Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1982 Feb 1;92(2):249–260. doi: 10.1083/jcb.92.2.249

Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate

PMCID: PMC2112085  PMID: 7037793

Abstract

The hypothesis is widely held that, in growth plate during endochondral ossification, proteoglycans in the extracellular matrix of the lower hypertrophic zone are degraded by proteases and removed before mineralization, and that this is the mechanism by which a noncalcifiable matrix is transformed into a calcifiable matrix. We have evaluated this hypothesis by examining the immunofluorescent localization and concentrations of proteoglycan monomer core protein and link protein, and the concentrations of glycosaminoglycans demonstrated by safranin 0 staining, in the different zones of the bovine fetal cartilage growth plate. Monospecific antibodies were prepared to proteoglycan monomer core protein and to link protein. The immunofluorescent localization of these species was examined in decalcified and undecalcified sections containing the zones of proliferating and hypertrophic chondrocytes and in sections containing the zones of proliferating and hypertrophic chondrocytes and the metaphysis, decalcified in 0.5 M EDTA, pH 7.5, in the presence of protease inhibitors. Proteoglycan monomer core protein and link protein are demonstrable without detectable loss throughout the extracellular matrix of the longitudinal septa of the hypertrophic zone and in the calcified cartilage of the metaphysis. In fact, increased staining is observed in the calcifying cartilage. Contrary to the prevailing hypothesis, our results indicate that there is no net loss of proteoglycans during mineralization and that the proteoglycans become entombed in the calcified cartilage which provides a scaffolding on which osteoid and bone are formed. Proteoglycans appear to persist unaltered in the calcified cartilage core of the trabeculae, until at last the entire trabeculae are eroded from their surfaces and removed by osteoclasts, when the primary spongiosa is replaced by the secondary spongiosa.

Full Text

The Full Text of this article is available as a PDF (3.1 MB).

