Purpose: Articular cartilage is a physiologically hypoxic tissue with a gradient of oxygen tension ranging from about 10% oxygen at the cartilage surface to less than 1% in the deepest layers. The overall goal of the study was to determine whether an injectable allogeneic/autologous fibrin scaffolds in combination with mesenchymal stem cells (MSCs) is suitable for articular cartilage tissue engineering, and to determine the effect of hypoxic culture conditions on the stability of cell-fibrin scaffolds. The secondary goal was to enhance the accumulation of extracellular matrix (ECM) inside the fibrin scaffold under these conditions.

Method: Chondroprogenitor clonal cell line RCJ3.1C5.18 (C5.18) and human mesenchymal stem cells (hMSCs) were encapsulated in fibrin hydrogel and fibrin glue scaffolds. The stabilization of fibrin scaffolds and development of ECM components were evaluated using zymography, SDS-polyacrylamide electrophoresis (SDS-PAGE), immunochemistry, spectrophotometry, RT-PCR including real time and histology (Ahmed TA., et al. Tissue Engineering2007;13(7): 1469–77).

Results: After encapsulation of C5.18 and hMSCs, fibrin gels quickly degraded under normoxic conditions (21 % oxygen) due to upregulation of plasminogen and matrix metalloproteinases (MMPs) genes especially MMP-2, -3, and -9. Protease inhibitors such as aprotinin and galardin (GM6001), in combination or separately, prevented the fibrin-C5.18 hydrogels breakdown for up to 5 weeks. Only a combination of aprotinin and galardin resulted in accumulation of ECM components such as collagen II and aggrecan. In contrast, fibrin-hMSCs hydrogels were found to be stable under hypoxic conditions (5% O2) for up to 4 weeks in the absence of inhibitors, suggesting that hypoxic conditions may downregulate the expression of the enzymes responsible for fibrin-hydrogel breakdown.

Conclusion: These results suggest that in C5.18 and MSCs cell lines, expression of matrix metalloproteinases (MMPs) and plasmin is upregulated under normoxic conditions and is responsible for fibrin-hydrogel breakdown. Moreover, inhibition of both proteases is required to enhance the accumulation of ECM. However, fibrin hydrogel scaffolds were stabilized under low oxygen tension, which is more physiological than normoxia and therefore these constructs may be stable after implantation in the absence of protease inhibitors.

Purpose: The objective of this project is to determine the suitability of modified fibrin hydrogels as scaffolds for articular cartilage tissue engineering. The attractive feature of the fibrin system is that the gel precursors are available in autologous form. We have previously demonstrated that genipin, a naturally occurring cross-linking agent, stabilizes the fibrin gel.

Methods: Human articular chondrocytes were isolated from articular cartilage harvested from consenting patients undergoing total knee arthroplasty. The human cells were encapsulated into fibrin gels where gelation was induced by combining fibrinogen, thrombin, and genipin. The resulting gels were evaluated for extracellular matrix (ECM) production, mechanical properties, cell viability, and biodegradation.

Results: No breakdown of the gels was detected during 5 weeks of cell culture. After several weeks in culture, histology indicates significant proteoglycan production by encapsulated cells, and collagen II and aggrecan were detected in this ECM by immunostaining. There was a greater accumulation of cartilage-like ECM in the gels cross-linked with genipin. Dynamic compression tests performed at 0.1 Hz for 10 cycles using an MTS machine indicate that accumulation of ECM was associated with increased stiffness of the material. Cell viability was assessed using live/dead staining, and was found to be > 50% after 24 hours and at 1 week in culture. The presence of genipin cross-linking did not significantly affect cell viability. Real-Time RT-PCR indicated that encapsulated chondrocytes show an increase in Sox9, collagen II and aggrecan expression over 5 weeks and that this is further increased in the presence of genipin. The gene expression results agreed with the enhanced ECM seen under these conditions by histology and immunostaining. The fibrin material was also implanted subcutaneously into rats and after 30 days the material was removed, sectioned and evaluated. Immunostaining indicated that while there was evidence of biodegradation, the material did not appear to cause an inflammatory response.

Conclusions: Modified fibrin hydrogels show potential as cellular scaffolds for articular cartilage tissue engineering. An in vivo orthopaedic model must now be developed to fully evaluate the potential of the fibrin gel. Funding: Other Education Grant