CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes T. Fujisawa, Ph.D., D.D.S.,

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CCN family 2/connective tissue growth factor (CCN2/CTGF) stimulates proliferation and differentiation of auricular chondrocytes T. Fujisawa, Ph.D., D.D.S., T. Hattori, Ph.D., M. Ono, Ph.D., D.D.S., J. Uehara, Ph.D., D.D.S., S. Kubota, Ph.D., D.D.S., T. Kuboki, Ph.D., D.D.S., M. Takigawa, Ph.D., D.D.S. Osteoarthritis and Cartilage Volume 16, Issue 7, Pages 787-795 (July 2008) DOI: 10.1016/j.joca.2007.11.001 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 rhCCN2/CTGF enhances the proliferation of rabbit auricular chondrocytes in a dose-dependent manner. (A) Auricular chondrocytes were grown in αMEM containing 10% FBS until sub confluence, and the medium was subsequently changed to αMEM containing 0.5% FBS. After 24h of incubation, the cells were treated with rhCCN2/CTGF at the indicated concentrations for 24h. PBS was added to control cultures. The cells were labeled with [3H] thymidine during the last 4h. (B) MTS assay for cell proliferation showing an enhanced number of rabbit auricular chondrocytes after 5 and 7 days of treatment with rhCCN2/CTGF. Auricular chondrocytes were grown in αMEM containing 10% FBS for 24h, and the medium was changed to αMEM containing 0.5% FBS with (black columns) or without (open columns) rhCCN2/CTGF (50ng/ml). Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 rhCCN2/CTGF enhances proteoglycan synthesis of rabbit auricular chondrocytes in a dose-dependent manner. Rabbit auricular chondrocytes were cultured until confluence in αMEM containing 10% FBS, changed to a medium with serum-free αMEM, and incubated for another 24h. rhCCN2/CTGF was added to the media at the indicated concentrations, and the cells were incubated for 22h and labeled with [35S] sulfate for the final 17h. [35S] sulfate was incorporated into CPC-precipitable materials and was measured after digestion with actinase E. Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 rhCCN2/CTGF enhances the expression of ccn2/ctgf, elastin, and col2a1, but does not enhance the expression of col10a1. (A–D) Rabbit auricular chondrocytes were treated with or without rhCCN2/CTGF (50ng/ml) for 48h in αMEM containing 0.5% FBS. The total RNA was isolated, and ccn2/ctgf (A), elastin (B), col2a1 (C), and col10a1 mRNA (D) were measured by real-time RT-PCR, and each value was standardized to 18S rRNA. (E) Elastic fibers were stained with Victoria Blue after 14 days of incubation with rhCCN2/CTGF (50ng/ml). (F) The cells were stained by Alizarin Red after 28 days of incubation with rhCCN2/CTGF (50ng/ml). Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 CCN2/CTGF stimulates MGP mRNA expression in auricular chondrocytes. (A) Auricular chondrocytes were infected with AdCCN2 or AdLacZ (10moi) for 96h, and total RNA was collected. RNAs were subjected to Northern blotting and hybridized with a probe for MGP. (B) Auricular chondrocytes were infected with AdCCN2 (10moi) or treated with rhCCN2/CTGF (50ng/ml). After 48h of incubation, the RNAs were collected and subjected to real-time PCR using MGP-specific primers. Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 rhCCN2/CTGF enhances the subcutaneous growth of auricular chondrocyte pellets and supports the maintenance of the phenotype. Rabbit auricular chondrocytes (2×106cells) were centrifuged, cultured as pellets in 15ml tubes in αMEM containing 10% FBS for 3 weeks, and immersed in PBS with or without rhCCN2/CTGF (10μg/ml) for 1h. The pellets were implanted in subcutaneous pockets in nude mice (4pellets/animal) and analyzed 4 weeks after implantation. This figure depicts sections of two representative ectopic cartilage specimens out of six samples (three controls and three rhCCN2/CTGF-treated samples) analyzed. (A) PBS-treated (left) and rhCCN2/CTGF-treated (right) harvested pellets from the implant cultures after 3 weeks in mice. Bar: 1mm. Histological evaluation by safranin O of the (B) PBS and (C) rhCCN2/CTGF-treated implant cultures. (D) Dry weight of the implant cultures (two controls and two rhCCN2/CTGF-treated samples). Alcian blue staining of PBS-treated (E) and rhCCN2/CTGF-treated (F) harvested implanted pellets. (G, H): High magnification of the inserts shown in (E) and (F), respectively. (I, J): Type II collagen staining of PBS-treated (I) and rhCCN2/CTGF-treated (J) harvested implant pellets. (K, L): Type X collagen staining of PBS-treated (K) and rhCCN2/CTGF-treated (L) harvested implant pellets. (M, N): Victoria blue staining of PBS-treated (M) and rhCCN2/CTGF-treated (N) harvested implant pellets. Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 rhCCN2/CTGF enhances the subcutaneous growth of auricular chondrocyte pellets and supports the maintenance of the phenotype. Rabbit auricular chondrocytes (2×106cells) were centrifuged, cultured as pellets in 15ml tubes in αMEM containing 10% FBS for 3 weeks, and immersed in PBS with or without rhCCN2/CTGF (10μg/ml) for 1h. The pellets were implanted in subcutaneous pockets in nude mice (4pellets/animal) and analyzed 4 weeks after implantation. This figure depicts sections of two representative ectopic cartilage specimens out of six samples (three controls and three rhCCN2/CTGF-treated samples) analyzed. (A) PBS-treated (left) and rhCCN2/CTGF-treated (right) harvested pellets from the implant cultures after 3 weeks in mice. Bar: 1mm. Histological evaluation by safranin O of the (B) PBS and (C) rhCCN2/CTGF-treated implant cultures. (D) Dry weight of the implant cultures (two controls and two rhCCN2/CTGF-treated samples). Alcian blue staining of PBS-treated (E) and rhCCN2/CTGF-treated (F) harvested implanted pellets. (G, H): High magnification of the inserts shown in (E) and (F), respectively. (I, J): Type II collagen staining of PBS-treated (I) and rhCCN2/CTGF-treated (J) harvested implant pellets. (K, L): Type X collagen staining of PBS-treated (K) and rhCCN2/CTGF-treated (L) harvested implant pellets. (M, N): Victoria blue staining of PBS-treated (M) and rhCCN2/CTGF-treated (N) harvested implant pellets. Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 rhCCN2/CTGF inhibits apoptotic cell death in auricular chondrocyte implants. Chondrocyte implants without (A) or with (B) treatment of rhCCN2/CTGF were prepared as in Fig. 5. The sections were analyzed by TUNEL assay, and positive cells were counted in 10 different fields of 400×300μm2 (C). Osteoarthritis and Cartilage 2008 16, 787-795DOI: (10.1016/j.joca.2007.11.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions