Medium Perfusion Enhances Osteogenesis by Murine Osteosarcoma Cells in Three-Dimensional Collagen Sponges

Oxford University Press (OUP) - Tập 14 Số 12 - Trang 2118-2126 - 1999
Stefan M. Mueller1, Shuichi Mizuno1, Louis C. Gerstenfeld2, Julie Glowacki3,4
1Department of Orthopedic Surgery, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A.
2Musculoskeletal Research Laboartory, Department of Orthopedic Surgery, Boston University Medical Center, Boston, Massachusetts, U.S.A.
3Department of Orthopedic Surgery, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts, U.S.A.
4Skeletal Biology Research Center, Massachusetts General Hospital, Harvard School of Dental Medicine, Boston, Massachusetts, U.S.A.

Tóm tắt

Abstract In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.

Từ khóa


Tài liệu tham khảo

Ecarot-Charrier, 1983, Osteoblasts isolated from mouse calvaria initiate matrix mineralization in culture, J Cell Biol, 96, 639, 10.1083/jcb.96.3.639

Bellows, 1990, Determination of the capacity of proliferation and differentiation for osteoprogenitor cells in the presence and absence of dexamethasone, Dev Biol, 140, 132, 10.1016/0012-1606(90)90060-V

Williams, 1980, Mineralization and metabolic response in serially passaged adult rat bone cells, Calcif Tissue Int, 30, 233, 10.1007/BF02408633

Whitson, 1984, Fetal bovine cell synthesize bone-specific matrix proteins, J Cell Biol, 99, 607, 10.1083/jcb.99.2.607

Schmidt, 1993, Long-term cultivation of human osteoblasts, Bone Miner, 20, 211, 10.1016/S0169-6009(08)80002-5

Shima, 1988, Microcarriers facilitate mineralization in MC3T3–E1 cells, Calcif Tissue Int, 43, 19, 10.1007/BF02555163

Glowacki, 1996, Cellular reactions to bone-derived material, Clin Orthop, 324, 47, 10.1097/00003086-199603000-00007

Sudo, 1986, Cell-Mediated Calcification and Matrix Vesicles, 291

Casser-Bette, 1990, Bone formation by osteoblast-like cells in a three-dimensional cell culture, Calcif Tissue Int, 46, 46, 10.1007/BF02555824

Schoeters, 1992, Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional sponges, Cell Prolif, 25, 587, 10.1111/j.1365-2184.1992.tb01462.x

Nishimoto, 1987, Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension culture, Calcif Tissue Int, 41, 274, 10.1007/BF02555229

Ernst, 1987, Osteoblastlike cells in a serum-free methylcellulose medium form colonies: Effects of insulin and insulin-like growth factor I, Calcif Tissue Int, 40, 27, 10.1007/BF02555725

Majmudar, 1991, 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, 869, 10.1002/jbmr.5650060813

Benayahu, 1994, Mineralization of marrow-stromal osteoblasts MBA-15 on three-dimensional carriers, Calcif Tissue Int, 55, 120, 10.1007/BF00297187

Inoue, 1997, The effect of aging on bone formation in porous hydroxyapatite: Biochemical and histological analysis, J Bone Miner Res, 12, 989, 10.1359/jbmr.1997.12.6.989

Gerstenfeld, 1996, Osteogenic potential of murine osteosarcoma cells: Comparison of bone-specific gene expression in in vitro and in vivo conditions, Lab Invest, 74, 895

Ducy, 1997, Osf2/Cbfa1: A transcriptional activator of osteoblast differentiation, Cell, 89, 747, 10.1016/S0092-8674(00)80257-3

Komori, 1997, Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts, Cell, 89, 755, 10.1016/S0092-8674(00)80258-5

Mizuno, 1996, Chondroinduction of human dermal fibroblasts by demineralized bone in three-dimensional culture, Exp Cell Res, 227, 89, 10.1006/excr.1996.0253

Mizuno, 1996, Three-dimensional composite of demineralized bone powder and collagen for in vitro analysis of chondroinduction of human dermal fibroblasts, Biomaterials, 17, 1819, 10.1016/0142-9612(96)00041-5

Schmidt, 1988, Establishment and characterization of osteogenic cell lines from a spontaneous murine osteosarcoma, Differentiation, 39, 151, 10.1111/j.1432-0436.1988.tb00090.x

West, 1985, A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue-cultured mammalian cells, Anal Biochem, 147, 289, 10.1016/0003-2697(85)90274-X

Lehrach, 1977, RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination, Biochemistry, 16, 4743, 10.1021/bi00640a033

Thomas, 1980, Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose, Proc Natl Acad Sci USA, 77, 5201, 10.1073/pnas.77.9.5201

Genovese, 1984, Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D, Biochemistry, 23, 6210, 10.1021/bi00320a049

Oldberg, 1986, Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell binding sequence, Proc Natl Acad Sci USA, 83, 8819, 10.1073/pnas.83.23.8819

Ogawa, 1993, PEBP2/PEA2 represents a new family of transcription factors homologous to the products of the Drosophila runt gene and the human AML1 gene, Proc Natl Acad Sci USA, 90, 6859, 10.1073/pnas.90.14.6859

Shih, 1999

Glowacki, 1998, Perfusion enhances functions of bone marrow stromal cells in three-dimensional culture, Cell Transplant, 7, 319, 10.1177/096368979800700310

Mizuno, 1998, Effects of physical stimulation on chondrogenesis in vitro, Mat Sci Engin C, 6, 301, 10.1016/S0928-4931(98)00068-X

Bujia, 1994, Culture of human cartilage tissue using a perfusion chamber, Laryngorhinootologie, 73, 577

Koller, 1993, Large-scale expansion of human stem and progenitor cels from bone marrow mononuclear cells in continuous perfusion cultures, Blood, 82, 378, 10.1182/blood.V82.2.378.378

Palsson, 1993, Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system, Biotechnology, 11, 368, 10.1038/nbt0393-368

Caldwell, 1991, Culture perfusion schedules influence the metabolic activity and granulocyte-macrophage colony-stimulating factor production rates of human bone marrow stromal cells, J Cell Physiol, 147, 344, 10.1002/jcp.1041470221

Merriman, 1995, The tissue-specific nuclear matrix protein, NMP-2, is a member of the AML/CBF/PEBP2/runt domain transcription factor family: Interactions with the osteocalcin gene promoter, Biochemistry, 34, 13125, 10.1021/bi00040a025

Schwartz, 1991, Rapid medium perfusion rate significantly increases the productivity and longevity of human bone marrow cultures, Proc Natl Acad Sci USA, 88, 6760, 10.1073/pnas.88.15.6760

Phillips, 1995, Genetic factors influencing murine hematopoietic productivity in culture, J Cell Physiol, 164, 99, 10.1002/jcp.1041640113

Van Zant, 1994, Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood, Blood Cells, 20, 482

Fung, 1993, Biomechanics: Mechanical Properties of Living Tissues, 2nd Ed., 66, 10.1007/978-1-4757-2257-4_3

Owan, 1997, Mechanotransduction in bone: Osteoblasts are more responsive to fluid forces than mechanical strain, Am J Physiol, 273, C810, 10.1152/ajpcell.1997.273.3.C810

Toma, 1997, 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, 1626, 10.1359/jbmr.1997.12.10.1626

Stanford, 1995, Proliferative and phenotypic responses of bone-like cells to mechanical deformation, J Orthop Res, 13, 664, 10.1002/jor.1100130505

Malekzadeh, 1998, Isolation of human osteoblast-like cells and in vitro amplification for tissue engineering, J Periodontol, 69, 1256, 10.1902/jop.1998.69.11.1256

Laurencin, 1996, A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal tissue regeneration, J Biomed Mater Res, 30, 133, 10.1002/(SICI)1097-4636(199602)30:2<133::AID-JBM1>3.0.CO;2-S

Thông tin
Thông tin xuất bản

Oxford University Press (OUP)

Tập 14 Số 12

2118-2126

Thông tin tác giả