Volume 12, Issue 2, Pages 229-238 (August 2005) Improved Tissue Repair in Articular Cartilage Defects in Vivo by rAAV-Mediated Overexpression of Human Fibroblast Growth Factor 2  Magali Cucchiarini, Henning Madry, Chunyan Ma, Tanja Thurn, David Zurakowski, Michael D. Menger, Dieter Kohn, Stephen B. Trippel, Ernest F. Terwilliger  Molecular Therapy  Volume 12, Issue 2, Pages 229-238 (August 2005) DOI: 10.1016/j.ymthe.2005.03.012 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Analysis of FGF-2 expression in vitro. (A) Western blotting of lysates from rabbit articular chondrocytes and rabbit bone marrow clots transduced by rAAV. Lane 1, extracts from rAAV-lacZ-transduced chondrocytes (10 μg); lane 2, extracts from rAAV-hFGF-2-transduced chondrocytes (10 μg); lane 3, extracts from rAAV-lacZ-transduced bone marrow clots (60 μg); lane 4, extracts from rAAV-hFGF-2-transduced bone marrow clots (60 μg). (B) Time course analysis of FGF-2 production in transduced alginate–chondrocyte constructs. Cells were transduced by rAAV-lacZ or rAAV-hFGF-2 and encapsulated in alginate 2 days after vector application. rAAV-lacZ- and rAAV-hFGF-2-transduced constructs were prepared and maintained in culture for 26 days. Conditioned medium was collected at the denoted time points after encapsulation (n = 9 per time point and condition) and FGF-2 production was measured by ELISA (±SD) with a detection limit of 3 pg/ml. Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Histological sections of transduced alginate–chondrocyte constructs. rAAV-lacZ- (A and B) and rAAV-hFGF-2-transduced constructs (C and D) were histologically processed at day 26 after encapsulation (n = 6 per condition) and analyzed for HE staining (A and C) and for immunohistological detection of type-II collagen with a mouse anti-type-II antibody (1:100), using a biotinylated goat anti-mouse antibody (1:200). Revelation was performed by the ABC method using DAB as the chromogen. Samples were examined under light microscopy. Original magnification, ×20. Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Representative analysis of lacZ expression in osteochondral cartilage defects in vivo. Transgene expression was detected by X-Gal staining in knees retrieved 10 days (A–D; C and D, original magnification, ×100) or 20 days (E and F, original magnification, ×100) after vector application and by immunohistochemistry using a mouse anti-β-gal antibody (1:50) in knees retrieved 4 months postadministration (G and H, original magnification, ×20), as described in the legend to Fig. 2 and under Materials and Methods. (A, C, E, and G) Application of rAAV-lacZ (10 μl); (B, D, F, and H) application of rAAV-hFGF-2 (10 μl). Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Representative analysis of FGF-2 expression in osteochondral cartilage defects in vivo. Transgene expression was detected by immunohistochemistry in sections from knees retrieved 10 days (A and B), 20 days (C and D), and 4 months (E and F) after vector application using a mouse anti-FGF-2 antibody (1:100), as described in the legend to Fig. 2 and under Materials and Methods. (A, C, and E) Application of rAAV-lacZ (10 μl); (B, D, and F) application of rAAV-hFGF-2 (10 μl). Original magnification, ×20. Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Analysis of type-II and type-I collagen expression in osteochondral cartilage defects in vivo. Immunostaining was performed in sections from knees retrieved 20 days (A, C, E, and G) and 4 months (B, D, F, and H) after vector application using a mouse anti-type-II collagen antibody (1:50) (A–D) and a mouse anti-type-I collagen antibody (1:100) (E–H), as described in the legend to Fig. 2 and under Materials and Methods. (A, B, E, and F) Application of rAAV-lacZ (10 μl); (C, D, G, and H) application of rAAV-hFGF-2 (10 μl). Original magnification, ×4. Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Histological sections of osteochondral cartilage defects. Safranin O staining was performed on sections from knees retrieved 20 days (A and D, original magnification, ×2) and 4 months after vector application (B and E, original magnification, ×2; C and F, original magnification ×4). (A–C) Application of rAAV-lacZ (10 μl); (D–F) application of rAAV-hFGF-2 (10 μl). Molecular Therapy 2005 12, 229-238DOI: (10.1016/j.ymthe.2005.03.012) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions