Controlled Release of Bioactive Transforming Growth Factor Beta-1 from Fibrin Gels <i>In Vitro</i>

This study analyzed the ability of fibrin gels to deliver added recombinant transforming growth factor beta-1 (TGF-beta1) in a controlled manner and biologically active form. First, the effects of the amount of TGF-beta1 on the release kinetics were analyzed using a single fibrin gel formulation (fibrinogen complex (FC) at 25 mg/mL, thrombin at 2 IU/mL). Then, the effects of FC and thrombin concentrations were analyzed. Finally, to test the biological activity of the released TGF-beta1 from the gels, medium supernatants taken from gels at day 3 were used as culture medium for human mesenchymal stem cell (HMSC) monolayers. Cell proliferation was analyzed after staining with calcein dye, and changes in cell morphology were observed under fluorescence microscopy at days 1, 4, and 7. At day 7, HMSC chondrogenic differentiation was assessed by Alcian Blue staining and osteogenic differentiation by alkaline phosphatase activity and Alizarin Red staining. Results showed that TGF-beta1 added to fibrin gels was gradually released from the gels and increased with the amount of TGF-beta1 initially seeded, with a total of approximately 50% of the initial amount released by day 10 (with gels containing 25 mg/mL of FC and 2 IU/mL of thrombin). The release was lower with increasing FC concentrations, suggesting a binding affinity of TGF-beta1 with the FC component. Varying the thrombin concentration had a lesser effect. HMSC monolayers cultured with medium supernatants collected from gels at day 3 and containing released TGF-beta1 showed a change in morphology (squared to polygonal), lower cell proliferation, positive Alcian Blue staining but low levels of osteogenic differentiation markers. These results demonstrated that released TGF-beta1 was still bioactive and tended to induce mainly chondrogenic differentiation of the HMSC. Overall, the present study demonstrated that fibrin gels could be used as a carrier matrix for controlled release of bioactive TGF-beta1 by adjusting the concentrations of FC and thrombin in the gels.