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.
Similar content being viewed by others
References
Aydelotte, M. B.; Kuettner, K. E. Differences between sub-populations of cultured bovine articular chondrocytes. Connect. Tissue Res. 18:205–222; 1988.
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.
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.
Bruckner, P.; Hörler, I.; Mendler, M., et al. Induction and prevention of chondrocyte hypertrophy in culture. J. Cell Biol. 109:2537–2545; 1989.
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.
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.
Gibson, G. J.; Lin, D. L. Type X collagen morphology in calf growth plate and articular cartilage. Trans. Orthop. Res. Soc. 20:28; 1995.
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.
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.
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.
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.
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.
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.
Lovell, T. P.; Eyre, D. R. Unique biochemical characteristics of the calcified zone of articular cartilage. Trans. Orthop. Res. Soc. 13:511; 1988.
Mitrovic, D. R.; Darmon, N. Characterization of proteoglycans synthesized by different layers of adult human femoral head cartilage. Osteoarthritis Cartilage 2:119–131; 1994.
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.
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.
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.
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.
Poole, R. A.; Matsui, Y.; Hinek, A., et al. Cartilage macromolecules and the calcification of cartilage matrix. Anat. Rec. 224:167–179; 1989.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Wardale, J. R.; Duance, V. C. Quantification and immunolocalisation of porcine articular and growth plate cartilage collagens. J. Cell Sci. 105:975–984; 1993.
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.
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.
Wuthier, R. E. Mechanism of matrix vesicle mediated mineralization of cartilage. ISI Atlas Sci. Biochem. 1:231–241; 1988.
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.
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.
Zimmermann, B.; Wachtel, H. C.; Somogyi, H. Endochondral mineralization in cartilage organoid culture. Cell Differ. Develop. 31:11–22; 1990.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kandel, R.A., Boyle, J., Gibson, G. et al. In vitro formation of mineralized cartilagenous tissue by articular chondrocytes. In Vitro Cell.Dev.Biol.-Animal 33, 174–181 (1997). https://doi.org/10.1007/s11626-997-0138-7
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s11626-997-0138-7