title: Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein.
abstract: Growth-plate chondrocytes undergo proliferation, maturation, hypertrophic differentiation, and calcification; and these processes can be reproduced in vitro in a chondrocyte culture system. Using this system, we have shown that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes all stages of proliferation, maturation, hypertrophic differentiation, and calcification, thus suggesting that CCN2 is a multifunctional growth factor for chondrocytes and plays important roles in chondrocyte proliferation and differentiation. In this chapter, we describe how to evaluate CCN2 functions in these processes occurring in cultured chondrocytes. Evaluation strategies for cell proliferation include measuring DNA synthesis by [3H]-thymidine incorporation, cellular metabolic activity, and cell number with a hemocytometer. Next, evaluation strategies to assess maturation are analysis of the gene expression of markers of mature chondrocytes, and examination of proteoglycan and collagen synthesis by using radioactive compounds. In addition, cytohistochemical detection of glycosaminoglycans (GAGs), such as chondroitin sulfate, by use of alcian blue and toluidine blue staining is useful to evaluate chondrocyte maturation. These methods can be also used for evaluation of physiological functions of CCN2 in permanent chondrocytes such as articular and auricular chondrocytes, which do not calcify under physiological conditions. Next, evaluation of hypertrophic differentiation is performed by detecting type X collagen, which is specific marker of hypertrophic chondrocytes, and by measuring alkaline phosphatase (ALP) activity. Finally, evaluation of calcification is performed by detecting matrix calcification by use of alizarin red staining and by examining the incorporation of45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.
authors.affiliation: Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. tnishida@md.okayama-u.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishida
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.|Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubota
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. takigawa@md.okayama-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D000818:Animals; D001681:Biological Assay; D015415:Biomarkers; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D020219:Chondrogenesis / Q000187:drug effects*; D055513:Connective Tissue Growth Factor / Q000378:metabolism* / Q000494:pharmacology*; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D009243:NAD / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins
chemical_list: D015415:Biomarkers; D011509:Proteoglycans; D011994:Recombinant Proteins; D009243:NAD; D055513:Connective Tissue Growth Factor
keywords: Alizarin red staining; Alkaline phosphatase; Chondrocyte calcification; Chondrocyte hypertrophy; Chondrocyte maturation; Chondrocyte proliferation; Chondrocytes; Collagen; Proteoglycan; Recombinant CCN2 protein (rCCN2)