Cell biological assays for measuring chondrogenic activities of CCN2 protein

Research output: Chapter in Book/Report/Conference proceedingChapter

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

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages219-237
Number of pages19
Volume1489
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1489
ISSN (Print)10643745

Fingerprint

Connective Tissue Growth Factor
Chondrocytes
Biological Assay
CCN Intercellular Signaling Proteins
Collagen Type X
Staining and Labeling
Tolonium Chloride
Alcian Blue
Growth Plate
Chondroitin Sulfates
Proteoglycans
Glycosaminoglycans
Thymidine
Alkaline Phosphatase
Intercellular Signaling Peptides and Proteins
Collagen
Cell Count
Joints
Cell Proliferation

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Nishida, T., Kubota, S., & Takigawa, M. (2017). Cell biological assays for measuring chondrogenic activities of CCN2 protein. In Methods in Molecular Biology (Vol. 1489, pp. 219-237). (Methods in Molecular Biology; Vol. 1489). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6430-7_21

Cell biological assays for measuring chondrogenic activities of CCN2 protein. / Nishida, Takashi; Kubota, Satoshi; Takigawa, Masaharu.

Methods in Molecular Biology. Vol. 1489 Humana Press Inc., 2017. p. 219-237 (Methods in Molecular Biology; Vol. 1489).

Research output: Chapter in Book/Report/Conference proceedingChapter

Nishida, T, Kubota, S & Takigawa, M 2017, Cell biological assays for measuring chondrogenic activities of CCN2 protein. in Methods in Molecular Biology. vol. 1489, Methods in Molecular Biology, vol. 1489, Humana Press Inc., pp. 219-237. https://doi.org/10.1007/978-1-4939-6430-7_21
Nishida T, Kubota S, Takigawa M. Cell biological assays for measuring chondrogenic activities of CCN2 protein. In Methods in Molecular Biology. Vol. 1489. Humana Press Inc. 2017. p. 219-237. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-6430-7_21
Nishida, Takashi ; Kubota, Satoshi ; Takigawa, Masaharu. / Cell biological assays for measuring chondrogenic activities of CCN2 protein. Methods in Molecular Biology. Vol. 1489 Humana Press Inc., 2017. pp. 219-237 (Methods in Molecular Biology).
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