High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development
Received: 04 November 1998 Accepted: 08 December 1998 DOI:
10.1007/s11626-999-0070-0 Cite this article as: Mello, M.A. & Tuan, R.S. In Vitro Cell.Dev.Biol.-Animal (1999) 35: 262. doi:10.1007/s11626-999-0070-0 Summary
To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.
Key words micromass cultures endochondral ossification cartilage maturation calcification apoptosis collagen type X References
Ahrens, P. B.; Solursh, M.; Reiters, R. Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60:69–82; 1977.
Alini, M.; Carey, D.; Hirata, S., et al. Cellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: arrest of Type X collagen synthesis and net loss of collagen when calcification is initiated. J. Bone Miner. Res. 9:1077–1087; 1994.
Alini, M.; Kofsky, Y.; Wu, W., et al. In serum-free culture thyroid hormones can induce full expression of chondrocyte hypertrophy leading to matrix calcification. J. Bone Miner. Res. 11:105–113; 1996.
Anderson, H. C. Molecular biology of matrix vesicles. Clin. Orthop. Relat. Res. 314:266–280; 1995.
Ballock, R.; Reddi, A. H. Thyroxine is the serum factor that regulates morphogenesis of columnar cartilage from isolated chondrocytes in chemically defined medium. J. Cell Biol. 126:1311–1318; 1994.
Ballock, R. T.; Reddy, A. H. Morphogenesis of columnar cartilage from isolated chondrocytes in chemically-defined media is thyroxine dependent. Trans. Orthop. Res. Soc. 19:124; 1994.
Benya, P. D.; Schaffer, J. D. Dedifferentiation chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 30:215–224; 1982.
Boskey, A. L.; Stiner, D.; Doty, S. B., et al. Studies of mineralization in tissue culture: optimal conditions for cartilage calcification. J. Bone Miner. Res. 16:11–36; 1991.
Farnum, C. E.; Wilsman, N. J. Histochemical evidence of DNA fragmentation characteristic of apoptosis in hypertrophic chondrocytes. Trans. Orthop. Res. Soc. 20:77; 1995.
Flechtenmacher, J.; Aydelotte, M. B.; Hauselmann, H. J., et al. Growth plate chondrocytes but not other chondrocytes form single cell-columns on a modified alginate gel system. Trans. Orthop. Res. Soc. 19:416; 1994.
Galotto, M.; Campanile, G.; Robino, G., et al. Hypertrophic chondrocytes undergo further differentiation to osteoblast-like cells and participate in the initial bone formation in developing chicken embryo. J. Bone Miner. Res. 9:1239–1249; 1994.
Gavrieli, Y.; Sherman, Y.; Ben-Sasson, S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119:493–501; 1992.
Gerstenfeld, L. C.; Shapiro, F. D. Expression of bone-specific genes by hypertrophic chrondrocytes: implication of the complex functions of the hypertrophic chrondrocyte during endochondral bone development. J. Cell. Biochem. 62:1–9; 1996.
Gibson, G. J.; Kohler, W. J.; Schaffler, M. B. Chondrocyte apoptosis in endochondral ossification of chick sterna. Dev. Dyn. 203:466–476; 1995.
Groessner-Schreiber, B.; Tuan, R. S. Enhanced extracellular matrix production and mineralization by osteoblasts cultured on titanium surfaces in vitro. J. Cell Sci. 101:209–217; 1992.
Groessner-Schreiber, B.; Kreitzer, D.; Tuan, R. S. Bone cell response to hydroxyapatite-coated titanium surfaces in vitro. Semin. Arthroplasty; 2:260–267; 1991.
Hatori, M.; Klatte, K. J.; Teixeira, C. C., et al. End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosis in terminally differentiated chondrocytes. J. Bone Miner. Res. 10:1960–1968; 1995.
Hunziker, E. B. Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc. Res. Tech. 28:505–519; 1994.
Hunziker, E. B.; Ludi, A.; Herrmann, W. Preservation of cartilage matrix proteoglycans using cationic dyes chemically related to ruthenium hexamine trichloride. J. Histochem. Cytochem. 40:909–917; 1992
Jacenko, O.; Tuan, R. S. Calcium deficiency induces expression of cartilage-like phenotype in chicken embryo calvaria. Dev. Biol. 115:215–232; 1986.
Kato, Y.; Iwamoto, M. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J. Biol. Chem. 265:5903–5909; 1990.
Kiernan, J. A. Histological & histochemical methods. 2nd ed. New York: Pergamon Press; 1990.
Lev, R.; Spicer, S. Specific staining of sulphate groups with alcian blue at low pH. J. Histochem. Cytochem. 12:309; 1964.
Linsenmayer, T. F.; Hendrix, M. J. C. Monoclonal antibodies to connective tissues macromolecules: type II collagen. Biochem. Biophys. Res. Commun. 92:440–446; 1980.
Loredo, G. A.; Koolpe, M.; Benton, H. P. Influence of alginate polysaccharide composition and culture conditions on chondrocytes in three-dimensional culture. Tissue Engineer. 2:115–125; 1996.
Mello, M. A.; Tuan, R. S. Growth cartilage maturation in micromass cultures. Mol. Biol. Cell Suppl. 6:392a; 1995.
Mello, M. A.; Tuan, R. S. Programmed cell death in micromass cultures of growth cartilage derived from embryonic limb mesenchyme. Mol. Biol. Cell. Suppl. 7:581a; 1996.
Oberlender, S.; Tuan, R. S. Expression and functional involvement of
N-cadherin in embryonic limb chondrogenesis. Development 120:177–197; 1990.
Pechak, D. G.; Ilujawa, M. J.; Caplan, A. L. Morphology of bone development and bone remodeling in embryonic chick limbs. Bone 7:459–472; 1986.
Ray, S.; Ponnathpur, V.; Huang, Y., et al. 1-β-
d-Arabinofuranosylcytosine-, mitoxantrone, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation. Cancer Chemotherap. Pharmacol. 34:356–371; 1994.
Reginato, A. M.; Tuan, R. S.; Ono, T., et al. Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum. Dev. Dyn. 189:284–295; 1993.
Roach, H. I.; Erenpreisa, J. The phenotypic switch from chrondrocytes to bone-forming cells involves asymmetric cell division and apoptosis. Connect. Tissue Res. 35:85–91; 1996.
Roach, I. New aspects of endochrondral ossification in the chick: chondrocyte apoptosis, bone formation by former chondrocytes, and acid phosphatase activity in the endochondral bone matrix. J. Bone Miner. Res. 12:795–805; 1997.
Roach, I.; Erenpreisa, J.; Aigner, T. Osteogenic differentiation of hypertrophic chondrocytes involve asymmetric cell divisions and apoptosis. J. Cell Biol. 131:483–494; 1995.
Roark, E. F.; Greer, K. Transforming growth factor-β and bone morphogenetic protein-2 act by distinct mechanisms to promote chick limb cartilage differentiation in vitro. Dev. Dyn. 200:103–116; 1994.
San Antonio, J. D.; Tuan, R. S. Chondrogenesis of limb mesenchyme
: stimulation by cations. Dev. Biol. 115:313–324; 1986.
Tilly, J. L.; Hsueh, A. J. W. Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation. J. Cell. Physiol. 154:519–526; 1993.
Wong, M.; Tuan, R. S. Nuserum, a synthetic serum replacement, supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass cultures. In vitro Cell. Dev. Biol. Animal 29:917–922; 1993.
Zenmyo, M.; Komiya, S.; Kawabata, R., et al. Morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. J. Pathol. 180:430–433; 1996.
PubMed CrossRef Copyright information
© Society for In Vitro Biology 1999