Transforming growth factor-beta inhibition of mineralization by neonatal rat osteoblasts in monolayer and collagen gel culture

  • Deanna J. Talley-Ronsholdt
  • Evelyn Lajiness
  • Kishan Nagodawithana
Cellular Models


The latent form of transforming growth factor-beta (TGF-β) is a component of the extracellular matrix of bone. The active form, when locally injected in vivo, stimulates both inflammation and ectopic bone formation. The present study was undertaken to determine if TGF-β also stimulated mineralization by isolated rat calvarial osteoblasts cultured in collagen gels. Gels were used because they should mimic in vivo conditions better than classical monolayer culture. Compared to cells in monolayers, osteoblasts cultured in collagen gels exhibited slower growth, but higher alkaline phosphatase activity and mineral deposition. Cultured cells also synthesized the osteoblast-specific marker, osteocalcin. The increase in osteocalcin in cell layers was parallel to the increase in mineral deposition. In the presence of TGF-β, neither cell growth nor alkaline phosphatase activity increased. Instead, a small decrease occurred in both parameters when compared to untreated cultures. Accumulation of collagen, the major component of the extracellular matrix where mineralization occurs, was similar in untreated and TGF-β1-treated cultures. However, 8 pM TGF-β1 dramatically suppressed mineral deposition in both types of cultures. Despite TGF-β1 stimulating a fourfold increase in lactic acid, the consequent increase in culture medium acidity did not account for the inhibitory effects of TGF-β1 on mineralization. These results demonstrate that collagen gel culture is an improved technique over conventional monolayer culture for demonstrating differentiated osteoblast function and sensitivity to TGF-β1. TGF-β1, at a concentration that has little effect on cell growth, alkaline phosphatase activity, or collagen accumulation, is a potent inhibitor of mineralization. The mechanism by which TGF-β1 inhibits mineralization remains to be determined.

Key words

osteoblasts bone collagen gels mineralization TGF-beta 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ahnoff, M.; Grundevik, I.; Arfwidsson, A., et al. Derivatization with 4-chloro-7-nitrobenzofurazan for liquid chromatographic determination of hydroxyproline in collagen hydrolysate. Anal. Chem. 53:485–489; 1981.PubMedGoogle Scholar
  2. 2.
    Assoian, R. K.; Komoriya, A.; Myers, C. A., et al. Transforming growth factor-beta in human platelets. J. Biol. Chem. 258:7155–7160; 1983.PubMedGoogle Scholar
  3. 3.
    Beck, L. S.; Deguzman, L.; Lee, W. P., et al. TGF-beta-1 induces bone closure of skull defects. J. Bone Miner. Res. 6:1257–1265; 1991.PubMedGoogle Scholar
  4. 4.
    Bellows, C. G.; Heersche, J. N. M.; Aubin, J. E. Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro. Bone Miner. 17:15–29; 1992.PubMedCrossRefGoogle Scholar
  5. 5.
    Berg, R. A. Determination of 3- and 4-hydroxyproline. In: Cunningham, L. W.; Frederiksen, D. W. eds. Methods in Enzymology. Vol. 82. New York: Academic Press; 1982:372–398.Google Scholar
  6. 6.
    Boerner, P.; Resnick, R. J.; Racker, E. Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factorβ and epidermal growth factors. Proc. Natl. Acad. Sci. USA 82:1350–1353; 1985.PubMedCrossRefGoogle Scholar
  7. 7.
    Bonewald, L. F.; Kester, M. B.; Schwartz, Z., et al. Effects of combining transforming growth factor-beta and 1,25-dihydroxyvitamin-D3 on differentiation of a human osteosarcoma (MG-63). J. Biol. Chem. 267:8943–8949; 1992.PubMedGoogle Scholar
  8. 8.
    Centrella, M.; Casinghino, S.; Ignotz, R., et al. Multiple regulatory effects by transforming growth factor-beta on type I-collagen levels in osteoblast-enriched cultures from fetal rat bone. Endocrinology 131:2863–2872; 1992.PubMedCrossRefGoogle Scholar
  9. 9.
    Centrella, M.; McCarthy, T. L.; Canalis, E. Transforming growth factor beta is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fetal rat bones. J. Biol. Chem. 262:2869–2874; 1987.PubMedGoogle Scholar
  10. 10.
    Centrella, M.; McCarthy, T. L.; Canalis, E. Skeletal tissue and transforming growth factorβ. FASEB J. 2:3066–3073; 1988.PubMedGoogle Scholar
  11. 11.
    Coe, M. R.; Summers, T. A.; Parsons, S. J., et al. Matrix mineralization in hypertrophic chondrocyte cultures-beta-glycerophosphate increases type X-collagen messenger RNA and the specific activity of pp60 (c-src) kinase. Bone Miner. 18:91–106; 1992.PubMedCrossRefGoogle Scholar
  12. 12.
    Elford, P. R.; Guenther, H. L.; Felix, R., et al. Transforming growth factor-β reduces the phenotypic expression of osteoblastic MC3T3-E1 cells in monolayer culture. Bone 8:259–262;1987.PubMedCrossRefGoogle Scholar
  13. 13.
    Feige, J. J.; Cochet, C.; Rainey, W. E., et al., Type beta transforming growth factor affects adrenocortical cell-differentiated functions. J. Biol. Chem. 262:13491–13495; 1987.PubMedGoogle Scholar
  14. 14.
    Florini, J. R.; Roberts, A. B.; Ewton, D. Z., et al. Transforming growth factor-β; a very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells. J. Biol. Chem. 261:16509–16513; 1986.PubMedGoogle Scholar
  15. 15.
    Galera, P.; Redini, F.; Vivien, D., et al. Effect of transforming growth factor-β1 (TGF-β1) on matrix synthesis by monolayer cultures of rabbit articular chondrocytes during the dedifferentiation process. Exp. Cell Res. 200:379–392; 1992.PubMedCrossRefGoogle Scholar
  16. 16.
    Geesin, J. C.; Brown, L. J.; Gordon, J. S., et al. Regulation of collagen synthesis in human dermal fibroblasts in contracted collagen gels by ascorbic acid, growth factors and inhibitors of lipid peroxidation. Exp. Cell Res. 206:283–290; 1993.CrossRefGoogle Scholar
  17. 17.
    Gundberg, C. M.; Hauschka, P. V.; Lian, J. B., et al. Osteocalcin: isolation, characterization and detection. In: Moldave, K., ed. Methods in Enzymology. Vol. 107. New York: Academic Press; 1984:516–544.Google Scholar
  18. 18.
    Harris, S. E.; Harris, M. A.; Feng, J. Q., et al. Expression of bone morphogenetic proteins (BMPs) during differentiation of fetal rat calvarial osteoblasts in vitro. J. Bone Miner. Res. 7:S112; 1992.Google Scholar
  19. 19.
    Harris, S. E.; Sabatini, M.; Harris, M. A., et al. Expression of bone morphogenetic protein messenger RNA in prolonged cultures of fetal rat calvarial cells. J. Bone Miner. Res. 9:389–394; 1994.PubMedGoogle Scholar
  20. 20.
    Hauschka, P. V.; Lian, J. B.; Cole, D. E. C., et al. Osteocalcin and matrix gla protein: vitamin K-dependent proteins in bone. Physiol. Rev. 69:990–1047; 1989.PubMedGoogle Scholar
  21. 21.
    Ibaraki, K.; Termine, J. D.; Whitson, S. W., et al. Bone matrix messenger RNA expression in differentiating fetal bovine osteoblasts. J. Bone Miner. Res. 7:743–754; 1992.PubMedGoogle Scholar
  22. 22.
    Inman, W. H.; Colowick, S. P. Stimulation of glucose uptake by transforming growth factorβ: evidence for the requirement of epidermal growth factor-receptor activation. Proc. Natl. Acad. Sci. USA 82:1346–1349; 1985.PubMedCrossRefGoogle Scholar
  23. 23.
    Inoue, H.; Kato, Y.; Iwamoto, M., et al. Stimulation of cartilage-matrix proteoglycan synthesis by morphologically transformed chondrocytes grown in the presence of fibroblast growth factor and transforming growth factor-beta. J. Cell. Physiol. 138:329–337; 1989.PubMedCrossRefGoogle Scholar
  24. 24.
    Izumi, T.; Scully, S. P.; Heydemann, A., et al. Transforming growth factor-beta-1 stimulates type II collagen expression in cultured periosteum-derived cells. J. Bone Miner. Res. 7:115–121; 1992.PubMedGoogle Scholar
  25. 25.
    Kato, Y.; Iwamoto, M.; Koike, T., et al. Terminal differentiation and calcification in rabbit chondrocyte cultures growth in centrifuge tubes: regulation by transforming growth factorβ and serum factors. Proc. Natl. Acad. Sci. USA 85:9552–9556; 1988.PubMedCrossRefGoogle Scholar
  26. 26.
    Kojima, S.; Nara, K.; Rifkin, D. B. Requirement for transglutaminase in the activation of latent transforming growth factor-β in bovine endothelial cells. J. Cell Biol. 121:439–448; 1993.PubMedCrossRefGoogle Scholar
  27. 27.
    Lian, J. B.; Coutts, M.; Canalis, E. Studies of hormonal regulation of osteocalcin synthesis in cultured fetal rat calvariae. J. Biol. Chem. 260:8706–8710; 1985.PubMedGoogle Scholar
  28. 28.
    Lowry, O. H.; Roberts, N. R.; Wu, M. L., et al. The quantitative histochemistry of brain. II. Enzyme measurement. J. Biol. Chem. 207:19–39; 1954.PubMedGoogle Scholar
  29. 29.
    Madri, J. A.; Pratt, B. M.; Tucker, A. M. Phenotypic modulation of endothelial cells by transforming growth factor-β depends upon the composition and organization of the extracellular matrix. J. Cell Biol. 106:1375–1384; 1988.PubMedCrossRefGoogle Scholar
  30. 30.
    Massague, J. The transforming growth factor-beta family. Annu. Rev. Cell Biol. 6:597–641; 1990.PubMedCrossRefGoogle Scholar
  31. 31.
    Massague, J.; Cheifetz, S.; Boyd, F. T., et al. TGF-β receptors and TGF-β binding proteoglycans: recent progress in identifying their functional properties. Ann. NY Acad. Sci. 593:59–72; 1990.PubMedCrossRefGoogle Scholar
  32. 32.
    Nichols, F. C.; Neuman, W. F. Lactic acid production in mouse calvariain vitro with and without parathyroid hormone stimulation: lack of acetazolamide effects. Bone 8:105–109; 1987.PubMedCrossRefGoogle Scholar
  33. 33.
    Noda, M.; Camilliere, J. J. In vivo stimulation of bone formation by transforming growth factor-β. Endocrinology 124:2991–2994; 1989.PubMedGoogle Scholar
  34. 34.
    Noda, M.; Rodan, G. A. Typeβ transforming growth factor (TGF-β) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells. J. Cell. Physiol. 133:426–437; 1987.PubMedCrossRefGoogle Scholar
  35. 35.
    Ohtsuki, M.; Massague, J. Evidence for the involvement of protein kinase activity in transforming growth factor-beta signal transduction. Mol. Cell. Biol. 12:261–265; 1991.Google Scholar
  36. 36.
    Oreffo, R. O. C.; Mundy, G. R.; Seyedin, S. M., et al. Activation of the bone-derived latent TGF beta complex by isolated osteoclasts. Biochem. Biophys. Res. Commun. 158:817–823; 1989.PubMedCrossRefGoogle Scholar
  37. 37.
    Pfeilschifter, J.; D’Souza, S. M.; Mundy, G. R. Effects of transforming growth factor-β on osteoblastic osteosarcoma cells. Endocrinology 121:212–218; 1987.PubMedCrossRefGoogle Scholar
