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 language | English |
|---|---|
| Title of host publication | Methods in Molecular Biology |
| Publisher | Humana Press Inc. |
| Pages | 219-237 |
| Number of pages | 19 |
| Volume | 1489 |
| DOIs | |
| Publication status | Published - 2017 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 1489 |
| ISSN (Print) | 10643745 |
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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
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 proceeding › Chapter
}
TY - CHAP
T1 - Cell biological assays for measuring chondrogenic activities of CCN2 protein
AU - Nishida, Takashi
AU - Kubota, Satoshi
AU - Takigawa, Masaharu
PY - 2017
Y1 - 2017
N2 - 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.
AB - 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.
KW - Alizarin red staining
KW - Alkaline phosphatase
KW - Chondrocyte calcification
KW - Chondrocyte hypertrophy
KW - Chondrocyte maturation
KW - Chondrocyte proliferation
KW - Chondrocytes
KW - Collagen
KW - Proteoglycan
KW - Recombinant CCN2 protein (rCCN2)
UR - http://www.scopus.com/inward/record.url?scp=84991677466&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84991677466&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-6430-7_21
DO - 10.1007/978-1-4939-6430-7_21
M3 - Chapter
C2 - 27734380
AN - SCOPUS:84991677466
VL - 1489
T3 - Methods in Molecular Biology
SP - 219
EP - 237
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -