Summary
The bone cells and fibroblasts from fetal rat calvaria can be isolated by collagenase digestion of the collagen matrix and separated into specific cell types by free-flow electrophoresis. The method involves injection of a specially prepared suspension of cells into a stream of buffer across which is maintained an electric field of 60 V/cm. The fetal bone cell types are differentially deflected toward the anode where they can be collected. Free-flow electrophoresis of this heterogenous cell preparation yields three distinguishable peaks which can be identified by morphologic, morphometric, and enzymatic characteristics. All three cell peaks have greater than 95% viability as judged by trypan blue exclusion and will grow to confluent monolayers in culture. The data indicate that these cell peaks may be comprised of osteoclasts and/or preosteoclasts, osteoblasts and/or preosteoblasts, and fibroblasts.
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References
Peck, W.A., Birge, S.J., Fedak, S.A.: Bone cells; biochemical and biological studies after enzymatic isolation, Science146:1476–1477, 1964
Dziak, R., Brand, J.S.: Calcium transport in isolated bone cells; bone cell isolation procedures, J. Cell Physiol.84:75–84, 1974
Peck, W.A., Carpenter, J., Messinger, K., DeBra, D.: Cyclic 3′5′ adenosine monophosphate in isolated bone cells: response to low concentrations of parathyroid hormone, Endocrinology92:692–697, 1973
Peck, W.A., Messinger, K., Carpenter, J.: Regulation of primidine ribonucleotide incorporation in isolated bone cells, J. Biol. Chem.246:4439–4446, 1971
Puzas, J.E., Brand, J.S.: Collagenolytic activity from isolated bone cells, Biochim. Biophys. Acta429:964–974, 1975
Smith, D.M., Johnston, C.C., Severson, A.R.: Studies of the metabolism of separated bone cells, Calcif. Tissue Res.11:56–69, 1973
Wong, G., Cohn, D.V.: Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells, Nature252:713–715, 1974
Yagiela, J.A., Woodbury, D.M.: Enzymatic isolation of osteoblasts from fetal rat calvaria, Anat. Rec.188:287–305, 1977
Peck, W.A., Burks, J.K., Wilkins, J., Rodan, S.B., Rodan, G.: Evidence for preferential effects of parathyroid hormone, calcitonin and adenosine on bone and periosteum, Endocrinology100:1357–1364, 1977
Hannig, K.: Free flow electrophoresis. In J.R. Norris, D.W. Ribbons, (eds.): Methods in Microbiology, Academic Press, London, 1971
Hannig, K.: Separation of cells and particles by continuous free flow electrophoresis. In D. Glick, R.M. Rosenbaum (eds.): Techniques of Biochemical and Biophysical Morphology, John Wiley & Sons, New York, 1972
Kono, T.: Purification and partial characterization of collagenolytic enzymes fromClostridium histolyticum, Biochemistry7:1106–1114, 1968
Boyle, W.: An extension of the51Cr-release assay for the estimation of mouse cytotoxins, Transplantation6:761–764, 1968
Bessey, O., Lowry, O., Brock, M.: A method for the rapid determination of alkaline phosphatase, J. Biol. Chem.164:321–329, 1946
Puzas, J.E., Goodman, D.B.P.: A rapid assay for cellular deoxyribonucleic acid, Anal. Biochem.86:50–55, 1978
Gay, C.V., Mueller, W.: Carbonic anhydrase and osteoclasts: localization by labeled inhibitor autoradiography, Science183:432–434, 1974
Wong, G., 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. U.S.A.72:3167–3171, 1975
Wergedal, J.E., Baylink, D.: Distribution of acid and alkaline phosphatase activity in undermineralized sections of the rat tibial diaphysis, J. Histochem. Cytochem.17:799–806, 1969
Fullmer, H.M.: Histochemical studies of the periodontium, J. Dent. Res. (Suppl. to No. 3)45:469–477, 1966
Owen, M.: Cell population kinetics of an osteogenic tissue; I and II, J. Cell Biol.19:19–44, 1963
Maren, T.H.: Carbonic anhydrase: chemistry, physiology, and inhibition, Physiol. Rev.47:595–781, 1967
Waite, L.C., Volkert, W., Kenney, A.: Inhibition of bone resorption by acetazolamide in the rat, Endocrinology87:1129–1139 1970
Minkin, C., Jennings, J.: Carbonic anhydrase and bone remodeling: sulfonamide inhibition of bone resorption in organ culture, Science176:1031–1033, 1972
Gay, C., Mueller, W.: Cellular localization of carbonic anhydrase in avian tissues by labeled inhibitor autoradiography, J. Histochem. Cytochem.21:693–702, 1973
Luben, R., Wong, G., Cohn, D.: Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts, Endocrinology99:526–534, 1976
Peck, W., Birge, S., Brandt, J.: Collagen synthesis by isolated bone cells: stimulation by ascorbic acidin vitro, Biochem. Biophys. Acta142:512–525, 1967
Dziak, R., Stern, P.: Responses of fetal rat bone cells and bone organ cultures to the ionophore, A23187, Calcif. Tissue Res.22:137–147, 1976
Wong, G., Luben, R., Cohn, D.: 1,25-Dihydroxycholecalciferol and parathormone: effects on isolated osteoclast-like and osteoblast-like cells, Science197:663–665, 1977
Luben, R., Wong, G., Cohn, D.: Parathormone-stimulated resorption of devitalised bone by cultured osteoclast-type bone cells, Nature265:629–630, 1977
Binderman, I., Duskin, D., Harrell, A., Katzir, E., Sachs, L.: Formation of bone tissue in culture from isolated bone cells, J. Cell Biol.61:427–439, 1974
Tonna, E.: The cellular complement of the skeletal system studied autoradiographically with tritiated thymidine during growth and aging, J. Biophys. Biochem. Cytol.9:813–824, 1961
Ham, A.: Joints. In Histology, pp. 448–465 J.B. Lippincott Co., Philadelphia, 1974
Mundy, G., Altman, A., Gondek, M., Bandelin, J.: Direct resorption of bone by human monocytes, Science196:1109–1111, 1977
Kahn, A., Stewart, C., Teitelbaum, S.: Contact-mediated bone resorption by human monocytes in vitro, Science199:988–990, 1978
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Puzas, J.E., Vignery, A. & Rasmussen, H. Isolation of specific bone cell types by free-flow electrophoresis. Calcif Tissue Int 27, 263–268 (1979). https://doi.org/10.1007/BF02441195
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DOI: https://doi.org/10.1007/BF02441195