Abstract
In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cellisolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.
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Gundle, R. and Beresford, J.N. (1995) The isolation and culture of cells from explants of human trabecular bone. Calcif. Tissue Int. 56, 8–10.
Mills, B.G., Singer, F.R., Weiner, L.P., and Holst, P.A. (1979) Long-term cultures of cells from bone affected by Paget’s disease. Calcif. Tissue Int. 29, 79–87.
Beresford, J.N., Gallagher, J.A., Poser, J.W., and Russell, R.G.G. (1984) Production of osteocalcin by human bone cells in vitro: Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, and glucocorticoids. Metab. Bone Dis. Relat. Res. 5, 229–234.
Ashton, B.A., Abdullah, F., Cave, J., et al. (1985) Characterization of cells with high alkaline phosphatase activity derived from human bone and marrow: Preliminary assessment of their osteogenicity. Bone 6, 313–319.
Beresford, J.N., Gallagher, J.A., and Russell, R.G.G. (1986) 1,25-dihydroxyvitamin D3 and human bone-derived cells in vitro: Effects on alkaline phosphatase, type I collagen and proliferation. Endocrinology 119, 1776–1785.
Weinreb, M., Shinar, D., and Rodan, G.A. (1990) Different patterns of alkaline phosphatase, osteopontin and osteocalcin expression in developing rat bone by in situ hybridization. J. Bone Miner. Res. 5, 831–842.
Gundle, R., Jouner, C., Bradley, J., Francis, M., Triffitt, J., and Beresford, J.N. (1994) Bone formation in vivo by cultured human marrow stromal and trabecular bone-derived cells. Bone 15, 230 abstract.
Robey, P. and Termine, J. (1985) Human bone cells in vitro. Calcif. Tissue Int. 37, 453–460.
Kirkpatrick, C.J., Wagner, M., Kohler, H., Bittinger, F., Otto, M., and Klein, C.L. (1997) The cell and molecular biological approach to biomaterial research: a perspective. J. Mater. Sci. Mater. Med. 8, 131–141.
Robey, P.G., Young, M.F., Flanders, K.C., et al. (1987) Osteoblasts synthesize and respond to TGF-β in vitro. J. Cell. Biol. 105, 457–463.
Grzesik, W.J. and Robey, P.G. (1994) Bone matrix RGD-glycoproteins: immunolocalization and their interaction with human primary osteoblastic bone cells in vitro. J. Bone Miner. Res. 9, 487–496.
Sinha, R.K., Morris, F., Shah, S.A., and Tuan, R.S. (1994) Surface composition of orthopaedic metals regulates cell attachment, spreading, and cytoskeletal organization of primary human osteoblasts in vitro. Clin. Orthop. 305, 258–272.
Noth, U., Osyczka, A.M., Tuli, R., Hickok, N.J., Danielson, K.G., and Tuan, R.S. (2002) Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells. J. Orthop. Res., 20, 1060–1069.
Hayflick, L. (1965) The limited in vitro lifetime of human diploid cell strains. Exp. Cell. Res. 37, 614–636.
Freshney, R.I. (1994) The culture environment: substrate, gas phase, medium and temperature. In Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, New York, pp. 71–101.
Caterson, E.J., Nesti, L.J., Danielson, K.G., and Tuan, R.S. (2002) Human marrow-derived mesenchymal progenitor cells. Mol. Biotechnol. 20, 245–256.
Sinha, R.K. and Tuan, R.S. (1996) Regulation of human osteoblast integrin expression by orthopaedic implant materials. Bone 18, 451–457.
Pittenger, M.F., Mackay, A.M., et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147.
Johnstone, B., Hering, M.H., Caplan, A.I., Goldberg, V.M., and Yoo, J.U. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp. Cell. Res. 238, 265–272.
Mackay, A.M., Beck, S.C., Murphy, J.M., Barry, F.P., Chichester, C.O., and Pittenger, M.F. (1998) Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4, 415–428.
Yoo, J.U., Barthel, T.S., Nishimura, K., et al. (1998) The chondrogenic potential of human bone marrow-derived mesenchymal progenitor cells. J. Bone Jt. Surg. 80, 1745–1757.
Wong, M.M., Rao, L.G., Ly, H., Hamilton, L., Tong, J., Aubin, J.E., Turksen, K., and Heersch, J.N.M. (1993) Osteoblastic cell lineage. In: Cellular and Molecular Biology of Bone (Noda, M., ed.), Academic Press, Tokyo, pp. 1–45.
Matsuyama, T., Lau, K-H.W., and Wergedal, J.E. (1990) Monolayer cultures of normal human bone cells contain multiple subpopulations of alkaline phosphatase positive cells. Calcif. Tissue Int. 47, 276–283.
Young, M.F., Kerr, J.M., Ibaraki, K., Heegaard, A.M., and Robey, P.G. (1992) Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. Clin. Orthop. 281, 275–294.
Mills, B.G., Singer, F.R., Weiner, L.P., and Holst, P.A. (1979) Long-term culture of cells from bone affected by Paget’s disease. Calcif. Tissue Int. 29, 79–87.
Gallagher, J.A., Beresford, J.N., Poser, J., Coulton, L.A., Kanis, J.A., and Russell, R.G.G. (1982) Human bone cell cultures—studies of steroid action. Calcif. Tissue Int. 34(Suppl), 33.
Beresford, J.N., Gallagher, J.A., Poser, J.W., and Russell, R.G.G. (1984) Production of osteocalcin by human bone cells in vitro. Effects of 1,25-(OH)2D3, 24,25-(OH)2D3, parathyroid hormone, and glucocorticoids. Metab. Bone Dis. Rel. Res. 5, 229–234.
Wergedal, J.E. and Baylink, D.J. (1984) Characterization of cells isolated and cultured from human bones. Proc. Soc. Exp. Biol. Med. 176, 27–31.
Bard, D.R., Dickens, M.J., Smith, A.U., and Zarek, J. M. (1972) Isolation of living cells from mature mammalian bone. Nature 236, 314.
Robey, P.G. (1995) Collagenase-treated trabecular bone fragments: A reproducible source of cells in the osteoblast lineage. Calcif. Tissue Int. 56(Suppl 1), S11-S12.
Brighton, C.T., Lorich, D.G., Kupcha, R., Reilly, T.M., Jones, A.R., and Woodbury, R.A. (1990) The pericyte as a possible osteoblast progenitor cell. Clin. Orthop. 275, 287–299.
Doherty, M.J., Ashton, B.A., Walsh, S., Beresford, J.N., Grant, M.E., and Canfield, A.E. (1998) Vascular pericytes express osteogenic potential in vitro and in vivo. J. Bone Miner. Res. 13, 828–838.
Reilly, T.M., Seldes, R., Luchetti, W., and Brighton, C.T. (1998) Similarities in the phenotypic expression of pericytes and bone cells. Clin. Orthop. 346, 95–103.
Diefenderfer, D.L. and Brighton, C.T. (2000) Microvascular pericytes express aggrecan message which is regulated by BMP-2. Biochem. Biophys. Res. Commun. 269, 172–178.
Manduca, P., Sanguineti, C., Pistone, M., et al. (1993) Differential expression of alkaline phosphatase in clones of human osteoblast-like cells. J. Bone Miner. Res. 8, 291–300.
Martin, T.J., Findlay, D.M., Heath, J.K., and Ng, K.W. (1993) Osteoblasts: differentiation and function. In: Handbook of Experimental Pharmacology (Mundy, J.R. and Martin, T.J., ed.), Springer, Berlin, pp. 149–183.
Stein, G.S. and Lian, J.B. (1995) Molecular mechanisms mediated proliferation-differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr. Rev. 4, 290–297.
Rodan, G.A. and Rodan, S.B. (1983) Expression of the osteoblastic phenotype. In: Annual Advances in Bone and Mineral Research, vol. 2 (Peck, W.A., ed.), Elsevier Science Publishers, Amsterdam, pp. 244–285.
Wong, M.M., Rao, L.G., Ly, H., et al. (1990) Long-term effects of physiologic concentrations of dexamethasone on human bone-derived cells. J. Bone Miner. Res. 5, 803–813.
Nakahara, H., Goldberg, V.M., and Caplan, A.I. (1991) Culture-expanded human periosteal-derived cells exhibit osteochondral potential in vivo. J. Orthop. Res. 9, 465–476.
Zuk, P.A., Zhu, M., Mizuno, H., et al. (2001) Multilineage Cells from Human Adipose Tissue: Implications for Cell-Based Therapies. Tissue Eng. 7, 211–228.
Wakitani, S., Goto, T., Pineda, S.J., Young, R.G., Mansour, J.M., Caplan, A.I., and Goldberg, V.M. (1994) Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone Joint Surg. Am. 76, 579–592.
Vogel, G. (2000) Cell biology. Stem Cells: New excitement, persistent questions. Science 290, 1672–1674.
Muschler, G.F., Boehm, C.A., and Easley, K.A. (1997) Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J. Bone Joint Surg. Am. 79-A, 1699–1709.
Muschler, G.F., Nitto, H., and Boehm, C.A. (2001) Age-and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors. J. Orthop. Res. 19, 117–125.
Iwamoto, I., Douchi, T., Kosha, S., Murakami, M., Fujino, T., and Nagata, Y. (2000) Relationship between serum leptin level and regional bone mineral density, bone metabolic markers in healthy women. Acta. Obstet. Gynecol. Scand. 79, 1060–1064.
Krischak, G.D., Augat, P., Wachter, N.J., Kinzl, L., and Claes, L.E. (1999) Predictive value of bone mineral density and Singh Index for the in vitro mechanical properties of cancellous bone in the femoral head. Clin. Biomech., 14, 346–351.
Chaffai, S., Peyrin, F., Nuzzo, S., Porcher, R., Berger, G., and Laugier, P. (2002) Ultrasonic characterization of human cancellous bone using transmission and backscatter measurements: relationships to density and microstructure. Bone 30, 229–237.
Hordon, L.D., Raisi, M., Aaron, J.E., Paxton, S.K., Beneton, M., and Kanis, J.A. (2000) Trabecular architecture in women and men of similar bone mass with and without vertebral fracture: I. Two-dimensional histology. Bone 27, 271–276.
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Tuli, R., Seghatoleslami, M.R., Tuli, S. et al. A simple, high-yield method for obtaining multipotential mesenchymal progenitor cells from trabecular bone. Mol Biotechnol 23, 37–49 (2003). https://doi.org/10.1385/MB:23:1:37
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DOI: https://doi.org/10.1385/MB:23:1:37