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Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins

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  • Cell and Tissue Models
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Summary

In vitro propagation of osteoblasts in three-dimensional culture has been explored as a means of cell line expansion and tissue engineering purposes. Studies investigating optimal culture conditions are being conducted to produced bone-like material. This study demonstrates the use of collagen microcarrier beads as a substrate for three-dimensional cell culture. We have earlier reported that microcarriers consisting of cross-linked type I collagen support chondrocyte proliferation and synthesis of extracellular matrix. In this study, we investigated the use of collagen microcarriers to propagate human trabecular bone-derived osteoblasts. Aggregation of cell-seeded microcarriers and production of extracellular matrix-like material were observed after 5 d in culture. Expression of extracellular matrix proteins osteocalcin, osteopontin, and type I collagen was confirmed by messenger ribonucleic acid analysis, radioimmunoassay, and Western blot analysis. The efficient recovery of viable cells was achieved by collagenase digestion of the cell-seeded microcarriers. The collagen microcarrier spinner culture system provides an efficient method to amplify large numbers of healthy functional cells that can be subsequently used for further in vitro or transplantation studies.

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References

  • Bellows, C. G.; Aubin, J. E.; Heershce, J. N. M.; Antosz, M. E. Mineralized bone nodules formed in vitro from enzymatically released rat calavaria cell population. Calcif. Tissue Int. 38:143–154; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Benayahu, D.; Kompier, R. Shamay, A.; Kadouri, A.; Zipori, D.; Wientroub, S. Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers. Calcif. Tissue Int. 55(2):120–127; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Botchwey, E. A.; Pollack, S. R.; Levine, E. M.; Laurencin, C. T. Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system. J. Biomed. Mater. Res. 55(2):242–253; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Bouchet, B. Y.; Colon, M.; Polotsky, A.; Shikani, A. H.; Hungerford, D. S.; Frondoza, C. G. Beta-1 integrin expression by human nasal chondrocytes in microcarrier spinner culture. J. Biomed. Mater. Res. 52(4):716–724; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Brewer, J. W.; Hendershot, L. M.; Sherr, C. J.; Diehl J. A. Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression. Proc. Natl. Acad. Sci. USA 96(15):8505–8510; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Buckley, M. J.; Banes, A. J.; Jordan, R. D. The effects of mechanical strain on osteoblasts in vitro. J. Oral Maxillofac. Surg. 48(3):276–282; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Campbell, T. M.; Wong, W. T.: Mackie, E. J. Establishment of a model of cortical bone repair in mice [epub ahead of print] Calcif Tissue Int. 8: 2003.

  • Carvalho, R. S.; Scott, J. E.; Suga, D. M.; Yen, E. H. Stimulation of signal transduction pathways in osteoblasts by mechanical strain potentiated by parathyroid hormone. J. Bone Miner. Res. 9(7):999–1011; 1994.

    PubMed  CAS  Google Scholar 

  • Casser-Bette, M.; Murray, A. B.; Closs, E. I.; Erfle, V.; Schmidt, J. Bone formation by osteoblast-like cells in a three-dimensional cell culture. Calcif. Tissue Int. 46(1):46–56; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Clark, J. M.; Hirtenstein, M. D. Optimizing culture conditions for the production of animal cells in microcarrier culture. Ann. NY Acad. Sci. 369:33–46; 1981.

    Article  PubMed  CAS  Google Scholar 

  • Ducheyne, P.; Qiu, Q. Bioactive ceramics: the effect of surface reactivity on bone formation and bone cell function. Biomaterials 20(23, 24):2287–2303; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Ferrera, D.; Poggi, S.; Biassoni, C., et al. Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice. Bone 30(5):718–725; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Frondoza, C.; Sohrabi, A.; Hungerford, D. Human chondrocytes proliferate and produce matrix components in microcarrier suspension culture. Biomaterials 17(9):879–888; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Goldstein, A. S.; Zhu, G.; Morris, G. E.; Meszlenyi, R. K.; Mikos, A. G. Effect of osteoblastic culture conditions on the structure of poly(Dl-lactic-co-glycolic acid) foam scaffolds. Tissue Eng. 5(5):421–434; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Gooch, K. J.; Kwon, J. H.; Blunk, T.; Langer, R.; Freed, L. E.; Vunjak-Novakovic, G. Effects of mixing intensity on tissue-engineered cartilage. Biotechnol. Bioeng. 72(4):402–407; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Granet, C.; Laroche, N.; Vico, L.; Alexandre, C.; Lafage-Proust, M. H. Rotating-wall vessels, promising bioreactors for osteoblastic cell culture: comparison with other 3D conditions. Med. Biol. Eng. Comput. 36(4):513–519; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Harter, L. V.; Hruska, K. A.; Duncan, R. L. Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation. Endocrinology 136(2):528–535; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Hench, L. L.; Splinter, R. J.; Allen, W. C., et al. Bonding mechanisms at the interface of ceramic prosthetic materials. J. Biomed. Mat. Res. 2(1):117–141; 1971.

    Article  Google Scholar 

  • Hoffman, R. M. To do tissue culture in two or three dimensions? That is the question. Stem Cells 11(2):105–111; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Howard, G. A.; Turner, R. T.; Puzas, J. E.; Nichols, F.; Baylink, D. J. Bone cells in microspheres. JAMA 249(2):258–259; 1983.

    Article  PubMed  CAS  Google Scholar 

  • Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds. J. Biomed. Mater. Res. 36(1):17–28; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers. Biomaterials 19(15):1405–1412; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Kwon, Y. J.; Peng, C. A. Calcium-alginate gel bead cross-linked with gelatin as microcarrier for anchorage-dependent cell culture. Biotechniques 33(1):212–214, 216, 218; 2002.

    PubMed  CAS  Google Scholar 

  • Lahiji, A.; Sohrabi, A.; Hungerford, D. S.; Frondoza, C. G. Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes. J. Biomed. Mater. Res. 51:586–595; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Lajeunesse, D.; Frondoza, C.; Schoffield, B.; Sacktor, B. Osteocalcin secretion by the human osteosarcoma cell line MG-63. J Bone Miner. Res. 5(9):915–922; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Laurencin, C. T.; Attawia, M. A.; Elgendy, H. E.; Herbert, K. M. Tissue engineered bone-regeneration using degradable polymers: the formation of mineralized matrices. Bone 19(Suppl. 1):93S-99S; 1996a.

    Article  PubMed  CAS  Google Scholar 

  • Laurencin, C. T.; El-Amin, S. F.; Ibim, S. E.; Willoughby, D. A.; Attawia, M.; Allcock, H. R.; Ambrosio, A. A. A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration. J. Biomed. Mater. Res. 30(2):133–138; 1996b.

    Article  PubMed  CAS  Google Scholar 

  • Levine, D. W.; Thilly, W. G.; Wang, D. I. Parameters affecting cell growth on reduced charge microcarriers. Dev. Biol. Stand. 42:159–163; 1979.

    PubMed  CAS  Google Scholar 

  • Levine, D. W.; Wong, J. S.; Wang, D. I.; Thilly, W. G. Microcarrier cell culture: new methods for research-scale application. Somat. Cell Genet. 3(2):149–155; 1977.

    Article  PubMed  CAS  Google Scholar 

  • Lian, J. B.; Stein, G. S. Osteoblast biology. In: Marcus, R.; Feldman, D.; Kelsey, J., ed. Osteoporosis. San Diego, CA: Academic Press; 1996: 23–59.

    Google Scholar 

  • Livingston, T.; Ducheyne, P.; Garino, J. In vivo evaluation of a bioactive scaffold for bone tissue engineering. J. Biomed. Mater. Res. 62(1):1–13; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Majmudar, G.; Bole, D.; Goldstein, S. A.; Bonadio, J. Bone cell culture in a three-dimensional polymer bead stabilizes the differentiated phenotype and provides evidence that osteoblastic cells synthesize type III collagen and fibronectin. J. Bone Miner. Res. 6(8):869–881; 1991.

    PubMed  CAS  Google Scholar 

  • Masi, L.; Franchi, A.; Santucci, M., et al. Adhesion, growth, and matrix production by osteoblasts on collagen substrata. Calcif. Tissue Int. 51(3):202–212; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Ontiveros, C.; McCabe, L. R. Simulated microgravity suppresses osteoblast phenotype, Runx2 levels and AP-I transactivation. J. Cell. Biochem. 88(3):427–437; 2003.

    Article  PubMed  CAS  Google Scholar 

  • Pollack, S. R.; Meaney, D. F.; Levine, E. M.; Litt, M.; Johnston E. D. Numerical model and experimental validation of microcarrier motion in a rotating bioreactor. Tissue Eng. 6(5):519–530; 2000.

    Article  PubMed  CAS  Google Scholar 

  • SQiu, Q. Q.; Ducheyne, P.; Ayyaswamy, P. S. Fabrication, characterization and evaluation of bioceramic hollow microspheres used as microcarriers for 3-D bone tissue formation in rotating bioreactors. Biomaterials 20(11):989–1001; 1999.