Selected References

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

  1. Baker J. R., Caterson B. The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage. J Biol Chem. 1979 Apr 10;254(7):2387–2393. [PubMed] [Google Scholar]
  2. Blumenthal N. C., Posner A. S., Silverman L. D., Rosenberg L. C. Effect of proteoglycans on in vitro hydroxyapatite formation. Calcif Tissue Int. 1979 Mar 13;27(1):75–82. doi: 10.1007/BF02441164. [DOI] [PubMed] [Google Scholar]
  3. Campo R. D. Soluble and resistant proteoglycans in epiphyseal plate cartilage. Calcif Tissue Res. 1974;14(2):105–119. doi: 10.1007/BF02060287. [DOI] [PubMed] [Google Scholar]
  4. Caterson B., Baker J. R. The link proteins as specific components of cartilage proteoglycan aggregates in vivo. Associative extraction of proteoglycan aggregate from swarm rat chondrosarcoma. J Biol Chem. 1979 Apr 10;254(7):2394–2399. [PubMed] [Google Scholar]
  5. Caterson B., Baker J. The interaction of link proteins with proteoglycan monomers in the absence of hyaluronic acid. Biochem Biophys Res Commun. 1978 Feb 14;80(3):496–503. doi: 10.1016/0006-291x(78)91596-6. [DOI] [PubMed] [Google Scholar]
  6. Christner J. E., Brown M. L., Dziewiatkowski D. D. Interaction of cartilage proteoglycans with hyaluronic acid. The role of the hyaluronic acid carboxyl groups. Biochem J. 1977 Dec 1;167(3):711–716. doi: 10.1042/bj1670711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cuervo L. A., Pita J. C., Howell D. S. Inhibition of calcium phosphate mineral growth by proteoglycan aggregate fractions in a synthetic lymph. Calcif Tissue Res. 1973;13(1):1–10. doi: 10.1007/BF02015390. [DOI] [PubMed] [Google Scholar]
  8. ENGFELDT B., HULTH A., WESTERBORN O. Effect of papain on bone. I. A histologic autoradiographic, and micro-radiographic study on young dogs. Arch Pathol. 1959 Dec;68:600–614. [PubMed] [Google Scholar]
  9. Gay S., Müller P. K., Lemmen C., Remberger K., Matzen K., Kühn K. Immunohistological study on collagen in cartilage-bone metamorphosis and degenerative osteoarthrosis. Klin Wochenschr. 1976 Oct 15;54(20):969–976. doi: 10.1007/BF01468947. [DOI] [PubMed] [Google Scholar]
  10. Granda J. L., Posner A. S. Distribution of four hydrolases in the epiphyseal plate. Clin Orthop Relat Res. 1971 Jan;74:269–272. [PubMed] [Google Scholar]
  11. HJERTQUIST S. O., WESTERBORN O. The effect of papain on epiphysial cartilage in rachitic rats: histologic, autoradiographic and microradiographic studies. Virchows Arch Pathol Anat Physiol Klin Med. 1962;335:143–158. doi: 10.1007/BF02438702. [DOI] [PubMed] [Google Scholar]
  12. Hardingham T. E., Muir H. The specific interaction of hyaluronic acid with cartillage proteoglycans. Biochim Biophys Acta. 1972 Sep 15;279(2):401–405. doi: 10.1016/0304-4165(72)90160-2. [DOI] [PubMed] [Google Scholar]
  13. Hardingham T. E. The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J. 1979 Jan 1;177(1):237–247. doi: 10.1042/bj1770237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hascall V. C., Heinegård D. Aggregation of cartilage proteoglycans. I. The role of hyaluronic acid. J Biol Chem. 1974 Jul 10;249(13):4232–4241. [PubMed] [Google Scholar]
  15. Hascall V. C., Heinegård D. Aggregation of cartilage proteoglycans. II. Oligosaccharide competitors of the proteoglycan-hyaluronic acid interaction. J Biol Chem. 1974 Jul 10;249(13):4242–4249. [PubMed] [Google Scholar]
  16. Hascall V. C. Interaction of cartilage proteoglycans with hyaluronic acid. J Supramol Struct. 1977;7(1):101–120. doi: 10.1002/jss.400070110. [DOI] [PubMed] [Google Scholar]
  17. Heinegård D. Extraction, fractionation and characterization of proteoglycans from bovine tracheal cartilage. Biochim Biophys Acta. 1972 Nov 28;285(1):181–192. doi: 10.1016/0005-2795(72)90190-0. [DOI] [PubMed] [Google Scholar]
  18. Hirschman A., Dziewiatkowski D. D. Protein-polysaccharide loss during endochondral ossification: immunochemical evidence. Science. 1966 Oct 21;154(3747):393–395. doi: 10.1126/science.154.3747.393. [DOI] [PubMed] [Google Scholar]
  19. Howell D. S., Pita J. C. Calcificaiton of growth plate cartilage with special reference to studies on micropuncture fluids. Clin Orthop Relat Res. 1976 Jul-Aug;(118):208–229. [PubMed] [Google Scholar]
  20. Howell D. S., Pita J. C., Marquez J. F., Gatter R. A. Demonstration of macromolecular inhibitors of calcification and nucleational factors in fluid from calcifying sites in cartilage. J Clin Invest. 1969 Apr;48(4):630–641. doi: 10.1172/JCI106021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jibril A. O. Proteolytic degradation of ossifying cartilage matrix and the removal of acid mucopolysaccharides prior to bone formation. Biochim Biophys Acta. 1967 Feb 7;136(1):162–165. doi: 10.1016/0304-4165(67)90335-2. [DOI] [PubMed] [Google Scholar]
  22. Kember N. F. Comparative patterns of cell division in epiphyseal cartilage plates in the rat. J Anat. 1972 Jan;111(Pt 1):137–142. [PMC free article] [PubMed] [Google Scholar]
  23. Kember N. F., Walker K. V. Control of bone growth in rats. Nature. 1971 Feb 5;229(5284):428–429. doi: 10.1038/229428a0. [DOI] [PubMed] [Google Scholar]
  24. Kempson G. E., Muir H., Pollard C., Tuke M. The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans. Biochim Biophys Acta. 1973 Feb 28;297(2):456–472. doi: 10.1016/0304-4165(73)90093-7. [DOI] [PubMed] [Google Scholar]
  25. Kempson G. E., Muir H., Swanson S. A., Freeman M. A. Correlations between stiffness and the chemical constituents of cartilage on the human femoral head. Biochim Biophys Acta. 1970 Jul 21;215(1):70–77. doi: 10.1016/0304-4165(70)90388-0. [DOI] [PubMed] [Google Scholar]
