Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1984 Dec 1;99(6):1960–1969. doi: 10.1083/jcb.99.6.1960

Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III. Effects of ascorbate

PMCID: PMC2113574  PMID: 6501411

Abstract

Chondrocytes isolated from bovine articular cartilage were plated at high density and grown in the presence or absence of ascorbate. Collagen and proteoglycans, the major matrix macromolecules synthesized by these cells, were isolated at times during the course of the culture period and characterized. In both control and ascorbate-treated cultures, type II collagen and cartilage proteoglycans accumulated in the cell-associated matrix. Control cells secreted proteoglycans and type II collagen into the medium, whereas with time in culture, ascorbate-treated cells secreted an increasing proportion of types I and III collagens into the medium. The ascorbate-treated cells did not incorporate type I collagen into the cell-associated matrix, but continued to accumulate type II collagen in this compartment. Upon removal of ascorbate, the cells ceased to synthesize type I collagen. Morphological examination of ascorbate-treated and control chondrocyte culture revealed that both collagen and proteoglycans were deposited into the extracellular matrix. The ascorbate-treated cells accumulated a more extensive matrix that was rich in collagen fibrils and ruthenium red-positive proteoglycans. This study demonstrated that although ascorbate facilitates the formation of an extracellular matrix in chondrocyte cultures, it can also cause a reversible alteration in the phenotypic expression of those cells in vitro.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BITTER T., MUIR H. M. A modified uronic acid carbazole reaction. Anal Biochem. 1962 Oct;4:330–334. doi: 10.1016/0003-2697(62)90095-7. [DOI] [PubMed] [Google Scholar]
  2. Benya P. D., Nimni M. E. The stability of the collagen phenotype during stimulated collagen, glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures. Arch Biochem Biophys. 1979 Feb;192(2):327–335. doi: 10.1016/0003-9861(79)90100-0. [DOI] [PubMed] [Google Scholar]
  3. Benya P. D., Padilla S. R., Nimni M. E. The progeny of rabbit articular chondrocytes synthesize collagen types I and III and type I trimer, but not type II. Verifications by cyanogen bromide peptide analysis. Biochemistry. 1977 Mar 8;16(5):865–872. doi: 10.1021/bi00624a009. [DOI] [PubMed] [Google Scholar]
  4. Benya P. D., Shaffer J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. 1982 Aug;30(1):215–224. doi: 10.1016/0092-8674(82)90027-7. [DOI] [PubMed] [Google Scholar]
  5. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  6. Capasso O., Gionti E., Pontarelli G., Ambesi-Impiombato F. S., Nitsch L., Tajana G., Cancedda R. The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes. Exp Cell Res. 1982 Nov;142(1):197–206. doi: 10.1016/0014-4827(82)90423-2. [DOI] [PubMed] [Google Scholar]
  7. Daniel J. C., Kosher R. A., Hamos J. E., Lash J. W. Influence of external potassium on the synthesis and deposition of matrix components by chondrocytes in vitro. J Cell Biol. 1974 Dec;63(3):843–854. doi: 10.1083/jcb.63.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dorfman A., Vertel B. M., Schwartz N. B. Immunological methods in the study of chondroitin sulfate proteoglycans. Curr Top Dev Biol. 1980;14(Pt 2):169–198. doi: 10.1016/s0070-2153(08)60194-5. [DOI] [PubMed] [Google Scholar]
  9. Dunn D. M., Franzblau C. Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures. Biochemistry. 1982 Aug 31;21(18):4195–4202. doi: 10.1021/bi00261a001. [DOI] [PubMed] [Google Scholar]
  10. Fessler J. H., Fessler L. I. Biosynthesis of procollagen. Annu Rev Biochem. 1978;47:129–162. doi: 10.1146/annurev.bi.47.070178.001021. [DOI] [PubMed] [Google Scholar]
  11. Kimura J. H., Caputo C. B., Hascall V. C. The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma. J Biol Chem. 1981 May 10;256(9):4368–4376. [PubMed] [Google Scholar]
