Chondrocyte biomechanics and chondrogenesis
Koay, Eugene J.
Athanasiou, Kyriacos A.
Doctor of Philosophy
This thesis contributes to two aspects of cartilage research: understanding the biomechanical characteristics of single chondrocytes, and tissue engineering musculoskeletal cartilages with human embryonic stem cells (hESCs). Both of these areas of investigation are motivated by the significant economic and social burdens of cartilage afflictions. Mechanical forces directly influence the biological processes of native chondrocytes; understanding and controlling this phenomenon begins at the single cell level. Thus, the first aspect of this thesis analyzes the biomechanical nature of the single chondrocyte by developing (1) a creep cytoindentation apparatus (CCA) to obtain the viscoelastic properties of single chondrocytes and (2) a new method to investigate the response of single chondrocytes to direct compression. The CCA was built, validated, and used to show that chondrocytes are three orders of magnitude less stiff than reported values for their surrounding extracellular matrix, suggesting that the cellular mechanical milieu is markedly different from the rest of the tissue. Chondrocytes were also found to alter their biomechanical properties in response to physicochemical stimuli while exhibiting a mechanical yield behavior. These results counter current theories of cartilage that assume fixed cellular properties and illuminate a possible threshold between physiological and detrimental mechanical stimuli. The second aspect of this thesis involves the design and application of a strategy to engineer musculoskeletal cartilages with hESCs. These cells, which have been scarcely studied to date, possess unique properties, such as pluripotency and self-renewal, that make them advantageous for tissue engineering. A novel modular experimental approach was established, involving hESC chondrogenic differentiation followed by a scaffold-less engineering strategy called self-assembly. Results showed that the properties of hESC-derived cartilage can be modulated through biochemical growth factors, differentiation timelines, and hypoxia, raising the possibility of engineering tissue with hESCs for various cartilage applications, including fibrocartilages and hyaline articular cartilage. Additionally, serum-free, chemically defined conditions were developed for the entire modular approach, aiding future translation of this research to a cartilage therapy. This thesis generates new directions for understanding and utilizing mechanical forces to stimulate chondrocytes and opens avenues to further investigate a powerful cell source for tissue engineering in hESCs.