onto fiber bonding
SPCL scaffolds (a blend of starch and poly-caprolactone), respectively.
BMSCs and AFSCs were successfully differentiated into the osteogenic lineage, and
developed mineralized ECM. Nevertheless, cells presented different expression patterns of
bone-related markers as well as different timings of differentiation, indicating that both cell
origins and the culturing environment have a significant impact in the progression of the
osteogenic phenotype in AFSCs and BMSCs.
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VI.2. Introduction
The human body has an endogenous system of regeneration and repair through stem cells,
which can be found in almost every living tissue[1-4]. Therefore, stem cells are an obvious
choice for regenerative medicine applications, including bone tissue engineering therapies.
Nevertheless, the selection of the most appropriate source of stem cells for efficient bone
regeneration is still a major issue to be considered in the tissue engineering field[3, 5].
The ideal cell source should combine insignificant donor morbidity or tissue scarcity with no
amount limitations, be easy to harvest, isolate and maintain in vitro, be available according to
patients needs, have no issues of immunogenicity, no diseases transmission risks and be of low
cost. So far, different cell sources match some of the parameters in this list, thus directing the
applicability of cell sources into more specific strategies, yet always with some limitations.
Among adult stem cells, bone marrow mesenchymal stem cells (BMSCs) are possibly the
ones most investigated. Their multipotential to differentiate into different lineages was
confirmed by several research groups, including a high potential for bone regeneration
strategies[5-8]. Nevertheless, BMSCs harvesting is an invasive and rather painful procedure to
the donor. Furthermore, their numbers, proliferation, and differentiation potential decline
with age[9, 10]. Some studies suggest that BMSCs are also sensitive to subculturing
methodologies, and their stability in long-term in vitro culture is controversial[11, 12].
Nevertheless, BMSCs represent a promising cell source for cell-based therapeutics showing
great promise in animal studies and some clinical trials[1, 13, 14].
Amniotic fluid derived stem cells (AFSCs) were recently introduced to bone TE [2, 15, 16]
whose behavior and characteristics are not so deeply investigated so far. Nevertheless, these
cells have demonstrated interesting characteristics, since they derive from embryonic and
extra-embryonic tissues during the process of foetal development and growth. In fact, AFSCs
have shown to be highly proliferative[12], with high self-renewal capability and have the
potential to differentiate into several lineages, including the osteogenic phenotype[2, 17].
AFSCs are easy to obtain, representing an an almost unlimited stem cell source with
immunosuppressive properties[18] and, whose harvesting procedure does not raise ethical
concerns, sometimes associated with human embryonic stem cell research[15, 16]. AFSCs have
been also described to be in an intermediate stage between embryonic stem cells and adult
stem cells due to their extensive ability for expansion and differentiation into functional cells
of the three embryonic germ layers[1, 2], and thus exhibiting important potential in future
regenerative strategies.
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Few studies[12, 18, 19] established a comparison between human BMSCs and AFSCs to
determine how the origin could influence the osteogenic differentiation potential of stem cells.
In mice, mesenchymal stem cells (MSCs) from different origins are known to vary in
clonogenic capacity, surface markers and differentiation potential[19]. Studies with human
cells indicate that MSCs isolated from fetal tissues are more plastic and grow faster than adult
MSCs, while evidencing similar immunophenotypic characteristics and maintaining long and
stable telomeres[12].
The culturing environment provided by the substrate/scaffold characteristics, such as the
structure and mechanical properties may also deeply affect cell behavior[20, 21]. In this sense,
we also propose to evaluate herein human AFSCs and BMSCs in 2D and 3D cultures by seeding
and culturing these cells onto tissue culture well plates or onto a fiber bonding mesh scaffold
made of a blend of starch and poly ε-caprolactone (SPCL). SPCL scaffold, obtained by a fiber
melt extrusion/fiber bonding process[22] were selected as 3D environment for osteogenic
differentiation of both types of cells as these scaffolds have been extensively studied and
characterized in the context of bone tissue engineering strategies [23-26].
The main objective of this study was to compare the osteogenic potential of marrow
derived mesenchymal stem cells (BMSCs) and amniotic fluid stem cells (AFSCs) aiming at bone
TE strategies. We proposed to evaluate osteogenic differentiation of these cells both on 2D
and 3D cultures, and to compare the development of the osteogenic phenotype for up to 3
weeks in conditioned osteogenic medium. Furthermore, we selected a well characterized fiber
bonding mesh scaffold made of a blend of starch and poly-caprolactone (SPCL) to analyse the
osteogenic phenotype of both BMSCs and AFSCs when cultured in a 3D matrix.
Cells in both 2D and 3D conditions were cultured in the presence of osteogenic media for 0,
7, 14 and 21 days and then characterized for cellular viability using a MTS assay while
osteogenic phenotypic expression and matrix formation were assessed by
immunofluorescence for RunX-2, collagen I and ALP activity, and by EDS and calcium
quantification, respectively.
This study may provide important cues of the cell behaviour of AFSCs and BMSCs when
differentiating into the osteogenic phenotype and the variations induced by 2D and 3D seeding
substrates.
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VI.3. Materials and Methods
VI.3.1. Cell culture
Human BMSCs, purchased from Lonza® (Walkersville, USA), were expanded in basal BMSCs
medium composed of α-MEM (HyClone Laboratories Inc.) supplemented with 10 % ES-FBS
(HyClone Laboratories Inc.) and 1 % penicillin/streptavidin solution.
hAFSCs were isolated as previously described[2], and cultured in basic amniotic fluid cell
(BAFC) medium composed of α-MEM (HyClone) with 18 % Chang B (Irvine Scientific) and 1 %
Chang C (Irvine Scientific) media as well as 2 % L-glutamine (HyClone Laboratories Inc.) and 15
% ES-FBS.
hBMSCs and hAFSCs were seeded onto tissue culture 6-well plates at passage 5 and
passage 24, respectively, at a concentration of 30,000 cells/well. Cells were cultured in the
well-plates for 3 days with basal medium, and then, exchanged to osteogenic medium,
composed of DMEM (HyClone Laboratories Inc.) supplemented with 10 % FBS (HyClone) and
100 nM dexamethasone (Sigma), 50 µM L-ascorbic acid (Sigma) and 10 mM glycerol 2phosphate disodium salt hydrate (Sigma), for up to 3 weeks (0, 7, 14 and 21 days). Before
osteogenic characterization, cells were briefly rinsed in PBS (HyClone Laboratories Inc.) and
fixed in 10 % neutral buffered formalin (Surgipath Medical Industries, Inc.).
In order to study the behavior of hBMSCs and hAFSCs in a 3D environment, both type of
cells were seeded onto SPCL scaffolds (7 mm x 4 mm cylinders) at a concentration of 1.2x106
cells/scaffold and cultured in basal medium for 3 days and then in osteogenic media for up to 3
weeks. SPCL scaffold is a blend of starch and polycaprolactone, 30:70 (wt %) (Novamont)
produced by fiber bonding technique as previously described[22].
Samples were retrieved after every 7 days in culture to be characterized for cellular viability
with Calcein AM and for assessing the presence of osteogenic markers and matrix formation by
alkaline phosphatase (ALP), Alizarin Red (AR) stainings as well as the presence of runx-2 and
collagen I in the matrix by immunofluorescence. Cell morphology and matrix formation were
also assessed by scanning electronic microscopy (SEM) in the 3D environment.
VI.3.2. Calcein AM assay
AFSCs- and hMSCs-SPCL constructs were rinsed in PBS and incubated in cell culture medium
with 3 µM Calcein AM (Molecular Probes, Invitrogen) for 30 minutes at 37 ºC in a 5 % CO2
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environment. Then, constructs were rinsed in PBS and fixed in 10 % buffered formalin
(Surgipath Medical Industries, Inc.) overnight at 4 ºC. AFSCs viability was observed under a
confocal microscope (Axiovert 100 M, Zeiss) equipped with argon/He-Ne laser sources. DAPI
(Molecular Probes, Invitrogen) was used as a nuclei marker in cells (live or dead).
VI.3.3. Alkaline Phosphatase (ALP) staining
ALP staining was performed in 2D cultures to evaluate the osteogenic differentiation
process of hAFSCs and hBMSCs in the tissue culture-well plates. Cells were fixed with 10 %
buffered formalin solution overnight at 4 ºC and then rinsed, and kept in PBS until incubating
the cells in a staining solution of 0.25 % Napthol AS-MX phosphate alkaline solution (SigmaAldrich) and Fast Violet B salt (Sigma) for 30 minutes. Afterwards, to remove excess of nonspecific staining, samples were rinsed in PBS and observed under an inverted microscope
(Leica, DMI4000B), and images acquired using a camera Q-Imaging (Retiga-2000RV).
VI.3.4. Alizarin Red Staining (AR)
Alizarin Red staining was performed in both 2D and 3D osteogenic constructs after fixing
the constructs as described above. For this purpose, a 2 % alizarin red solution (Sigma-Aldrich)
was prepared (pH adjusted to 4.1-4.3) and samples stained by immersion for 2 minutes. After
image acquisition of the stained cells and cells-scaffold constructs, AR staining was solubilized
in cetylpyridinium chloride (Sigma) at pH 7.0 for 15 minutes under milt agitation and calciumbounded to AR quantified at 562 nm, using a plate reader (SpectraMax MS, Molecular
Devices).
VI.3.5. Immunofluorescence
Immunofluorescence analysis was performed in samples retrieved from 2D and 3D
conditions in order to assess the presence of bone related proteins, namely RunX-2 and
Collagen type I. RunX-2 and Collagen type I (1:100 and 1:20 dilution, respectively), diluted in
antibody diluent with background reducing components from Dako, were assessed by a
regular immunofluorescence procedure using AlexaFluor 488 or 594 (Molecular Probes,
Invitrogen, 1:200 dilution) as a secondary antibody. Protein-block serum free (Dako, Denmark)
was used in the blocking step.
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VI.3.6. Scanning electronic microscopy (SEM)
Cell seeded scaffolds (3D condition) were observed by scanning electron microscopy
(Hitacho S-2600N, Hitachi Science Systems, Ltd) to assess cell morphology, proliferation as well
as the presence/absence of a mineralized ECM matrix produced by either AFSCs or BMSCs.
SEM was performed in fixed constructs, after dehydrating the samples in a series of ethanol
concentrations followed by drying in a critical point drying equipment (EMS850X, Electron
Microscopy Sciences), and gold sputtered (Hummer 6.2 sputtering system, Anatech Ltd).
VI.4. Results and Discussion
VI.4.1. Osteogenic differentiation of AFSCs and BMSCs in a 2D culture environment
Immunofluorescence imaging (Calcein and DAPI staining) showed that both AFSCs and
BMSCs attached and proliferated for up to 3 weeks in 2D cultures (Figure VI.4.1).
Figure VI.4.1 – Viability assay (green) and Alizarin Red (red) staining of hAFSCs and hBMSCs
cultured in 2D environment for 0, 7, 14 or 21 days in osteogenic medium.
The same pictures suggest an increase in AFSCs viability for 2 weeks in osteogenic medium
while BMSCs maintained high viability levels during the 3 weeks of experiment (Figure VI.4.1).
Alizarin red staining was detected in both cell types after 14 days in culture (Figure VI.4.1),
which tends to become more intense in hAFSCs by the end of week 3 in osteogenic medium.
The increase in staining intensity is not so evident for hBMSCs for the same period of time.
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Concerning the ALP staining that is associated to ECM maturation, hBMSCs cells express the
highest ALP levels of the experiment after 2 weeks in osteogenic culture, indicating an active
phase for the development of ECM. In contrast, AFSCs do not express significant amounts of
ALP during the 2D experimental study (Figure VI.4.2).
Figure VI.4.2- ALP staining of hAFSCs and hBMSCs cultured in 2D environment for 0, 7, 14 or 21
days in osteogenic medium.
RunX-2 is an important transcription factor (TF) involved in the osteogenic development
which was also considered for the characterization of the osteogenic differentiation of AFSCs
and BMSCs. In cell culture plates, RunX-2 is expressed by AFSCs and BMSCs at day 0. After 7
days, a weak signal is also detected in osteogenic culture, being more evident in BMSCs. After
14 days in culture, the presence of RunX-2 is hardly registered (data not showed).
Immunofluorescence for collagen I, an important protein in bone ECM, was not detected
either in AFSCs or BMSCs (data not showed) for up to 3 weeks in 2D cultures.
Calcium levels were quantified using a microplate reader after solving alizarin red staining
(Figure VI.4.3).
Up to week 2, calcium deposition by both cell types followed a similar pattern, exhibiting
relatively low values during the first 14 days in osteogenic medium. After 21 days of culturing,
the calcium amounts deposited by AFSCs increased dramatically (Figure VI.4.3), showing that
AFSCs produce a higher amount of mineralized matrix in the 2D culturing environment when
compared to hBMSCs.
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Figure VI.4.3 – Calcium quantification (g/ml/well) of AFSCs and BMSCs in 2D cultures in
osteogenic medium for 0, 7, 14 or 21 days.
VI.4.2. Osteogenic differentiation of hAFSCs and hBMSCs seeded on SPCL scaffolds (3D
environment)
According to immunofluorescence observations, both stem cell types kept high viability
when cultured on SPCL scaffolds (3D cultures) for up to 3 weeks (Figure VI.4.4).
Figure VI.4.4 – Viability assay of AFSCs and BMSCs seeded onto SPCL scaffolds and cultured in
osteogenic medium for 0, 7, 14 or 21 days.
SEM micrographs revealed a good AFSCs and BMSCs proliferation and distribution on the
SPCL scaffolds, clearly evidenced by the thick layer of cells throughout the entire scaffold
(Figure VI.4.5). The same images suggest the presence of extracellular matrix in BMSCs-SPCL
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constructs after 2 weeks in culture as well as some mineralization nodules (white dots in the
SEM images), which increased in number after 3 weeks in osteogenic supplemented medium.
In AFSCs constructs these nodules are only observed at week 3.
Figure VI.4.5 - Scanning electron microscopy of AFSCs and BMSCs seeded onto SPCL scaffolds
in osteogenic culture for 0, 7, 14 or 21 days.
The formation of mineralization nodules was also evaluated in BMSCs- and AFSCs-SPCL
constructs by Alizarin Red staining (Figure VI.4.6) and in order to determine the presence of
CaP in these aggregates.
Figure VI.4.6 - Calcium quantification (g/ml/construct) of AFSCs- and BMSCs-SPCL constructs
in osteogenic medium for 0, 7, 14 or 21 days.
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Concerning Alizarin Red staining, AFSCs required more calcium in the first days for
metabolic processes, but this tendency changes after 2 weeks in osteogenic medium and by 3
weeks in culture, the calcium concentration produced by BMSCs in SPCL scaffolds is
dramatically higher than in AFSCs-SPCL constructs (Figure VI.4.6).
SEM data are in agreement with results obtained from the calcium quantification assay in
AFSCs- and BMSCs-SPCL constructs, showing that BMSCs are faster in achieving a calcified
matrix in 3D fiber bonding SPCL scaffolds.
In SPCL scaffolds, BMSCs and AFSCs expressed similar RunX2 for the first week of
osteogenic differentiation (Figure VI.4.7).
