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PAPER 007: DEVELOPMENT OF A GENIPIN CROSS-LINKED AUTOLOGOUS HUMAN FIBRIN GLUE SCAFFOLD FOR IN VITRO ARTICULAR CARTILAGE REGENERATION



Abstract

Purpose: The objective of this project is to evaluate the human fibrin glue, CryoSeal®, as a scaffold for articular cartilage tissue engineering. An autologous system would eliminate risks associated with biocompatibility and virus transmission.

Method: Human articular chondrocytes were isolated from articular cartilage harvested from consenting patients undergoing total knee arthroplasty. The cells were encapsulated into CryoSeal® fibrin glue – which is derived from a single patient’s plasma using the Cryo-Seal® Fibrin Sealant System (Thermogenesis Corp.) – and cross-linked with genipin, which is a natural cross-linking agent with anti-inflammatory activity. The resulting gels were cultured in vitro for up to 7 weeks under either normal (21%) or low oxygen (5%) conditions and were evaluated for mechanical properties, extracellular matrix (ECM) production, viability, and biodegradation. Fibrin glue components were isolated from either fresh or frozen plasma.

Results: The dynamic compression modulus of the genipin cross-linked fresh plasma (FSP) CryoSeal® gels increased by ~4.4-fold over 5 weeks in culture. The glycosaminoglycan (GAG) content of the FSP gels increased by 4.7-fold over 5 weeks in low oxygen (LO) culture, which was 1.7-fold greater than in normal oxygen (NO) culture. The total collagen content of the FSP cultures increased by 6.0-fold over 5 weeks in LO culture, which was 2.2-fold greater than in NO culture. These changes in ECM were confirmed by histology (Alcian Blue) and immunostaining (to detect collagen II, collagen I, aggrecan, Sox9) of gel cryosections. After 5 weeks in LO culture the FSP CryoSeal®-encapsulated chondrocytes expressed a 6.4 ± 1.1 fold increase in collagen II gene expression, which was 5.8 ± 1.0 fold greater than in NO cultures. In addition, chondrocyte viability within the FSP and frozen plasma (FZP) CryoSeal® gels was ~90% at both 24 hours and 2 weeks after gelation. When fibrin hydrogels were implanted subcutaneously into rats it was found that inflammation was inhibited with increasing genipin and when the material origin was species-specific.

Conclusion: The CryoSeal® fibrin gel system demonstrates promise for autologous human articular cartilage tissue engineering. An in vivo orthopaedic implantation model must be developed for further testing.

Correspondence should be addressed to Meghan Corbeil, Meetings Coordinator Email: meghan@canorth.org