  38. 38.
    Rizzino, A. Transforming growth factor-β: multiple effects on cell differentiation and extracellular matrices. Dev. Biol. 130:411–422; 1988.PubMedCrossRefGoogle Scholar
  39. 39.
    Roberts, A. B.; Anzano, M. A.; Lamb, L. C., et al. A new class of transforming growth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissue. Proc. Natl. Acad. Sci. USA 78:5339–5343; 1981.PubMedCrossRefGoogle Scholar
  40. 40.
    Rosen, D. M.; Stempien, S. A.; Thompson, A. Y., et al. Transforming growth factor-beta modulates the expression of osteoblast and chondroblast phenotypes in vitro. J. Cell Physiol. 134:337–346; 1988.PubMedCrossRefGoogle Scholar
  41. 41.
    Schultzcherry, S.; Murphyullrich, J. E. Thrombospondin causes activation of latent transforming growth factor-beta secreted by endothelial cells by a novel mechanism. J. Cell Biol. 122:923–932; 1993.CrossRefGoogle Scholar
  42. 42.
    Seitz, P. K.; Zhu, B. T.; Cooper, C. W. Effect of transforming growth factor-beta on parathyroid hormone receptor binding and cAMP formation in rat osteosarcoma cells. J. Bone Miner. Res. 7:541–546; 1992.PubMedGoogle Scholar
  43. 43.
    Seyedin, S. M.; Thompson, A. Y.; Bentz, H., et al. Cartilage-inducing factor-A. J. Biol. Chem. 261:5693–5695; 1986.PubMedGoogle Scholar
  44. 44.
    Sparks, R. L.; Scott, R. E. Transforming growth factor typeβ is a specific inhibitor of 3T3 mesenchymal stem cell differentiation. Exp. Cell Res. 165:345–352; 1986.PubMedCrossRefGoogle Scholar
  45. 45.
    Sporn, M. B.; Roberts, A. B. The transforming growth factor-betas: past, present and future. Ann. NY Acad. Sci. 593:1–6; 1990.PubMedCrossRefGoogle Scholar
  46. 46.
    Talley, D. J.; Roy, W. A.; Li, J. J. Behavior of primary and serially transplanted estrogen-dependent renal carcinoma cells in monolayer and in collagen gel culture. In Vitro 18:149–156; 1982.PubMedCrossRefGoogle Scholar
  47. 47.
    Tanaka, T.; Taniguchi, Y.; Gotoh, K., et al. Morphological study of recombinant human transforming growth factor beta 1-induced intramembranous ossification in neonatal rat parietal bone. Bone 14:117–123; 1993.PubMedCrossRefGoogle Scholar
  48. 48.
    Tashjian, Jr., A. H.; Voelkel, E. F.; Lazzaro, M., et al.α andβ human transforming growth factors stimulate postaglandin production and bone resorption in cultured mouse calvaria. Proc. Natl. Acad. Sci. USA 82:4535–4538; 1985.PubMedCrossRefGoogle Scholar
  49. 49.
    Tschan, T.; Bohme, K.; Conscience-Egli, M., et al. Autocrine or paracrine transforming growth factor-β modulates the phenotype of chick embryo sternal chondrocytes in serum-free agarose culture. J. Biol. Chem. 268:5156–5161; 1993.PubMedGoogle Scholar
  50. 50.
    Vivien, D.; Galera, P.; Lebrun, E., et al. TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease—possible involvement of pertussis toxin-sensitive pathway. J. Cell. Physiol. 150:291–298; 1992.PubMedCrossRefGoogle Scholar
  51. 51.
    Vivien, D.; Redini, F.; Galera, P., et al. Rabbit articular chondrocytes (RAC) express distinct transforming growth factor-β receptor phenotypes as a function of cell cycle phases. Exp. Cell Res. 205:165–170; 1993.PubMedCrossRefGoogle Scholar
  52. 52.
    Whitson, S. W.; Whitson, M. A.; Bowers, D. E., et al. Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells growth in vitro. J. Bone Miner. Res. 7:727–741; 1992.PubMedCrossRefGoogle Scholar
  53. 53.
    Wong, G. L.; Cohn, D. V. Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc. Natl. Acad. Sci. USA 72:3167–3171; 1975.PubMedCrossRefGoogle Scholar
  54. 54.
    Wu, L. N. Y.; Genge, B. R.; Ishikawa, Y., et al. Modulation of cultured chicken growth plate chondrocytes by transforming growth factor-beta-1 and basic fibroblast growth factor. J. Cell. Biochem. 49:181–198; 1992.PubMedCrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 1995

Authors and Affiliations

  • Deanna J. Talley-Ronsholdt
    • 1
  • Evelyn Lajiness
    • 1
  • Kishan Nagodawithana
    • 1
  1. 1.Department of Basic Health SciencesMarquette University School of DentistryMilwaukee

Personalised recommendations