Auswahl der wissenschaftlichen Literatur zum Thema „Cartilage cells – Transplantation“

Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.

This review addresses the progress in cartilage repair technology over the decades with an emphasis on cartilage regeneration with cell therapy. The most abundant cartilage is the hyaline cartilage that covers the surface of our joints and, due to avascularity, this tissue is unable to repair itself. The cartilage degeneration seen in osteoarthritis causes patient suffering and is a huge burden to society. The surgical approach to cartilage repair was non-existing until the 1950s when new surgical techniques emerged. The use of cultured cells for cell therapy started as experimental studies in the 1970s that developed over the years to a clinical application in 1994 with the introduction of the autologous chondrocyte transplantation technique (ACT). The technology is now spread worldwide and has been further refined by combining arthroscopic techniques with cells cultured on matrix (MACI technology). The non-regenerating hypothesis of cartilage has been revisited and we are now able to demonstrate cell divisions and presence of stem-cell niches in the joint. Furthermore, cartilage derived from human embryonic stem cells and induced pluripotent stem cells could be the base for new broader cell treatments for cartilage injuries and the future technology base for prevention and cure of osteoarthritis.

Cartilage injuries are typically caused by trauma, chronic overload, and autoimmune diseases. Owing to the avascular structure and low metabolic activities of chondrocytes, cartilage generally does not self-repair following an injury. Currently, clinical interventions for cartilage injuries include chondrocyte implantation, microfracture, and osteochondral transplantation. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Stem cell therapies, especially mesenchymal stem cell (MSCs) therapies, were found to be a feasible strategy in the treatment of cartilage injuries. MSCs can easily be isolated from mesenchymal tissue and be differentiated into chondrocytes with the support of chondrogenic factors or scaffolds to repair damaged cartilage tissue. In this review, we highlighted the full success of cartilage repair using MSCs, or MSCs in combination with chondrogenic factors and scaffolds, and predicted their pros and cons for prospective translation to clinical practice.

The aim of the study was to verify whether there is a difference in the lengthwise growth of the femurs and in their angular deformity when comparing preventive vs. therapeutic transplantation of allogeneic mesenchymal stem cells (MSCs) to an iatrogenic defect in the distal physis of femur. Modified composite chitosan/collagen type I scaffold with MSCs was transplanted to an iatrogenically created defect of the growth cartilage in the lateral condyle of the left femur in 20 miniature male pigs. In Group A of animals (n = 10) allogeneic MSCs were transplanted immediately after creating the defect in the distal physis of femur (preventive transplantation). In Group B of animals (n = 10) the same defect of the growth cartilage was treated by transplantation of allogeneic MSCs four weeks after its creation (therapeutic transplantation), after the excision of the bone bridge that had formed in it. On average, left femurs with a damaged distal physis and preventively transplanted allogeneic MSCs (Group A) grew during 4 months from transplantation by 0.56 ± 0.44 cm more than right femurs without the transplantation of MSCs, whereas left femurs with physeal defects treated with a therapeutic transplantation of allogeneic MSCs (Group B) by 0.14 ± 0.72 cm only, compared to right femurs without transplanted MSCs. Four months after preventive transplantation of MSCs (Group A), valgus deformity of the distal part of left femur with the defect was 0.78 ± 0.82°; in the control group (right femur in the same animal without MSCs transplantation) it was 3.7 ± 0.82°. After therapeutic transplantation of MSCs (Group B) 0.6 ± 3.4°, in the control group (right femur in the same animal without MSCs transplantation) it was 2.1 ± 2.9°. In all the animals of Groups A and B, the presence of newly formed hyaline cartilage was confirmed histologically and immunohistochemically. In the distal physis of right femurs with an iatrogenic defect of the growth cartilage without the transplantation of MSCs (control) bone bridge was formed. Preventive transplantation of allogeneic MSCs into the defect of the distal growth zone of femur appears more suitable compared to the therapeutic transplantation, with regard to the more pronounced lengthwise bone growth. Differences found in the extent of valgus deformity were non-significant comparing preventive and therapeutic transplantations of MSCs.

Regeneration of articular cartilage is of great interest in cartilage tissue engineering since articular cartilage has a low regenerative capacity. Due to the difficulty in obtaining healthy cartilage for transplantation, there is a need to develop an alternative and effective regeneration therapy to treat degenerative or damaged joint diseases. Stem cells including various adult stem cells and pluripotent stem cells are now actively used in tissue engineering. Here, we provide an overview of the current status of cord blood cells and induced pluripotent stem cells derived from these cells in cartilage regeneration. The abilities of these cells to undergo chondrogenic differentiation are also described. Finally, the technical challenges of articular cartilage regeneration and future directions are discussed.

A fresh osteochondral allograft is one of the most effective treatments for cartilage defects of the knee. Despite the clinical success, fresh osteochondral allografts have great limitations in relation to the short storage time that cartilage tissues can be well-preserved. Fresh osteochondral grafts are generally stored in culture medium at 4°C. While the viability of articular cartilage stored in culture medium is significantly diminished within 1 week, appropriate serology testing to minimize the chances for the disease transmission requires a minimum of 2 weeks. (–)-Epigallocatechin-3- O-gallate (EGCG) has differential effects on the proliferation of cancer and normal cells, thus a cytotoxic effect on various cancer cells, but a cytopreservative effect on normal cells. Therefore, a storage solution containing EGCG might extend the storage duration of articular cartilages. Rabbit osteochondral allografts were performed with osteochondral grafts stored at 4°C in culture medium containing EGCG for 2 weeks and then the clinical effects were examined with macroscopic and histological assessment after 4 weeks. The cartilaginous structure of an osteochondral graft stored with EGCG was well-preserved with high cell viability and glycosaminoglycan (GAG) content of the extracellular matrix (ECM). After an osteochondral allograft, the implanted osteochondral grafts stored with EGCG also provided a significantly better retention of the articular cartilage with viability and metabolic activity. These data suggest that EGCG can be an effective storage agent that allows long-term preservation of articular cartilage under cold storage conditions.

Isolated syngeneic epiphyseal chondrocytes transplanted into a muscle formed cartilage in which matrix resorption and endochondral ossification began at the end of the second week after transplantation. After 56 days cartilage was converted into an ossicle. In 7-day-old intrarenal transplants, epiphyseal chondrocytes formed nodules of cartilage. In 10-day-old transplants, islands of bone appeared. Slight resorption of cartilage was first noted in 14-day-old transplants of chondrocytes. After eight weeks, transplants contained mainly bone. Intramuscularly transplanted rib chondrocytes formed cartilage which did not ossify. Nevertheless, bone islands appeared in intrarenal transplants of rib chondrocytes. Bone was not formed in allogeneic intrarenal transplants of epiphyseal or rib chondrocytes, but appeared in such transplants in animals immunosuppressed by anti-thymocyte serum and procarbazine. When spleen cells from animals immunized with allogeneic chondrocytes were transferred to immunosuppressed chondrocyte recipients two weeks after intrarenal chondrocyte transplantation, the majority of osteocytes in bone islands was dead. On the other hand, endochondral bone formed in intramuscular transplants of allogenic epiphyseal chondrocytes in immunosuppressed recipients was not damaged by sensitized spleen cells. This suggested that bone in 10- to 14-day-old intrarenal transplants of chondrocytes arose from injected cells and not by induction. To see whether bone was formed by chondrocytes or by some cells contaminating the chondrocyte suspension, the superficial layer of rib cartilage was removed by collagenase digestion and only more central chondrocytes were used for transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper reviews our research in developing novel matrices for cell transplantation using bioresorbable polymers. We focus on applications to liver and cartilage as paradigms for regeneration of metabolic and structural tissue, but review the approach in the context of cell transplantation as a whole. Important engineering issues in the design of successful devices are the surface chemistry and surface microstructure, which influence the ability of the cells to attach, grow, and function normally; the porosity and macroscopic dimensions, which affect the transport of nutrients to the implanted cells; the shape, which may be necessary for proper function in tissues like cartilage; and the choice of implantation site, which may be dictated by the total mass of the implant and which may influence the dimensions of the device by the available vascularity. Studies show that both liver and cartilage cells can be transplanted in small animals using this approach.

Cartilage defects represent a common problem in orthopaedic practice. Predisposing factors include traumas, inflammatory conditions, and biomechanics alterations. Conservative management of cartilage defects often fails, and patients with this lesions may need surgical intervention. Several treatment strategies have been proposed, although only surgery has been proved to be predictably effective. Usually, in focal cartilage defects without a stable fibrocartilaginous repair tissue formed, surgeons try to promote a natural fibrocartilaginous response by using marrow stimulating techniques, such as microfracture, abrasion arthroplasty, and Pridie drilling, with the aim of reducing swelling and pain and improving joint function of the patients. These procedures have demonstrated to be clinically useful and are usually considered as first-line treatment for focal cartilage defects. However, fibrocartilage presents inferior mechanical and biochemical properties compared to normal hyaline articular cartilage, characterized by poor organization, significant amounts of collagen type I, and an increased susceptibility to injury, which ultimately leads to premature osteoarthritis (OA). Therefore, the aim of future therapeutic strategies for articular cartilage regeneration is to obtain a hyaline-like cartilage repair tissue by transplantation of tissues or cells. Further studies are required to clarify the role of gene therapy and mesenchimal stem cells for management of cartilage lesions.

[Truncated abstract] Articular cartilage (AC) covers the surface of synovial joints providing a nearly frictionless bearing surface and distributing mechanical load. Joint trauma can damage the articular surface causing pain, loss of mobility and deformation. Currently there is no uniform treatment protocol for managing focal cartilage defects, with most treatment options targeted towards symptomatic relief but not limiting the progression into osteoarthritis (OA). Autologous chondrocyte implantation (ACI) and more recently matrix-induced autologous chondrocyte implantation (MACI), have emerged as promising methods for producing hyaline or hyaline-like repair tissue, however there remains some controversy regarding the exact histological nature of the tissue formed. Histological characterisation of AC repairs requires destructive tissue biopsy potentially inducing further joint pathology thereby negating the treatment effect. OA is recognised as a major cause of pain, loss of function and disability in Western populations, however the exact aetiology is yet to be elucidated. The assessment of both OA and cartilage repair has been limited to macroscopic observation, radiography, magnetic resonance imaging (MRI) or destructive biopsy. The development of non-destructive AC assessment modalities will facilitate further development of AC repair techniques and enable early monitoring of OA changes in both experimental animal models and clinical subjects. Confocal laser scanning microscopy (CLSM) is a type of fluorescence microscopy that generates high-resolution three-dimensional images from relatively thick sections of tissue. ... Biomechanical analysis suggested that the mechanical properties of MACI tissue remain inferior for at least three months. This study showed the potential of a multi-site sheep model of articular cartilage defect repair and validated its assessment via LSCA. Finally, the LSCA was used to arthroscopically image the cartilage of an intact fresh frozen cadaveric knee from a patient with clinically diagnosed OA. Images were correlated to ICRS (Outerbridge) Grades I-IV and histology. The LSCA gave excellent visualization of cell morphology and cell density to a depth of up to 200'm. Classical OA changes including clustering chondrocytes, surface fibrillation and fissure formation were imaged. Fair to moderate agreement was demonstrated with statistically significant correlations between all modalities. This study confirmed the viability of the LSCA for non-destructive imaging of the microstructure of the OA cartilage. In conclusion, the LSCA identified histological features of orthopaedic tissues, accurately quantified chondrocyte morphology and demonstrated classical OA changes. While the development and investigation of an ovine model of cartilage repair showed the treatment benefit of MACI, some biomechanical issues remain. Ultimately, the LSCA has been demonstrated as a reliable nondestructive imaging modality capable of providing optical histology without the need for mechanical biopsy. Medical Subject Headings (MESH): articular cartilage; autologous chondrocyte implantation; matrix-induced autologous chondrocyte implantation; biomechanics; cartilage; confocal microscopy; diagnosis; histology; image analysis; immunohistochemistry; magnetic resonance imaging; microscopy; osteoarthritis

Les affections articulaires touchant le cartilage, telles que les lésions focales et l’arthrose, correspondent aux principales causes de baisse de performance et d’arrêt prématuré de la carrière sportive du cheval. Ainsi, le traitement des affections du cartilage représente un enjeu vétérinaire majeur dans le monde équin, du fait des importantes pertes financières qu’elles occasionnent à la filière. Les faibles capacités de réparation intrinsèque du cartilage, ainsi que l’absence de thérapie à long terme des dommages cartilagineux, nécessitent le recours à des thérapies de nouvelles générations telle que l’ingénierie tissulaire du cartilage. Dans ce cadre, notre étude s’est attachée à comparer différents types cellulaires pour la génération de cartilage in vitro, afin d’envisager une implantation pour traiter les atteintes cartilagineuses chez le cheval. Une technique initialement développée chez l’Homme, la transplantation de chondrocytes autologues, représente toujours un « gold standard » en ingénierie tissulaire du cartilage. Dans ce travail de thèse, après avoir développé une nouvelle génération de substitut cartilagineux de haute qualité biologique, à partir de chondrocytes articulaires équins, des limites techniques et biologiques inhérentes au type cellulaire persistent. Ainsi, nos travaux se sont tournés vers la recherche de types cellulaires alternatifs. Les cellules souches/stromales mésenchymateuses (CSM) néonatales issues de cordon ombilical telles que les CSM de sang placentaire (CSM-SPL) et les CSM de gelée de Wharton (CSM-GW) pourraient représenter un avantage thérapeutique du fait de leur isolement non-invasif, de leur forte prolifération cellulaire et de leur capacité de différenciation en chondrocyte. Il est néanmoins indispensable de définir le meilleur candidat thérapeutique, parmi ces deux sources cellulaires, pour l’obtention d’un substitut cartilagineux de qualité biologique optimale. Ces résultats de thèse ont montré d’importantes différences dans le processus de chondrogenèse de ces deux sources de CSM néonatales et plaident en faveur de l’utilisation des CSM-SPL dans le cadre d’une stratégie thérapeutique d’ingénierie tissulaire du cartilage équin. Ces travaux ont permis une meilleure compréhension de la biologie du chondrocyte et des CSM. De surcroît, ces travaux permettent d’envisager de futurs essais cliniques chez le cheval, afin de traiter les affections articulaires de ce modèle gros animal
Articular cartilage disorders, such as focal defects and osteoarthritis, are the main causes of decreased performance or early retirement of sport- and racehorses. Thus, cartilage disorders represent a major veterinary issue in the equine industry, due to significant financial losses. Poor intrinsic cartilage repair properties and the absence of long- term therapy for cartilage defects lead to the development and use of new generation therapies such as autologous chondrocytes implantation. In this context, our study aimed to compare different cell types for the in vitro cartilage generation, in order to implant the biological substitute to treat cartilage defects in the horse. A therapeutic strategy initially developed in human medicine, the autologous chondrocytes transplantation, always represents a "gold standard" in cartilage tissue engineering. In the present study, after developing a new generation of cartilaginous substitute of high biological quality, composed of equine articular chondrocytes, technical and biological limits inherent to the cell type persist. Thus, we have used alternative cell types such as neonatal mesenchymal stem/stromal cells (MSCs) from umbilical cord, such as umbilical cord blood MSC (UCB-MSCs) and umbilical cord matrix or Wharton jelly MSCs (UCM- MSCs). These MSCs sources could represent a therapeutic advantage due to their non-invasive isolation, their high cell proliferation and their ability to differentiate into chondrocytes. Nevertheless, it is essential to define the best therapeutic candidate between these two MSCs sources, to obtain an optimal quality for the neocartilaginous substitute. Our data highlighted important differences in the chondrogenesis process of these two neonatal MSCs sources, allowing us to consider UCB-MSCs as the best therapeutic candidate for equine cartilage tissue engineering. This work allows a better understanding of the chondrocyte and MSCs biology. Moreover, this work leads the way to setting-up future clinical trials in the horse, in order to treat articular defects of this large animal model

The extracellular matrix is (ECM) is a network of large, structural proteins and polysaccharides, important for cellular behavior, tissue development and maintenance. Present thesis describes work exploring ECM as scaffolds for tissue engineering by manipulating cells cultured in vitro or by influencing ECM expression in vivo. By culturing cells on polymer meshes under dynamic culture conditions, deposition of a complex ECM could be achieved, but with low yields. Since the major part of synthesized ECM diffused into the medium the rate limiting step of deposition was investigated. This quantitative analysis showed that the real rate limiting factor is the low proportion of new proteins which are deposited as functional ECM. It is suggested that cells are pre-embedded in for example collagen gels to increase the steric retention and hence functional deposition. The possibility to induce endogenous ECM formation and tissue regeneration by implantation of growth factors in a carrier material was investigated. Bone morphogenetic protein-2 (BMP-2) is a growth factor known to be involved in growth and differentiation of bone and cartilage tissue. The BMP-2 processing and secretion was examined in two cell systems representing endochondral (chondrocytes) and intramembranous (mesenchymal stem cells) bone formation. It was discovered that chondrocytes are more efficient in producing BMP-2 compared to MSC. The role of the antagonist noggin was also investigated and was found to affect the stability of BMP-2 and modulate its effect. Finally, an injectable gel of the ECM component hyaluronan has been evaluated as delivery vehicle in cartilage regeneration. The hyaluronan hydrogel system showed promising results as a versatile biomaterial for cartilage regeneration, could easily be placed intraarticulary and can be used for both cell based and cell free therapies.

Cheuk, Yau Chuk.
Thesis submitted in: Dec 2008.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 132-144).
Abstracts in English and Chinese.
ABSTRACT --- p.i
論文摘要 --- p.iii
PUBLICATIONS --- p.v
ACKNOWLEDGEMENT --- p.vi
LIST OF ABBREBIVIATIONS --- p.vii
INDEX FOR FIGURES --- p.x
INDEX FOR TABLES --- p.xiv
TABLE OF CONTENTS --- p.xv
Chapter CHAPTER ONE - --- INTRODUCTION
Chapter 1.1 --- "Joint function, structure and biochemistry"
Chapter 1.1.1 --- Function of joint --- p.1
Chapter 1.1.2 --- Types of cartilage --- p.1
Chapter 1.1.3 --- Composition and structure of articular cartilage --- p.2
Chapter 1.1.4 --- The subchondral bone --- p.3
Chapter 1.1.5 --- Maturation of articular cartilage and subchondral bone --- p.3
Chapter 1.2 --- Osteochondral defect
Chapter 1.2.1 --- Clinical problem --- p.6
Chapter 1.2.2 --- Spontaneous repair --- p.7
Chapter 1.2.3 --- Current treatment strategies --- p.7
Chapter 1.2.4 --- Limitations of current treatment strategies --- p.8
Chapter 1.2.5 --- Treatments under development --- p.11
Chapter 1.2.6 --- Potential and limitations in cell therapies --- p.14
Chapter 1.3 --- The 3-D scaffold-free cartilage
Chapter 1.3.1 --- Fabrication of scaffold-free cartilage --- p.16
Chapter 1.3.2 --- Scaffold-free cartilage for chondral / osteochondral defect repair --- p.18
Chapter 1.3.3 --- Scaffold-free bioengineered chondrocyte pellet from our group --- p.20
Chapter 1.3.4 --- BCP as a possible treatment for OCD --- p.21
Chapter 1.4 --- The objectives of the study --- p.22
Chapter 1.5 --- The study plan
Chapter 1.5.1 --- Design of the study --- p.23
Chapter 1.5.2 --- Choice of animal model --- p.23
Chapter 1.5.3 --- Selection of evaluation time points --- p.24
Chapter 1.5.4 --- Choice and modification of histological scoring system --- p.24
Chapter CHAPTER TWO - --- METHODOLOGY
Chapter 2.1 --- Preparation of reagents and materials for tissue culture and histology --- p.26
Chapter 2.2 --- Creation of osteochondral defect model --- p.28
Chapter 2.3 --- Synthesis of scaffold-free cartilage using 3-D chondrocyte pellet culture
Chapter 2.3.1 --- Isolation of rabbit costal chondrocytes --- p.31
Chapter 2.3.2 --- Three-dimensional chondrocyte pellet culture --- p.31
Chapter 2.3.3 --- BrdU labeling for cell fate tracing --- p.32
Chapter 2.4 --- Further characterization of the 3-D scaffold-free chondrocyte pellet
Chapter 2.4.1 --- Gross appearance --- p.35
Chapter 2.4.2 --- Cell viability
Chapter 2.4.2.1 --- Alamar blue reduction assay --- p.35
Chapter 2.4.3 --- Preparation of samples for histology --- p.36
Chapter 2.4.4 --- General morphology and histomorphology
Chapter 2.4.4.1 --- H&E staining --- p.36
Chapter 2.4.5 --- Cartilage properties
Chapter 2.4.5.1 --- Safranin O /Fast Green staining --- p.37
Chapter 2.4.5.2 --- Immunohistochemistry of type II collagen --- p.37
Chapter 2.4.5.3 --- Immunohistochemistry of type I collagen --- p.38
Chapter 2.4.6 --- Angiogenic properties
Chapter 2.4.6.1 --- Immunohistochemistry of VEGF --- p.40
Chapter 2.4.7 --- Osteogenic properties
Chapter 2.4.7.1 --- ALP staining --- p.40
Chapter 2.5 --- Implantation of scaffold-free cartilage into osteochondral defect model
Chapter 2.5.1 --- Surgical procedures --- p.41
Chapter 2.5.2 --- Experimental groups --- p.42
Chapter 2.6 --- Assessment of osteochondral defect healing
Chapter 2.6.1 --- Macroscopic evaluation --- p.43
Chapter 2.6.2 --- Preparation of samples for histology --- p.43
Chapter 2.6.3 --- Histology for general morphology
Chapter 2.6.3.1 --- H&E staining --- p.45
Chapter 2.6.4 --- Histological scoring
Chapter 2.6.4.1 --- Modification of the scoring system --- p.45
Chapter 2.6.4.2 --- Procedures of scoring and validation --- p.45
Chapter 2.6.5 --- Cell proliferation
Chapter 2.6.5.1 --- Immunohistochemistry of PCNA --- p.49
Chapter 2.6.6 --- Cartilage regeneration
Chapter 2.6.6.1 --- Safranin O /Fast Green staining --- p.49
Chapter 2.6.6.2 --- Immunohistochemistry of type II collagen --- p.49
Chapter 2.6.6.3 --- Immunohistochemistry of type I collagen --- p.50
Chapter 2.6.6.4 --- Polarized light microscopy --- p.50
Chapter 2.6.7 --- Expression of angiogenic factor
Chapter 2.6.7.1 --- Immunohistochemistry of VEGF --- p.50
Chapter 2.6.8 --- Bone regeneration
Chapter 2.6.8.1 --- μCT analysis --- p.50
Chapter 2.6.9 --- Histomorphometric analysis of cartilage and bone regeneration --- p.53
Chapter 2.6.10 --- BrdU detection for cell fate tracing --- p.55
Chapter 2.6.11 --- Statistical analysis --- p.55
Chapter CHAPTER THREE - --- RESULTS
Chapter 3.1 --- Further characterization of the 3-D chondrocyte pellet culture
Chapter 3.1.1 --- Gross examination --- p.57
Chapter 3.1.2 --- Cell viability --- p.57
Chapter 3.1.3 --- Cartilage properties --- p.61
Chapter 3.1.4 --- Angiogenic properties --- p.63
Chapter 3.1.5 --- Osteogenic properties --- p.64
Chapter 3.2 --- Implantation of scaffold-free cartilage and assessment
Chapter 3.2.1 --- Gross examination --- p.65
Chapter 3.2.2 --- General morphology --- p.67
Chapter 3.2.3 --- Histological scores --- p.71
Chapter 3.2.4 --- Cell proliferation --- p.75
Chapter 3.2.5 --- Cartilage regeneration --- p.78
Chapter 3.2.6 --- Expression of angiogenic factor --- p.90
Chapter 3.2.7 --- Bone regeneration --- p.93
Chapter 3.2.8 --- Histomorphometric analysis on cartilage and bone regeneration --- p.96
Chapter 3.2.9 --- Cell fate tracing --- p.100
Chapter CHAPTER FOUR - --- DISCUSSION
Chapter 4.1 --- Summary of key findings
Chapter 4.1.1 --- Further characterization of BCP and determination of implantation time --- p.102
Chapter 4.1.2 --- Implantation of BCP in OCD --- p.102
Chapter 4.2 --- Spontaneous healing in osteochondral defect
Chapter 4.2.1 --- Findings from the current study --- p.104
Chapter 4.2.2 --- Comparison with other studies --- p.104
Chapter 4.2.3 --- Factors affecting spontaneous healing --- p.105
Chapter 4.3 --- Fabrication and further characterization of the 3-D chondrocyte pellet
Chapter 4.3.1 --- Comparison of different methods of producing scaffold-free cartilage construct --- p.106
Chapter 4.3.2 --- Cartilage phenotype of the BCP --- p.107
Chapter 4.3.3 --- Angiogenic and osteogenic potential of the BCP --- p.108
Chapter 4.3.4 --- Role of mechanical stimulation on tissue-engineered cartilage --- p.109
Chapter 4.4 --- Repair of osteochondral defect with allogeneic scaffold-free cartilage
Chapter 4.4.1 --- Advantages of the current scaffold-free chondrocyte pellet --- p.111
Chapter 4.4.2 --- Remodeling of BCP after implantation --- p.111
Chapter 4.4.3 --- Effect of BCP on cartilage repair --- p.112
Chapter 4.4.4 --- Effect of BCP on bone regeneration
Chapter 4.4.4.1 --- Findings in the present study --- p.113
Chapter 4.4.4.2 --- Possible reasons of slow bone repair --- p.114
Chapter 4.4.4.3 --- Effect of BCP on bone region peripheral to defect --- p.115
Chapter 4.4.5 --- Immunorejection-free properties of the BCP --- p.116
Chapter 4.4.6 --- Comparison with other animal studies using scaffold-free cartilage --- p.117
Chapter 4.4.7 --- Possibility of implanting a BCP cultured for shorter or longer period --- p.118
Chapter 4.4.8 --- Scaffold-free cartilage construct and construct with scaffold for OCD repair --- p.119
Chapter 4.4.9 --- Chondrocytes and stem cells for OCD repair --- p.120
Chapter 4.5 --- Limitations of the study
Chapter 4.5.1 --- Animal model --- p.122
Chapter 4.5.2 --- Histomorphometric analysis --- p.122
Chapter 4.5.3 --- Lack of quantitative data analysis --- p.122
Chapter 4.5.4 --- BrdU labeling of cells --- p.123
Chapter 4.5.5 --- Lack of biomechanical test --- p.123
Chapter 4.5.6 --- Small sample size --- p.123
Chapter CHAPTER FIVE - --- CONCLUSION --- p.124
Chapter CHAPTER SIX - --- FUTURE STUDIES
Chapter 6.1 --- Identification of factors affecting bone repair after OCD treatment --- p.125
Chapter 6.2 --- Modifications of BCP treatment --- p.125
Chapter 6.3 --- Alternative cell source --- p.126
Chapter 6.4 --- Alternative cell tracking methods --- p.126
Chapter 6.5 --- Inclusion of biomechanical test --- p.126
APPENDICES
Appendix 1. Conference paper 1 --- p.129
Appendix 2: Conference paper 2 --- p.130
Appendix 3: Animal experimentation ethics approval --- p.131
BIBLIOGRAPHY --- p.132

