Effects of single rAAV-mediated TGF-beta overexpression over time in human osteoarthritic articular cartilage

Abstract

Purpose: rAAV vectors are promising tools to directly apply candidate genes in osteoarthritic (OA) cartilage. Here, we analyzed the effects of TGF-b overexpression via rAAV upon the structure of human OA cartilage in situ using one single application over a prolonged period of time. Methods: The vectors were packaged, purified, and titrated using standard protocols. Human normal articular cartilage was obtained from unaffected areas in knee joints removed during tumor surgery (n 1⁄4 10) and human OA cartilage from joints undergoing total knee arthroplasty (n 1⁄4 12) (Mankin score 7-9). Explants and chondrocytes were prepared using standard protocols. Cells and explants were transduced with 40 ml vectors for up to 30 days. The DNA contents were assayed using Hoechst 33258, the proteoglycan contents by binding to DMMB, and the type-II and type-X collagen contents by ELISA. Paraffinembedded sections were stained with safranin O/H&E and to detect TGF-b, type-II and type-X collagen. TGF-b was also monitored by ELISA. Proliferation was evaluated by immunolabeling following BrdU incorporation. Morphometric measurements were performed at 3 standardized sites along the explant surface by image analysis. Each condition was performed in duplicate in 3 independent experiments. The t-test and Mann-Whitney Rank Sum Test were employed with P 0.05 considered statistically significant. Results: TGF-b production was sustained in rAAV-hTGF-b chondrocytes compared with rAAV-lacZ (184.2 vs.11.3 pg/ml/24 h in normal cells, 219.4 vs. 10.6 pg/ml/24 h in OA cells, up to 21-fold difference, always P 0.001). The % of cells positive for BrdU uptake (Fig. 1) and the contents of DNA, proteoglycans, and type-II collagen were always higher in the rAAV-hTGF-b normal and OA cells compared with rAAVlacZ (up to 12-fold, always P 0.001). The type-X collagen contents significantly decreased in OA cells with rAAV-hTGF-b (1.6-fold, P 0.001). TGF-b production was also sustained in rAAV-hTGF-b explants compared with rAAV-lacZ (724.5 vs. 92.3 pg/ml/24 h in normal cartilage, 987.7 vs. 83.4 pg/ml/24 h in OA cartilage, up to 12-fold difference, always P 0.001) (Fig. 2). The cell densities (Fig. 3), % of cells positive for BrdU uptake (Fig. 4), DNA contents, proteoglycan contents and matrix staining intensity (Fig. 3), and type-II contents and immunoreactivity (insets of Fig. 3) were always higher in the rAAV-hTGFb normal and OA explants compared with rAAV-lacZ (up to 23-fold, always P 0.001). The type-X collagen contents and immunoreactivity significantly decreased in OA cartilage with rAAV-hTGF-b (4fold, P 0.001) (Fig. 5). Conclusions: TGF-b can be overexpressed in human normal and OA chondrocytes via rAAV in vitro and in situ, leading to enhanced cell proliferation and matrix synthesis and to OA cartilage remodelling with reduced hypertrophic type-X collagen expression. Administration of the vector in vivo, probably combined with other favorable candidates, will allow to determine the beneficial effects of the approach on reconstructing OA cartilage to avoid osteophyte formation. Fig. 1. Immunocytochemical detection of BrdU in transduced human normal and OA chondrocytes (day 30). Magnification x10.