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CCN Proteins pp 219-237 | Cite as

Cell Biological Assays for Measuring Chondrogenic Activities of CCN2 Protein

  • Takashi NishidaEmail author
  • Satoshi Kubota
  • Masaharu TakigawaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1489)

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 of 45Ca into cartilaginous matrix. These methods would be useful for the evaluation not only of CCN2 but also of its derivatives and other CCN proteins.

Key words

Recombinant CCN2 protein (rCCN2) Chondrocytes Chondrocyte proliferation Chondrocyte maturation Proteoglycan Collagen Chondrocyte hypertrophy Alkaline phosphatase Chondrocyte calcification Alizarin red staining 

Notes

Acknowledgments

This work was supported in part by grants from the programs Grants-in-Aid for Scientific Research (C) to T.N. (#JP26462810) and to S.K. (#JP25462886) and Scientific Research (B) to M.T. (#JP15H05014) from the Japan Society for the Promotion of Sciences, Japan.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular DentistryOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  2. 2.Advanced Research Center for Oral and Craniofacial SciencesOkayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  3. 3.Department of Membrane BiochemistryOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan
  4. 4.Advanced Research Center for Oral and Craniofacial SciencesOkayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan

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