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This thesis uses a multiscale approach to identify and manipulate physiologic and in vitro developmental milieus towards the functional repair of articular cartilage. The overarching goals of this work are to improve knowledge of cartilage physiology and to enhance functional engineering of biologic cartilage replacements. Towards this end, assessment and modulation of cartilage phenotype were undertaken at multiple levels of complexity: gene transcription, cytoskeletal architecture, ion channels, single cells, extracellular matrix, intact tissue, and the whole joint. The first part of this thesis focused on probing cartilage phenotype at the single cell level. A quantitative single cell gene expression assay was developed and used to quantify cell-to-cell variability and the chondrocyte response to growth factors. Next, the viscoelastic compressive properties of single chondrocytes were measured and compared to cytoskeleton organization before and after growth factor exposure. It was found that growth factors increased matrix gene expression and induced cell stiffening in a time- and cartilage zone-dependent manner. The second part of this thesis investigated the modulation of the chondrocyte microenvironment for enhanced cartilage tissue engineering. Tissue constructs were grown in vitro using a chondrocyte self-assembly process. In one study, it was found that TRPV4 ion channel activation significantly increased cartilage matrix production and improved tensile properties in self-assembled constructs. In a second study, constructs were exposed to static or dynamic application of hypo-osmotic and hyper-osmotic stress. Static application of hyper-osmotic stress was found to improve construct compressive and tensile properties, and their corresponding biochemical mediators, significantly. A third study showed that treatment of constructs with ribose, an agent used for non-enzymatic glycation, produced enhanced tissue mechanics and biochemistry in a time-dependent manner. The third part of this thesis describes efforts to improve the potential clinical translatability of in vitro cartilage repair strategies. A technique was developed to decellularize xenogenic self-assembled constructs. Decellularization resulted in histologic and biochemical cell depletion with maintenance of tissue mechanical properties. Additionally, a comprehensive characterization of the major tissues of the immature knee joint revealed and reinforced important structure-function relationships that will inform future cartilage repair strategies. The total body of work contained in this thesis contributes significantly both to a basic understanding of cartilage physiology as well as to evolving strategies for cartilage repair. This thesis advances the field of cartilage tissue engineering by examining chondrocyte phenotype, the cell and tissue microenvironment, and avenues for clinical translation.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13068号 / 論医博第2123号 / 新制||医||1019(附属図書館) / 33219 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 教授 戸口田 淳也 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Os estudos na área de engenharia de tecidos aplicada à reparação da cartilagem articular estão voltados ao desenvolvimento de uma matriz biocompatível que permita a diferenciação, proliferação e manutenção de células para produção de cartilagem hialina. A quitosana é um biomaterial e vem sendo estudada como suporte para condrócitos e para liberação controlada de substâncias. O objetivo deste trabalho foi estudar a biocompatibilidade do gel de quitosana associada ao fosfato de glicerol para reparação de defeitos osteocondrais induzidos experimentalmente na tróclea do talus de eqüinos. Foram utilizados cinco cavalos da raça Mangalarga, de três anos de idade, e por artroscopia foi criado um defeito osteocondral na tróclea lateral do talus de cada articulação. De forma aleatória um defeito foi escolhido para implante do gel de quitosana - fosfato de glicerol, e o defeito da articulação contralateral foi mantido vazio, servindo como controle. Para acompanhamento da evolução do processo de reparação da cartilagem articular foram realizados os exames físico, radiográfico e ultrassonográfico; análise do líquido sinovial (física, celularidade, quantificação de proteína, condroitim sulfato e ácido hialurônico); e análise da cartilagem articular (histológica e produção de proteoglicanos). Os resultados obtidos de todas as avaliações realizadas foram semelhantes entre os defeitos tratados e controle. O gel de quitosana fosfato de glicerol é biocompatível com o ambiente articular e pode ser indicado para futuras aplicações como suporte de células e para liberação controlada de medicamentos. / The tissue engineering studies applied to articular cartilage repair are focused on the development of scaffold biocompatibility allowing the differentiation, proliferation and cells maintenance providing production of the hyaline cartilage. Chitosan is a biomaterial that has been evaluated as a scaffold for chondrocyts implant and also as a drug-delivery control material. The aim of this work was to evaluate the chitosan glycerol phosphate gel biocompatibility in experimentally induced equine talus osteochondral defect. Five three years old Mangalarga breed horses were submitted to arthroscopy for osteochondral defect production on the lateral troclea of the talus in both tibiotarsal joints by arthroscopy. In a random form one defect was chosen for chitosan-glycerol phosphate gel implant, and the defect of the opposed joint was kept empty and used as a control. For the assessment of the articular cartilage repair process was performed the physic, radiographic and ultrassonographic exams; the synovial fluid analyze (physic, cellularity, protein quantification, chondroitin sulphate and hialuronan); and the articular cartilage analyze (hystologic and proteoglicans production). The results obtained in all evaluations performed were similar between the treated and control defects. The chitosan glycerol phosphate gel is biocompatible with the articular environment and can be indicate for future applications as an scaffold for cells support and drug-delivery control system.

Articular cartilage represents a primary target for tissue engineering strategies as it does not functionally regenerate within the joint. Many tissue engineering approaches have focused on the in vitro generation of neo-cartilage using chondrocyte-seeded scaffolds. Several studies have reported the morphological appearance of native cartilage, although its functional competence has not been demonstrated. Accordingly, mechanical conditioning has often been introduced to enhance biosynthetic activity of chondrocytes within 3D constructs. However although this strategy has significantly up-regulated proteoglycan synthesis, its effects on the synthesis of the other major solid constituent, type II collagen, has been modest. Analyses of normal joint activities reveal that cartilage is subjected to shear superimposed on uniaxial compression. This complex mechanical state has motivated the design of a biaxial loading system intended for use in vitro to stimulated bovine chondrocytes seeded in agarose constructs. This necessitated the redesign of the construct from cylindrical morphology to accommodate shear loading. The experimental approach was complemented with the development of computational models, which permitted prediction of both cell distortion under biaxial loading regimens and nutrient diffusion within the 3D constructs. An initial study established the profile of proteoglycan and collagen synthesis in free swelling cultures up to day 12. The introduction of dynamic compression (15% strain, 1 Hz for 48 h) enhanced proteoglycan synthesis significantly. In addition, when dynamic shear (10%, 1 Hz) was superimposed on dynamic compression, total collagen synthesis was also up-regulated, within 3 days of culture, without compromising proteoglycan synthesis. Histological analysis revealed marked collagen deposition around individual chondrocytes. However, a significant proportion (50%) of collagen was released into the culture medium, suggesting that it was not fully processed. The overall biosynthetic activity was enhanced more when the biaxial stimulation was applied in a continuous mode as opposed to intermittent loading. The present work offers the potential for a more effective preconditioning of cell-seeded constructs with functional integrity intended for use to resolve defects in joint cartilage.

Articular cartilage damage associated with joint trauma seldom heals and often leads to osteoarthritis (OA). Current treatment often fails to regenerated functional cartilage close to native tissue. We previously identified a migratory chondrogenic progenitor cell (CPC) population that responded chemotactically to cell death and rapidly repopulated the injured cartilage matrix, which suggested their potential for cartilage repair. To test that potential we filled experimental full thickness chondral defects with an acellular hydrogel containing SDF-1α. We expect that SDF-1α can increase the recruitment of CPCs, and then promote the formation of a functional cartilage matrix with chondrogenic factors. Full-thickness bovine chondral defects were filled with hydrogel comprised of fibrin and hyaluronic acid and containing SDF-1α. Cell migration was monitored, followed by chondrogenic induction. Regenerated tissue was evaluated by histology, immunohistochemistry, and scanning electron microscopy. Push-out tests were performed to assess the strength of integration between regenerated tissue and host cartilage. Significant numbers of progenitor cells were recruited by SDF-1α within 12 days. By 5 weeks chondrogenesis, repair tissue cell morphology, proteoglycan density and surface ultrastructure were similar to native cartilage. SDF-1α treated defects had significantly greater interfacial strength than untreated controls. However, regenerated neocartilage had relatively inferior mechanical properties compared with native cartilage. In addition to that, we developed a 3D bioprinting platform, which can directly print chondrocytes as well as CPCs to fabricated articular cartilage tissue in vitro. We successfully implanted the printed tissue into an osteochondral defect, and observed tissue repair after implantation. The regerated tissue has biochemical and mechanical properties within the physiological range of native articular cartilage. This study showed that, when CPC chemotaxis and chondrogenesis are stimulated sequentially, in situ full thickness cartilage regeneration and bonding of repair tissue to surrounding cartilage could occur without the need for cell transplantation from exogenous sources. This study also demonstrated the potential of using 3D bioprinting to engineer articular cartilage implants for repairing cartilage defect.

Articular cartilage is a complex soft tissue covering the end of moving bones in joints which provide pressure load distribution over the joint surface and smooth lubrication with little friction for establishing movement. Articular cartilage has an intrinsically limited capacity for self-repair when injured due to the lack of nerve and blood supply. Considered that injured cartilage is left untreated, it is likely to undergo progressive cartilage degeneration without pain which may lead to posttraumatic osteoarthritis. Therefore functional and physiologic restoration of injured cartilage back to a normal condition has long been in demand, yet current available repairing methods in clinics have met with limited success. Mechanically applied loads to articular cartilage is necessary for chondrocytes, cartilage cells, since they are responsible for cartilage matrix turnover by synthesizing extracellular matrix (ECM) molecules in response to bio- chemical and mechanical changes in ECM. Ultrasound has emerged as an anabolic stimulator over the past few decades and a number of studies have proven that ultrasound therapy is beneficial for cartilage repair by synthesizing cartilage ECM components such as type II collagen and proteoglycan. Ultrasound therapy has also proven its potential for the attenuation of progressive cartilage degradation and induction of chondrogenic differentiation of mesenchymal stem cells. The use of ultrasound as an anabolic stimulator would be valuable with respect to cartilage repair since ultrasound as a form of mechanical energy can be non-invasively transferred into a human body. However, understanding the underlying mechanisms has been slow and the mechanisms have been roughly classified into thermal and non-thermal effects. Biologically detailed underlying mechanisms have not been sufficiently studied. That might be the reason why the application of ultrasound as a therapeutic tool has been limitedly available in clinics. In this study, mechanism involved biophysical effects of low intensity ultrasound has been studied for cartilage regeneration. First of all, the effect of ultrasound therapy as a mechanical stimulator on chondrogenic progenitor cell homing toward injured sites in cartilage was investigated with underlying biologic mechanisms. And the feasibility of ultrasound therapy for reactive oxygen species production mediated cartilage energy modulation was evaluated. There have been extensive preclinical studies about the effects of microbubble mediated ultrasound therapy on the targeted drugs or gene delivery into tissues of interest. Mechanical shock waves are released during ultrasound mediated microbubble destruction and the waves facilitate drug delivery into target tissues through transient blood vessel disruption. However, the clinical use of this technique has been limited through vascular system. In this study, the effects of microbubble mediated low intensity ultrasound therapy on directly delivered mechanical shock waves and controlled drug release were investigated. In conclusion, low intensity ultrasound therapy accelerates the homing of chondrogeic progenitor cells toward injured sites in cartilage via triggering mechanotransductive cell signaling pathways. This may result in speed up the return to normal cellularity and cartilage integrity by accelerating cartilage matrix repair. Low intensity ultrasound therapy was investigated as an energy modulator for chondrocytes via reactive oxygen species production in articular cartilage; however, little effects of ultrasound therapy driven cartilage energy modulation were found. The strong relationship between microbubbles mediated low intensity ultrasound therapy and the controlled release of drugs and mechanical shock waves was found. This strongly suggests that low intensity ultrasound therapy can play a role as a non-invasive controller for the release of drugs and lethal shock waves upon request.

Background: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair.

Co-delivery of the embedded growth factor-loaded microspheres and adult stem cells in a hydrogel matrix was studied for its potential as a cell-based therapeutic strategy for cartilage regeneration in partial thickness chondral defects. A photopolymerizable N-methacrylate glycol chitosan (MGC) was employed to form an in situ gel that was embedded with two formulations of growth factor-loaded microspheres and human adipose-derived stem cells (ASC). The polymeric microspheres were used as a delivery vehicle for the controlled release of growth factors to stimulate differentiation of the ASC towards the chondrocyte lineage. The microspheres were made of amphiphilic low molecular weight (Mn < 10,000 Da) poly(1,3-trimethylene carbonate-co--caprolactone)-b-poly(ethylene glycol)-b-poly(1,3-trimethylene carbonate-co--caprolactone) (P(TMC-CL)2-PEG)). This triblock copolymer is solid below 100C, but liquid with a low degree of crystallinity at physiological temperature and degrades slowly, and so acidic degradation products do not accumulate locally. Bone morphogenetic protein-6 (BMP-6) and transforming growth factor-3 (TGF-3) were delivered at 5 ng/day with initial bursts of 14.3 and 23.6%, respectively. Both growth factors were highly bioactive when released, retaining greater than 95% bioactivity for 33 days as measured by cell-based assays. To improve ASC viability within the MGC vehicle, an RGD-containing ligand was grafted to the MGC backbone. Prior to chondrogenic induction within the MGC gel, ASC viability was assessed and greater than 90% of ASC were viable in the gel grafted with cell-adhesive RGD peptides as compared to that in non-RGD grafted gels. For ASC chondrogenesis induced by the sustained release of BMP-6 and TGF-3 in MGC gels, the ASC cellularity and glycosaminosglycan production were similar for 28 days. The ratio of collagen type II to I per cell (normalized to deoxyribonucleic acid content) in the microsphere delivery group was significantly higher than that of non-induced ASC or with soluble growth factor administration in the culture media, and increased with time. Thus, the co-delivery of growth factor-loaded microspheres and ASC in MGC gels successfully induced ASC chondrogenesis and is a promising strategy for cartilage repair. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2011-06-07 19:32:50.94