Transglutaminases as bonding agents for use in cartilage repair

A consistent failure of cartilage repair technologies has been the secure anchoring of repair materials into defect sites. Until this is overcome, cartilage repair will continue to be compromised. The protein cross-linking, calcium-dependent enzyme tissue transglutaminase (tTG) has been proposed as an adhesive for binding cartilagecartilage interfaces. Work presented in this thpsis aimed to investigate the potential of tTG to act as a bonding agent for use in cartilage repair. An in vitro test system was developed to measure tTG-catalysed bonding at cartilagecartilage interfaces in response to tensile stress. tTG bond strength was found to be dependent on enzyme concentration, increasing incubation time and was consistent over a humidity range of 20-100%. Methods of pre-treating the tissue surfaces to improve bonding were investigated and enzymatic digestion of cartilage glycosaminoglycans (GAG's) proved most successful. To determine why this was the case, cartilage surfaces were analysed using various microscopy techniques. ATM analysis revealed GAG-digestion to be reducing the microscopic topography of the tissue surfaces, possibly allowing for more points of direct contact when opposed at an interface. Fluorescence microscopy showed that the digestion also exposes more potential TG-substrate, peptide-bound γ-glutamyl residues on the tissue surfaces. These may contribute to the increased bond strength of tTG at GAG-digested interfaces by allowing for a greater degree of cross-linking between the tissue surfaces. Biomaterials are increasingly important in cartilage repair and the present study demonstrates that tTG has the capacity to bond synthetic polymer biomaterials to GAG-digested cartilage surfaces. The materials were optimised for tTG-catalysed bonding by the inclusion of TG-substrate residues/proteins, indicating that cartilage repair materials may now be designed and optimised for maximum TG-catalysed retention at implant sites. A method was also developed for measuring chondrocyte viability at experimental wound edges in cartilage explants cultured in vitro. The loss of cell viability observed at the wound edges was decreased when the edge was cultured in contact with surrounding tissue, and increased by GAG-digestion of the tissue. It was unaffected by tTG-catalysed cross-linking of the wound edge interface, suggesting that tTG application to cartilage interfaces in vivo will not have a detrimental effect on cell viability at the interface surfaces.