Arthritis includes many conditions of the joints characterized by inflammation, pain, and loss of joint function that affect 66 million people in the U.S. alone. During arthritic degeneration, chondrocytes exhibit downregulated synthesis of extracellular matrix molecules and upregulation of proteolytic enzymes. Fibrochondrocytes, found in meniscal fibrocartilage, appear to behave in a similar way. Metalloproteinases, including matrix metalloproteinases (MMP) and a disintegrin and metollproteinase with thrombospondin motif (ADAMTS) class enzymes have demonstrated efficient, distinct aggrecan degradation in models of arthritis. ADAMTS-4 and ADAMTS-5 are thought to be primary mediators of pathologic aggrecan catabolism, while MMP-17 may be involved in ADAMTS activation. There is also growing evidence of metalloproteinase-independent mechanisms in aggrecan catabolism. The cysteine endopeptidase m-calpain has been detected in cartilage from arthritic joints, and chondrocytes can secrete this protease. The overall objective of this work was to investigate metalloproteinases and m-calpain as comediators of aggrecan turnover in articular cartilage and meniscal fibrocartilage. The central hypothesis was that metalloproteinases cooperate with m-calpain to mediate cytokine-induced aggrecan turnover and associated changes in tissue mechanics. Experiments involved using inhibitors to perturb protease systems, antibodies targeting aggrecan neoepitopes to characterize enzyme activity, and established methods of evaluating tissue compressive and shear properties. Models of degradation and de novo tissue assembly were used to investigate tissue-specific differences in aggrecan turnover. The results of this work demonstrate tissue-specific differences in the abundance and structure of aggrecan, and indicate that the mechanisms and extent of aggrecan processing in the meniscus is dependent on location within the tissue. The relationships between aggrecan structure and tissue material properties are discussed, along with implications for development, disease, and repair.

Cartilage repair and/or replacement is necessary for many orthopaedic conditions including fissures from blunt trauma, autograft or allograft transplantation, and replacement of focal defects with biological or synthetic constructs. In cartilage repair, initial integration between the host tissue and repair site is desirable to allow for nutrient transport, molecular deposition to enhance fixation, and eventual stress transmission across the interface. It has been postulated that effective transport and crosslinking of newly synthesized collagen molecules across a repair site may be vital to the process of integrative repair, and recent experiments have correlated collagen deposition with the strength of such repair. Other investigations have shown that enzymatic degradation of the cartilage surface may enhance integrative repair and can increase bond strength of an adhesive to cartilage. This study explored a novel approach involving photochemical bonding of cartilage tissue samples through collagen crosslinking as a means to achieve rapid and effective initial fixation, with the goal of enhancing biological integration. Photosensitized collagen gels were first analyzed via FTIR to determine the crosslinking effects with respect to collagen type and photochemical mechanism. Using the photogellation FTIR results as a parametric guide, in vitro mechanical testing of photochemically bonded meniscal fibrocartilage and hyaline articular cartilage tissues was performed using a modified single-lap shear test. Finally, the cellular viability and bond stability of a photochemically bonded cartilage interface was evaluated over seven days of in vitro culture, where the bond strength was assessed by pushout of cores from annular defects. Results of this study have demonstrated the potential of combining enzymatic surface modification with photodynamic techniques to directly bond cartilage tissues for initial fixation.

Disease and degeneration of articular cartilage and fibrocartilage tissues severely compromise the quality of life for millions of people. Although current surgical repair techniques can address symptoms in the short term, they do not adequately treat degenerative joint diseases such as osteoarthritis. Thus, novel tissue engineering strategies may be necessary to combat disease progression and repair or replace damaged tissue. Both articular cartilage and the meniscal fibrocartilage in the knee joint are subjected to a complex mechanical environment consisting of compressive, shear, and tensile forces. Therefore, engineered replacement tissues must be both mechanically and biologically competent to function after implantation. The goal of this work was to investigate the effects of oscillatory tensile loading on three dimensional engineered cartilaginous tissues in an effort to elucidate important aspects of chondrocyte and fibrochondrocyte mechanobiology. To investigate the metabolic responses of articular chondrocytes and meniscal fibrochondrocytes to oscillatory tensile loading, various protocols were used to identify stimulatory parameters. Several days of continuously applied tensile loading inhibited extracellular matrix metabolism, whereas short durations and intermittently applied loading could stimulate matrix production. Subpopulations of chondrocytes, separated based on their zonal origin within the tissue, differentially responded to tensile loading. Proteoglycan synthesis was enhanced in superficial zone cells, but the molecular structure of these molecules was not affected. In contrast, neither total proteoglycan nor protein synthesis levels of middle and deep zone chondrocytes were substantially affected by tensile loading; however, the sizes of these new matrix molecules were altered. Up to 14 days of intermittently applied oscillatory tensile loading induced modest increases in construct mechanical properties, but longer durations adversely affected these mechanical properties and increased degradative enzyme activity. These results provide insights into cartilage and fibrocartilage mechanobiology by elucidating cellular responses to tensile mechanical stimulation, which previously had not been widely explored for these tissues. Understanding the role that mechanical stimuli such as tension can play in the generation of engineered cartilaginous tissues will further the goal of developing successful treatment strategies for degenerative joint diseases.

Articular cartilage provides a low-friction surface during normal joint motion and distributes forces to the underlying bone. The extracellular matrix (ECM) composition of healthy cartilage has previously been shown to be an excellent predictor of its mechanical properties. Changes in ECM composition and loss of mechanical function are known to occur with degenerative conditions such as osteoarthritis. Identifying differences in the composition-function relationships of cartilage under different anabolic, catabolic, and homeostatic conditions may thus be a useful approach for identifying factors (e.g. ECM content, distribution, and structure) which are critical to mechanical function. In addition, diagnostic tools capable of monitoring changes in the cartilage ECM may increase our understanding of the effects of ECM changes on composition-functions relationships. The goals of this work were to investigate composition-function relationships in healthy, degraded, and engineered cartilage and to develop a microcomputed tomography-based approach to analyze changes in matrix composition and morphology in articular cartilage. In healthy explants, compressive and shear properties were dependent on both sGAG and collagen content. In contrast, the compressive properties of IL-1stimulated cartilage were dependent on sGAG but not collagen content. To assess changes in sGAG content, EPIC-microcomputed tomography, a 3D contrast-enhanced microcomputed tomography technique was developed. EPIC-microcomputed tomography attenuation was found to be an excellent predictor of sGAG content in IL-1-stimulated cartilage explants and engineered cartilage. In addition, analytical approaches were developed to use EPIC-microcomputed tomography for the in situ analysis of cartilage morphology. EPIC-microcomputed tomography was also used to analyze spatial differences in sGAG accumulation in bilayer engineered cartilage for comparison with the local strain profile. This work underscores the significance of ECM composition and structure in regulating cartilage mechanical properties and validates the use of EPIC-microcomputed tomography as a diagnostic for monitoring sGAG content and potentially for assessing mechanical function in models of degeneration and regeneration.

Bioreactors are devices used for the growth of tissues in a laboratory environment. They exist in many different forms, each designed to enable the production of high-quality tissues. The dynamic environment within bioreactors is known to significantly affect the growth and development of the tissue. Chondrocytes, the building blocks of articular cartilage, for example, are stimulated by mechanical stresses such as shear, as compared with those in tissues grown under static incubation conditions. On the other hand, high shear can damage cells. Consequently the shear-stress level has to be controlled in order to optimize the design and the operating conditions of bioreactors. Spinner flasks have been used for the production of articular cartilage in vitro. Assuming the existence of a relation between the cellular glycosaminoglycan (GAG) synthesis and the local shear stresses on the construct surfaces, this research focuses on the development of a model for cartilage growth in such devices. The flow produced in a model spinner flask is characterized experimentally using particle-image velocimetry (PIV). A computational fluid dynamic (CFD) model validated with respect to the laboratory measurements is constructed in order to predict the local shear stresses on the construct surfaces. Tissue growth experiments conducted in the prototype bioreactor permit construct histologies and GAG contents to be analyzed and then correlated with the shear-stress predictions. The integration of this relation into the CFD model enables the prediction of GAG synthesis through convective effects. Coupling this convective model to an existing diffusive model produces a complete cartilage-growth model for use in aiding the optimization of existing bioreactors, and in the design of new ones.

[Truncated abstract] An important field of research in orthopaedic biomechanics is the elucidation and mathematical modelling of the mechanical response of cartilaginous tissues. Such research has applications in the understanding of joint function and degenerative processes, as well as in surgical planning and simulation, and engineering of tissue replacements. In the case of surgical and tissue engineering applications especially, patient-specific mechanical properties are highly desirable. Unfortunately, obtaining such information would generally involve destructive mechanical testing of patient tissue, thus rendering the tissue functionally unusable. Development of a laser scanning confocal arthroscope (LSCA) within our School will soon allow non-invasive extraction of 3D microstructural images of cartilaginous tissues in vivo. It is also envisaged that, linked to a suitably formulated constitutive formulation, such information could allow estimation of tissue mechanical response without physical biopsy. This thesis describes the development of techniques to potentially allow non-invasive patient-specific estimation of tissue mechanical response based on confocal arthroscopy data. A microstructural constitutive model is developed which is capable of directly incorporating LSCA-derived patient-specific structural information. A fibre composite type homogenisation approach is used as the basis for the model. ... The result is a series of orientation tensors describing the 3D orientation of linear features in the image stack. The developed analysis techniques are used to estimate fibre volume fraction and orientation distribution for each of the meniscal specimens. The developed constitutive model and image-derived structural parameters are finally used to estimate the reaction force history of two meniscal cartilage specimens subjected to partially confined compression. The predictions are made on the basis of the specimens? individual structural condition as assessed by confocal microscopy and involve no tuning of material parameters. Although the model does not reproduce all features of the experimental curves, as an unfitted estimate of mechanical response the prediction is quite accurate. In light of the obtained results it is judged that more general non-invasive estimation of tissue mechanical properties is possible using the developed framework. The likely limitations and potential areas of improvement are discussed.