Figure VI.4.7 – Immunofluorescence of RunX2 expression in AFSCs- and BMSCs-SPCL
constructs in osteogenic medium for 0, 7, 14 or 21 days.
The intensity decreases from day 0 to day 7, increasing again after 2 weeks in osteogenic
medium, especially for AFSCs. By the end of the third week, RunX2 expression is kept high with
a bright green intensity, evidencing that the osteogenic process continues in the 3D constructs
throughout a 3 week-time line and even in the presence of mineralized matrix.
Collagen I was strongly stained in cells seeded onto SPCL scaffolds during the experimental
time (Figure VI.4.8). At day 0, an undifferentiated stage, similar detection levels were
observed for AFSCs and BMSCs. After 7 and 14 days of culture in osteogenic media, a more
intense fluorescence is observed in AFSCs constructs that tends to decrease on week 3, while
in BMSCs-SPCL scaffolds, collagen I showed an increment in fluorescence intensity.
Furthermore, collagen fibers seem to be somehow oriented in some directions over the
SPCL fibers, forming a dense ECM.
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Figure VI.4.8 – Immunofluorescence of Collagen I expression in AFSCs- and BMSCs-SPCL
constructs in osteogenic medium for 0, 7, 14 or 21 days.
VI.5. Discussion
In a 2D culturing environment (tissue culture plates), both AFSCs and BMSCs were able to
differentiate into the osteogenic lineage and produce mineralized ECM, even in the absence of
collagen I and RunX2 expression. Although collagen I accounts for the majority of the organic
matrix of bone tissues, collagen I cannot initiate tissue mineralization since is not responsible
for nucleation and post-nucleation growth of apatite crystallites[27]. Osteogenic cells can
produce a layer of non-collagenous matrix in vitro, at the surface of nonbiological substrata,
prior to a calcium and phosphorus rich ECM. This layer is described to be similar to the cement
line in vivo[28]. In summary, despite some differences found between cells, AFSCs showed
higher proliferation rates and enhanced mineralization of the ECM in 2D cultures when
compared to BMSCs. However, the mineralization seems to compromise the viability of AFSCs
in 2D cultures.
In a 3D environment, i.e., when cells are cultured onto the SPCL scaffolds, the
mineralization observed at 14 days and 21 days for BMSCs and AFSCs, respectively, seems not
to influence cell viability. AFSCs and BMSCs also showed changes in the expression of bone
related markers from 2D to 3D cultures, indicating that culture environments play an
important role in cellular response during the osteogenic differentiation process. Stem cells
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have been described[20, 29, 30] to be quite sensitive to microenvironments and to respond to
topographical patterns suggesting a physical contribution to the cellular expansion and
differentiation process. Some osteoblast responses seem also to be rather substratedependent[31], evidencing the importance of culture surface in modulating cell proliferation,
matrix and inflammatory factor expression.
Unlike the results registered for 2D cultures, in 3D scaffolds collagen I is present before the
mineralization occurs in both AFSCs and BMSCs-SPCL scaffolds, providing the framework and
spatial constraint for ordered crystal deposition and thus assisting formation of calcium
phosphate nodules. The collagen fibers covering the scaffolds seem to be aligned, showing
some degree of organization, which may be important in the process, since a natural
regeneration typically involves an organized distribution of ECM rather than a random
dispersed matrix, which, in the in vivo scenario, is associated to scar or fibrotic tissue
formation.
Despite similar viability and RunX2 levels during the experimental study, as well as collagen
I levels after 21 days in osteo culture, BMSCs and AFSCs showed a different behavior in terms
of mineralization; not only mineralization occurs latter in AFSCs constructs but BMSCs also
produced more mineralized matrix, when seeded onto SPCL scaffolds.
Besides culture substrate-ECM interactions, inductive factors also influence the
programming of MSCs by matrix[20]. Multiple in vitro and in vivo studies confirmed that bonespecific growth factors exert autocrine and paracrine effects on the proliferation,
differentiation, and maturation of osteoprogenitor cells. In this study, the participation of
growth factors in the culture medium is quite evident, due to their pivotal role in achieving the
osteogenic differentiation process. The continuous expression of RunX-2 in constructs also
indicates that osteoblast differentiation process is likely to continue in time, reinforcing the
ECM production and maturation.
In both environments, AFSCs and BMSCs were able to proliferate and differentiate into the
osteogenic phenotype with the production of a mineralized matrix. The final goal was achieved
although differences in osteo markers expression, such as ALP activity, RunX2 and collagen I
were clearly observed. These differences may be related to different contribution from the
external environment, as well as the influence of stem cell source in cellular responses towards
the osteogenic differentiation process, which directly affects ECM production and
composition.
AFSCs higher proliferative capacity and their ability to maintain their pluripotency at higher
passages could be exploitable as a ready available source for large numbers of osteo
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progenitor cells with applicability for allogenous strategies, a broader spectrum than BMSCs,
which may be limited to autologous interventions and to donor’s age. Despite the limitations,
BMSCs represent a promising cell source for cell-based therapeutics, especially in
orthopaedics, since BMSCs are predisposed in a certain extent to follow the osteogenic
phenotype due to their natural commitment to the mesenchymal lineage. The hBMSCs used in
this study were at passage 5, as hBMSC gene expression profiles were demonstrated[32] to
remain stable in vitro between passage 3 and passage 6, thus validating results obtained in this
study from BMSCs. AFSCs share embryonic stem cells and adult stem cells characteristics, and
described to be genetically and phenotypically stable pluripotent cells[11, 18]. Furthermore,
these cells can be extensively expanded, while keeping long telomers and a normal kariotype
to over 250 population doublings, and differentiated into functional cells[2].
The origin of stem cells may also influence cellular response towards external signals,
causing changes in ECM production and composition. Studies describe stem cells differences in
proliferation[15] and differentiation[3, 15] potential, survival capacity[15], cytokines
expression[16] and immunomodulatory properties[19], depending which tissues cells were
obtained from. This is also observed for lineage specific markers such as ALP activity and RunX2 expression[15]. Our data follows this trend, as cells from bone marrow and amniotic fluid
derived stem cells act differently to different substrate matrices, affecting the timing of
differentiation and bone related protein synthesis.
Animal studies are envisioned to understand the full potential and behaviour of both types
of cells while observing the influence of cell source and 3D scaffold when implanted in vivo in a
pre-clinical situation.
VI.6. Conclusions
The application of stem cells might be an adequate alternative for translational practice,
since these cells can be used to enhance the body’s own regenerative potential or developing
new therapies.
The present data showed cellular proliferation of AFSCs and BMSCs, and subsequent
colonization of the substrate. In fact, in both 2D and 3D cultures, the surface was covered with
adherent cells, forming a network that followed the osteogenic differentiation process for up
to 3 weeks in inductive osteo medium. Osteogenic specific markers were detected and
mineralized matrix was formed in 2D as well as 3D substrates seeded with AFSCs and BMSCs.
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Interestingly, cells from different origins seem to express different bone-related markers at
different end points, which may correlate to cell origin and to substrate properties. The
interplay of ECM molecules, cells and culture surface results in a dynamic and balanced system
with different possible outcomes.
In the adaptative process of osteogenic differentiation, the cell source origin seems to have
an important role in selecting pathways involved in ECM synthesis, resulting in different cell
responses to time frames, protein synthesis and culture substrate variations. Thus, AFSCs
might be an interesting alternative to BMSCs for bone tissue engineering applications.
VI.7. References
1. Bajada S, Mazakova I, Richardson JB, Ashammakhi N: Updates on stem cells and their
applications in regenerative medicine. J Tissue Eng Regen Med 2008, 2: 169-183.
2. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC,
Snyder EY, Yoo JJ, et al.: Isolation of amniotic stem cell lines with potential for therapy. Nat
Biotechnol 2007, 25: 100-106.
3. Peng L, Jia Z, Yin X, Zhang X, Liu Y, Chen P, Ma K, Zhou C: Comparative analysis of
mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev
2008, 17: 761-773.
4. Ringe J, Leinhase I, Stich S, Loch A, Neumann K, Haisch A, Haupl T, Manz R, Kaps C, Sittinger
M: Human mastoid periosteum-derived stem cells: promising candidates for skeletal tissue
engineering. J Tissue Eng Regen Med 2008, 2: 136-146.
5. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem
cells. Science 1999, 284: 143-147.
6. Krampera M, Pizzolo G, Aprili G, Franchini M: Mesenchymal stem cells for bone, cartilage,
tendon and skeletal muscle repair. Bone 2006, 39: 678-683.
7. Mauney JR, Volloch V, Kaplan DL: Role of adult mesenchymal stem cells in bone tissue
engineering applications: current status and future prospects. Tissue Eng 2005, 11: 787-802.
8. Tuan RS, Boland G, Tuli R: Adult mesenchymal stem cells and cell-based tissue engineering.
Arthritis Res Ther 2003, 5: 32-45.
9. Stolzing A, Jones E, McGonagle D, Scutt A: Age-related changes in human bone marrowderived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 2008, 129:
163-173.
Chapter VI – Amniotic fluid stem cells versus bone marrow mesenchymal stem cells as a source for bone tissue
engineering
- 145 -
10. Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, Baggett LS, Mikos AG, Cao Y: Donor
age and cell passage affects differentiation potential of murine bone marrow-derived stem
cells. BMC Cell Biol 2008, 9: 60.
11. Rosland GV, Svendsen A, Torsvik A, Sobala E, McCormack E, Immervoll H, Mysliwietz J,
Tonn JC, Goldbrunner R, Lonning PE, et al.: Long-term cultures of bone marrow-derived human
mesenchymal stem cells frequently undergo spontaneous malignant transformation. Cancer
Res 2009, 69: 5331-5339.
12. Poloni A, Maurizi G, Babini L, Serrani F, Berardinelli E, Mancini S, Costantini B, Discepoli G,
Leoni P: Human Mesenchymal Stem Cells from chorionic villi and amniotic fluid are not
susceptible to transformation after extensive in vitro expansion. Cell Transplant 2010.
13. Wakitani S, Mitsuoka T, Nakamura N, Toritsuka Y, Nakamura Y, Horibe S: Autologous bone
marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in
human patellae: two case reports. Cell Transplant 2004, 13: 595-600.
14. Kuroda R, Ishida K, Matsumoto T, Akisue T, Fujioka H, Mizuno K, Ohgushi H, Wakitani S,
Kurosaka M: Treatment of a full-thickness articular cartilage defect in the femoral condyle of
an athlete with autologous bone-marrow stromal cells. Osteoarthritis Cartilage 2007, 15: 226231.
15. Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M: Oct-4-expressing cells in
human amniotic fluid: a new source for stem cell research? Hum Reprod 2003, 18: 1489-1493.
16. Fauza D: Amniotic fluid and placental stem cells. Best Pract Res Clin Obstet Gynaecol 2004,
18: 877-891.
17. Kim J, Lee Y, Kim H, Hwang KJ, Kwon HC, Kim SK, Cho DJ, Kang SG, You J: Human amniotic
fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif 2007, 40: 7590.
18. Sessarego N, Parodi A, Podesta M, Benvenuto F, Mogni M, Raviolo V, Lituania M, Kunkl A,
Ferlazzo G, Bricarelli FD, et al.: Multipotent mesenchymal stromal cells from amniotic fluid:
solid perspectives for clinical application. Haematologica 2008, 93: 339-346.
19. Nadri S, Soleimani M: Comparative analysis of mesenchymal stromal cells from murine
bone marrow and amniotic fluid. Cytotherapy 2007, 9: 729-737.
20. Discher DE, Mooney DJ, Zandstra PW: Growth factors, matrices, and forces combine and
control stem cells. Science 2009, 324: 1673-1677.
21. Khatiwala CB, Kim PD, Peyton SR, Putnam AJ: ECM compliance regulates osteogenesis by
influencing MAPK signaling downstream of RhoA and ROCK. J Bone Miner Res 2009, 24: 886898.
22. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2: 243-252.
Chapter VI – Amniotic fluid stem cells versus bone marrow mesenchymal stem cells as a source for bone tissue
engineering
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23. Gomes ME, Bossano CM, Johnston CM, Reis RL, Mikos AG: In vitro localization of bone
growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and
cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12: 177-188.
24. Gomes ME, Holtorf HL, Reis RL, Mikos AG: Influence of the porosity of starch-based fiber
mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal
cells cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12: 801-809.
25. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5: 41-49.
26. Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, Kirkpatrick CJ: Response of micro- and
macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.
Biomaterials 2007, 28: 240-248.
27. Gu LS, Kim J, Kim YK, Liu Y, Dickens SH, Pashley DH, Ling JQ, Tay FR: A chemical
phosphorylation-inspired design for Type I collagen biomimetic remineralization. Dent Mater
2010, 26: 1077-1089.
28. Davies JE: In vitro modeling of the bone/implant interface. Anat Rec 1996, 245: 426-445.
29. Park JY, Takayama S, Lee SH: Regulating microenvironmental stimuli for stem cells and
cancer cells using microsystems. Integr Biol (Camb) 2: 229-240.
30. Choi CK, Breckenridge MT, Chen CS: Engineered materials and the cellular
microenvironment: a strengthening interface between cell biology and bioengineering. Trends
Cell Biol 2010, 20: 705-714.
31. Tonnarelli B, Manferdini C, Piacentini A, Codeluppi K, Zini N, Ghisu S, Facchini A, Lisignoli G:
Surface-dependent modulation of proliferation, bone matrix molecules, and inflammatory
factors in human osteoblasts. J Biomed Mater Res A 2009, 89: 687-696.
32. Tsai MS, Hwang SM, Chen KD, Lee YS, Hsu LW, Chang YJ, Wang CN, Peng HH, Chang YL,
Chao AS, et al.: Functional network analysis of the transcriptomes of mesenchymal stem cells
derived from amniotic fluid, amniotic membrane, cord blood, and bone marrow. Stem Cells
2007, 25: 2511-2523.
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SECTION V
IN VIVO STUDIES
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Chapter VII
TISSUE ENGINEERED CONSTRUCTS BASED ON SPCL SCAFFOLDS
CULTURED WITH GOAT MARROW CELLS: FUNCTIONALITY IN FEMORAL
DEFECTS
This chapter is based on the following publication:
Rodrigues MT, Gomes ME, Viegas CAA, Azevedo JT, Dias IR, Guzón F, and Reis RL,
Tissue Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow
Cells: Functionality in Femoral Defects. Journal of Tissue Engineering and Regenerative
Medicine, Journal of Tissue Engineering and Regenerative Medicine, 5: 41-49 (2011).
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Chapter VII
TISSUE ENGINEERED CONSTRUCTS BASED ON SPCL SCAFFOLDS
CULTURED WITH GOAT MARROW CELLS: FUNCTIONALITY IN FEMORAL DEFECTS
VII.1. Abstract
This study aims to assess in vivo performance of cell-scaffold constructs of goat marrow
mesenchymal stromal cells (gBMSCs) and SPCL (a blend of starch with polycaprolactone) fiber
mesh scaffolds at different stages of development, using an autologous model. gBMSCs, from
iliac crests, were seeded onto SPCL scaffolds and in vitro cultured for 1 or 7 days in osteogenic
media. After 1 and 7 days, constructs were characterized for proliferation and initial
osteoblastic expression by alkaline phosphatase (ALP) activity. Scanning electron microscopy
analysis was performed to investigate cellular morphology and adhesion to SPCL scaffolds.
Non-critical defects (6 mm diameter, 3 mm depth) were drilled in posterior femurs of 4
adult goats, from which bone marrow and serum were previously collected. Drill defects alone
and defects filled with scaffolds without cells were used as controls. After implantation,
intravital fluorescence markers: xylenol orange, calcein green and tetracycline were injected
subcutaneously after 2, 4 and 6 weeks, respectively for bone formation and mineralization
monitoring. Subsequently, samples were stained with Lévai Laczkó for bone formation and
histomorphometric analysis.
gBMSCs adhered and proliferated on SPCL scaffolds and an initial differentiation into preosteoblasts was detected by an increasing level of ALP activity with the culturing time. In vivo
experiments indicated that bone neoformation occurred in all femoral defects.
Results obtained provided important information about the performance of gBMSC-SPCL
constructs in an orthotopic goat model that enable to design future studies to investigate in
vivo functionality of gBMSC-SPCL constructs in more complex models, namely critical sized
defects and evaluate the influence of in vitro cultured autologous cells in the healing and bone
regenerative process.
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VII.2. Introduction
Skeletal regeneration is initiated by a traumatic episode involving bone damage that often
includes the periosteum, bone marrow spaces, and surrounding soft tissues. Trauma, such as
fracture or surgical cutting and drilling, causes a physical disruption of the mineralized tissue
matrix, death of many cell types, and interruption of the local blood supply. A key stage in
bone regeneration is the differentiation of pluripotential mesenchymal cells from the initial
granuloma containing several cellular types, towards cartilage, fibrous cartilage, fibrous tissue
or bone[1].
Bone marrow mesenchymal cells (BMCs) have been widely used in studies involving TE
strategies for bone and cartilage as they provide a potential autologous source of cells[2-5]
that are able to differentiate into chondrogenic and osteogenic lineages in the presence of
specific differentiation supplements such as transforming growth factor-beta and
dexamethasone, respectively[6].
Though BMCs are abundant in skeletal tissues, damaged bone may fail to heal
spontaneously and, in most cases, the use of marrow cells alone is not ideal to accomplish the
necessary requirements for the repair and or replacement of injured tissues. In order to
overcome the limitations of current treatments, the TE field proposes the use of bioactive or
inductive factors as well as a scaffold structure to support and complement the role of
reparative cells (differentiated or non-differentiated) when implanted on injured or
dysfunctional areas.
The selection of a scaffold material for bone TE purposes is therefore of extreme
significance[7]. Scaffolds must be biocompatible, biodegradable and, simultaneously, promote
the easy diffusion of nutrients and cellular waste products as well as present suitable
mechanical properties for cell support and new tissue ingrowth. Furthermore, the scaffold
must possess adequate porosity, good interconnectivity and a degradation rate[8] adapted to
the time required for tissue regeneration.
Several biodegradable polymers have been proposed to obtain three-dimensional scaffolds
for bone TE, including a new range of natural origin polymers based on starch[8-10]. Starchbased polymers, such as starch-polycaprolactone blends (SPCL), are degradable and
biocompatible polymers with distinct structural forms which had shown to be suitable for
bone TE applications in several previous studies[11-15].
The seeding and extended in vitro culturing of cells within a biodegradable scaffold before
implantation is a widespread TE approach. Some studies also report the importance of cell in
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vitro pre-culture period onto scaffolds as it may influence the osteogenic potential of bone
marrow cells[16]. During the in vitro culturing, seeded cells are expected to proliferate and
secrete growth factors and matrix proteins that possibly will stimulate other cells to accelerate
or recover and, in more dramatic situations, to stimulate the natural regenerative functionality
when transferred to the in vivo environment.
Ultimately and despite the relevance of achieved results from in vitro studies, the use of
animal models is an essential step to evaluate TE constructs prior to their clinical application.
Different animal models, such as rat, rabbit, dog, goat, sheep or monkey have been projected
with the purpose of fairly accurate human model requests[17] and guide potential clinical
applications.
In the last few years, goats are becoming increasingly popular as a valid animal subject in
this research field. In fact, due to its nature of a higher level vertebrate and the non pet status
when compared to dogs, goats play a significant role in the orthopaedics field as a feasible
model for orthotopic applications. In addition, goats not only have metabolic and bone
remodelling rates similar to that of humans but also comparable sequence of events in bone
graft incorporation and healing capacities[18], which explains the fact that this model has been
frequently used in studies of bone formation and regeneration[19-21], biocompatibility[10],
and osteochondral[22, 23] regeneration.
Cell based strategies sustained by a support material have been applied to generate ectopic
or orthotopic bone[24-27]. Although the latter presents a major potential for skeletal
regeneration procedures, most of the in vivo studies are conducted using an ectopic approach
and/or performed in small animal models, such as mice or rats[14, 26, 28-31]. Although noncritical sized defects are usually evaluated in ectopic models, orthotopic location provides a
more accurate idea of the influence or local effects of implanted cells or cell-scaffold
constructs where they were initially designed to be functional.
Autologous approaches have also been considered in recent studies[19, 21, 23] avoiding
immune complex problems that interfere with the regenerative process as well as with the
patient follow up.
The present work describes the assessment of the in vivo osteogenic ability of cell-scaffold
constructs based on seeding marrow mesenchymal stromal cells onto SPCL fiber mesh
scaffolds and in vitro cultured during different periods of time (using an autologous goat
model). As mentioned before, these scaffolds have demonstrated a very good in vitro
functionality in several studies performed by our group. Therefore, the aim of this work is to
obtain the first data concerning the in vivo functionality of SPCL scaffolds and gBMSCs
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constructs, in new bone formation of a orthotopic non critical defect in order to better
understand the behaviour of cell-scaffold constructs implanted in femurs and to design future
in vivo studies to be performed in the critical size defect model, which will be the ultimate
applications for such TE strategies.
VII.3. Materials and Methods
VII.3.1. Production of SPCL scaffolds
The polymer scaffolds used in this study are based on a blend of corn starch and polycaprolactone) (SPCL, 30/70 % wt) and were produced by a fiber bonding method into
mesh structures with a porosity of around 75 % and cut into discs (6 mm diameter, 2 mm
height) as described previously[12, 13]. All samples were sterilized using ethylene oxide and,
prior to cell seeding, the scaffolds were immersed in 20 mL of serum free medium in 50 mL
tubes for 30 minutes.
VII.3.2. gBMSCs harvesting
In order to harvest the gBMSCs, the animals were placed under general anaesthesia and
iliac regions were shaved and disinfected. Animals were submitted to a preanesthetic
medication with acepromazine maleate (5 mg EV, Calmivet, Vetóquinol, France) and placed
under general anaesthesia by induction with thiopenthal sodium (20-25 mg/Kg EV, Pentothal
sodium, Abbott Labs, USA), maintained by inhalation of a mixture of 1.5 % isoflurane (IsoFlo,
Abbott Labs, USA) and oxygen for a maximum of 30 minutes.
From each iliac crest of the goats, 10 mL samples of bone marrow aspirate were obtained,
using a bone marrow aspiration needle (Inter.V, Medical Device Technologies, Inc.) and a 10
mL syringe, containing 1 mL heparin (5,000 U.I., Heparin sodium, B. Braun Medical, Inc.) to
avoid marrow coagulation. The content of each syringe was then transferred into sterile 50 mL
tubes and mixed with 30 mL of RPMI-1640 culture medium (Sigma-Aldrich), containing 1 %
penicillin/streptomycin (Gibco) and an additional 1 mL of heparin (5,000 U.I.). Afterwards,
gBMSCs were centrifuged for 10 minutes at 1200 rpm and a dense cellular pellet was collected
and cultured in 75 cm2 flasks (Corning) using basic culture medium – DMEM (Dulbecco’s
Modified Eagle’s Medium) (Sigma-Aldrich) supplemented with 10 % of autologous serum
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isolated from goat peripheral blood and 1 % antibiotic/antimicotic solution (A/B, Invitrogen).
Four days after the harvesting procedure, the medium containing non-adherent cells was
removed and the adherent cells were rinsed with a sterile phosphate buffered saline (PBS,
Sigma) solution and fresh medium was added. Cells were expanded in basic culture medium
until about 80 % of confluence before being seeded onto SPCL scaffolds at 3 Pa.
During this study, every 3 weeks, goat peripheral blood samples were collected from
jugular vein of each animal, in order to obtain autologous serum, and collected into serum
tubes without anticoagulant (Sarstedt – Monovette® - Serum Gel S). Thirty minutes after
collection, blood samples were centrifuged at 3,000 rpm for 10 minutes. Harvested sera was
immediately stored in appropriate tubes and preserved at -20 ºC until usage.
VII.3.3. In vitro cell seeding and culture
In order to cell seed the scaffolds, gBMSCs cells were thawed and expanded until 90 %
confluence. Afterwards, cells were enzymatically lifted with 0.05 % trypsin-EDTA (Invitrogen)
and at 2 Pa, a cell suspension was prepared (2,500,000 cells/mL) and seeded onto the SPCL
porous scaffolds in a drop-wise manner, at a cellular density of 500,000 cells per scaffold and
using seeding chambers in order to improve cell seeding efficiency by avoiding cellular
dispersion.
After in vitro seeding, cell-scaffold constructs were cultured in non-adherent 12-well plates
(Costar, Becton Dickinson), to avoid cellular adhesion to the bottom of the plates, and using
alpha-MEM (Minimal Essential Medium Eagle alpha modification, Sigma-Aldrich), autologous
sera (10 %), A/B (1 %) and osteogenic supplements, namely, dexamethasone (10-8 M) (SigmaAldrich), ascorbic acid (50 g/mL) (Sigma), and -glycerophosphate (10 mM) (Sigma) for 1 and
7 days prior to implantation. An in vitro control of the experiment was kept, consisting of cellscaffold constructs seeded and cultured under the same conditions and for the same periods
of time. Autologous culture medium was changed twice a week in all cell cultures.
VII.3.4. In vitro characterization of cells-scaffold constructs
Samples were collected on day 1 and day 7 after seeding for the assessment of proliferation
by DNA quantification and initial osteogenic differentiation studies by ALP activity analysis. For
these purposes, samples removed from culture were rinsed twice in a PBS solution and
transferred into 1.5 mL microtubes containing 1 mL of ultra-pure water. Then, gBMSC-SPCL
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constructs were incubated for 1 hour at 37 ºC in a water-bath and stored in a -80 ºC freezer,
promoting a thermal shock variation and thus inducing cell lysis, until testing. Before assessing
DNA and ALP levels, constructs were thawed and sonicated for 15 minutes.
A fluorimetric dsDNA quantification kit (PicoGreen, Molecular Probes) was used to
determine the proliferation of cells in gBMSC-SPCL constructs. Samples and standards (ranging
between 0 and 2 µg.mL-1) were prepared. Triplicates were made for samples and standards.
Afterwards, the 96 well white plate (Costar, Becton Dickinson) was incubated for 10 minutes in
the dark and the fluorescence was read using a microplate ELISA reader (BioTek, USA) at an
excitation of 485/20 nm and an emission of 528/20 nm. A standard curve was developed in
order to read DNA values of samples from the standard graph.
ALP activity was measured to detect initial osteogenic differentiation on day 1 and day 7.
For this purpose, to each well of a 96-well plate (Costar, Becton Dickinson) were added 20 µl of
sample plus 60 µl of substrate solution consisting of 0.2 % (wt/v) p-nytrophenyl phosphate
(Sigma) in a substrate buffer with 1 M diethanolamine HCl (Merck), at pH 9.8. The plate was
then incubated in the dark for 45 minutes at 37 ºC. After the incubation period, 80 µl of a stop
solution (2 M NaOH (Panreac) plus 0.2 mM EDTA (Sigma), was added to each well. Standards
were prepared with p-nytrophenol (10 µmol.mL-1) (Sigma) in order to achieve final
concentrations ranging between 0 and 0.3 µmol.mL-1. Samples and standards were prepared in
triplicates. Absorbance was read at 405 nm and sample concentrations were read off from
standard graph.
gBMSCs adhesion and morphology was also investigated using scanning electronic
microscopy (SEM) by previously fixing cells-scaffold constructs in a 2.5 % glutaraldehyde
solution (Sigma), rinsing and dehydrating through a series of ethanol concentrations, before
coating them in a gold sputter.
VII.3.5. Animals Study
Four skeletally adult female goats weighting 30-45 kg were used in this study. The housing
care and experimental protocol were performed according to the national guidelines, after
approval by the National Ethical Committee for Laboratory Animals (2007-07-27, document
number 018939) and conducted in accordance with international standards on animal welfare
as defined by the European Communities Council Directive of 2 November 1986 (86/609/EEC).
During the entire study, adequate measures were taken to minimize any pain and discomfort.
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Animals were kept in light and temperature controlled rooms and health parameters, such
as appetite, weight maintenance or signs of infection were monitored at a daily basis.
VII.3.5.1. Implantation procedures
Surgical procedures were performed under standard conditions and the drill-hole technique
selected was based on the one described by Hallfeldt et al.[32].
After general anaesthesia each goat was positioned in lateral recumbency, prepared and
draped in a sterile manner to perform a surgical access to the lateral diaphysis of femur. A skin
incision was then performed from the greater throcanter and continued distally to the lateral
femoral condyle. The subcutaneous tissue, tensor fascia lata and lateral fascia of the vastus
muscle were incised. Biceps femoris muscles were retracted posteriorly, and the vastus muscle
was retracted anteriorly, after being detached from the linea aspera and femora shaft, like the
periosteum. Non-critical size defects (6 mm diameter and 3 mm depth) were drilled in the
lateral diaphysis of both posterior femurs of the 4 adult goats with a bone drill (Synthes,
Switzerland). Eight drills were made in each posterior femur, with a separation distance
between drills of 3 cm, in two non parallel sections in order to avoid fracture tension in the
bone and also to avoid new bone formation among drills. Two of those were left empty and
two were filled with scaffolds without cells, which were the controls for this experiment. The
remaining drills were filled in with cells-scaffold constructs cultured for 1 day (2 defects) and 7
days (2 defects) (Figure VII.3.1).
Figure VII.3.1 - Diagram of the implantation site: A) empty drill defects, B) defects filled with
SPCL (no cells), defects filled with cell-SPCL constructs after C) 1 day of culture and D) 7 days of
culture in osteogenic medium. Implants were placed in the same anatomical site relative to
both posterior femurs in each animal.
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The implants were carefully pressed fit and placed into the bilateral defects. The muscle
was replaced over the bone, and the fascia lata and skin were closed with resorbable and nonresorbable sutures, respectively.
After implantation, intravital fluorescence markers, namely, xylenol orange (90 mg/kg,
Aldrich), calcein green (10 mg/kg, Sigma) and tetracycline (25 mg/kg, Sigma) were injected
subcutaneously (after 2, 4 and 6 weeks, respectively) for bone formation and mineralization
monitoring, along with the implantation period.
In the ambulation, animals were observed at a daily basis and signs of infection or pain
were monitored. During the first post-operative week, the animals were subject to an
analgesic medication with flunixin meglumin (1 mg/kg IM, each 24 hours, Finadyne P.A.,
Schering-Plough II) for two days and to an antibiotic therapy with amoxicillin (15 mg/kg IM,
each 24 hours, Clamoxyl L.A., Pfizer) for 7 days.
After implantation, animals were kept in a 25 m2 room, with freedom of movement and full
weight-bearing of the posterior limbs during the complete post-operative period.
VII.3.5.2. Harvesting samples after implantation
Six weeks after the implantation procedure, and 24 hours after tetracycline injection,
animal euthanasia was performed using an overdose of pentobarbital sodium (Eutasil,
Sanofi, France). The femurs were then removed and cut into single defect-sections. Sections
were fixed in a 4 % formaldehyde solution (pH 7.2) (Sigma) and embedded in glycol
methacrylate (Technovit 7200® VLC–Heraus Kulzer GmbH, Germany) blocks. Thin sections with
about 30 µm were prepared using a special microtome, according to the Donath et al.
technique[33] using an Exakt-Cutting® System (Aparatebau GMBH, Germany) in order to slide
calcified bone. Only the mid section of each block was used for observation at the fluorescence
microscope (Olympus BX51, Germany) and for histomorphometric analysis.
Additional sections were also stained with Lévai-Laczkó[34] to observe the new bone
formation using a stereo microscope (Olympus SZX9, Germany). Quantitative measurement for
bone neoformation was carried out after selecting relevant drill surrounding areas where
neobone was marked and quantified using Microimage 4.0 software.
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VII.3.6. Statistical Analysis
Statistical analysis was carried out by mean  standard error of mean using T-Test and 2way ANOVA for in vitro and for in vivo measurements, respectively. At least 4 samples were
considered in the in vitro assays (DNA, ALP, SEM) while 16 samples were considered in vivo for
each condition; A) empty drill, B) drill with SPCL scaffold, drills with SPCL seeded with gBMSCs
for either C) 1 or D) 7 days in osteogenic medium).
In the present study, controls of the experiment were considered; indirectly by in vitro
assessments of DNA and ALP activity studies and directly by the histometric analysis of induced
drills performed in the posterior femurs of each animal.
VII.4. Results and Discussion
VII.4.1. In vitro characterization of autologous gBMSCs-SPCL constructs
SEM micrographs (Figure VII.4.1) indicated that gBMSCs attached to the SPCL fiber meshes,
presented a typical elongated morphology and were homogeneously distributed throughout
the surface of the SPCL scaffold.
These pictures also show an increase in gBMSCs proliferation with the culturing time, as
observed by the cell layer formed on top of the fibers, when compared to cells cultured for 1
day in the same conditions.
Figure VII.4.1 - SEM micrographs of SPCL scaffolds seeded with gBMSCs and in vitro cultured in
osteogenic culture for 1 day (A) or 7 days (B).
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Regarding cell proliferation results, data obtained from the DNA content test (Figure VII.4.2)
indicated that proliferation of gBMSCs seeded onto SPCL scaffolds seemed to slightly decrease
after 7 days in culture although no statistically differences were found (p<0.05, T-Test) to
support these results. Nevertheless, the tendency to cell proliferation decrease could be
directly associated to the increment of ALP activity of gBMSCs.
Figure VII.4.2 - In vitro double strand DNA concentration in SPCL scaffolds seeded with gBMSCs
cultured in osteogenic culture for 1 and 7 days.
Results obtained from the ALP assay revealed that, after 7 days in culture with osteogenic
medium, there was a significant increment in ALP activity levels (Figure VII.4.3) (p<0.05, T-Test)
when compared to levels obtained for 1 day of culture as expected, since these cells were
biochemically stimulated towards the osteogenic pathway[6, 35, 36].
Figure VII.4.3 - In vitro ALP activity in SPCL scaffolds seeded with gBMSCs cultured in
osteogenic culture for 1 or 7 days.
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VII.4.2. In vivo studies
All animals completed the study. No weight differences were observed and no signs of
infection or inflammation were found nearby the implantation areas after 6 weeks.
In this pilot study, orthotopic defects were drilled to determine the influence of SPCL
scaffolds alone and osteogenic differentiation stage of gBMSCs seeded onto SPCL scaffolds in
bone neoformation. In this way, cells were cultured on SPCL scaffolds, after 1 day in
osteogenic culture, practically undifferentiated, or after 7 days in culture when cells have
already initiated the osteogenic process as indicated by in vitro ALP activity levels and by
previous studies performed at our group.
Prior to constructs characterization, femurs were cleaned from muscle as the induced drills
were not easily detected 6 weeks after implantation (both controls and drills containing the
cell-SPCL constructs) due to an excellent regeneration process, which occurred in all cases. In
order to expose the drills and access the inner region, bone was longitudinally cut.
Under macroscopic observation of femoral defects, defects filled with cell-SPCL constructs
seemed to have higher bone growth than empty drill defects or defects filled with SPCL
materials alone.
VII.4.2.1. Histologic and Fluorescence analysis
The observation of sections stained with Lévai-Laczkó, a specific neobone marker (Figure
VII.4.4), shows that there was bone formation in all drill-defects as expected in non-critical
defects. Nevertheless, there was an enhanced neobone formation in defects containing SPCL
scaffolds seeded with gBMSCs, which suggest the importance of the presence of these cells in
the constructs to stimulate bone formation. Giant cells were present in one of the studied
samples, which indicated that after 6 weeks, scaffold materials are being absorbed by the
body, according to some research works[37].
The sequentially administration of fluorescent dyes allowed to monitor bone ingrowth
during the overall period of implantation.
Again, the presence of gBMSCs seems to positively influence bone ingrowth with the time,
especially two weeks after implantation when calcein green was subcutaneously injected
(Figure VII.4.5).
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Figure VII.4.4 - Drill sections marked with Lévai-Laczkó staining. In A) Control 1 – empty drill
defects, B) Control 2 – defects filled with SPCL (no cells), C) defects filled with cells-SPCL
constructs after C1) 1 day of culture and C2) 7 days of culture in osteogenic medium.
Figure VII.4.5 - Drill sections marked with Xylenol Orange (red), Calcein Green (green) and
Tetracycline (not observed) fluorescence stainings. In A) Control 1 – empty drill defects, B)
Control 2 – defects filled with SPCL (no cells), C) defects filled with cell-SPCL.
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However, the degradation rate of each staining was faster than expected, likely because of
the fast metabolism of these animals. Due to this, in some section regions, bone formation was
dark just before the subsequent fluorescent stain was injected. In some pictures is still possible
to observe dark neobone areas, as the new bone was formed. As tetracycline dye was injected
just 24 hours before samples retrieval, the fluorescence mark was too light to be easily
observed in the drill defect images. Even though the stain has reached the bone drills, 24 hours
was probably an insufficient period of time for the fluorescence to be imprinted in the fresh
bone.
Besides a qualitative analysis, histomorphological parameters of bone neoformation were
measured, to obtain quantitative data regarding the percentage of new bone formation and
new bone roundness. The percentage of neobone present in each drill was compared with the
remaining drills and statistically analyzed (Figure VII.4.6).
The amount of new bone formation tended to increase in the presence of cell seeded
scaffolds although the quantitative analysis performed did not reveal significant differences
between the values measured for new bone formation (%) between defects with and without
gBMSCs.
Figure VII.4.6 – New bone formation percentage in the different induced drills: A) empty drill
defects, B) defects filled with SPCL (no cells), defects filled with cell-SPCL constructs after C) 1
day of culture and D) 7 days of culture in osteogenic medium.
Other parameters namely, roundness of new bone formation, were also considered in
order to evaluate the spreading of neobone tissue into the induced defects.
As it can be observed in Figure VII.4.7, there was a tendency to an increment in new bone
roundness obtained in defects filled with cell seeded scaffolds (with increasing culturing times)
Chapter VII – Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in
femoral defects
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when compared to empty defects. This fact may be associated to a cellular stimulation
provided by autologous cells implanted in the animals.
Figure VII.4.7 - New bone roundness measured for the different induced drills: A) empty drill
defects, B) defects filled with SPCL (no cells), defects filled with cell-SPCL constructs after C) 1
day of culture and D) 7 days of culture in osteogenic medium.
Neither inflammatory response after 6 weeks of implantation nor animal behaviour
modifications in terms of food intake, movement or general health was detected. The
presence of fiber mesh SPCL scaffolds allowed for neobone ingrowth in the induced defects.
Furthermore, traces of cell-mediated absorption were observed in one of the retrieved
samples by the presence of fibrous tissue containing an occasional multinucleated giant cell on
the implant surface. This may indicate that locally, material absorption can initiate at an early
stage of bone regeneration.
VII.5. Conclusions
In the present study it was possible to observe the neoformation of bone in all orthotopic
induced drills in the goat femurs, as expected for non-critical defects. Nevertheless, it was
found an increased neobone formation as well as cellular distribution into the defect where
the gBMSC-SPCL constructs were implanted. This increment is enhanced with the in vitro
culturing time which indicates that in vitro culturing time of gBMSCs onto the SPCL constructs
may also play an important role in new bone growth. The data obtained concerning in vitro
Chapter VII – Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in
femoral defects
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proliferation and differentiation of these constructs suggest that, the in vitro culturing time of
gBMSCs in osteogenic medium, though still in a very early stage, is likely to play an important
role in bone growth onto these defects.
However, further studies should provide better understanding of the importance of the
number of cells and their differentiation stage as well as the time needed for osteogenic
supplementation to induce significant changes in bone formation in vivo. Nevertheless, SPCL
fiber mesh scaffolds showed a great potential for the development of adequate tissue 3D
support for the regeneration of bone in non-critical defects.
Thus, results obtained provided important information about the performance of gBMSCsSPCL constructs in an orthotopic goat that enable to design future studies to investigate the in
vivo functionality of gBMSC-SPCL constructs in more complex models, namely in induced
critical sized defects and evaluate the influence of in vitro cultured autologous cells in the
healing and bone regenerative process.
VII.6. References
1. Dennis R, Carter GSB: Chapter 7 - Skeletal Tissue Regeneration. In Skeletal Function and
Form - Mechanobiology of Skeletal Development, Aging and Regeneration. Edited by:
Cambridge University Press; 2001:161-200.
2. Mauney JR, Volloch V, Kaplan DL: Role of adult mesenchymal stem cells in bone tissue
engineering applications: current status and future prospects. Tissue Eng 2005, 11: 787-802.
3. Muraglia A, Cancedda R, Quarto R: Clonal mesenchymal progenitors from human bone
marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000, 113 (Pt 7):
1161-1166.
4. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA,
Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem
cells. Science 1999, 284: 143-147.
5. Tuan RS, Boland G, Tuli R: Adult mesenchymal stem cells and cell-based tissue engineering.
Arthritis Res Ther 2003, 5: 32-45.
6. Oliveira JM, Rodrigues MT, Silva SS, Malafaya PB, Gomes ME, Viegas CA, Dias IR, Azevedo JT,
Mano JF, Reis RL: Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissueengineering applications: Scaffold design and its performance when seeded with goat bone
marrow stromal cells. Biomaterials 2006, 27: 6123-6137.
C
Chapter VII – Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in
femoral defects
- 166 -
7. Hutmacher DW, Schantz JT, Lam CX, Tan KC, Lim TC: State of the art and future directions of
scaffold-based bone engineering from a biomaterials perspective. J Tissue Eng Regen Med
2007, 1: 245-260.
8. Gomes ME, Reis RL: Biodegradable polymers and composites in biomedical applications:
from catgut to tissue engineering - Part 1 - Available systems and their properties.
International Materials Reviews 2004, 49: 261-273.
9. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2: 243-252.
10. Mendes SC, Reis RL, Bovell YP, Cunha AM, van Blitterswijk CA, de Bruijn JD:
Biocompatibility testing of novel starch-based materials with potential application in
orthopaedic surgery: a preliminary study. Biomaterials 2001, 22: 2057-2064.
11. Gomes ME, Sikavitsas VI, Behravesh E, Reis RL, Mikos AG: Effect of flow perfusion on the
osteogenic differentiation of bone marrow stromal cells cultured on starch-based threedimensional scaffolds. J Biomed Mater Res A 2003, 67: 87-95.
12. Gomes ME, Holtorf HL, Reis RL, Mikos AG: Influence of the porosity of starch-based fiber
mesh scaffolds on the proliferation and osteogenic differentiation of bone marrow stromal
cells cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12: 801-809.
13. Gomes ME, Bossano CM, Johnston CM, Reis RL, Mikos AG: In vitro localization of bone
growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and
cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12: 177-188.
14. Mendes SC, Bezemer J, Claase MB, Grijpma DW, Bellia G, Degli-Innocenti F, Reis RL, de
Groot K, van Blitterswijk CA, de Bruijn JD: Evaluation of two biodegradable polymeric systems
as substrates for bone tissue engineering. Tissue Eng 2003, 9 Suppl 1: S91-101.
15. Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, Kirkpatrick CJ: Response of micro- and
macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.
Biomaterials 2007, 28: 240-248.
16. Sikavitsas VI, van den Dolder J, Bancroft GN, Jansen JA, Mikos AG: Influence of the in vitro
culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs
using a rat cranial critical size defect model. J Biomed Mater Res A 2003, 67: 944-951.
17. Buma P, Schreurs W, Verdonschot N: Skeletal tissue engineering-from in vitro studies to
large animal models. Biomaterials 2004, 25: 1487-1495.
18. Pearce AI, Richards RG, Milz S, Schneider E, Pearce SG: Animal models for implant
biomaterial research in bone: a review. Eur Cell Mater 2007, 13: 1-10.
19. Kruyt MC, Dhert WJ, Yuan H, Wilson CE, van Blitterswijk CA, Verbout AJ, de Bruijn JD: Bone
tissue engineering in a critical size defect compared to ectopic implantations in the goat. J
Orthop Res 2004, 22: 544-551.
Chapter VII – Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in
femoral defects
- 167 -
20. Li X, Feng Q, Liu X, Dong W, Cui F: Collagen-based implants reinforced by chitin fibres in a
goat shank bone defect model. Biomaterials 2006, 27: 1917-1923.
21. Zhu L, Liu W, Cui L, Cao Y: Tissue-engineered bone repair of goat-femur defects with
osteogenically induced bone marrow stromal cells. Tissue Eng 2006, 12: 423-433.
22. Lane JG, Massie JB, Ball ST, Amiel ME, Chen AC, Bae WC, Sah RL, Amiel D: Follow-up of
osteochondral plug transfers in a goat model: a 6-month study. Am J Sports Med 2004, 32:
1440-1450.
23. Niederauer GG, Slivka MA, Leatherbury NC, Korvick DL, Harroff HH, Ehler WC, Dunn CJ,
Kieswetter K: Evaluation of multiphase implants for repair of focal osteochondral defects in
goats. Biomaterials 2000, 21: 2561-2574.
24. Giannoni P, Mastrogiacomo M, Alini M, Pearce SG, Corsi A, Santolini F, Muraglia A, Bianco
P, Cancedda R: Regeneration of large bone defects in sheep using bone marrow stromal cells. J
Tissue Eng Regen Med 2008, 2: 253-262.
25. Kirker-Head C, Karageorgiou V, Hofmann S, Fajardo R, Betz O, Merkle HP, Hilbe M, von
Rechenberg B, McCool J, Abrahamsen L, et al.: BMP-silk composite matrices heal critically sized
femoral defects. Bone 2007, 41: 247-255.
26. Kruyt MC, Dhert WJ, Oner FC, van Blitterswijk CA, Verbout AJ, de Bruijn JD: Analysis of
ectopic and orthotopic bone formation in cell-based tissue-engineered constructs in goats.
Biomaterials 2007, 28: 1798-1805.
27. Meinel L, Betz O, Fajardo R, Hofmann S, Nazarian A, Cory E, Hilbe M, McCool J, Langer R,
Vunjak-Novakovic G, et al.: Silk based biomaterials to heal critical sized femur defects. Bone
2006, 39: 922-931.
28. Livingston T, Ducheyne P, Garino J: In vivo evaluation of a bioactive scaffold for bone tissue
engineering. Journal of Biomedical Materials Research 2002, 62: 1-13.
29. Mauney JR, Jaquiery C, Volloch V, Herberer M, Martin I, Kaplan DL: In vitro and in vivo
evaluation of differentially demineralized cancellous bone scaffolds combined with human
bone marrow stromal cells for tissue engineering. Biomaterials 2005, 26: 3173-3185.
30. Mastrogiacomo M, Papadimitropoulos A, Cedola A, Peyrin F, Giannoni P, Pearce SG, Alini
M, Giannini C, Guagliardi A, Cancedda R: Engineering of bone using bone marrow stromal cells
and a silicon-stabilized tricalcium phosphate bioceramic: evidence for a coupling between
bone formation and scaffold resorption. Biomaterials 2007, 28: 1376-1384.
31. Trojani C, Boukhechba F, Scimeca JC, Vandenbos F, Michiels JF, Daculsi G, Boileau P, Weiss
P, Carle GF, Rochet N: Ectopic bone formation using an injectable biphasic calcium
phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal
cells. Biomaterials 2006, 27: 3256-3264.
32. Hallfeldt KK, Stutzle H, Puhlmann M, Kessler S, Schweiberer L: Sterilization of partially
demineralized bone matrix: the effects of different sterilization techniques on osteogenetic
properties. J Surg Res 1995, 59: 614-620.
Chapter VII – Tissue engineered constructs based on SPCL scaffolds cultured with goat marrow cells: functionality in
femoral defects
- 168 -
33. Donath K: Preparation of histologic sections by the cutting-grinding technique for hard
tissue and other material not suitable to be sectioned by routine methods. In Equipment and
methodical performance. Edited by: Exakt-Kulzer-Publication; 1995.
34. Jeno L, Geza L: A simple differential staining method for semi-thin sections of ossifying
cartilage and bone tissues embedded in epoxy resin. Mikroskopie 1975, 31: 1-4.
35. Rodrigues MT, Oliveira JM, Gomes ME, Viegas CA, Dias IR, Mano JF, Reis RL: Novel
hydroxyapatite/chitosan bilayer scaffolds for the regeneration of osteochondral defects using
a tissue engineering approach based on culturing and differentiation of goat marrow cells into
osteoblasts or chondrocytes. In World Congress on Tissue Engineering and Regenerative
Medicine April Pittsburg, Pennsylvania, USA: 2006.
36. Rodrigues MT, Leonor I, Tuzlakoglu K, Gomes ME, Viegas CA, Dias IR, Reis RL: Novel In Situ
Approach for the Design of Osteoconductive and Osteoinductive 3D Wet-Spun Fibre Mesh
Scaffolds for Bone Tissue Engineering. In Tissue Engineering International and Regenerative
Medicine Society - Asia Pacific Chapter Meeting 2007; Tokyo, Japan: 2007.
37. Pego AP, Van Luyn MJ, Brouwer LA, van Wachem PB, Poot AA, Grijpma DW, Feijen J: In vivo
behavior of poly(1,3-trimethylene carbonate) and copolymers of 1,3-trimethylene carbonate
with D,L-lactide or epsilon-caprolactone: Degradation and tissue response. J Biomed Mater Res
A 2003, 67: 1044-1054.
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Chapter VIII
THE EFFECT OF THE DIFFERENTIATION STAGE OF AMNIOTIC FLUID
STEM CELLS SEEDED ONTO BIODEGRADABLE SCAFFOLDS IN THE
REGENERATION OF NON-UNION DEFECTS
This chapter is based on the following publication:
Rodrigues MT, Lee BK, Shiner T, Lee SJ, Gomes ME, Reis RL, Atala A, and Yoo J, The effect of the
differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds in the
regeneration of non-union defects, submitted
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Chapter VIII
THE STAGE OF DIFFERENTIATION OF AMNIOTIC FLUID STEM CELLS
SEEDED ONTO BIODEGRADABLE SCAFFOLDS IN THE REGENERATION OF NON-UNION DEFECTS
VIII.1. Abstract
Bone tissue engineering (TE) strategies mainly require cells with high proliferative and
osteogenic potential and a suitable scaffold to support cellular development towards new
bone tissue formation. This study aimed at evaluating the effect of the differentiation stage of
human amniotic fluid stem cells (hAFSCs) in the regeneration of femoral critical sized defects in
a nude rat model. For this purpose, hAFSCs were seeded onto a starch-polycaprolactone (SPCL)
scaffold and in vitro cultured for different periods of time in osteogenic medium in order to
obtain: i) undifferentiated cells, ii) cells committed to the osteogenic phenotype and iii)
“osteoblastic-like” cells. In vitro results indicated that hAFSCs kept a high viability for up to 3
weeks seeded onto SPCL scaffolds. Furthermore, hAFSCs were considered to be osteogenically
committed by the end of week 2 and osteoblastic-like after 3 weeks in culture.
The constructs, obtained by in vitro culturing hAFSCs onto SPCL scaffolds until reaching
these three differentiation stages, were then implanted in femoral critical sized defects
induced in nude rats for 4 or 16 weeks. Empty defects and defects filled with scaffold alone
were used as controls. The quality of new tissue formed in the defects was evaluated based on
micro-CT and histological analysis of samples retrieved at 4 and 16 weeks of implantation. In
vivo neoformation of bone was observed in all conditions. Nevertheless, the best bridging
between the two sections of the defect was observed in the presence of SPCL scaffolds seeded
with osteogenically committed AFSCs after 16 weeks. Furthermore, the presence of blood
vessels in the inner sections of the SPCL scaffold seeded with hAFSCs provides further evidence
of the great potential of SPCL scaffolds combined with hAFSCs for bone regeneration and
angiogenesis in non-union defects.
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
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VIII.2. Introduction
Fracture healing is a complex physiological phenomenon involving the spatial and temporal
coordinated action of different cell types, proteins and the expression of several genes working
towards the restoration of tissue structure and function in a proper mechanical environment.
Age, gender, mechanism of injury and type of fracture (e.g. force impact, stress or negligible
injuries combined with particular medical conditions, such as osteoporosis, bone cancer or
osteogenesis imperfecta), associated injuries, comorbidities, lifestyle and pharmacological
agents are all factors that could interfere with the fracture healing response and contribute to
the development of non-union of fractures[1]. Of all the fracture injuries, up to 10 % will
progress into complicated fractures, which may result in non-union defects[2]. This represents
a contemporary problem associated with an increasing clinical burden. Despite the bone
innate fracture repair mechanisms, non-unions or critical size segmental defects are still a
major challenge in reconstructive orthopedic surgery[3].
The current clinical strategy to treat fracture non-union is autologous bone grafting, where
bone chips are removed from a secondary site into the fracture to participate in the healing
process. Although bone graft remains the gold standard treatment of non-unions[4], some
problems may be associated to, such as bone graft tissue site morbidity and limited quantity or
quality of autologous bone graft material. In order to find an alternative solution, synthetic
materials have been developed with suitable degradation kinetics and interesting properties to
match the requirements in bone applications. Nevertheless, a highly osteogenic cell source is
required, together with a 3D scaffold, to improve the bone regenerative potential. This need is
even more important in larger injuries to establish cellular and molecular communications in
situ, promoting a bond between the implant and the native systems. Several studies
emphasized the importance of implanting cells into the defect to ensure the bone regenerative
process[5-7]. Undifferentiated stem cells are multipotent and, once at the injury site, they can
differentiate into the type of cell necessary according to the regenerative natural timing, while
recruiting important growth factors[8] that stimulate in situ repair.
Among the sources of stem cells, amniotic fluid arises as an attractive source of pluripotent
stem cells without raising the ethical concerns associated with the use of human embryonic
stem cells[9]. Additionally, AFSCs have a highly self-renewal capability and have the potential
to differentiate along several lineages[10, 11], including bone[10, 12], due to their origin from
embryonic and extra-embryonic tissues. AFSCs also present advantages compared to other
primary cells; unlike osteoblasts or bone marrow cells, AFSCs are not limited by tissue
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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insufficient supply and morbidity, host immune responses and disease transmission risks.
Furthermore, due to AFSCs high proliferative capacity and their ability to maintain their
pluripotency at higher passages, these cells may be a readily available source for large
numbers of osteo progenitor cells aimed at bone TE strategies.
Despite the potential of cell application in a scaffold to promote the regenerative process in
locus, few studies addressed the effect of the cell differentiation stage in the regenerative
process of bone[13, 14]. Since the bony milieu is a rich cocktail of growth factors, and
molecular signals, where diverse cell types are present in several stages of differentiation, it is
likely that the number or concentration of a particular molecule or cell type might result in a
different role in promoting bone healing. Furthermore, undifferentiated or partially
differentiated cells, due to their plasticity potential may participate in an immediate response
towards an injury or trauma event, and activate signaling pathways that may not be naturally
triggered by differentiated cells, such as osteoblasts.
In this work, we hypothesize that the stage of differentiation of human AFSCs into the
osteogenic lineage may affect differently the evolution of the regenerative process in nonunion defects of bone. The pre-culture period of hAFSCs in osteogenic supplemented medium
may be a critical factor, affecting the osteoinductive potential and consequently, the new bone
formation when orthotopically implanted.
Therefore, the main purpose of this study was to evaluate the influence of the
differentiation stage of human amniotic fluid stem cells (hAFSCs) cultured onto starch and
polycaprolactone (SPCL) scaffolds, and in the regeneration of bone critical sized defects.
In order to achieve our goals, stem cells from amniotic fluid were expanded in basic
amniotic fluid cell medium, seeded onto SPCL scaffolds and cultured in osteogenic medium for
different periods of time in order to have i) stem cells, ii) cells committed to the osteogenic
phenotype and iii) “osteoblastic” cells. After reaching each stage of differentiation, samples
were implanted in critical sized defects in the right femur of nude rats and kept for 4 or 16
weeks. In vivo bone regeneration was monitored through micro-CT and histological analysis
was performed after each end point.
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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VIII.3. Materials and Methods
VIII.3.1. In vitro Study
SPCL scaffolds were produced by a fiber bonding technique described previously[15, 16,
17], cut into cylinders with 3.8 mm diameter and 6.0 mm length and sterilized by ethylene
oxide.
Human AFSCs were isolated and characterized as previously described[10] and cultured in
basic amniotic fluid cell (BAFC) medium containing α-MEM (HyClone Laboratories IncUSA), 2 %
Chang B (Irvine Scientific, USA) and 18 % Chang C (Irvine Scientific, USA) media as well as 1 % Lglutamine (HyClone Laboratories Inc., USA) and 15 % embryonic stem screened fetal bovine
serum (ES-FBS, HyClone Laboratories Inc., Logan, Utah, USA). Then, hAFSCs were seeded onto
SPCL scaffolds at a density of 1.2x106 cells/scaffold and cultured in BAFC medium for 3 days.
hAFSCs were characterized in terms of the osteogenic differentiation process. Osteogenic
commitment was related to hAFSCs ability to express markers associated to the osteo lineage
while osteoblastic stage was to be accomplished with the production of a mineralized ECM.
Following 3 days in BAFC medium, hAFSCs seeded onto the SPCL scaffolds were cultured in
osteogenic medium, composed of DMEM (HyClone Laboratories Inc., Logan, Utah, USA)
supplemented with 10 % FBS (HyClone Laboratories Inc., USA) and osteogenic supplements;
100 nM dexamethasone (Sigma, USA), 50 µM L-ascorbic acid (Sigma, USA) and 10 mM glycerol
2-phosphate disodium salt hydrate (Sigma, USA) for different periods of time (0, 1, 2 and 3
weeks), determined in a preliminary study, so as to obtain i) undifferentiated cells or stem
cells, ii) cells committed to the osteogenic phenotype and iii) “osteoblastic-like” cells. After
each culturing period, hAFSCs-SPCL samples were characterized for osteogenic phenotypic
expression and matrix formation by alkaline phosphatase (ALP) and Alizarin Red (AR) stainings
as well as the presence of collagen I in the matrix. The detection of a mineralized matrix was
further analyzed by Fourier transformed infra red attenuated spectroscopy (FTIR-ATR). Cell
morphology and matrix formation were also assessed by scanning electronic microscopy
(SEM).
VIII.3.1.1. Alizarin Red (AR) and Alkaline phosphatase (ALP) staining
Alizarin Red and ALP staining were performed in hAFSCs-SPCL constructs cultured in either
BAFC (0 days) or osteogenic medium after each culturing period namely, 1, 2 and 3 weeks.
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
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Macroscopic sections of hAFSCs-SPCL construct, approximately 2 mm x 5 mm each, were
considered for ALP and AR stainings, where constructs were fixed with 10 % buffered formalin
solution overnight at 4 ºC and then rinsed and kept in PBS until usage.
AR: Samples were stained with a photosensitive solution of AR (0.2 %, Sigma-Aldrich, USA),
previously prepared (pH adjusted to 4.1-4.3), for about 2 minutes at room temperature using a
bench shaker. Then, samples were rinsed in PBS and AR staining was solubilized in a
cetylpyridinium chloride (Sigma, USA) solution at pH 7.0 for 15 minutes using a bench shaker.
Calcium-bound AR was quantified by spectrophotometric measurements in a plate reader
(SpectraMax MS, Molecular Devices, Sunnyvale, CA, USA) at 562 nm.
ALP: constructs were incubated in a staining solution of 0.25 % Napthol AS-MX phosphate
alkaline solution (Sigma-Aldrich, USA) and Fast Violet B salt (Sigma, USA) for 30 minutes.
Samples were rinsed in PBS to remove excess of non-specific staining.
VIII.3.1.2. SEM (scanning electronic microscopy)
Human AFSC-SPCL constructs were rinsed in PBS and fixed in 10 % buffered formalin
overnight, dehydrated in a series of ethanol concentrations and kept in absolute ethanol until
critical point drying (EMS850X, Electron Microscopy Sciences, USA). Afterwards, constructs
were gold sputtered (Hummer 6.2 sputtering system, Anatech Ltd, USA), before SEM (Hitacho
S-2600N, Hitachi Science Systems, Ltd, Japan) observation of the morphology of hAFSCs
seeded SPCL scaffolds after 0, 7, 14 and 21 days in osteogenic culture.
Also, in order to detect calcium (Ca) and phosphorus (P) ions present in the ECM matrix, an
energy dispersive spectroscopy (EDS) equipment (Leica Cambridge S360, UK) was used.
VIII.3.1.3. FTIR-ATR (Fourier transform attenuated total reflectance infrared
spectroscopy)
hAFSCs-SPCL samples, obtained from each end point, were dehydrated and air dried,
before proceeding to a FT-IR analysis. FT-IR was performed with an attenuated total
reflectance[18] in the spectral range of 1800-600 cm-1 using the Spectrum 400 FT-IR/FT-NIR
spectrometer (Perkin Elmer, USA).
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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VIII.3.1.4. Immunofluorescence
hAFSC-SPCL constructs were rinsed in PBS and fixed in formalin prior to the detection of
mouse monoclonal to collagen type I (Abcam, 1:100 dilution in antibody diluent with
background reducing components from Dako, Denmark) by a regular immunofluorescence
procedure using anti-mouse AlexaFluor 594 (Molecular Probes, Invitrogen; 1:200 dilution) as a
secondary antibody. Instead of animal serum, blocking step was performed using protein block
serum free (Dako, Denmark). Constructs were then observed under a confocal microscope
(Axiovert 100 M, Zeiss, Germany) equipped with argon/He-Ne laser sources.
VIII.3.2. In vivo Study
All surgical procedures were performed in accordance with Wake Forest University Animal
Care and Use Committee (ACUC) approved protocols (protocol number; A07-063/A10-032). In
this study, male athymic nude rats (36-40 week old) weighing 420-560 g (n=60) were
purchased from Charles River. Animals were randomly assigned into 5 groups (n=6 / group) as
follows: 1) no-repair group (empty defects, no implant), 2) scaffold-only group, and 3) scaffold
with undifferentiated hAFSCs cell group, 4) scaffold with hAFSC osteogenically committed
group, and 5) scaffold with hAFSCs differentiated into osteoblast like cells group.
Anesthesia was induced with 3 % isoflurane (USP, Novaplus) prior to surgical procedures.
The dorsolateral side of the right leg was shaved and sterilized with routine aseptic agent.
Under aseptic conditions, a 20 mm long incision was made on the skin over the femur of the
right hind limb. The skin and the gluteus muscle were dissected to approach the femur. A
periosteal incision was made on the periosteum of the femur, and the periosteum and the
attached muscles were elevated to expose the femur. Retracting adjacent tissues, a 3.0 mm
thick and 2.2 cm long custom-made bone plate was placed along the intact femur and was
fixed with 4 stainless steel screws using a micro drill system (BS72-4950, Harvard apparatus,
USA) to stabilize the femur after scaffold implantation. A 5 mm bone segment in between
screwed areas at both ends was removed from the femur using a bone cutting bur. The
created defects were thoroughly irrigated with sterile saline to avoid residual bone particles at
the site. Subsequently, the scaffolds and cell-scaffold constructs from the different study
groups, according to our experimental design, were implanted to the bony defects and fixed
with a press-fit manner. After inserting the scaffolds/constructs, the muscle and the skin were
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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closed layer by layer using 5-0 Vicryl sutures (Ethicon, USA). The animals were maintained
postoperatively according to the guideline of the ACUC.
Radiographic 2D images of the femoral defects were obtained from each animal every 4
weeks, using C-arm equipment (Siremobil Compact L, Siemens) to monitor plate stability and
select animals for micro-CT analysis. Animals were kept under general anaesthesia during both
(x-rays and micro-CT) procedures.
Rats were euthanized 4 or 16 weeks after implantation accordingly to ACUC guidelines.
Right femurs were retrieved, rinsed in PBS, placed in 10 % buffered formalin for 96 h and then
kept in 60 % ethanol until histological processing.
VIII.3.2.1. µ-CT scanning
Real time live imaging of the rat femoral defects was obtained using an μ-CT equipment,
Siemens MicroCAT II (Siemens Preclinical Solutions, USA) and MicroCAT II version 1.9d
software was used to acquire raw data.
Scans were performed with an x-ray voltage of 70 kVp, anode current of 500 μA, and an
exposure time of 1600 ms (BIN Factor of 2, 360 ° rotation, 360 steps, 36 micron isotropic voxel
dimension).
RVA version 4.2.9 and COBRA EXXIM version 4.9.52 were used to reconstruct the raw data
into raw slice images, and then Amira version 3.1 was used for conversion to DICOM images.
Analysis was done after transfer of images to TeraRecon AquariusNET Server (TeraRecon, Inc.,
USA) using TeraRecon software AquariusNET version 1.8.1.6. Another software packages used
for image analysis and volumetric measurements included Mimics version 13 (Materialise,
Belgium).
VIII.3.2.2. Tissue processing
Explants were decalcified using an acid solution of Immunocal® (Decal Chemical Corp, USA),
and tissue processed in a graded series of ethanol, xylene and embedded in paraffin blocks.
Sections of 10 m were obtained for histological characterization by Hematoxylin/Eosin (H&E)
staining and for immunocytochemistry (ICC) for goat anti-Collagen I (1310-01, Southern
Biotech; 1:20 dilution), goat polyclonal anti- Osteocalcin (OC) (V-19, Santa Cruz Biotechnology,
1:100) and rabbit polyclonal anti-VEGF (147, Santa Cruz Biotechnology, 1:100) antibodies. All
primary antibodies were diluted in antibody diluent with background reducing components
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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from Dako (Denmark). Biotinylated secondary antibodies were purchased to Vector Lab
(Burlingame, CA, USA) as well as R.T.U. HRP/Straptavidin (SA-5704). Immunohistochemistry
visualization was assessed by NovaRED substrate kit (SK-4800), followed by a brief
counterstain with Gill’s hematoxylin. Specimen slides were observed under a microscope
(Imager Z1m, Zeiss, Germany) equipped with a digital camera (AxioCam MRc5).
VIII.3.2.3. Histomorphometrical Analysis
Quantitative measurement for bone related markers, namely osteocalcin and collagen I as
well as VEGF vascular marker were carried out for the in vivo study. After selecting an interest
area within the defect, the protein expression was detected and quantified by Cell D software
(analysis image processing) and MicroImage software from Olympus Optical Co.
VIII.3.3. Statistical Analysis
Statistical analysis was carried out by mean  standard deviation for in vitro assays, while
histomorphometric analysis was carried out by mean  standard error of mean. Two Way
ANOVA test was also applied to check the existence of statistical differences between sample
groups followed by Bonferroni’s Multiple Comparison test (* = p<0.05).
VIII.4. Results and Discussion
VIII.4.1. In vitro study
hAFSCs adhered and maintained viability when seeded onto the SPCL scaffolds during the
experimental time up to 3 weeks (data not showed). According to SEM pictures (Figure
VIII.4.1), cells, independently of their differentiation stage, bridged between scaffold
microfibers covering the scaffold surface, without obstructing most pores, randomly
distributed throughout the scaffold.
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Figure VIII.4.1 - SEM micrographs of hAFSC-SPCL constructs after 0, 7, 14 or 21 days in
osteogenic medium.
When constructs were sectioned, the presence of cells was also observed inside the
scaffold (Figure VIII.4.1; SEM insets), showing that the seeding was efficient and the scaffold is
suitable for nourish human AFSCs.
Furthermore, after 2 weeks in osteogenic culture medium, cells were positively stained for
ALP (Figure VIII.4.2) and for immunofluorescence of collagen type I (Figure VIII.4.3).
Figure VIII.4.2 – Characterization of AFSC-SPCL constructs for ALP staining after 0, 7, 14 or
21 days in osteogenic culture.
Figure VIII.4.3 - Characterization of AFSC-SPCL constructs for collagen I by
immunofluorescence, after 0, 7, 14 or 21 days in osteogenic culture.
Osteoblastic differentiation is usually accompanied by an initial decrease in the cellular
proliferation rate, changes in gene and protein expression of several osteogenic markers, such
as ALP and type I collagen, and later on by the deposition of minerals on the ECM matrix.
The production of organic ECM by AFSCs after 2 weeks in osteogenic medium shows a
cellular commitment towards the osteogenic lineage, characterized by an ALP and collagen I
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strong expression. ALP is a glycoprotein associated with the formation and maturation of the
ECM[19], while collagen type I is the major constituent of bone inorganic matrix. The presence
of both markers in AFSCs-SPCL constructs point out a rich collagen-based ECM (Figure VIII.4.2
and VIII.4.3). Besides the structural support, ECM plays a critical role in cellular interactions
and sustains mineral nucleation, critical for bone functional properties.
Furthermore, after 14 days in osteo culture, thin, nano sized fibers surrounding the cells
that may represent matrix, were found at the surface and in inner sections of the construct in
SEM micrographs. Although cells are producing an ECM by 2 weeks in osteogenic culture, the
ECM mineralization only occurs after 3 weeks in osteogenic supplemented culture, with the
deposition of calcium and phosphate ions. In SEM images from 3 weeks in osteo medium,
calcium phosphate (CaPs) aggregates are found throughout the construct (Figure VIII.4.1 and
Figure VIII.4.4), confirmed by EDS analysis, calcium quantification, and FTIR (Figure VIII.4.4,
Figure VIII.4.5 and Figure VIII.4.6, respectively).
Figure VIII.4.4 – Characterization of AFSC-SPCL constructs by SEM and EDS analysis. SEM
magnified images of calcium phosphate nodules at the surface and inside (inset) of these
constructs after 21 days in osteogenic medium. The presence of calcium and phosphorus
atoms was detected by EDS analysis after 0, 7, 14 and 21 days in osteogenic culture. SPCL
spectrum represents a SPCL scaffolds without seeded cells.
Calcium quantification in the constructs indicated that the presence of calcium is kept at a
basal level from 0 to 14 days in culture and, at the third week in culture, this value increases
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about 50 %, which indicates that calcium is being added to the matrix, inducing the formation
of a calcified matrix produced by AFSCs.
Figure VIII.4.5 - Calcium content measurement in AFSC-SPCL constructs after 0, 7, 14 or 21
days in osteogenic culture.
Vibrational spectroscopy in the mid-infrared region can provide molecular structure
information about mineralized tissue[18]. The infrared spectrum in Figure VIII.4.6 showed the
presence of specific types of chemical bonds, associated to the major molecular species found
in bone; phosphate (bands ~900-1100 cm-1), carbonate (from carbonate substitution for
hydroxyl and phosphate groups, bands ~850 cm-1), and amide I, II, and III (bands ~1650 cm-1,
1550 cm-1 and 1250 cm-1, respectively) from the protein constituents (mainly type I collagen).
The spectrum of unseeded SPCL scaffold does not exhibit any of these bands, and the intensity
of these bands increases with the time in osteogenic culture. An increase in the intensity
values may be associated to the number of chemical bonds, and thus, molecular species,
present at the surface of the scaffold.
The presence of calcium phosphate (CaPs) aggregates in SEM micrographs correspond to
ECM mineralization, either at the surface and inner section of AFSC-SPCL constructs (Figure
VIII.4.1 and Figure VIII.4.4 – SEM after 21 days in culture), pointing toward the osteoblasticlike status of AFSCs after 3 weeks under osteogenic supplements.
The time frame of the osteogenic process confirms these results as mineral deposition is
accomplished largely by the precipitation of hydroxyapatite, which requires the presence of
collagen fibrils.
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Figure VIII.4.6 - Characterization of AFSC-SPCL constructs by FT-IR analysis. The presence of
calcium and phosphorus groups was detected after 0, 7, 14 or 21 days in osteogenic culture.
SPCL spectrum represents a SPCL scaffolds without seeded cells.
VIII.4.2. In vivo study
No animal subjected in this study showed any infection (Figure VIII.4.7), however some
rats (n= 14) showed bone plate loosening or small cracks during the surgery and were excluded
from this study.
Figure VIII.4.7 - Picture of dissected femurs after an end point. Femur on the left represents a
femur post-implantation and on the right, the left rear femur, control.
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VIII.4.2.1. Neobone formation assessment
Neobone formation to some extent was observed in all experimental groups, as expected,
both at 4 and 16 weeks by micro-CT analysis (Figure VIII.4.8).
Figure VIII.4.8 - m-CT images obtained from all defect conditions after 4 and 16 weeks of
implantation.
The formation of bone is observed 4 weeks after implantation at the segment ends of the
defect. In empty defects, new bone formation resulted in an increased appositional growth of
the bone in the border areas of bony segments, which could have resulted as a response of the
organism to the trauma in the femur during surgery (Figure VIII.4.8 and Figure VIII.4.9).
In all other conditions, interstitial bone growth was observed, and bridging bone was
evident in the presence of SPCL and SPCL seeded with cells (Figure VIII.4.9). The micro-CT
images also suggest that the presence of osteogenically committed hAFSCs seeded in the SPCL
scaffolds induced the best bridging with bone formation between the two sections of the
defect after 16 weeks of implantation. In the case of undifferentiated cells, bridging structures
were only observed in half of the specimens after 16 weeks.
Small particles of bone were also detected within the defect gap in SPCL and SPCL seeded
with cells conditions, suggesting that an initial new bone forming process starts as early as 4
weeks after implantation (Figure VIII.4.9). Although this bone ingrowth is reduced, when
compared to the 16 week group, these results also support the relevance of the SPCL scaffold
in new bone development with the time of implantation.
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Figure VIII.4.9 – Volumetric measurements of the defect section obtained from mCT
analysis using Mimics software, representing the bone neoformation area.
At 16 weeks, the volumetric measurements of bone increase in all conditions, but the
attempt of new bone to penetrate into the defect gap, and establish a link between the native
bone segments is only detected in the presence of SPCL and SPCL seeded with cells.
Furthermore, in most specimens analysed a lateral mineralized structure was formed and
observed in all conditions after 16 weeks but not detected in animals with empty defects by
the end of the first end point (4 weeks) (Figure VIII.4.8). This formation tends to increase in
length with the implantation time and is not typically described in non-union defects studies.
We believe that this new bone formation is associated to a secondary healing process
frequently observed in human fractures of skeletally mature or elder individuals. Not only
marrow bone formation is more limited in adult organisms, but bone marrow cells tends to
decrease in number and functionality with aging, and so does their stemness potential,
compromising the bone regeneration capacity[20-22].
In young individuals, bone growth through medulary influence is frequent and results in a
complete restoration of the affected tissue. In senior organisms, this route tends to be
replaced by alternative regeneration processes in order to create stability in motion and
sustain load bearing forces under a partial or absent medulary influence. Since the animals of
our study are geriatric rats, the secondary healing is the most likely process of bone
regeneration. Nevertheless, in the presence of SPCL scaffolds, the medulary effect seems to be
stimulated by the bridging formation in the femur defect. SPCL scaffolds are likely to indirectly
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participate in the hemostasis process that initiates the healing cascade by entrapping small
blood clots, resultant from the initial injury (defect surgery), that together with cells, are
important mediators for new bone formation. Thus, we believe that SPCL scaffolds assist the
molecular communication between the segmental areas leading to a bridging effect, not
observed in empty defects, the negative control of our experience.
VIII.4.2.2. Histology characterization
In general, H&E staining showed that cells were able to migrate into all induced defects,
colonizing between fibers of the SPCL scaffolds with matrix production at any time points. The
matrix formed by cells also increased with the implantation time. Empty defects, in particular,
showed a more irregular, dispersed and disorganized distribution of the cells and matrix.
At 4 weeks of implantation, cartilage tissue was also observed in sections of the implanted
area of several conditions, namely, empty defects, SPCL alone, SPCL-undifferentiated hAFSCs
and SPCL seeded with osteogenic differentiated hAFSCs. After 16 weeks of implantation, only
SPCL seeded with undifferentiated hAFSCs showed cartilagineous areas. The cartilage seems to
be associated to the endochondral ossification process, one of the two bone formation
processes, in which cartilage is formed and ultimately develops into new bone tissue.
Furthermore, bone remodeling at the end of bone segments is also observed (Figure VIII.4.10),
especially in empty, SPCL alone and SPCL seeded with undifferentiated hAFSCs by 4 weeks of
implantation.
Figure VIII.4.10 – Detailed image of native bone and SPCL scaffold interface in vivo after 4
weeks showing the native bone remodeling process aiming at defect regeneration (200x
magnified).
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In the scaffold implanted groups, general inflammatory response appeared to be moderate
at 4 weeks group and to be little at 16 week group which imply that our scaffold is
biocompatible. Additionally, in analyzed sections, some giant cells were also detected in the
defects implanted with SPCL scaffolds. The foreign body reaction, composed of macrophages
and foreign body giant cells, is the end-stage response of the inflammatory and wound healing
responses following implantation of a medical device, prosthesis or biomaterial[23]. The
presence of giant cells may also be a good indicator for SPCL in vivo degradation. As previously
showed[15, 16, 17], SPCL is a biodegradable scaffold and the adhesion of these cells to some
surface areas of the scaffold may stimulate the releasing of degradation mediators, such as
reactive oxygen intermediates, and expose SPCL to a higher concentration of these factors,
increasing scaffold susceptibility to degradation[23, 24].
VIII.4.2.2.1. Histomorphometric Analysis
Histomorphometric analysis was performed for collagen I, osteocalcin, and VEGF antibodies
so as to quantify the expression of these proteins in the regenerative process of our strategy
for bone non-unions regeneration.
Considering collagen I expression (Figure VIII.4.11), higher values were found in empty
defects although no statistical significance was found when compared to all other conditions.
Figure VIII.4.11 – Histometric analysis for collagen I expression (%) in the studied conditions.
Values are represented by mean  standard error of mean. Symbol * denote study groups with
statistically significant differences (p<0.05), as using Two Way ANOVA method.
Although collagen I is the major component of bone, it is also present in scar tissue, when
tissue heals by repair, and at the proliferation and extracellular matrix maturation during the
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osteoblast differentiation process. In defects filled with SPCL scaffolds, the expression of
collagen I decreases over time (p<0.05), while in SPCL with hAFSCs conditions, the expression
seems to be stable during the experiment.
Osteocalcin is a non-collagenous protein only secreted by osteoblasts, described to be
involved in the pro-osteoblastic process of bone, and often associated to bone
mineralization[25]. As observed in Figure VIII.4.12, osteocalcin expression tends to increase
from 4 to 16 weeks of implantation in all induced defects except the empty condition.
Figure VIII.4.12 – Histometric analysis for osteocalcin expression (%) in the studied conditions.
Values are represented by mean  standard error of mean. Symbol * denote study groups with
statistically significant differences (p<0.05), as using Two Way ANOVA method.
Moreover, the percentage of osteocalcin is higher in defects implanted with SPCL alone and
SPCL seeded with cells than in empty defect condition, which seems to indicate that tissue
engineered implants may participate in the osteogenic mechanism for bone mineralization.
Increasing values found in SPCL seeded with osteogenic like hAFSCs were found to be
statistically relevant (p<0.05) with the implantation time.
In the literature[26], higher levels of collagen I are found in osteoprogenitor to preosteoblast (osteogenic committed) cells while in mature osteoblasts, collagen I levels
decreases and osteocalcin protein increases significantly to promote mineralization. Our
results follow this tendency showing a higher percentage of collagen I in constructs of SPCL
seeded with hAFSCs or osteogenic committed hAFSCs, while SPCL with hAFSCs
(undifferentiated) express lower levels of osteocalcin, conversely to SPCL seeded with osteo
committed or osteoblast like hAFSCs. In this sense, implanted hAFSCs seem to get involved,
and somehow participate in the regenerative process.
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However, as observed by micro-CT analysis, bone formation (calcified matrix) is formed in
all conditions where SPCL is present, mainly around or at the scaffold periphery, but it does
not seem to occur inside the scaffold likewise. This demonstrates that SPCL seem to promote
the communication between the 2 bone segments of the femur as bone is formed at the
boundaries of the defects but degradation time of the scaffold should be optimized to
facilitate new bone formation inside of the scaffold.
As new bone was less formed inside the implanted scaffold, and cells were expressing
collagen I and osteocalcin, a possible explanation could reside on the influence of
environmental growth factors to the bone that are more widely accessible in peripheral
regions of the scaffold.
Since the process of a successful bone healing requires a good vascular network, as bone is
a highly vascularised tissue, angiogenesis plays a pivotal role in skeletal development and bone
fracture repair[27, 28]. VEGF is a signal protein associated to vasculogenesis and angiogenesis.
During bone repair, VEGF is required not only for blood vessel formation, but also for normal
callus volume and mineralization[29]. Furthermore, VEGF was described to inhibit osteoblast
apoptosis and stimulate osteogenic differentiation, indicating that VEGF may be the major
signal to couple angiogenesis and osteogenesis during bone repair[29, 30].
In our study, VEGF expression showed some variation among different animals from the
same condition, especially after 16 weeks of implantation (Figure VIII.4.13).
Figure VIII.4.13 – Histometric analysis for VEGF expression (%) in the studied conditions.
Values are represented by mean  standard error of mean.
Again, a basal expression of around 0.15 % was detected in empty controls, in which no
vascularization was significantly observed. VEGF expression tends to increase from 4 to 16
weeks in all conditions except SPCL with osteoblastic like cells.
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In this study, blood vessels were also microscopically visualized in all retrieved samples,
corresponding to all the different conditions analyzed, at both implantation periods (Figure
VIII.4.14).
Figure VIII.4.14 – Detection of blood vessel formation in H&E stained sections for all conditions
studied after 4 or 16 weeks of implantation. Pictures were 200x magnified.
In accordance to VEGF histometric data, after 4 weeks of implantation, the smallest vessels
were found in the empty defects while the largest sized vessels were found in defects filled
with SPCL scaffolds cultured with undifferentiated hAFSCs. After 16 weeks of implantation,
blood vessels in empty defects are only observed close to the bone segment top. The largest
vessels were found in seeded SPCL scaffolds with undifferentiated cells, followed by SPCL
seeded with osteogenic-like cells. These observations are in accordance to VEGF
histomorphometical data as the highest levels of VEGF are expressed in SPCL-hAFSCs
condition.
These findings suggest that hAFSCs may have a role in the angiogenic/inflammatory
processes, particularly the undifferentiated hAFSCs seeded on the SPCL scaffolds.
The innate stem cell nature of hAFSCs, as compared to the hAFSCs osteogenically
committed, can induce the production or migration of secreting factors, or responding to the
presence of local environmental contributions, towards angiogenesis[27] and consequently
bone formation. The fact that SPCL-osteoblast-like hAFSCs present larger vessels in Figure
VIII.4.14 but low percentage of VEGF in Figure VII.4.13 can be explained together with the data
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
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obtained for collagen I and osteocalcin. In SPCL-osteoblast-like hAFSC constructs,
mineralization is being achieved and the blood vessels have been already form, as observed in
Figure 12 so VEGF expression decreases, and a bigger input is given to the mineralization stage
of regeneration. Nonetheless, the presence and distribution of blood vessels in all SPCL
conditions indicated that SPCL scaffolds assist a good angiogenesis of the defect area, since an
active blood vessel network is an essential pre-requisite for constructs maintenance and
integration with existing host tissue.
VIII.5. Conclusions
In vitro results indicated that hAFSCs proliferated and differentiated into the osteogenic
lineage, when seeded onto SPCL scaffolds. hAFSCs were considered to be osteogenically
committed after 2 weeks in osteogenic medium with the expression of some bone related
markers and a protein rich extracellular matrix. By the end of week 3, cells became osteoblastlike with the production of a mineralized matrix.
After assessing in vitro functionally, in vivo studies were performed and, accordingly to
micro-CT analysis, all defect conditions showed new bone formation to some extent from 4 to
16 weeks of implantation. The amount of new bone was increased over time. No bridging
formation was observed in empty defects, and despite the fact that the bridging process was
not complete in any of the other conditions by 16 weeks; the presence of osteogenically
committed hAFSCs in the SPCL scaffolds showed the shortest gap (bridging) between the
femoral bone segments, after 16 weeks of implantation.
The presence of vascular vessels in the middle of the SPCL or hAFSCs-SPCL constructs also
indicates that SPCL scaffolds and hAFSCs participate in the angiogenesis in the defect, showing
that SPCL scaffolds are an interesting support for bone regeneration and angiogenesis in nonunion defects, especially when seeded with human amniotic fluid stem cells committed to the
osteogenic phenotype.
We conclude that our SPCL scaffold system can be a useful candidate for reconstructing a
long bone defect in the near future. Furthermore, the presence of hAFSCs can induce a
synergistic effect in this system, but additional studies will be needed to optimize degradation
time of the scaffold and use of hAFSCs condition.
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in the regeneration of non-union defects
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VIII.6. References
1. Calori GM, Albisetti W, Agus A, Iori S, Tagliabue L: Risk factors contributing to fracture nonunions. Injury 2007, 38 Suppl 2: S11-18.
2. Giannoudis PV, Atkins R: Management of long-bone non-unions. Injury 2007, 38 Suppl 2: S12.
3. Tseng SS, Lee MA, Reddi AH: Nonunions and the potential of stem cells in fracture-healing. J
Bone Joint Surg Am 2008, 90 Suppl 1: 92-98.
4. Mahendra A, Maclean AD: Available biological treatments for complex non-unions. Injury
2007, 38 Suppl 4: S7-12.
5. Petite H, Viateau V, Bensaid W, Meunier A, de Pollak C, Bourguignon M, Oudina K, Sedel L,
Guillemin G: Tissue-engineered bone regeneration. Nat Biotechnol 2000, 18: 959-963.
6. Liu X, Li X, Fan Y, Zhang G, Li D, Dong W, Sha Z, Yu X, Feng Q, Cui F, et al.: Repairing goat tibia
segmental bone defect using scaffold cultured with mesenchymal stem cells. J Biomed Mater
Res B Appl Biomater. 2010, 94(1): 44-52.
7. Dupont KM, Sharma K, Stevens HY, Boerckel JD, Garcia AJ, Guldberg RE: Human stem cell
delivery for treatment of large segmental bone defects. Proc Natl Acad Sci U S A 107: 33053310.
8. Jager M, Degistirici O, Knipper A, Fischer J, Sager M, Krauspe R: Bone healing and migration
of cord blood-derived stem cells into a critical size femoral defect after xenotransplantation. J
Bone Miner Res 2007, 22: 1224-1233.
9. Prusa AR, Marton E, Rosner M, Bernaschek G, Hengstschlager M: Oct-4-expressing cells in
human amniotic fluid: a new source for stem cell research? Hum Reprod 2003, 18: 1489-1493.
10. De Coppi P, Bartsch G, Jr., Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC,
Snyder EY, Yoo JJ, et al.: Isolation of amniotic stem cell lines with potential for therapy. Nat
Biotechnol 2007, 25: 100-106.
11. Kim J, Lee Y, Kim H, Hwang KJ, Kwon HC, Kim SK, Cho DJ, Kang SG, You J: Human amniotic
fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif 2007, 40: 7590.
12. Sun H, Feng K, Hu J, Soker S, Atala A, Ma PX: Osteogenic differentiation of human amniotic
fluid-derived stem cells induced by bone morphogenetic protein-7 and enhanced by
nanofibrous scaffolds. Biomaterials 31: 1133-1139.
13. Rodrigues MT, Gomes ME, Viegas CA, Azevedo JT, Dias IR, Guzón F, Reis RL: Tissue
Engineered Constructs based on SPCL Scaffolds Cultured with Goat Marrow Cells: Functionality
in Femoral Defects. J Tissue Eng Regen Med 2011, 5: 41-49.
14. Peters A, Toben D, Lienau J, Schell H, Bail HJ, Matziolis G, Duda GN, Kaspar K: Locally
applied osteogenic predifferentiated progenitor cells are more effective than undifferentiated
C
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
- 192 -
mesenchymal stem cells in the treatment of delayed bone healing. Tissue Eng Part A 2009, 15:
2947-2954.
15. Gomes ME, Bossano CM, Johnston CM, Reis RL, Mikos AG: In vitro localization of bone
growth factors in constructs of biodegradable scaffolds seeded with marrow stromal cells and
cultured in a flow perfusion bioreactor. Tissue Eng 2006, 12: 177-188.
16. Gomes ME, Azevedo HS, Moreira AR, Ella V, Kellomaki M, Reis RL: Starch-poly(epsiloncaprolactone) and starch-poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering
applications: structure, mechanical properties and degradation behaviour. J Tissue Eng Regen
Med 2008, 2: 243-252.
17. Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, Kirkpatrick CJ: Response of micro- and
macrovascular endothelial cells to starch-based fiber meshes for bone tissue engineering.
Biomaterials 2007, 28: 240-248.
18. Boskey A, Pleshko Camacho N: FT-IR imaging of native and tissue-engineered bone and
cartilage. Biomaterials 2007, 28: 2465-2478.
19. Lian JB, Stein GS: Concepts of osteoblast growth and differentiation: basis for modulation
of bone cell development and tissue formation. Crit Rev Oral Biol Med 1992, 3: 269-305.
20. Stolzing A, Jones E, McGonagle D, Scutt A: Age-related changes in human bone marrowderived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev 2008,
129:163-73.
21. Kretlow JD, Jin YQ, Liu W, Zhang WJ, Hong TH, Zhou G, Baggett LS, Mikos AG, Cao Y: Donor
age and cell passage affects differentiation potential of murine bone marrow-derived stem
cells. BMC Cell Biol 2008, 9: 60.
22. Yu JM, Wu X, Gimble JM, Guan X, Freitas MA, Bunnell BA: Age-related changes in
mesenchymal stem cells derived from rhesus macaque bone marrow. Aging Cell. 2011, 10: 6679.
23. Anderson JM, Rodriguez A, Chang DT: Foreign body reaction to biomaterials. Semin
Immunol 2008, 20: 86-100.
24. Pego AP, Van Luyn MJ, Brouwer LA, van Wachem PB, Poot AA, Grijpma DW, Feijen J: In vivo
behavior of poly(1,3-trimethylene carbonate) and copolymers of 1,3-trimethylene carbonate
with D,L-lactide or epsilon-caprolactone: Degradation and tissue response. J Biomed Mater Res
A 2003, 67: 1044-1054.
25. Neve A, Corrado A, Cantatore FP: Osteoblast physiology in normal and pathological
conditions. Cell Tissue Res 343: 289-302.
26. Lian JB, Stein GS: Development of the osteoblast phenotype: molecular mechanisms
mediating osteoblast growth and differentiation. Iowa Orthop J. 1995, 15: 118-40.
27. Kanczler JM, Oreffo RO: Osteogenesis and angiogenesis: the potential for engineering
bone. Eur Cell Mater 2008, 15: 100-114.
Chapter VIII – The effect of the differentiation stage of amniotic fluid stem cells seeded onto biodegradable scaffolds
in the regeneration of non-union defects
- 193 -
28. Carano RA, Filvaroff EH: Angiogenesis and bone repair. Drug Discov Today 2003, 8: 980989.
29. Filvaroff EH: VEGF and bone. J Musculoskelet Neuronal Interact 2003, 3: 304-307;
discussion 320-301.
30. Wernike E, Montjovent MO, Liu Y, Wismeijer D, Hunziker EB, Siebenrock KA, Hofstetter W,
Klenke FM: VEGF incorporated into calcium phosphate ceramics promotes vascularisation and
bone formation in vivo. Eur Cell Mater 19: 30-40.
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SECTION VI
GENERAL CONCLUSIONS
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Chapter IX
FINAL REMARKS AND FUTURE STUDIES
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Chapter IX
CONCLUSIONS AND FINAL REMARKS
Theory guides. Experiment decides.
(an old saying in science)
IX.1. General Conclusions
The broad subject of this Thesis permitted to reach further insights in some key issues of
tissue engineering strategies for the regeneration of bone and osteochondral tissues, namely
on scaffold development, stem cells sources and synergies between scaffold design, stem cells,
and culturing conditions. Additionally, in vivo studies allowed obtaining important data
regarding the relevance of using scaffolds alone or scaffolds with stem cells at different stages
of osteogenic differentiation in the treatment of bone defects. The results obtained are
described below:
i) Scaffold design and composition for bone and osteochondral defects regeneration
The development of multi-layered PCL-TCP scaffolds was successfully achieved, as well as
the in vitro assessment of their application aiming at bone tissue engineering (TE) strategies. In
the study described in chapter III, a PCL polymer was used as a nanofiber mesh substrate.
Despite the fact that PCL is a well described polymer for TE strategies, the relevance of this
study is not so much the polymer by itself but the nano structure obtained by means of using
electrospinning. Considering that bone extracellular matrix (ECM) is essentially an organicinorganic composite and nano-scaled organized, the integration of nano-structures in 3D
scaffolds could be an interesting structural approach. The main issue in this approach is the
fact that nano-structures do not support the mechanical needs of bone as for weight loading
or other mechanical stresses. To fulfill this gap, the study describes the idea of developing
multi-layered structures to adjust to the dimension of bone defects, and sustain body weight
whenever necessary during the regenerative process. Furthermore, to induce some osteogenic
activity, and eventually stimulate a bond between construct and native bone in an in vivo
situation, TCP granules were added, due to their osteoconductive properties. Thus, the
Chapter IX – Conclusions and Final Remarks
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combination of PCL with calcium phosphates was shown to be more efficient to generate
scaffolds with higher osteoconductive ability, but this was an expected result since TCP is an
osteoconductive material. However, particularly interesting was to conclude also that PCL-TCP
scaffolds did not required osteogenic supplements in the culture medium to induce the
osteogenic phenotype of gBMSCs, as long as constructs were cultured in dynamic conditions.
Furthermore, the detection of a calcified ECM in the absence of osteogenic medium indicated
an excellent cellular response to the scaffold and to mechanical stimuli. This study
demonstrated for the first time a composite multi-layered structure inspired in the natural
organic-inorganic nanostructure of bone with interesting results for bone applications.
SPCL is a blend of PCL with starch, a natural polymer, which has been widely studied at the
3Bs Research Group. SPCL scaffolds can be obtained by several processing methodologies as
described in this Thesis. The processability of SPCL using different techniques allows achieving
constructs with different designs and porous structures. For instance, with a fiber bonding
procedure, using melt spun fibers, it is easier to obtain 3D scaffolds with higher mechanical
properties, while wet-spinning technology produces a fiber mesh scaffold, where functional
groups can be incorporated in a single step procedure, and thus tailor the SPCL surface
composition. In chapter IV, the bioactivity behavior of wet-spun SPCL scaffold with silanol
groups (SPCL-Si) was demonstrated in the presence of a SBF solution. The presence of the
silanol groups showed to stimulate the osteogenic differentiation of bone marrow stromal
cells in vitro, but the ability to form an apatite layer together with the osteoconductive and
osteogenic properties might also have a significant impact in vivo, as the ideal implanted
construct should stimulate damaged tissue regeneration and restore its functionality.
However, presently, this process does not allow obtaining scaffolds with larger dimensions
very easily, and their mechanical properties are slightly lower than those for SPCL meshes
obtained from melt spun fibers. Nevertheless, SPCL-Si scaffolds might offer an alternative in
flat bone regeneration strategies, fitting the anatomically leveled areas such as calvarian bone.
SPCL scaffolds obtained by bonding of melt spun fibers are well characterized, and were
described in previous studies, particularly for bone engineered strategies. Thus, in chapter VI,
VIII and V, fiber bonding SPCL scaffolds were used in osteogenic and osteochondral studies,
respectively.
The application of a bilayered scaffold is quite attractive for osteochondral interfaces due
to the natural differences in cellular and tissue needs between cartilage and bone tissues.
Osteochondral studies from chapter V revealed that the assembly of a bilayered scaffold is
possible, and after AFSCs differentiation is achieved, cell viability is kept high, and both osteoChapter IX – Conclusions and Final Remarks
- 201 -
or chondro-genic differentiation are maintained in the osteochondral bilayered system.
Although not essential, after assembling the bilayer system, the presence of osteochondral
medium seems to maintain the osteogenic and chondrogenic phenotypes of human AFSCs.
The advantages of this system relay on the individual culture layers for selective
differentiation, which can be quickly and easily assembled, and maintained in basal culture
medium. Besides the simplification for the maintenance of differentiated cells in culture, a coculture medium would assist the communication between osteoblast- or chondrocyte-like cells
to be maintained in a bilayered system until construct implantation. With this strategy, time
and cost of a tissue engineered product could be eventually reduced, and become closer to a
clinical application. Again, SPCL suitability for bone applications is confirmed with great
potential to spread out into osteochondral strategies.
ii) Stem cells sources and relevance of the differentiation stage – in vitro studies
Although stem cells can be found in almost every tissue, cell origin can influence their
response either mechanically, physically or chemically. Bone marrow stem cells (BMSCs) are
the most studied, and frequently associated to autologous approaches regardless of the donor
age-affected and self-renewal limitations. Other stem cells sources, such as amniotic fluid
could overcome these limitations. Despite the potential of human amniotic stem cells (AFSCs),
few studies assessed AFSCs behavior both in vitro and implanted in an animal model. Since no
comparative analysis has been done to assess possible differences between BMSCs and AFSCs
due to origin outcomes, in chapter VI, a comparative study was performed to find out the cell
response to substrate and media supplementation. Results indicated that both cell types were
able to proliferate and differentiate into the osteogenic phenotype in 2D and 3D substrates.
Nevertheless, cells from different origins expressed different bone-related markers at different
end points, which may be related to cellular origin and substrate properties. In fact, besides
the influence of cell origin, stem cells response can be further tailored by scaffold materials,
namely by the design and chemical composition. Features, such as stiffness/softness or
elasticity, can be tailored to induce a determined cell response, as these characteristics are
known to be detected and understood differently by the ECM of the cells. The physical contact
between cells-scaffold will result in a series of cellular signals, through which cells attempt to
adapt to the surrounding environment. Additional stimulus, such as dynamic culturing
conditions can also act synergistically with the scaffold material, as showed in chapter III, thus
enhancing a specific cell response. Despite the fact that some stem cell sources might be more
Chapter IX – Conclusions and Final Remarks
- 202 -
sensitive to certain stimuli than others, it is likely that the combination of different physical
and chemical signals, similarly to those present in their native environment, will lead cells
more easily to a specific target response. Thus, the selection of a particular stem cell type may
not be a simple and direct process and relies on the TE strategy to be assessed, firstly in vitro
and later in an in vivo environment.
iii) Functionality assessment of scaffold-stem cells based strategies in bone tissue
engineering: in vivo studies
Cell-scaffold constructs were evaluated in vitro in terms of proliferative and selective
differentiation potential, following the evolution of the sequential stages of the differentiation
process from stem cells into osteoblast-like cells. After in vitro assessment of the promising
systems of SPCL-AFSCs or SPCL-BMSCs, the functionality of these constructs was evaluated in
vivo under two different studies considered in chapter VII and VIII.
Non-critical sized defects were induced in the rear femurs of skeletally mature goats using
an autologous cellular approach. One of the greatest advantages of autologous approaches is
the fact that xenogenic animal cells and medium supplements are avoided, thought to be
critical for successful future clinical procedures with human patients. Thus, several conditions
were acknowledged so as to comprehend the influence of scaffold, and scaffold seeded with
bone marrow cells (BMSCs) or with osteogenic committed BMSCs in the regeneration of bone.
This is particularly important since the stage of osteogenic differentiation can be critical for
achieving tissue-like functional substitutes. As expected, the neoformation of bone occurred in
all orthotopic induced defects. Nevertheless, results obtained indicated that neobone
formation and cellular distribution increased in the presence of cell seeded SPCL scaffolds,
which strongly shows the influence of implanted cells in the bone regeneration process. In
addition, the pre-differentiation of goat BMSCs into the osteogenic phenotype seems to
increase the neobone ingrowth into the defects, revealing not only the importance of
implanted cells but also of their differentiation stage in the communication process between
implant and native bone environment. This study also evaluated the orthotopic behaviour of
SPCL biomaterial revealing a biocompatible scaffold that does not contribute to an
overreaction from the immune response against the implant. Additionally, SPCL scaffolds
showed a great potential for the development of adequate tissue 3D support for the
regeneration of bone.
Chapter IX – Conclusions and Final Remarks
- 203 -
Although autologous approaches are the perfect tailored TE solution for individual needs,
they are quite time demanding, and some patients may not have a healthy and available stem
cell pool to harvest from, due to age or disease limitations. Thus, researchers have been
looking for a novel cell source that could be used for a wide range of therapies suiting patients
worldwide. Amniotic fluid has been studied aiming at a non-immunogenic and universal stem
cell source. These are advantageous features that brought AFSCs into highly complex bone
defects - non-unions or critical sized defects- to validate our strategy. Again, results indicated
that a greater amount of neobone in the presence of SPCL scaffolds and a bridging effect is
more prominent in the defects filled with scaffolds seeded with cells committed to the
osteogenic phenotype. The presence of large blood vessels in the defect areas, especially in
SPCL scaffolds seeded with undifferentiated cells, followed by SPCL seeded with osteogeniclike cells, also indicates the suitability of these constructs for the regeneration of non-union
defects as vascularization is important to assure local transport of nutrients, oxygen and
growth factors to the cells.
In summary, biomaterials described in this Thesis showed an interesting potential in
different strategies aiming at bone and osteochondral TE applications. Composite scaffolds
showed prospective interest under dynamic conditions for strategies involving flat bones and
cell culturing media without osteogenic supplements. Bioactive wet-spun fiber meshes of
SPCL-Si scaffolds also showed intrinsic properties able to sustain in vitro osteogenic features of
goat BMSCs. Nevertheless, SPCL meshes obtained from melt spun fiber are one step ahead as
scaffold structures, showing to provide the necessary support to novel and under development
bone and osteochondral TE strategies using different sources of stem cells and animal models.
The combination of cells and SPCL scaffolds showed the best results in pre-clinical models,
either in autologous (goat BMSCs) or heterologous (human AFSCs) cell sources. Moreover, the
stage of osteogenic differentiation seems to be an important factor to consider in the complex
process of bone regeneration.
Chapter IX – Conclusions and Final Remarks
- 204 -
Research is to see what everybody else has seen,
and to think what nobody else has thought.
Albert Szent-Györgi (1893-1986)
IX.2. Final Remarks and Future Studies
It would be exciting and very promising to develop a scaffold to stimulate osteogenic
differentiation and new bone formation in the absence of specific osteogenic supplements.
The cost of the tissue engineered products would decrease significantly, the culture conditions
simplified and short-termed, and some risks concerning the use of animal origin supplements
avoided. Herein we reported the development of multi-layered nanofiber scaffolds that may
fulfill these requirements, yet their mechanical properties may limit their application to flat
bone strategies.
We also reported here the advantageous features of wet spun SPCL fiber meshes that can
be in situ functionalized in one-step processing methodology, thus enabling a cost-effective
technology to produce scaffolds with an osteogenic stimulating surface, onto which BMSCs
differentiate into osteoblast-like cells, while producing a mineralized extracellular matrix. Thus,
future studies should address the influence of SPCL-Si scaffold in osteogenic differentiation in
basal culture medium. Since these scaffolds are bioactive, as TCP granules from composite
scaffolds of chapter III, perhaps silanol groups could provide a similar motivation as TCPs
towards the osteogenic differentiation of BMSCs, with or without the synergistic effect of a
dynamic environment. Additionally, the combination of starch with PCL could accelerate the in
vivo degradation rate, while bone tissue was naturally being regenerated.
Furthermore, in vivo studies are envisioned to understand SPCL-Si scaffolds functionality in
vivo, which is likely to be favorable for bone regeneration due to its intrinsic bioactivity.
A new approach for the treatment of osteochondral interfaces was also assessed,
demonstrating promising results. In these studies, described in chapter V, the selected
osteochondral medium showed to maintain the phenotypic expression of pre-differentiated
cells; future studies should investigate the influence of this medium in promoting simultaneous
osteo- or chondro-genic differentiation of stem cells seeded onto SPCL scaffolds or
encapsulated in agarose gels, without employing differentiation media. Also, more
chondrogenic related markers should be assessed, especially after cell maintenance in
Chapter IX – Conclusions and Final Remarks
- 205 -
osteochondral media, to determine how medium supplements are affecting the chondrogenic
process.
Concerning cell culturing, stem cells from different sources were differentiated into the
osteogenic phenotype, although cell response variations were found in commonly associated
bone markers. It is important to understand the mechanisms associated to cellular
communication between cells and substrate or culture medium. It is very likely that different
molecular pathways may be involved and stimuli triggered accordingly to cell origin and
scaffold structural and chemical properties. In addition, could osteogenic supplements be
radically eliminated from the medium? That seems to be possible for composite scaffolds, and
can be enlarged to other scaffolds playing with the unique properties of each type of scaffold
as well as all the other factors (such as lineage specific supplements, dynamic environment,
scaffold tri-dimensionality) known to participate in the progression into the osteogenic lineage
and, consequently in bone regeneration.
The experimental study comparing bone marrow and amniotic fluid stem cells showed
some interesting results and rose novel questions that need to be addressed in future studies.
Since the ideal cell source is yet to be found, currently available sources should be evaluated
considering the aimed application. Also, animals are known to be more resilient to diseases, to
immune reactions and to healing abilities than humans. It should be interesting to evaluate if
different animal sources of stem cells behave similarly to human correspondent ones.
As mentioned before, both critical and non-critical defects were studied for SPCL scaffolds
as well as the influence of cell seeded scaffolds in the regeneration of bone. Results
demonstrated that SPCL might fasten the repair of non-critical defects as well as to promote
the healing of non-union defects. The presence of cells and their differentiation stage also
impacts the new bone tissue formation. Yet, further studies are required to understand the
importance of the number of implanted cells versus their differentiation stage and to
determine the mechanisms that illicit such different responses in bone metabolism. It is likely
that cells undergoing different stages of differentiation may communicate differently with the
in vivo environment through the release of distinct cytokines and growth factors inducing
variations in the organism healing response. Understanding how these processes occur may
provide important cues to match the advantageous and promising features of the
experimental setups to a fully successful route towards human clinical therapies.

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