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Establishment of Periodontal Ligament Cell Lines from Temperature-Sensitive Simian Virus 40 Large T-antigen Transgenic Rats

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Abstract

Orthodontic tooth movement is controlled by various cell types in the periodontal ligament (PDL). Mechanical stresses, such as orthodontic force, are thought to induce differentiation of the mesenchymal cells in the PDL into osteoblasts and cementoblasts. The details of the process of differentiation, however, are not known, in part because adequate in vitro systems for their study do not yet exist. The purpose of this study was to establish and characterize immortalized PDL cell lines derived from the PDL of transgenic rats harboring the temperature-sensitive simian virus 40 T-antigen gene (TG rats). The PDL was removed from the molar roots of TG rats and incubated in tissue culture. Outgrowth cells from the PDL explant were passaged and cloned, depending on the shape of the colonies formed. The cell lines thus established were analyzed by reverse transcription–polymerase chain reaction for expression of type-I collagen, osteopontin, fibronectin, alkaline phosphatase (bone type), bone sialoprotein, the receptor activator of NF-κ B ligand, and osteoprotegerin. In addition, the capacity for formation of mineralized nodules was assessed by incubating cells in calcification-promoting medium at 37 °C. A total of 15 stable cell lines were successfully established and characterized. These cell lines were classified into six groups based on their pattern of gene expression at 33°C. Moreover, three of these clones were capable of forming calcified nodules. In conclusion, differential gene expression was demonstrated in 15 established PDL cell lines. Some cells had the potential to differentiate into cell types found in mineralized tissues, such as osteoblasts and cementoblasts, as well as cells expressing molecules that regulate osteoclast differentiation.

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References

  • Aronow M.A., Gerstenfeld L.C., Owen T.A., Tassinari M.S., Stein G.S. and Lian J.B.1990. Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvaria cells. J Cell Physiol 143: 213–221.

    Google Scholar 

  • Aubin J.E. and Triffitt T.J. 2002. Mesenchymal stem cells and osteoblast differentiation. In: Bilezikian P.J., Raisz G.L. and Rodan A.G. (eds), Principles of Bone Biology, 2nd edn, Vol. 1, Academic Press, CA, pp.59–81.

    Google Scholar 

  • Aubin J.E. 1998. Bone stem cells. J. Cell Biochem. Suppl 30/31: 73–82.

    Google Scholar 

  • Autillo-Touati A., Mallat M., Araud D., Moura Neto V., Vuillet J., Glowinski J., Seite R. and Prochiantz A. 1986. Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. III. A light and electron microscopic study. Brain Res 391: 33–47.

    Google Scholar 

  • Bellows C.G., Heersche J.N. and Aubin J.E.1990. Determination of the capacity for proliferation and differentiation of osteoprogenitor cells in the presence and absence of dexamethasone. Dev. Biol. 140: 132–138.

    Google Scholar 

  • Bosshardt D.D., Zalzal S., McKee M.D. and Nanci A. 1998. Developmental appearance and distribution of bone sialoprotein and osteopontin in human and rat cementum. Anat. Rec. 250: 13–33.

    Google Scholar 

  • Chen J.J., Jin H., Ranly D.M., Sodek J. and Boyan B.D. 1999. Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. J Bone Miner Res 14: 221–229.

    Google Scholar 

  • D'Errico J.A., Ouyang H., Berry J.E., MacNeil R.L., Strayhorn C., Imperiale M.J., Harris N.L., Goldberg H. and Somerman M.J.1999. Immortalized cementoblasts and periodontal ligament cells in culture. Bone 25: 39–47.

    Google Scholar 

  • Hou L.T. and Yaeger J.A.1993. Cloning and characterization of human gingival and periodontal ligament fibroblasts. J Periodontol 64: 1209–1218.

    Google Scholar 

  • Hunter G.K., Hauschka P.V., Poole A.R., Rosenberg L.C. and Goldberg H.A. 1996. Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 317: 59–64.

    Google Scholar 

  • Ikeda T., Nomura S., Yamaguchi A., Suda T. and Yoshiki S.1992. In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections. J Histochem Cytochem 40: 1079–1088.

    Google Scholar 

  • Ikegame M., Ishibashi O., Yoshizawa T., Shimomura J., Komori T., Ozawa H. and Kawashima H.2001. Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture. J Bone Miner Res 16: 24–32.

    Google Scholar 

  • Jat P.S. and Sharp P.A. 1989. Cell lines established by a temperature-sensitive simian virus 40 large-T-antigen gene are growth restricted at the nonpermissive temperature. Mol Cell Biol 9: 1672–1681.

    Google Scholar 

  • Kameoka J., Yanai N. and Obinata M. 1995. Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors. J Cell Physiol 164: 55–64.

    Google Scholar 

  • Kanzaki H., Chiba M., Shimizu Y. and Mitani H. 2001. Dual regulation of osteoclast differentiation by periodontal ligament cells through RANKL stimulation and OPG inhibition. J Dent Res 80: 887–891.

    Google Scholar 

  • Kapila Y.L., Lancero H. and Johnson P.W.1998. The response of periodontal ligament cells to fibronectin. J Periodontol 69: 1008–1019.

    Google Scholar 

  • Kataoka M., Shimizu Y., Kunikiyo K., Asahara Y., Yamashita K., Ninomiya M., Morisaki I., Ohsaki Y., Kido J.I. and Nagata T. 2000. Cyclosporin A decreases the degradation of type I collagen in rat gingival overgrowth. J Cell Physiol 182: 351–358.

    Google Scholar 

  • Kato K., Ishiguro S., Yamamoto H., Yanai N., Obinata M. and Tamai M. 1996. A retinal pigment epithelium-derived cell line from transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gen. Cell Struct Funct 21: 459–468.

    Google Scholar 

  • Kudo Y., Hiraoka M., Kitagawa S., Miyauchi M., Kakuo S., Zhao M., Ide T. and Takata T. 2002. Establishment of human cementifying fibroma cell lines by transfection with temperature-sensitive simian virus-40 T-antigen gene and hTERT gene. Bone 30: 712–717.

    Google Scholar 

  • Lekic P. and McCulloch C.A. 1996. Periodontal ligament cell population: The central role of fibroblasts in creating a unique tissue. Anat Rec 245: 327–341.

    Google Scholar 

  • Lukinmaa P.L., Mackie E.J. and Thesleff I. 1991. Immunohistochemical localization of the matrix glycoproteins-tenascin and the ED-sequence-containing form of cellular fibronectin-in human permanent teeth and periodontal ligament. J Dent Res 70: 19–26.

    Google Scholar 

  • MacLeod J.N., Burton-Wurster N., Gu D.N. and Lust G. 1996. Fibronectin mRNA splice variant in articular cartilage lacks bases encoding the V, III-15, and I-10 protein segments. J Biol Chem 271: 18954–18960.

    Google Scholar 

  • MacNeil R.L., Berry J., Strayhorn C. and Somerman M.J.1996. Expression of bone sialoprotein mRNA by cells lining the mouse tooth root during cementogenesis. Arch Oral Biol 41: 827–835.

    Google Scholar 

  • McCarthy T.L., Ji C. and Centrella M. 2000. Links among growth factors, hormones, and nuclear factors with essential roles in bone formation. Crit Rev Oral Biol Med 11: 409–422.

    Google Scholar 

  • McCulloch C.A. and Tenenbaum H.C. 1986. Dexamethasone induces proliferation and terminal differentiation of osteogenic cells in tissue culture. Anat Rec 215: 397–402.

    Google Scholar 

  • Merry K., Dodds R., Littlewood A. and Gowen M.1993. Expression of osteopontin mRNA by osteoclasts and osteoblasts in modelling adult human bone. J Cell Sci 104: 1013–1020.

    Google Scholar 

  • Myers D.E., Collier F.M., Minkin C., Wang H., Holloway W.R., Malakellis M. and Nicholson G.C.1999. Expression of functional RANK on mature rat and human osteoclasts. FEBS Lett 463: 295–300.

    Google Scholar 

  • Noda M. and Denhardt T.D. 2002. Osteopontin. In: Bilezikian P.J., Raisz G.L. and Rodan A.G. (ed.), Principles of Bone Biology, 2nd edn, Vol. 1, Academic Press, CA, pp.239–250.

    Google Scholar 

  • Nohutcu R.M., McCauley L.K., Koh A.J. and Somerman M.J. 1997. Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro. J Periodontol 68: 320–327.

    Google Scholar 

  • Okuyama R., Yanai N. and Obinata M. 1995. Differentiation capacity toward mesenchymal cell lineages of bone marrow stromal cells established from temperature-sensitive SV40 T-antigen gene transgenic mouse. Exp Cell Res 218: 424–429.

    Google Scholar 

  • Parkar M.H., Kuru L., O'Hare M., Newman H.N., Hughes F. and Olsen I. 1999. Retroviral transduction of human periodontal cells with a temperature-sensitive SV40 large T antigen. Arch Oral Biol 44: 823–834.

    Google Scholar 

  • Paul D., Hohne M., Pinkert C., Piasecki A., Ummelmann E. and Brinster R.L. 1988. Immortalized differentiated hepatocyte lines derived from transgenic mice harboring SV40 T-antigen genes. Exp Cell Res 175: 354–362.

    Google Scholar 

  • Roberts W.E. and Chase D.C. 1981. Kinetics of cell proliferation and migration associated with orthodontically-induced osteogenesis. J Dent Res 60: 174–181.

    Google Scholar 

  • Satomura K., Krebsbach P., Bianco P. and Gehron R.P. 2000. Osteogenic imprinting upstream of marrow stromal cell differentiation. J Cell Biochem 78: 391–403.

    Google Scholar 

  • Sugiyama N., Tabuchi Y., Horiuchi T., Obinata M. and Furusawa M. 1993. Establishment of gastric surface mucous cell lines from transgenic mice harboring temperature-sensitive simianvirus40largeT-antigengene.ExpCellRes209:382–387.

    Google Scholar 

  • Tso J.Y., Sun X.H., Kao T.H., Reece K.S. and Wu R. 1985. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: Genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13: 2485–2502.

    Google Scholar 

  • Yanai N., Satoh T., Kyo S., Abe K., Suzuki M. and Obinata M. 1991. A tubule cell line established from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene. Jpn J Cancer Res 82: 1344–1348.

    Google Scholar 

  • Yee J.A.1979. Response of periodontal ligament cells to orthodontic force: Ultrastructural identification of proliferating fibroblasts. Anat Rec 194: 603–614.

    Google Scholar 

  • Zohar R., Lee W., Arora P., Cheifetz S., McCulloch C. and Sode J.1997. Single cell analysis of intracellular osteopontin in osteogenic cultures of fetal rat calvarial cells. J Cell Physiol 170: 88–100.

    Google Scholar 

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Kubota, M., Chiba, M., Obinata, M. et al. Establishment of Periodontal Ligament Cell Lines from Temperature-Sensitive Simian Virus 40 Large T-antigen Transgenic Rats. Cytotechnology 44, 55–65 (2004). https://doi.org/10.1023/B:CYTO.0000043412.08814.80

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  • DOI: https://doi.org/10.1023/B:CYTO.0000043412.08814.80

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