Transport of GAG digesting enzymes in engineered cartilage tissues
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Osteoarthritis (OA) is a debilitating condition of articular cartilage that leads to pain and severe limitations in mobility. The generation of functional tissue-engineered cartilage in vitro that can be used for cartilage repair is a growing and promising OA treatment strategy. However, there exists a fundamental challenge in generating engineered cartilage which possesses native levels of cartilaginous extracellular matrix constituents (glycosaminoglycans [GAG] and type-II collagen) and mechanical properties, leaving engineered tissues inferior to their native counterparts and inherently more vulnerable to degeneration upon implantation in the mechanical environment of the synovial joint. In particular, while engineered cartilage tissues generally synthesize GAG at a rapid rate, the content of collagen is far more limited, compromising the tensile stiffness and long-term stability of engineered cartilage. A promising strategy has recently been developed to promote collagen enhancements in engineered cartilage. Here, GAG-degrading enzymes (e.g. chondroitinase, hyaluronidase) are administered to the tissues, which digest and suppress the accumulation of abundantly synthesized GAG matrix molecules, providing more room in the tissue for collagen deposition. While the short term exposure of high doses of these enzymes has exhibited measured success in enhancing tissue collagen levels, it is further associated with considerable limitations such as limited tissue penetration and significant loss of cell viability. A major goal of this research project is to optimize the delivery of GAG-depleting enzymes to achieve sufficient levels of GAG depletion without loss of cell viability. However, these enzymes exhibit highly complex transport kinetics into engineered tissues, influenced by ECM binding interactions and activity kinetics. As such, the methodology to optimize the concentration and temporal exposure of these enzymes remains quite complex. In the first study of the thesis, the effect of different doses of hyaluronidase supplement in tissue engineering is investigated. In the following chapter, an optimized fluorescent conjugation to hyaluronidase to maintain functionality is studied, then the transport distributions of the hyaluronidase in live tissue constructs are observed. The results demonstrate that fluorophore labeled hyaluronidase with a degree of labeling of 1 still remain 90% functionality. And the GAG content and cell viability in constructs are vary after being treated with hyaluronidase of different concentration. These results pave the path for future development of the application of hyaluronidase in cartilage tissue engineering.