Journal of Periodontal and Implant Science

Korean Academy of Periodontology (대한치주과학회)
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The effect of rhBMP-2 on the osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts in vitro

치주인대세포와 치은섬유아세포의 분화에 미치는 rhBMP-2의 효과에 대한 연구

  • Kim, Hyeon-Jong (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Choi, Sang-Mook (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Ku, Young (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Rhyu, In-Chul (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Chung, Chong-Pyoung (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Han, Soo-Boo (Department of Periodontology, College of Dentistry, Seoul National University) ;
  • Lee, Yong-Moo (Department of Periodontology, College of Dentistry, Seoul National University)
  • 김현종 (서울대학교 치과대학 치주과학교실) ;
  • 최상묵 (서울대학교 치과대학 치주과학교실) ;
  • 구영 (서울대학교 치과대학 치주과학교실) ;
  • 류인철 (서울대학교 치과대학 치주과학교실) ;
  • 정종평 (서울대학교 치과대학 치주과학교실) ;
  • 한수부 (서울대학교 치과대학 치주과학교실) ;
  • 이용무 (서울대학교 치과대학 치주과학교실)
  • Published : 2002.06.30
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Abstract

BMP can induce ectopic bone formation when implanted into sites such as rat muscle and can greatly enhance healing of bony defects when applied exogenously. In addition, BMP stimulated osteoblastic differentiation in vitro in various types of cells. The aim of this study was to investigate the effect of recombinant human bone morphogenetic protein(rhBMP-2) on the proliferation and osteoblastic differentiation of human periodontal ligament cells and gingival fibroblasts. The cell number and alkaline phosphatase activity were measured in 3 experimental groups of human periodontal ligament cells and gingival fibroblasts (control group, rhBMP-2 50ng/ml group, and rhBMP-2 100ng/ml group) at 1 and 2 weeks after culture. At the same time, total RNA of cultured cells were extracted and reverse trascription polymerase chain reaction(RT-PCR) was performed to determine the expression of mRNA of bone matrix protein. RhBMP-2 had no effect on the cell proliferation of human periodontal ligament cells and gingival fibroblasts. Alkaline phosphatase activity was elevated significantly by rhBMP-2 in both cells. And periodontal ligament cells showed significantly higher alkaline phosphatase activity than gingival fibroblasts. ${\beta}$-actin, type I collagen, alkaline phosphatase, BMP-2 mRNA were expressed in all of the samples. Osteopontin, osteocalcin mRNA were expressed in all periodontal ligament cell groups, and rhBMP-2 50ng/ml group, rhBMP-2 100ng/ml group of 2 week culture period of gingival fibroblasts. Bone sialoprotein mRNA was only expressed in rhBMP-2 50ng/ml group and rhBMP-2 100ng/ml group of 2-week culture period. These results suggest that rhBMP-2 stimulates osteoblastic differentiation in human periodontal ligament cells and gingival fibroblasts in vitro.

Keywords

References

  1. Gottlow J. Principles of regener-ative therapy. In: Molecular pathogenesis of periodontal disease. ASM Press pp.403-414. 1994
  2. Cho MI, Matsuda N, Ramakrishnan PR. Differential regulation of periodontal ligament cell activities by platelet-derived growth factor, insulin-like growth factor I, and epidermal growth factor. In: Molecular pathogenesis of periodontal disease. ASM Press pp.403-414. 1994
  3. Otsuga K, Pitaru S, Overall CM, Aubin JE, Sodek J. Biochemical comparison of fibroblast populations from different periodontal tissues : characterization of matrix protein and collagenolytic enzyme synthesis. Biochem Cell Biol. 1988;66:167-176 https://doi.org/10.1139/o88-023
  4. Sommerman MJ, Archer SY, Imm GR, Foster RA. A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro. J Dent Res. 1988;67:66-70 https://doi.org/10.1177/00220345880670011301
  5. Mariotti A, Cochran DL. Characterization of fibroblasts derived from human periodontal ligament and gingival. J Periodontol. 1990;60:103-111
  6. Hou LT, Yaeger JA. Cloning and characterization of human gingival and periodontal ligament fibroblasts. J Periodontol. 1993;64:1209-1218 https://doi.org/10.1902/jop.1993.64.12.1209
  7. Giannopoulou C, Ciamasoni G. Functional characteristics of gingival and peri- odontal ligament fibroblasts. J Dent Res. 1996;75:895-902 https://doi.org/10.1177/00220345960750030601
  8. Wozney JM. The potential role of bone morphogenetic proteins in periodontal reconstruction. J Periodontol. 1995;66(6):506-510 https://doi.org/10.1902/jop.1995.66.6.506
  9. Cochran DL, Wozney JM. Biological mediators for periodontal regeneration. Periodontol 2000. 1999;19:40-58 https://doi.org/10.1111/j.1600-0757.1999.tb00146.x
  10. King GN, King N, Cruchhley AT, Wozney JM, Hughes FJ. Recombinat human bone morphogenetic protein-2 promotes wound healing in rat periodontal fenestration defects. J Dent Res. 1997;76:1460-1470 https://doi.org/10.1177/00220345970760080801
  11. Kuboki Y, Sasaki M, Saito A, Takita H, Kato H. Regeneration of periodontal ligament and cementum by BMP-applied tissue engineering. Eur J Oral Sci. 1998;106Suppl 1:197-203 https://doi.org/10.1111/j.1600-0722.1998.tb02176.x
  12. Ripamonti U, Reddi AH. Tissue engineering,morphogenesis and regeneration of the periodontal tissues by bone morphogenetic proteins. Crit Rev Oral Biol Med. 1997;8(2):154-163 https://doi.org/10.1177/10454411970080020401
  13. Higuchi T, Kinoshita A, Takahashi K, Oda S, Ishikawa I. Bone regeneration by recombinant human bone morphogenetic protein-2 in rat mandibular defects. An experimental model of defect filling. J Periodontol. 1999;70(9): 1026-1031 https://doi.org/10.1902/jop.1999.70.9.1026
  14. Urist MR, Strates BS. Bone mor-phogenetic protein. J Dent R. 1971;50:1392-1406 https://doi.org/10.1177/00220345710500060601
  15. Kobayashi M, Takicuchi T, Suzuki R, Yamaguchi A, Deguchi K, Shionome M, Miyazawa Y, Nishihara T, Nagumo M, Hasegawa K. Recombinant human bone morphog-enetic protein-2 stimulates osteo-blastic differentiation in cells isolated from human periodontal ligament. J Dent Res. 1999;78(10): 1624-1633 https://doi.org/10.1177/00220345990780100701
  16. Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, et al. The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2. Biochem Biophys Res Commun. 1990;172:295-299 https://doi.org/10.1016/S0006-291X(05)80208-6
  17. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, Ikeda T, et al. Bone morphogenetic protein-2 converts the differentiation path-way of C2C12 myoblasts into the osteoblast lineage. J Cell Biol. 1994;127:1755-1766 https://doi.org/10.1083/jcb.127.6.1755
  18. Hiraki Y, Inoue H, Shigeno C, Sanma Y, Bentz H, Rosen DM, et al. Bone morphogenetic proteins (BMP-2 and BMP-3) promote growth and expression of the differentiated phenotype of rabbit chondrocytes and osteoblastic MC3T3-E1 cells in vitro. J Bone Miner Res. 1991;6:1373-1385 https://doi.org/10.1002/jbmr.5650061215
  19. Takuwa y, Ohse C, Wang EA, Wozney JM, Yamashita K. Bone morphogenetic protein-2 stimu-lates alkaline phosphatase activity, and collagen synthesis in cultured osteblastic cells, MC3T3-E1. Biochem Biophys Res Commun. 1991;174:96-101 https://doi.org/10.1016/0006-291X(91)90490-X
  20. Yamaguchi A, Katagiri T, Ikeda T, Wozney JM, Rosen V, Wang EA, et al. Recombinant human bone morphogenetic protein-2 stimu-lates osteoblastic maturation and inhibits myogenic differentiation in vitro. J Cell Biol. 1991;113:681-687 https://doi.org/10.1083/jcb.113.3.681
  21. Yamaguchi A, Ishizuya T, Kintou N, Wada Y, Katagiri T, Wozney JM et al. Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differen-tiation of bone marrow-derived stromal cell line, ST2 and MC 3T3-G2/PA6. Biochem Biophys Res Commun. 1996;220:366-371 https://doi.org/10.1006/bbrc.1996.0411
  22. Thies RS, Bauduy M, Ashton BA, Kurtzberg L, Wozney JM, Rosen V. Recombinant human bone morpho-genetic protein-2 induces osteob-lastic differentiation in W-20-17 stromal cells. Endorinology. 1992; 130:1318-1324 https://doi.org/10.1210/en.130.3.1318
  23. Knutsen R, Wergedal JE, Sampath TK, Baylink DJ, Mohan S. Osteogenic protein-1 stimulates proliferation and differentiation of human bone cells in vitro. Biochem Biophys Res Commun. 1993;194:1352-1358 https://doi.org/10.1006/bbrc.1993.1973
  24. Wang EA, Israel DI, Kelly S, Luxenberg DP. Recombinant human bone morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth factors. 1993;9:57-71 https://doi.org/10.3109/08977199308991582
  25. Barboza EP, Duarte ME, Geolas L, Sorensen RG, Riedel GE, Wikesjo UM. Ridge augm entation following implantation of recombinant human bone morphogenetic protein-2 in the dog. J Periodontol. 2000;71(3): 488-486 https://doi.org/10.1902/jop.2000.71.3.488
  26. Howell TH, Fiorellini J, Jones A, Alder M, Nummikoski P, Lazaro M, Lilly L, Cochran D. A feasibility study evaluating rhBMP-2/absor- bable collagen sponge device for local alveolar ridge preservation or augmentation. Int J Periodontics Restorative Dent. 1997;17(2):124-139
  27. Cochran D. Jones A, Lilly L, Fiorellini J, Howell TH. Evaluation of recombinant human bone mor-phogenetic protein-2 in oral applications including the use of endosseous implants; 3-year results of a pilot study in humans. J Periodontol. 2000;71(8);1241-1257 https://doi.org/10.1902/jop.2000.71.8.1241
  28. Terheyden H, Jepsen H, Moller B, Tucker MM, Rueger DC. Sinus floor augmentation with simultaneous placement of dental implants using a combination of deproteinized bone xenografts and recombinant human osteogenic protein-1. A histometric study in miniature pigs. Clin Oral Implants Res. 1999; 10(6):510-521 https://doi.org/10.1034/j.1600-0501.1999.100609.x
  29. Bronckers ALJJ, Farach-Carson MC, van Waeren E, Burtler WT. Immunolocalization of osteopontin, osteocalcin and dentin sialoprotein during dental root formation and early cementogenesis in the rat. J Bone Miner Res. 1994;9:833-841 https://doi.org/10.1002/jbmr.5650090609
  30. MacNeil RL, Berry J, D'Errico J, Strayhorn C, Piotrowski B, Some-rman MJ. Role of two mineral-associated adhesion molecules, osteopontin and bone sialoprotein, during cementogenesis. Connect Tissue Res. 1995;33:1-7 https://doi.org/10.3109/03008209509016974
  31. Kagayama M, Li HC, Zhu J, Sasano Y, Hatakeyama Y, Mizoguchi I. Expression osteocalcin in cemen-toblasts forming acellular cemen-tum. J Periodont Res. 1997;32: 273-278 https://doi.org/10.1111/j.1600-0765.1997.tb00534.x
  32. Boskey AL. Matrix proteins and mineralization: an overview. Connect Tissue Res. 1996;35: 357-363 https://doi.org/10.3109/03008209609029212
  33. Chen TL, Bates RL, Dudley A, Hammonds RG, Amento EP. Bone morphogenetic protein-2b stimul-ation of growth and osteogenic phenotypes in rat osteblast-like cells: comparison with TGF-$\beta$1. J Bone Miner Res. 1991;6:1387-1393 https://doi.org/10.1002/jbmr.5650061216
  34. Hughes FJ, Collyer J, Stanfield M, Goodman SA. The effects of bone morphogenetic protein-2, -4, and -6 on differentiation of rat osteoblast cells in vitro. Endo-crinology. 1995;136:2671-2677
  35. Zheng MH, Wood DJ, Wysocki S, Papadimitriou JM, Wang EA. Recombinant human bone morpho-genetic protein-2 enhances expr-ession of interleukin-6 and transforming growth factor-$\beta$1 genes in normal human osteoblast-like cells. J Cell Physiol. 1994;159:76-82 https://doi.org/10.1002/jcp.1041590111
  36. Ahrens M, Ankenbauer T, Schroder D, Hollnagel A, Mayer H, Gross G. Expression of human bone morpho-genetic protein-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages. DNA Cell Biol. 1993;12:871-880
  37. Takiguchi T, Kobayashi M, Suzuki R, Yamaguchi A, Isatsu K, Nishihara T, et al. J Periodont Res. 1998;33:476-485
  38. Chen J, Shapiro HS, Sodek J. Developmental expression of bone sialoprotein mRNA in rat miner-alized connective tissues. J Bone Miner Res. 1992;7:987-997 https://doi.org/10.1002/jbmr.5650070816
  39. Chen J, McCulloch CA, Sodek J. Bone sialoprotein in developing porcine dental tissues; cellular expression and comparison of tissue localization with osteopontin and osteonectin. Arch Oral Biol. 1993;38:241-249 https://doi.org/10.1016/0003-9969(93)90034-J
  40. Lian J, Stein GS. Concepts of osteoblast growth and differentiation; Basic for modulation of bone cell development and tissue formation. Crit Rev Oral Biol Med. 1992;3: 269-305 https://doi.org/10.1177/10454411920030030501
  41. Yao KL, Todescan R, Sodek J. Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture. J Bone Miner Res. 1994;9:231-240 https://doi.org/10.1002/jbmr.5650090212
  42. Lecanda F, Avioli LV, Cheng SL. Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphorgenetic protein-2. J Cell Biochem. 1997;67:386-396 https://doi.org/10.1002/(SICI)1097-4644(19971201)67:3<386::AID-JCB10>3.0.CO;2-B
  43. Shibano K ,Watanabe J, Iwamoto M, Ogawa R, Kanamura S. Culture of stromal cells derived from medullary cavity of human long bone in the presence of 1,25-dihydroxyvitamin $D_3$, recombinant human bone morphorgenetic protein 2, or ipriflavone. Bone 1998;22:251-258 https://doi.org/10.1016/S8756-3282(97)00274-3
  44. Ivanovski S, Li H, Haase HR, Bartold PM. Expression of bone associated macromolecules by gingival and periodontal ligament fibroblasts. J Periodont Res. 2001;36(3):131-141 https://doi.org/10.1034/j.1600-0765.2001.360301.x
  45. Piche JE, Carnes DL Jr, Graves DT. Initial characterization of cells derived from human periodontia. J Dent Res. 1989;68:761767
  46. Abe T, Hara Y, Abe Y, Aida Y, Maeda K. Isolation of alkaline phosphatasepositive gingival fibroblasts from patients with chronic inflammatory periodontal disease. J Periodont Res. 1996; 31:285-293 https://doi.org/10.1111/j.1600-0765.1996.tb00495.x
  47. Carnes DL, Maeda CL, Graves DT. Cells with osteoblastic phenotypes can be explanted from human gingival and periodontal ligament. J Periodontol. 1997;68:701-70 https://doi.org/10.1902/jop.1997.68.7.701