In vitro formation of mineralized cartilagenous tissue by articular chondrocytes

  • R. A. Kandel
  • J. Boyle
  • G. Gibson
  • T. Cruz
  • M. Speagle
Cellular Models

Summary

Study of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CMR filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.

Key words

chondrocytes calcification articular cartilage 

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References

  1. 1.
    Aydelotte, M. B.; Kuettner, K. E. Differences between sub-populations of cultured bovine articular chondrocytes. Connect. Tissue Res. 18:205–222; 1988.PubMedGoogle Scholar
  2. 2.
    Boyle, J.; Luan, B.; Cruz, T. F., et al. Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro. Osteoarthritis Cartilage 3:117–125; 1995.PubMedCrossRefGoogle Scholar
  3. 3.
    Boskey, A. L.; Stiner, D.; Doty, S. B., et al. Studies of mineralization in tissue culture: optimal conditions for cartilage calcification. Bone Miner. 16:11–36; 1992.PubMedCrossRefGoogle Scholar
  4. 4.
    Bruckner, P.; Hörler, I.; Mendler, M., et al. Induction and prevention of chondrocyte hypertrophy in culture. J. Cell Biol. 109:2537–2545; 1989.PubMedCrossRefGoogle Scholar
  5. 5.
    Chung, C.-H.; Golub, E. E.; Forbes, E., et al. Mechanism of action of beta-glycerophosphate on bone cell mineralization. Calcif. Tissue Int. 51:305–311; 1992.PubMedCrossRefGoogle Scholar
  6. 6.
    Gannon, J. M.; Walker, G.; Fischer, M., et al. Localization of type X collagen in canine growth plate and adult articular cartilage. J. Orthop. Res. 9:485–494; 1991.PubMedCrossRefGoogle Scholar
  7. 7.
    Gibson, G. J.; Lin, D. L. Type X collagen morphology in calf growth plate and articular cartilage. Trans. Orthop. Res. Soc. 20:28; 1995.Google Scholar
  8. 8.
    Hascall, V. C.; Oegema, T. R.; Brown, M., et al. Isolation and characterization of proteoglycans from chick limb bud chondrocytes grown in vitro. J. Biol. Chem. 251:3511–3519; 1976.PubMedGoogle Scholar
  9. 9.
    Iwamoto, M.; Sato, K.; Nakashima, K., et al. Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor. Dev. Biol. 136:500–507; 1989.PubMedCrossRefGoogle Scholar
  10. 10.
    Kato, Y.; Iwamoto, M.; Koike, T., et al. Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor β and serum factors. Proc. Nat. Acad. Sci. 85:9552–9556; 1988.PubMedCrossRefGoogle Scholar
  11. 11.
    Kimata, K.; Oike, Y.; Tani, K., et al. A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo. J. Biol. Chem. 261:13517–13525; 1986.PubMedGoogle Scholar
  12. 12.
    Korver, G. H. V.; van de Stadt, R. J.; van Kampen, G. P. J., et al. Composition of proteoglycans synthesized in different layers of cultured anatomically intact articular cartilage. Matrix 10:394–401; 1990.PubMedGoogle Scholar
  13. 13.
    Lemperg, R. The subchondral bone plate of the femoral head in adult rabbits. I. Spontaneous remodelling studied by microradiography and tetracycline labelling. Virchows Arch. Abt. A. Path. Anat. 352:1–13; 1971.CrossRefGoogle Scholar
  14. 14.
    Lovell, T. P.; Eyre, D. R. Unique biochemical characteristics of the calcified zone of articular cartilage. Trans. Orthop. Res. Soc. 13:511; 1988.Google Scholar
  15. 15.
    Mitrovic, D. R.; Darmon, N. Characterization of proteoglycans synthesized by different layers of adult human femoral head cartilage. Osteoarthritis Cartilage 2:119–131; 1994.PubMedCrossRefGoogle Scholar
  16. 16.
    Nakagawa, Y.; Shimizu, K.; Hamamoto, T., et al. Electron microscopy of calcification during high-density suspension culture of chondrocytes. Calcif. Tissue Int. 53:127–134; 1993.PubMedCrossRefGoogle Scholar
  17. 17.
    Oegema, T. R., Jr.; Thompson, R. C., Jr. The zone of calcified cartilage. Its role in osteoarthritis. In: Kuettner, K., ed. Articular cartilage and osteoarthritis. New York: Raven Press; 1992:319–331.Google Scholar
  18. 18.
    Oegema, T. R., Jr.; Thompson, R. C., Jr. Cartilage-bone interface (Tidemark). In: Brandt, K., ed. Cartilage changes in osteoarthritis. Indiana School of Medicine publication. Basel: Ciba-Geigy; 1990:43–52.Google Scholar
  19. 19.
    Plaas, A. H. K.; Sandy, J. D. A cartilage explant system for studies on aggrecan structure, biosynthesis and catabolism in discrete zones of the mammalian growth plate. Matrix 13:135–147; 1993.PubMedGoogle Scholar
  20. 20.
    Poole, R. A.; Matsui, Y.; Hinek, A., et al. Cartilage macromolecules and the calcification of cartilage matrix. Anat. Rec. 224:167–179; 1989.PubMedCrossRefGoogle Scholar
  21. 21.
    Poole, R. A. Cartilage in health and disease. In: McCarty, D.; Koopman, W., ed. Arthritis and allied conditions. A textbook of rheumatology. 12th ed. Malvern: Lea and Febiger; 1992:279–333.Google Scholar
  22. 22.
    Quarto, R.; Dozin, B.; Tacchette, C., et al. In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells. J. Cell Biol. 110:1379–1386; 1990.PubMedCrossRefGoogle Scholar
  23. 23.
    Schumacher, B. L.; Block, J. A.; Schmid, T. M., et al. A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage. Arch. Biochem. Biophys. 311:144–152; 1994.PubMedCrossRefGoogle Scholar
  24. 24.
    Siczkowski, M.; Watt, F. M. Subpopulation of chondrocytes from different zones of pig articular cartilage: isolation, growth and proteoglycan synthesis in culture. J. Cell. Sci. 97:349–360; 1990.PubMedGoogle Scholar
  25. 25.
    Silbermann, M.; Tenenbaum, H.; Livne, E., et al. The in vitro behavior of fetal condylar cartilage in serum-free hormone-supplemented medium. Bone 8:117–126; 1987.PubMedCrossRefGoogle Scholar
  26. 26.
    Shaklee, P. N.; Conrad, H. E. Structural changes in the large proteoglycan in differentiating chondrocytes from the chick embryo tibiotarsus. J. Biol. Chem. 260:16064–16067; 1985.PubMedGoogle Scholar
  27. 27.
    Thompson, R. C.; Oegema, T. R., Jr.; Lewis, J. L., et al. Osteoarthrotic changes after acute transarticular load. J. Bone Joint Surg. 73A:990–1001; 1991.Google Scholar
  28. 28.
    Vellet, A. D.; Marks, P. H.; Fowler, P. J., et al. Occult posttraumatic osteochondral lesions of the knee: prevalence, classification, and short-term sequelae evaluated with MR imaging. Radiology 178:271–276; 1991.PubMedGoogle Scholar
  29. 29.
    Walker, G.; Carpenter, R. J.; Oegema, T. R., Jr., et al. Evidence for activity in the tidemark in normal articular cartilage. Trans. Orthop. Res. Soc. 15:182; 1990.Google Scholar
  30. 30.
    Wardale, J. R.; Duance, V. C. Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens. J. Cell Sci. 105:975–984; 1993.PubMedGoogle Scholar
  31. 31.
    Whyte, M. P. Alkaline phosphatase: physiological role explored in hypophosphatasia. In: Peck, W. A., ed. Bone and mineral research. 6th ed. Amsterdam: Elsevier Science Publishers; 1989:175–218.Google Scholar
  32. 32.
    Wu, L. N. Y.; Sauer, G. R.; Genge, B. R., et al. Induction of mineral deposition by primary cultures of chicken growth plate chondrocytes in ascorbate-containing media. J. Biol. Chem. 264:21346–21355; 1989.PubMedGoogle Scholar
  33. 33.
    Wuthier, R. E. Mechanism of matrix vesicle mediated mineralization of cartilage. ISI Atlas Sci. Biochem. 1:231–241; 1988.Google Scholar
  34. 34.
    Xu, Y.; Pritzker, K. P. H.; Cruz, T. Characterization of chondrocyte alkaline phosphatase as a potential mediator in the dissolution of calcium pyrophosphate dihydrate crystals. J. Rheumatol. 21:912–919; 1994.PubMedGoogle Scholar
  35. 35.
    Yoon, K.; Golub, E. E.; Rodan, G. A. Alkaline phosphatase cDNA transfected cells promote calcium and phosphate deposition. In: Glimcher, M. J.; Lian, J. B., ed. Proceedings of the Third International Conference on the Chemistry and Biology of Mineralized Tissues. New York: Gordon and Breach Science Publishers; 1989:643–652.Google Scholar
  36. 36.
    Zimmermann, B.; Wachtel, H. C.; Somogyi, H. Endochondral mineralization in cartilage organoid culture. Cell Differ. Develop. 31:11–22; 1990.CrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 1997

Authors and Affiliations

  • R. A. Kandel
    • 1
  • J. Boyle
    • 1
  • G. Gibson
    • 2
  • T. Cruz
    • 1
  • M. Speagle
    • 1
  1. 1.Department of Pathology, Connective Tissue Research Group, Mount Sinai HospitalUniversity of TorontoTorontoCanada
  2. 2.Henry Ford HospitalDetroit

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