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Abstract: This book describes how bioprinting emerged from 3D printing and details the accomplishments and challenges in bioprinting tissues of cartilage, skin, bone, muscle, neuromuscular junctions, liver, heart, lung, and kidney. It explains how scientists are attempting to provide these bioprinted tissues with a blood supply and the ability to carry nerve signals so that the tissues might be used for transplantation into persons with diseased or damaged organs. The book presents all the common terms in the bioprinting field and clarifies their meaning using plain language. Readers will learn about bioink—a bioprinting material containing living cells and supportive biomaterials. In addition, readers will become at ease with concepts such as fugitive inks (sacrificial inks used to make channels for blood flow), extracellular matrices (the biological environment surrounding cells), decellularization (the process of isolating cells from their native environment), hydrogels (water-based substances that can substitute for the extracellular matrix), rheology (the flow properties of a bioink), and bioreactors (containers to provide the environment cells need to thrive and multiply). Further vocabulary that will become familiar includes diffusion (passive movement of oxygen and nutrients from regions of high concentration to regions of low concentration), stem cells (cells with the potential to develop into different bodily cell types), progenitor cells (early descendants of stem cells), gene expression (the process by which proteins develop from instructions in our DNA), and growth factors (substances—often proteins—that stimulate cell growth, proliferation, and differentiation). The book contains an extensive glossary for quick reference.

It is an impressive spectacle. Multicellular organisms—from fruitflies to humans—emerge from a single cell through a coordinated sequence of cell division, movement, and specialization. Many of the fundamental mechanisms of animal development are known: differentiated cells arise from less specialized precursor or stem cells, cells organize into functional units by migration and selective adhesion, and cell-secreted growth factors stimulate growth or differentiation in other cells. Despite extensive progress in acquiring basic knowledge, however, therapeutic opportunities for patients with tissue loss due to trauma or disease remain extremely limited. Degeneration within the nervous system can reduce the quality and length of life for individuals with Parkinson’s disease. Inadequate healing can cause various problems, including liver failure after hepatitis infections, as well as chronic pain from venous leg ulcers and severe infections in burn victims. The symphony of development is difficult to conduct in adults. Tissue or whole-organ transplantation is one of the few options currently available for patients with many common ailments including excessive skin loss and artery occlusion. During the past century, many of the obstacles to transplantation were cleared: immunosuppressive drugs and advanced surgical techniques make liver, heart, kidney, blood vessel, and other major organ transplantations a daily reality. But transplantation technology has encountered another severe limitation. The number of patients requiring a transplant far exceeds the available supply of donor tissues. New technology is needed to reduce this deficit. Some advances will come from individuals trained to synthesize basic scientific discoveries (for example, in developmental biology) with modern bioengineering principles. Tissue engineering grew from the challenge presented by tissue shortage. Tissue engineers are working to develop new approaches for encouraging tissue growth and repair; these approaches are founded on basic science of organ development and wound healing. A few pioneering efforts are already being tested in patients; these include engineered skin equivalents for wound repair, transplanted cells that are isolated from the immune system by encapsulation in polymer membranes for treatment of diabetes, and chondrocyte implantation for repair of articular cartilage defects.

Abstract: Bioprinting: To Make Ourselves Anew describes how bioprinting emerged from 3D printing and details the accomplishments and challenges in bioprinting tissues of cartilage, skin, bone, muscle, neuromuscular junctions, liver, heart, lung, and kidney. It explains how scientists are attempting to provide these bioprinted tissues with a blood supply and the ability to carry nerve signals so that the tissues might be used for transplantation into persons with diseased or damaged organs. The book presents all the common terms in the bioprinting field and clarifies their meaning using plain language. The reader will learn about bioink—a bioprinting material containing living cells and supportive biomaterials. Additionally, readers will become at ease with concepts such as fugitive inks (sacrificial inks used to make channels for blood flow), extracellular matrices (the biological environment surrounding cells), decellularization (the process of isolating cells from their native environment), hydrogels (water-based substances that can substitute for the extracellular matrix), rheology (the flow properties of a bioink), bioreactors (containers to provide the environment cells need to thrive and multiply). Further vocabulary that will become familiar includes diffusion (passive movement of oxygen and nutrients from regions of high concentration to regions of low concentration), stem cells (cells with the potential to develop into different bodily cell types), progenitor cells (early descendants of stem cells), gene expression (the process by which proteins develop from instructions in our DNA), and growth factors (substances—often proteins—that stimulate cell growth, proliferation, and differentiation). The book contains an extensive glossary for quick reference.

The previous chapter provided some examples of tissue engineering, in which cells that were isolated and engineered outside of the body are introduced into a patient by direct injection of a cell suspension, typically into the circulatory system. But the field of tissue engineering also points to treatments that are conceptually different from variations on cell transfusion technology; tissue engineering promises the regrowth of adult tissue structure through application of engineered cells and synthetic materials. In support of this broad claim, the field of tissue engineering can point to some initial successes. For example, synthetic materials are now available that accelerate healing of burns and skin ulcers. In addition, in vitro cell culture methods now allow the amplification of a patient’s own cells for cartilage repair or bone marrow transplantation. But major obstacles to the widespread application of tissue engineering remain. Tissue engineers have not yet learned how to reproduce complex tissue architectures, such as vascular networks, which are essential for the normal function of many tissues. In fact, the tissue engineering concepts that have been demonstrated in the laboratory to date involve arrangements of cells and materials into precursor tissues (or neotissues) that develop according to natural processes that are already present within the cells or the materials at the time of implantation. These methods may be suitable for production of some tissues in which either the structure is relatively homogeneous (such as cartilage, in which a tissue structure can reform after the implantation of chondrocytes into a tissue defect) or the structure develops naturally (such as in some tissue-engineered skin, in which the stratified epithelium develops naturally by culturing at an air–liquid interface). The engineering of many tissue structures—such as the branching architectures found in many tissues or the intricate network architecture of the nervous system—will probably require methods for introducing and changing molecular signals during the process of neo-tissue development. For example, it is well known that chemical gradients of factors known as morphogens induce the formation of structures during development; some of the attributes of morphogens were introduced in Chapter 3.

While hyaluronic acid (HA) hydrogels provide a stable 3D environment that is conducive to the chondrogenesis of mesenchymal stem cells (MSCs) in the presence of growth factors [1], the neocartilage that is formed remains inferior to native tissue, even after long culture durations. Additionally, MSCs eventually transit into a hypertrophic phenotype after chondrogenic induction, resulting in the calcification of the ECM after ectopic transplantation [2]. From a material design perspective, variation in the HA hydrogel scaffold crosslinking density via changes in the HA macromer concentration can influence chondrogenesis of MSCs and neocartilage formation [3]. Recent studies have also demonstrated that dynamic compression enhances the expression of chondrogenic markers and cartilage matrix synthesis by MSCs encapsulated in various hydrogels, including agarose [4], alginate [5] and fibrin [6]. Furthermore, mechanical signals also regulate growth plate and articular cartilage chondrocyte hypertrophy via the IHH-PTHrP (India hedgehog, Parathyroid hormone-related protein) pathway [7]. In contrast to biologically inert scaffold materials, HA is capable of interacting with cells (including MSCs) via cell surface receptors (CD44, CD54, and CD168) [8; 9]. Therefore the objectives of this study were to (i) evaluate the effects of both hydrogel crosslinking and dynamic compressive loading on (i) chondrogenesis and cartilage matrix production/distribution of human MSCs encapsulated in HA gels and (ii) hypertrophic differentiation of human MSCs using an in vitro MSC hypertrophy model [10].