    Article  Google Scholar 

  • Qiu, Q. Q.; Ducheyne, P.; Ayyaswamy, P. S. New bioactive, degradable composite microspheres as tissue engineering substrates. J. Biomed. Mater. Res. 52(1):66–76; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Qiu, Q. Q.; Ducheyne, P.; Ayyaswamy, P. S. 3D bone tissue engineered with bioactive microspheres in simulated microgravity. In Vitro Cell. Dev. Biol. 37A(3):157–165; 2001.

    Article  Google Scholar 

  • Qiu, Q.; Ducheyne, P.; Gao, H.; Ayyaswamy, P. Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel. Tissue Eng., 4(1):19–34; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Rattner, A.; Sabido, O.; Massoubre, C.; Rascle, F.; Frey, J.; Characterization of human osteoblastic cells: influence of the culture conditions. In Vitro Cell. Dev. Biol. 33A(10):757–762; 1997.

    Google Scholar 

  • Rucci, N.; Migliaccio, S.; Zani, B. M. Taranta, A.; Teti, A. Characterization of the osteoblast-like cell phenotype under microgravity conditions in the NASA-approved rotating wall vessel bioreactor (RWV). J. Cell. Biochem. 85(1):167–179; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Sautier, J. M.; Nefussi, J. R.; Forest, N. Mineralization and bone formation on microcarrier beads with isolated rat calvaria cell population. Calcif. Tissue Int. 50(6):527–532; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Service, R. F. Tissue engineers build new bone. Science 289(5484):1498–1500; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Sherr, C. J. D-type cyclins. Trends Biochem. Sci. 20(5):187–190; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Shima, M.; Seino, Y.; Tanaka, H.; Kurose, H.; Ishida, M.; Yabuuchi, H.; Kodama, H. Microcarriers facilitate mineralization in MC3T3-E1 cells. Calcif. Tissue Int 43(1):19–25; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Sikavitsas, V. I.; Bancroft, G. N.; Mikos, A. G. Formation of three-dimensional cell/polymer constructs for bone tissue engineering in a spinner flask and a rotating wall vessel bioreactor. J. Biomed. Mater. Res. 62(1):136–148; 2002.

    Article  PubMed  CAS  Google Scholar 

  • Tang, J. S.; Chao, C. F.; Au, M. K. Growth and metabolism of cultured bone cells using microcarrier and monolayer techniques. Clin. Orthop. 300:254–258; 1994.

    PubMed  Google Scholar 

  • Toma, C. D.; Ashkar, S.; Gray, M. L.; Schaffer, J. L.; Gerstenfeld, L. C. Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts. J. Bone Miner. Res. 12(10):1626–1636; 1997.

    Article  PubMed  CAS  Google Scholar 

  • van Wezel, A. L. Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature 216(110):64–65; 1967.

    Article  PubMed  Google Scholar 

  • Varani, J.; Bendelow, M. J.; Chun, J. H.; Hillegas, W. A. Cell growth on microcarriers: comparison of proliferation on and recovery from various substrates. J. Biol. Stand. 14(4):331–336; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki, M.; Nakajima, F.; Ogasawara, A.; Moriya, H.; Majeska, R. J.; Einhorn, T. A. Spatial and temporal distribution of CD44 and osteopontin in fracture callus. J. Bone Joint Surg. Br. 81(3):508–515; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Zaman, G.; Suswillo, R. F.; Cheng, M. Z.; Tavares, I. A.; Lanyon, L. E. Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture. J. Bone Miner. Res. 12(5):769–777; 1997.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Division of Arthritis Surgery, Department of Orthopaedic Surgery, The Good Samaritan Hospital, The Johns Hopkins University, Professional Office Building, Suite G-1, 5601 Loch Raven Boulevard, Baltimore, Maryland, 21239

    Michael Overstreet, Afshin Sohrabi, Anna Polotsky, David S. Hungerford & Carmelita G. Frondoza

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  1. Michael Overstreet
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  2. Afshin Sohrabi
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  3. Anna Polotsky
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  4. David S. Hungerford
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  5. Carmelita G. Frondoza
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Correspondence to Carmelita G. Frondoza.

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Overstreet, M., Sohrabi, A., Polotsky, A. et al. Collagen microcarrier spinner culture promotes osteoblast proliferation and synthesis of matrix proteins. In Vitro Cell.Dev.Biol.-Animal 39, 228–234 (2003). https://doi.org/10.1290/1543-706X(2003)039<0228:CMSCPO>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1543-706X(2003)039<0228:CMSCPO>2.0.CO;2

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