Effects of Porphyromonas gingivalis extracts on the function of mouse calvarial primary osteoblastic cells

Porphyromonas gingivalis 추출물이 마우스 두개골 일차 조골세포의 기능에 미치는 효과

  • Yun, Jeong-Ho (Department of Periodontology, Oral Science Research Institute, College of Dentistry, Yonsei University) ;
  • Choi, Seong-Ho (Department of Periodontology, Oral Science Research Institute, College of Dentistry and Brain Korea 21 project for Medical Science, Yonsei University) ;
  • Cho, Kyoo-Sung (Department of Periodontology, Oral Science Research Institute, College of Dentistry and Brain Korea 21 project for Medical Science, Yonsei University) ;
  • Chai, Jung-Kiu (Department of Periodontology, Oral Science Research Institute, College of Dentistry, Yonsei University) ;
  • Kim, Chong-Kwan (Department of Periodontology, Oral Science Research Institute, College of Dentistry and Brain Korea 21 project for Medical Science, Yonsei University) ;
  • Kim, Chang-Sung (Department of Periodontology, Oral Science Research Institute, College of Dentistry, Yonsei University)
  • 윤정호 (연세대학교 치과대학 치주과학교실, 구강과학연구소) ;
  • 최성호 (연세대학교 치과대학 치주과학교실, 구강과학연구소, BK21 의과학사업단) ;
  • 조규성 (연세대학교 치과대학 치주과학교실, 구강과학연구소, BK21 의과학사업단) ;
  • 채중규 (연세대학교 치과대학 치주과학교실, 구강과학연구소) ;
  • 김종관 (연세대학교 치과대학 치주과학교실, 구강과학연구소, BK21 의과학사업단) ;
  • 김창성 (연세대학교 치과대학 치주과학교실, 구강과학연구소)
  • Published : 2003.12.31

Abstract

Porphyromonas gingivalis has been implicated as an important periodontophathic bacterium in the etiology and progression of periodontal diseases. It has been reported that P.gingivalis may mediate periodontal destruction not only directly through its virulence factors, but also indirectly by including complex host mediated inflammatory reponses. The purpose of this study was t o evaluate the effects of P.gingivalis on the bone formation and resorption by osteoblasts. For this purpose, after determining the concentration below which sonicated P.gingivalis extracts (SPEs) have no cytotoxicity on mouse calvarial primary osteoblastic (POB) cells, we investigated the effects of SPEs on the alkaline phosphatase (ALP) activity, matrix metalloproteinase (MMP) expression (MMP-2, -9, 13), and prostaglandin $E_2$ ($PGE_2$) release in POB cells by treatment with SPEs below that concentration. The results were as follows; 1. SPEs showed no cytotoxic effect on POB cells up to a concentration of 1 ${\mu}m$/ml. 2. The treatment with SPEs reduced ALP activity in a dose-dependent manner in POB cells, In addition, when we investigated the effect of SPEs (1 ${\mu}m$/ml) on ALP activity for different exposure periods, statistically significant inhibition of ALP activity was shown at 2 days of exposure, and further significant inhibition occurred by extending the periods of exposure. 3. The treatment with SPEs stimulated the gene expression of MMP-9 in POB cells. 4. The pre-treatment with SPEs increased the amount of $PGE_2$ released in POB cells. In summary, the present study shows that P.gingivalis could inhibit osteogenesis and stimulate bone resorption not only by reducing ALP activity but also by increasing MMP-9 mRNA expression in osteoblasts, possibly through an endogenous $PGE_2$ pathway. In addition, our results suggest that if P.gingivalis affects osteoblasts in early differentiation stage, such effects by P. gingivalis could be irreversible.

