Summary
One of the most important indicators in vitro of the bone-cell phenotype is the synthesis of mineralized bone-like tissue. This has been achieved by supplementing isolated bone-cell and tissue cultures with organic phosphates, in particular, β-glycerophosphate. To analyze the effects of β-glycerophosphate on bone-cell metabolism and osteogenesis in vitro, both biochemical analyses and computer-assisted morphometry were used. Simultaneous autoradiographic and histochemical analyses of proliferating and alkaline phosphatase-positive cells were used to measure osteogenic events at the cellular level. Morphometric data showed that β-glycerophosphate-treated cultures mineralized, but exhibited significantly less bone matrix (P < 0.05) than non-mineralizing controls. Cultures treated with inorganic phosphate failed to mineralize. Cellular proliferation was unaffected by β-glycerophosphate; however, there was a decrease in the amount of 3H-thymidine incorporation into the DNA of β-glycerophosphate-treated cells as detected by autoradiography. The percentage of alkaline phosphatase-positive cells was identical in β-glycerophosphate-treated or control cultures. In agreement with previous biochemical results, there was a decrease in the amount of alkaline phosphatase enzyme activity per cell. The kinetics of alkaline phosphatase enzymes were measured on individual cells by microdensitometry. β-Glycerophosphate-treated cultures exhibited more rapid reaction rates than control cultures (p < 0.05). Taken together, the results suggest that β-glycerophosphate has global effects on bone-cell metabolism in vitro including its importance in mineralization.
Similar content being viewed by others
References
Bellows CG, Aubin JE, Heersche JNM, Antosz ME (1986) Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations. Calcif Tissue Int 38:143–154
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein binding. Anal Biochem 72:248–254
Cleaver JE (1967) Thymidine metabolism and cell kinetics. North Holland, Amsterdam
Drury RAB, Wallington EA, Cameron R (1973) Carleton's histological technique, 4th Ed., Oxford University Press, London, p 150
Ecarot-Charrier B, Glorieux FH, Van der Rest M, Pereira G (1983) Osteoblasts isolated from mouse calvaria initiate matrix mineralization. J Cell Biol 96:639–643
Elias L (1988) Stimulation by tumor necrosis factor of HL-60 thymidine salvage pathway metabolism dissociated from proliferation. J Cell Physiol 136:95–102
Gehron-Robey P, Termine JD (1985) Human bone cells in vitro. Calcif Tissue Int 37:453–460
McComb RB, Bowers GN, Posen S (1979) Alkaline Phosphatase. Plenum Press New York London
McCulloch CAG, Tenenbaum HC (1986) Dexamethasone induces proliferation and terminal differentiation of osteogenic cells in tissue culture. Anat Rec 215:397–402
McCulloch CAG, Tenenbaum HC, Fair CA, Birek C (1989) Site specific regulation of osteogenesis: Maintenance of discrete levels of phenotypic expression in vitro. Anat Rec 223:27–34
Nefussi J-R, Boy-Lefevre ML, Boulekbach H, Forest N (1985) Mineralization in vitro of matrix formed by osteoblasts isolated by collagenase digestion. Differentiation 29:160–168
Nijweide PJ (1975) Embryonic chicken periosteum in tissue culture: Osteoid formation and calicum uptake. Proc Kon Ned Akad Wet C78:410–417
Roach HI (1987) Does β-glycerophosphate induce calcification in vitro by affecting cellular viability? [Abstr]. Calcif Tissue Int Suppl # 2 41:43
Shimizu N, Vieth R, Reimers S, Heersche JNM (1988) The effects of vitamin D restriction on bone and dentine apposition in the rat [Abstr]. J Dent Res 67:148
Tanaka H, Seino Y, Shima M, Yamaoka K, Yabuuchi H, Yoshikawa H, Masuhara K, Takaoka K, Ono K (1988) Effect of phosphorous supplementation on bone formation induced by osteosarcoma-derived bone inducing substance in X-linked hypophosphatemic mice. Bone and Mineral 4:237–246
Tenenbaum HC (1981) Role of organic phosphate in mineralization of bone in vitro. J Dent Res 60(C):1586–1589
Tenenbaum HC (1987) Levamisole and inorganic pyrophosphate inhibit β-glycerophosphate induced mineralization of bone formed in vitro. Bone and Mineral 3:13–26
Tenenbaum HC, Heersche JNM (1982) Differentiation of osteoblasts and formation of mineralized bone in vitro. Calcif Tissue Int 34:76–79
Tenenbaum HC, Hunter GK (1987) Chondroitin sulfate inhibits calcification of bone formed in vitro. Bone and Mineral 2:43–51
Tenenbaum HC, Palangio K (1987) Phosphoethanolamine and fructose 1,6-diphosphate-induced calcium uptake in bone formed in vitro. Bone and Mineral 2:201–210
Tenenbaum HC, McCulloch CAG, Palangio K (1986a) Simultaneous autoradiographic and histochemical analysis of bone formed in vitro. J Histochem Cytochem 34:769–773
Tenenbaum HC, Palangio K, Holmyard DP, Pritzker KPH (1986b) An ultrastructural study of osteogenesis in vitro. Bone 7:295–302
Whitson SW, Harrison W, Dunlap MK, Bowers DE Jr, Fisher LW, Gehron-Robey P, Termine JD (1984) Fetal bovine bone cells synthesize bone-specific matrix proteins. J Cell Biol 99:607–614
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tenenbaum, H.C., McCulloch, C.A.G., Fair, C. et al. The regulatory effect of phosphates on bone metabolism in vitro. Cell Tissue Res. 257, 555–563 (1989). https://doi.org/10.1007/BF00221466
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00221466