Hydrogel-Guided, rAAV-Mediated IGF-I Overexpression Enables Long-Term Cartilage Repair and Protection against Perifocal Osteoarthritis in a Large-Animal Full-Thickness Chondral Defect Model at One Year In Vivo

Abstract

The regeneration of focal articular cartilage defects is complicated by the reduced quality of the repair tissue and the potential development of perifocal osteoarthritis (OA). Biomaterial-guided gene therapy may enhance cartilage repair by controlling the release of therapeutic sequences in a spatiotemporal manner. Here, the benefits of delivering a recombinant adeno-associated virus (rAAV) vector coding for the human insulin-like growth factor I (IGF-I) via an alginate hydrogel (IGF-I/AlgPH155) to enhance repair of full-thickness chondral defects following microfracture surgery after one year in minipigs versus control (lacZ/AlgPH155) treatment are reported. Sustained IGF-I overexpression is significantly achieved in the repair tissue of defects treated with IGF-I/AlgPH155 versus those receiving lacZ/AlgPH155 for one year and in the cartilage surrounding the defects. Administration of IGF-I/AlgPH155 significantly improves parameters of cartilage repair at one year relative to lacZ/AlgPH155 (semiquantitative total histological score, cell densities, matrix deposition) without deleterious or immune reactions. Remarkably, delivery of IGF-I/AlgPH155 also significantly reduces perifocal OA and inflammation after one year versus lacZ/AlgPH155 treatment. Biomaterial-guided rAAV gene transfer represents a valuable clinical approach to promote cartilage repair and to protect against OA.

Keywords: IGF-I; alginate hydrogel; cartilage repair; perifocal osteoarthritis; rAAV.

Figure 1

Detection of transgene (IGF‐I) expression in MSCs treated with the rAAV/AlgPH155 hydrogel system. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were directly applied to cultures of MSCs (25 µL for 2×10⁴ cells, i.e., 3.6 × 10⁵ transgene copies) for up to 21 days as described in the Experimental Section. Controls included the application of hydrogel without rAAV (AlgPH155), the administration of hydrogel‐free rAAV vectors at similar vector doses (IGF‐I/‐ or lacZ/‐ treatments), and the absence of both hydrogel and rAAV (‐/‐ treatment). Cumulative IGF‐I expression was measured at the denoted time points by ELISA as described in the Experimental Section. Statistically significant relative to lacZ/AlgPH155 (a), AlgPH155 (b), IGF‐I/‐ (c), lacZ/‐ (d), and ‐/‐ (e) treatment.

Figure 2

Detection of transgene (IGF‐I) expression in the repair tissue of the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems (25 µL per defect; 3.6 × 10⁵ transgene copies) were implanted for one year in chondral defects (4 mm diameter, 5 mm depth) created in the lateral, distal femur trochlea of minipigs following standardized microfracture as described in the Experimental Section. The osteochondral units containing the defects were retrieved after one year and processed to monitor IGF‐I expression by immunohistochemistry as described in the Experimental Section (insets: detection of IGF‐I in the cartilage surrounding the defects; magnification ×10; all representative data).

Figure 3

Macroscopic analysis of the repair tissue in the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were implanted in the minipig chondral defects as shown in Figure 2 and in the Experimental Section. The osteochondral units containing the defects were retrieved after one year for macroscopic evaluation as described in the Experimental Section (all representative data).

Figure 4

Histological analyses of the repair tissue in the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were implanted in the minipig chondral defects as shown in Figures 2 and 3 and in the Experimental Section. The osteochondral units containing the defects were retrieved after one year and processed for safranin O/H&E staining as described in the Experimental Section. The (*) indicate the integration sites and the boxes in (A) present the zones depicted in (B–D) (A: magnification ×2; B: magnification ×10; C: magnification ×20; D: magnification ×100; all representative data).

Figure 5

Immunohistochemical analyses of the expression of ECM components in the repair tissue of the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were implanted in the minipig chondral defects as shown in Figures 2, 3, 4 and in the Experimental Section. A,B) The osteochondral units containing the defects were retrieved after one year and processed to evaluate the deposition of type‐II collagen (A) and type‐I collagen (B) by immunohistochemistry as described in the Experimental Section (magnification ×4; all representative data).

Figure 6

Histological analyses of the cartilage adjacent to the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were implanted in the minipig chondral defects as shown in Figures 2, 3, 4, 5 and in the Experimental Section. The osteochondral units containing the defects were retrieved after one year and processed for safranin O staining as described in the Experimental Section. The (*) indicate the integration sites and the boxes in (A) present the zones depicted in (B–D) (A: magnification ×4; B: magnification ×10; C: magnification ×20; D: magnification ×40; all representative data).

Figure 7

Immunohistochemical analyses of the inflammatory responses in the repair tissue and in the surrounding cartilage of the rAAV/AlgPH155‐treated minipig chondral defects. The IGF‐I/AlgPH155 and lacZ/AlgPH155 hydrogel systems were implanted in the minipig chondral defects as shown in Figures 2, 3, 4, 5, 6 and in the Experimental Section. A,B) The osteochondral units containing the defects were retrieved after one year and processed to evaluate the expression of IL‐1β (A) and TNF‐α (B) by immunohistochemistry in the repair tissue of the defects and in the surrounding cartilage (insets) as described in the Experimental Section (magnification ×40; all representative data).