Direct FGF-2 Gene Transfer via Recombinant Adeno-Associated Virus Vectors Stimulates Cell Proliferation, Collagen Production, and the Repair of Experimental Lesions in the Human ACL

Abstract

Background: Basic fibroblast growth factor (FGF-2) is a powerful stimulator of fibroblast proliferation and type I/III collagen production. Hypothesis: Overexpression of FGF-2 via direct recombinant adeno-associated virus (rAAV) vector–mediated gene transfer enhances the healing of experimental lesions to the human anterior cruciate ligament (ACL). Study Design: Controlled laboratory study. Methods: rAAV vectors carrying a human FGF-2 sequence or the lacZ marker gene were applied to primary human ACL fibroblasts in vitro and to intact or experimentally injured human ACL explants in situ to evaluate the efficacy and duration of transgene expression and the potential effects of FGF-2 treatment upon the proliferative, metabolic, and regenerative activities in these systems. Results: Sustained, effective dose-dependent lacZ expression was achieved in all systems tested (up to 96% ± 2% in vitro and 80%-85% in situ for at least 30 days). rAAV allowed for continuous FGF-2 production both in vitro and in the intact ACL in situ (32.7 ± 1.4 and 33.1 ± 0.8 pg/mL/24 h, respectively, ie, up to 41-fold more than in the controls at day 30; always P ≤ .001), leading to significantly and durably enhanced levels of proliferation and type I/III collagen production vis-à-vis lacZ (at least 3- and 4-fold increases at day 30, respectively; always P ≤ .001). Most notably, rAAV FGF-2 promoted a significant, long-term production of the factor in experimental ACL lesions (92.7 ± 3.9 pg/mL/24 h, ie, about 5-fold more than in the controls; P ≤ .001) associated with enhanced levels of proliferation and type I/III collagen synthesis (at least 2- and 4-fold increases at day 30, respectively; always P ≤ .001). Remarkably, the FGF-2 treatment allowed for a decrease in the amplitude of such lesions possibly because of the increased expression in contractile α–smooth muscle actin, ligament-specific transcription factor scleraxis, and nuclear factor–κB for proliferation and collagen deposition, which are all markers commonly induced in response to injury. Conclusion: Efficient, stable FGF-2 expression via rAAV enhances the healing of experimental human ACL lesions by activating key cellular and metabolic processes. Clinical Relevance: This approach has potential value for the development of novel, effective treatments for ligament reconstruction.