  26. Kimura J. H., Hardingham T. E., Hascall V. C. Assembly of newly synthesized proteoglycan and link protein into aggregates in cultures of chondrosarcoma chondrocytes. J Biol Chem. 1980 Aug 10;255(15):7134–7143. [PubMed] [Google Scholar]
  27. Kimura J. H., Hardingham T. E., Hascall V. C., Solursh M. Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma. J Biol Chem. 1979 Apr 25;254(8):2600–2609. [PubMed] [Google Scholar]
  28. Larsson S. E., Ray R. D., Kuettner K. E. Microchemical studies on acid glycosaminoglycans of the epiphyseal zones during endochondral calcification. Calcif Tissue Res. 1973 Dec 31;13(4):271–285. doi: 10.1007/BF02015417. [DOI] [PubMed] [Google Scholar]
  29. Lindenbaum A., Kuettner K. E. Mucopolysaccharides and mucoproteins of calf scapula. Calcif Tissue Res. 1967;1(2):153–165. doi: 10.1007/BF02008085. [DOI] [PubMed] [Google Scholar]
  30. Lohmander S., Hjerpe A. Proteoglycans of mineralizing rib and epiphyseal cartilage. Biochim Biophys Acta. 1975 Sep 8;404(1):93–109. doi: 10.1016/0304-4165(75)90151-8. [DOI] [PubMed] [Google Scholar]
  31. Matukas V. J., Krikos G. A. Evidence for changes in protein polysaccharide associated with the onset of calcification in cartilage. J Cell Biol. 1968 Oct;39(1):43–48. doi: 10.1083/jcb.39.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pal S., Tang L. H., Choi H., Habermann E., Rosenberg L., Roughley P., Poole A. R. Structural changes during development in bovine fetal epiphyseal cartilage. Coll Relat Res. 1981 Feb;1(2):151–176. doi: 10.1016/s0174-173x(81)80017-9. [DOI] [PubMed] [Google Scholar]
  33. Pita J. C., Cuervo L. A., Madruga J. E., Muller F. J., Howell D. S. Evidence for a role of proteinpolysaccharides in regulation of mineral phase separation in calcifying cartilage. J Clin Invest. 1970 Dec;49(12):2188–2197. doi: 10.1172/JCI106437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Poole A. R., Pidoux I., Reiner A., Tang L. H., Choi H., Rosenberg L. Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study. J Histochem Cytochem. 1980 Jul;28(7):621–635. doi: 10.1177/28.7.6156200. [DOI] [PubMed] [Google Scholar]
  35. Poole A. R., Reiner A., Tang L. H., Rosenberg L. Proteoglycans from bovine nasal cartilage. Immunochemical studies of link protein. J Biol Chem. 1980 Oct 10;255(19):9295–9305. [PubMed] [Google Scholar]
  36. Remberger K., Gay S. Immunhistochemical demonstration of different collagen types in the normal epiphyseal plate and in benign and malignant tumors of bone and cartilage. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol. 1977 Oct;90(1):95–106. doi: 10.1007/BF00306024. [DOI] [PubMed] [Google Scholar]
  37. Rosenberg L. C., Pal S., Beale R. J. Proteoglycans from bovine proximal humeral articular cartilage. J Biol Chem. 1973 May 25;248(10):3681–3690. [PubMed] [Google Scholar]
  38. Rosenberg L. Chemical basis for the histological use of safranin O in the study of articular cartilage. J Bone Joint Surg Am. 1971 Jan;53(1):69–82. [PubMed] [Google Scholar]
  39. Rosenberg L., Hellmann W., Kleinschmidt A. K. Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J Biol Chem. 1975 Mar 10;250(5):1877–1883. [PubMed] [Google Scholar]
  40. Rosenberg L., Pal S., Beale R., Schubert M. A comparison of proteinpolysaccharides of bovine nasal cartilage isolated and fractionated by different methods. J Biol Chem. 1970 Aug 25;245(16):4112–4122. [PubMed] [Google Scholar]
  41. Rosenberg L., Wolfenstein-Todel C., Margolis R., Pal S., Strider W. Proteoglycans from bovine proximal humeral articular cartilage. Structural basis for the polydispersity of proteoglycan subunit. J Biol Chem. 1976 Oct 25;251(20):6439–6444. [PubMed] [Google Scholar]
  42. Roughley P., Dickson I. Factors influencing proteoglycan size in rachitic-chick growth cartilage. Biochem J. 1980 Jan 1;185(1):33–39. doi: 10.1042/bj1850033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schenk R. K., Spiro D., Wiener J. Cartilage resorption in the tibial epiphyseal plate of growing rats. J Cell Biol. 1967 Jul;34(1):275–291. doi: 10.1083/jcb.34.1.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Tang L. H., Rosenberg L., Reiner A., Poole A. R. Proteoglycans from bovine nasal cartilage. Properties of a soluble form of link protein. J Biol Chem. 1979 Oct 25;254(20):10523–10531. [PubMed] [Google Scholar]
  45. Thyberg J., Lohmander S., Friberg U. Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage. J Ultrastruct Res. 1973 Dec;45(5):407–427. doi: 10.1016/s0022-5320(73)80070-x. [DOI] [PubMed] [Google Scholar]
  46. WEATHERELL J. A., WEIDMANN S. M. THE DISTRIBUTION OF ORGANICALLY BOUND SULPHATE IN BONE AND CARTILAGE DURING CALCIFICATION. Biochem J. 1963 Nov;89:265–267. doi: 10.1042/bj0890265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wuthier R. E. A zonal analysis of inorganic and organic constituents of the epiphysis during endochondral calcification. Calcif Tissue Res. 1969 Aug 11;4(1):20–38. doi: 10.1007/BF02279103. [DOI] [PubMed] [Google Scholar]
  48. von der Mark H., von der Mark K., Gay S. Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo. Dev Biol. 1976 Feb;48(2):237–249. doi: 10.1016/0012-1606(76)90088-9. [DOI] [PubMed] [Google Scholar]
  49. von der Mark K., von der Mark H. The role of three genetically distinct collagen types in endochondral ossification and calcification of cartilage. J Bone Joint Surg Br. 1977 Nov;59-B(4):458–464. doi: 10.1302/0301-620X.59B4.72756. [DOI] [PubMed] [Google Scholar]

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

RESOURCES