Retroviral transduction with SOX9 enhances re-expression of the chondrocyte phenotype in passaged osteoarthritic human articular chondrocytes  Simon R. Tew, Ph.D., Ying Li, Ph.D., Peraphan Pothacharoen, M.Sc., Lisa M. Tweats, Robert E. Hawkins, Ph.D., Timothy E. Hardingham, Ph.D.  Osteoarthritis and Cartilage  Volume 13, Issue 1, Pages 80-89 (January 2005) DOI: 10.1016/j.joca.2004.10.011 Copyright © 2004 Terms and Conditions

Fig. 1 (A) Real time PCR analysis of cDNA derived from monolayer and pellet cultures of untransduced human OA chondrocytes. The cells were examined between passages 7 and 10 as described in the text and pellet cultures were conducted for 14 days. Black bars represent expression levels for comparable monolayer cultures (n=11), grey bars represent pellet cultures grown in serum free medium (n=4) and white bars represent pellet cultures grown in medium containing 10% FCS (n=3). *=P<0.05 in pellet vs monolayer, Student's unpaired t test. (B) Real time PCR analysis of cDNA derived from monolayer and pellet cultures of human OA chondrocytes transduced with SOX9 retrovirus. The black bars represent expression levels for comparable monolayer cultures (n=9), the grey bars represent pellet cultures grown in serum free medium (n=4) and white bars represent pellet cultures grown in medium containing 10% FCS (n=3). *=P<0.01 in pellet vs monolayer, Student's unpaired t test. (C) Wet weight of GFP and SOX9 transduced human OA chondrocytes grown for 14 days as pellets in the presence or absence of 10% FCS. *=P<0.05. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions

Fig. 2 Matrix accumulation in pellet cultures of late passage (passage 10) human OA chondrocytes transduced with SOX9 or GFP and early passage (passage 4) untransduced OA chondrocytes. Cultures were grown for 14 days in the presence or absence of growth factors. (A) Pellet wet weight, (B) GAG content of pellet normalised to DNA content (weight per weight). Pellets were grown without growth factors (black bars) or in the presence of 100ng/ml IGF-1 (dark grey bars), 10ng/ml TGFβ-3 (light grey bars) or both IGF-1 and TGFβ-3 together (white bars). *=P<0.05 in growth factor treatment vs no growth factors, †=P<0.05 in SOX9 transduced vs GFP transduced grown under the same conditions. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions

Fig. 3 (A) Histological analysis of SOX9 and GFP transduced late passage human OA chondrocytes grown as pellets for 14 days. Paraffin wax sections from pellets treated with or without growth factors were stained with safranin-O. GFP transduced (i–iv) and SOX9 transduced (v–viii) cell pellets were treated with no growth factor (i and v), 100ng/ml IGF-1 (ii and vi), 10ng/ml TGFβ-3 (iii and vii) or both IGF-1 and TGFβ-3 together (iv and viii). Scale bar=100μm. (B) Real time PCR analysis of cDNA derived from growth factor stimulated pellets of late passage OA HAC grown for 14 days. SOX9 transduced and GFP transduced cells were examined for mRNA levels of COL1A1, COL2A1 and SOX9. Pellets were grown without growth factors (black bars), in the presence of 100ng/ml IGF-1 (dark grey bars), 10ng/ml TGFβ-3 (light grey bars) or both IGF-1 and TGFβ-3 together (white bars). (n=3 for all pellets). *=P<0.05 in growth factor treatment vs no growth factors. †=P<0.05 in SOX9 transduced vs GFP transduced grown under the same conditions. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions

Fig. 4 Real time PCR analysis of cDNA derived from growth factor stimulated pellets of late passage human OA chondrocytes grown for 14 days. SOX9 and GFP transduced cells were examined for mRNA levels of L-SOX5, SOX6, aggrecan, decorin, biglycan, and fibromodulin as indicated. GFS indicates treatment with 100ng/ml IGF-1 and 10ng/ml TGFβ-3 together. *=P<0.05 in growth factor treatment vs no growth factors. n=3 in all instances except aggrecan and SOX6 analysis of GFP no-growth factor pellets (n=7) and SOX9 no-growth factor pellets (n=5) **=P<0.05 in SOX9 transduced vs GFP transduced grown under the same conditions. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions

Fig. 5 Immunohistochemical analysis of 14-day OA chondrocyte pellet cultures grown in the presence of 100ng/ml IGF-1 and 10ng/ml TGFβ-3. Chondrocytes were transduced with GFP (A, C) or SOX9 (B, D). Sections were labelled with antibodies to collagen II (A and B) or collagen I (C and D). GFP transduced sections labelled using no primary antibody (E) or control IgG (F) are shown as examples of controls. Scale bars=100μm. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions

Fig. 6 Normal HAC or human dermal fibroblasts transduced with SOX9 and cultured as pellets for 14 days in media supplemented with 100ng/ml IGF-1 and 10ng/ml TGFβ-3. (A) Real time PCR analysis of pellets formed from normal chondrocytes, SOX9 transduced (white bars) and GFP transduced cells (black bars) were examined for mRNA levels for COL1A1, COL2A1 and SOX9. The scale is the same as that in Fig. 3 and the data are comparable to the OA chondrocyte expression levels shown in Fig. 3(B). (B) Immunohistochemical analysis of collagen type II and type I in 14-day normal chondrocyte pellet cultures, either GFP transduced or SOX9 transduced as indicated. Pellets were grown in the presence of 100ng/ml IGF-1 and 10ng/ml TGFβ-3. Scale bar=100μm. (C) Real time PCR analysis of 14-day NHDF pellet cultures transduced with GFP (black bars) or SOX9 (white bars) and grown in the presence of 100ng/ml IGF-1 and 10ng/ml TGFβ-3. (D) Safranin-O staining of sections from SOX9 or GFP transduced NHDF cell pellets grown for 14 days in the presence of 100ng/ml IGF-1 and 10ng/ml TGFβ-3. Scale bar=100μm. Osteoarthritis and Cartilage 2005 13, 80-89DOI: (10.1016/j.joca.2004.10.011) Copyright © 2004 Terms and Conditions