rAAV TGF-β and FGF-2 Overexpression via pNaSS-Grafted PCL Films Stimulates the Reparative Activities of Human ACL Fibroblasts

Abstract

Lesions in the human anterior cruciate ligament (ACL) are frequent, unsolved clinical issues due to the limited self-healing ability of the ACL and lack of treatments supporting full, durable ACL repair. Gene therapy guided through the use of biomaterials may steadily activate the processes of repair in sites of ACL injury. The goal of the present study was to test the hypothesis that functionalized poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can effectively deliver recombinant adeno-associated virus (rAAV) vectors as a means of overexpressing two reparative factors (transforming growth factor beta-TGF-β and basic fibroblast growth factor-FGF-2) in primary human ACL fibroblasts. Effective, durable rAAV reporter red fluorescent protein and candidate TGF-β and FGF-2 gene overexpression was achieved in the cells for at least 21 days, especially when pNaSS-grafted PCL films were used versus control conditions, such as ungrafted films and systems lacking vectors or films (between 1.8- and 5.2-fold differences), showing interactive regulation of growth factor production. The expression of TGF-β and FGF-2 from rAAV via PCL films safely enhanced extracellular matrix depositions of type-I/-III collagen, proteoglycans/decorin, and tenascin-C (between 1.4- and 4.5-fold differences) in the cells over time with increased levels of expression of the specific transcription factors Mohawk and scleraxis (between 1.7- and 3.7-fold differences) and without the activation of the inflammatory mediators IL-1β and TNF-α, most particularly with pNaSS-grafted PCL films relative to the controls. This work shows the value of combining rAAV gene therapy with functionalized PCL films to enhance ACL repair.