gms | German Medical Science

Objectives: Anterior cruciate ligament (ACL) ruptures are highly prevalent knee injuries that may lead to osteoarthritis. Gene therapy using clinically adapted recombinant adeno-associated virus (rAAV) vectors has strong value to treat ACL lesions. Here, we tested the potential of poly(sodium styrene sulfonate) (pNaSS)-grafted poly(ε-caprolactone) (PCL) film-guided gene therapy to apply a biologically active rAAV TGF-β gene vector to human ACL explant cultures ex vivo.

Methods: Human ACL (hACL) samples were obtained from donors undergoing total knee arthroplasty (n= 3). rAAV vectors were prepared as previously described, including the reporter rAAV-lacZ control vector and the rAAV-hTGF-β candidate vector, with both transgenes controlled by the CMV-IE promoter/enhancer. PCL films (Figure 1A [Fig. 1]) were fabricated by spin-coating, grafted by ozonation with 0.7 M pNaSS, and incubated with 0.002% poly-L-lysine to immobilize rAAV (40μ l). pNaSS-grafted (or ungrafted) PCL films coated with rAAV (or uncoated) were inserted in lesions made with a scalpel in hACL explants that were maintained in DMEM, 10% bovine calf serum, 1% penicillin-streptomycin at 37°C under 5% CO2 for up to 21 days. Reporter lacZ expression was monitored by X-Gal staining and the expression of TGF-β and type-I and -III collagen (COL1A1, COL3A1) was assessed by ELISA (Abbexa, BioCat, R&D Systems). An ordinary one-way ANOVA was used for statistical analyses, with P ≤ 0.05 considered statistically significant.

Results: Treatment of hACL lesions with rAAV-lacZ-coated pNaSS-grafted PCL films (LacZ/G) led to the durable lacZ expression in the lesions after 21 days, with a stronger signal than when using ungrafted films (LacZ/-) of films without rAAV (-/-, -/G) (Figure 1A [Fig. 1]). Delivery of rAAV-hTGF-β via pNaSS-grafted PCL films (TGF-β/G) in the lesions led to a significant, durable TGF-β overexpression in the lesions after 21 days relative to all other conditions (TGF-β/-, -/-, -/G) (P ≤ 0.03) (Figure 1B [Fig. 1]). While rAAV-hTGF-β had no effect on type-III collagen expression regardless of the films employed and relative to controls without rAAV (P ≤ 0.05) (Figure 1C [Fig. 1]), delivery of this vector via pNaSS-grafted PCL films (TGF-β/G) significantly enhanced type-I collagen expression on days 14 and 21 relative to all other conditions (P ≤ 0.001).

Conclusion: Delivery of the candidate rAAV-hTGF-β via pNaSS-grafted PCL films has value to stimulate the production of specific ACL matrix component for future improved approaches at ACL repair