  12. Kuettner K. E., Memoli V. A., Pauli B. U., Wrobel N. C., Thonar E. J., Daniel J. C. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. II. Maintenance of collagen and proteoglycan phenotype. J Cell Biol. 1982 Jun;93(3):751–757. doi: 10.1083/jcb.93.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kuettner K. E., Pauli B. U., Gall G., Memoli V. A., Schenk R. K. Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology. J Cell Biol. 1982 Jun;93(3):743–750. doi: 10.1083/jcb.93.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Layman D. L., Sokoloff L., Miller E. J. Collagen synthesis by articular in monolayer culture. Exp Cell Res. 1972 Jul;73(1):107–112. doi: 10.1016/0014-4827(72)90107-3. [DOI] [PubMed] [Google Scholar]
  16. Lichtenstein J. R., Byers P. H., Smith B. D., Martin G. R. Identification of the collagenous proteins synthesized by cultured cells from human skin. Biochemistry. 1975 Apr 22;14(8):1589–1594. doi: 10.1021/bi00679a007. [DOI] [PubMed] [Google Scholar]
  17. Mayne R., Vail M. S., Mayne P. M., Miller E. J. Changes in type of collagen synthesized as clones of chick chondrocytes grow and eventually lose division capacity. Proc Natl Acad Sci U S A. 1976 May;73(5):1674–1678. doi: 10.1073/pnas.73.5.1674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mayne R., Vail M. S., Miller E. J. Analysis of changes in collagen biosynthesis that occur when chick chondrocytes are grown in 5-bromo-2'-deoxyuridine. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4511–4515. doi: 10.1073/pnas.72.11.4511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meier S., Solursh M. Ultrastructural analysis of the effect of ascorbic acid on secretion and assembly of extracellular matrix by cultured chick embryo chondrocytes. J Ultrastruct Res. 1978 Oct;65(1):48–59. doi: 10.1016/s0022-5320(78)90021-7. [DOI] [PubMed] [Google Scholar]
  20. Miller E. J., Rhodes R. K. Preparation and characterization of the different types of collagen. Methods Enzymol. 1982;82(Pt A):33–64. doi: 10.1016/0076-6879(82)82059-4. [DOI] [PubMed] [Google Scholar]
  21. Murad S., Grove D., Lindberg K. A., Reynolds G., Sivarajah A., Pinnell S. R. Regulation of collagen synthesis by ascorbic acid. Proc Natl Acad Sci U S A. 1981 May;78(5):2879–2882. doi: 10.1073/pnas.78.5.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peterkofsky B. Regulation of collagen secretion by ascorbic acid in 3T3 and chick embryo fibroblasts. Biochem Biophys Res Commun. 1972 Dec 4;49(5):1343–1350. doi: 10.1016/0006-291x(72)90614-6. [DOI] [PubMed] [Google Scholar]
  23. Sajdera S. W., Hascall V. C. Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J Biol Chem. 1969 Jan 10;244(1):77–87. [PubMed] [Google Scholar]
  24. Schiltz J. R., Ward S. Effects of chick embryo extract fractions on collagen and glycosaminoglycan metabolism by chick chondroblasts. Biochim Biophys Acta. 1980 Mar 20;628(3):343–354. doi: 10.1016/0304-4165(80)90384-0. [DOI] [PubMed] [Google Scholar]
  25. Schmid T. M., Conrad H. E. A unique low molecular weight collagen secreted by cultured chick embryo chondrocytes. J Biol Chem. 1982 Oct 25;257(20):12444–12450. [PubMed] [Google Scholar]
  26. Schwartz E., Bienkowski R. S., Coltoff-Schiller B., Goldfischer S., Blumenfeld O. O. Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding. J Cell Biol. 1982 Feb;92(2):462–470. doi: 10.1083/jcb.92.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Tajima S., Pinnell S. R. Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA. Biochem Biophys Res Commun. 1982 May 31;106(2):632–637. doi: 10.1016/0006-291x(82)91157-3. [DOI] [PubMed] [Google Scholar]
  28. von der Mark H., von der Mark H. Isolation and characterization of collagen A and B chains from chick embryos. FEBS Lett. 1979 Mar 1;99(1):101–105. doi: 10.1016/0014-5793(79)80258-6. [DOI] [PubMed] [Google Scholar]
  29. von der Mark K., Gauss V., von der Mark H., Müller P. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature. 1977 Jun 9;267(5611):531–532. doi: 10.1038/267531a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES