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 hyperosmotic 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.