Keywords

References

  1. Loesche WJ, Syed SA, Schmidt ES, Morrison EC. Bacterial profiles of subgingival plaques in periodontitis. J Periodontol 1985;447-456
  2. Dahlen GG. Black-pigmented gram-negative anaerobes in periodontitis. FEMS Immunol Med Microbiol 1993;6:181-192 https://doi.org/10.1111/j.1574-695X.1993.tb00323.x
  3. Tanner AC, Socransky SS, Goodson JM. Microbiota of periodontal pockets losing crestal alveolar bone. J Periodont Res 1984;19:279-291 https://doi.org/10.1111/j.1600-0765.1984.tb00819.x
  4. Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev 1988;52:134-152
  5. Rodenburg JP, van Winkelhoff AJ, Winkel EG, et al. Occurence of Bacteroides gingivalis, Bacteroides intermedius, and Actinobacillus actinomycetemcomitans in severe periodontitis in relation to age and treatment history. J Clin Periodontol 1990;17:392-399 https://doi.org/10.1111/j.1600-051X.1990.tb00036.x
  6. Christersson LA, Rosling BG, Dunford RG, et al. Monitoring of subgingival Bacteroides gingivalis and Actinobaillus actinomycetemcomitans in the management of advanced periodontitis. Adv Dent Res. 1988;2:382-388 https://doi.org/10.1177/08959374880020023301
  7. Uitto VJ, Larjava H, Heino J, Sorsa T. A protease of Bacteroides gingivalis degrades cell surface and matrix glycoproteins of cultured gingival fibroblasts and induces secretion of collagenase and plasminogen activator. Infect Immun 1989;57:213-218
  8. Meghji S, Henderson B, Nair S, Wilson M. Inhibition of bone DNA and collagen production by surface-associated material from bacteria implicated in the pathology of periodontal disease. J Periodontol 1992;63:736-742 https://doi.org/10.1902/jop.1992.63.9.736
  9. Morioka M, Hinode D, Nagata A, et al. Cytotoxicity of Porphyromonas gingivalis toward cultured human gingival fibroblasts. Oral Microbial Immunol 1993;8:203-207 https://doi.org/10.1111/j.1399-302X.1993.tb00560.x
  10. Birkedal-Hansen H. Role of cytokines and inflammatory mediators in tissue destruction. J Periodont Res 1993;28:500-510 https://doi.org/10.1111/j.1600-0765.1993.tb02113.x
  11. Sismey-Durrant HJ, Hopps RM. Effect of lipopolysaccharide from Porphyromonas gingivalis on prostaglandin E2 and interleukin-l-$\beta$ release from rat periosteal and human gingival fibroblasts in vitro. Oral Microbiol Immunol 1991;6:378-380 https://doi.org/10.1111/j.1399-302X.1991.tb00510.x
  12. Steffen MJ, Holt SC, Ebersole JL. Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts. Oral Microbiol Immunol 2000;15:172-180 https://doi.org/10.1034/j.1399-302x.2000.150305.x
  13. Noguchi K, Yanai M, Shitashige M, Nishihara T, Ishikawa I. Cyclooxygenase-2-dependent prostaglandin production by peripheral blood monocytes stimulated with lipopolysaccharides isolated from periodontopathogenic bacteria. J Periodontol 2000;71:1575-1582 https://doi.org/10.1902/jop.2000.71.10.1575
  14. Loomer PM, Sigusch B, Sukhu B, Ellen RP, Tenenbaum HC. Direct effects of metabolic products and sonicated extracts of Porphyromonas gingivalis 2561 on osteogenesis in vitro. Infect Immun 1994;62:1289-1297
  15. Loomer PM, Ellen RP, Tenenbaum HC. Characterization of inhibitory effects of suspected periodontopathogens on osteogenesis in vitro. Infect Immun 1995;63:3287-3296
  16. Kadono H, Kido J, Kataoka M, Yamauchi N, Nagata T. Inhibition of osteoblastic cell differentiation by lipopolysaccharide extract from Porphyromonas gingivslis. Infect Immun 1999;67:2841-2846
  17. Sorsa T, Ingman T, Suomalainen K, et al. Identification of proteases from periodontopathogenic bacteria as activators of latent human neutrophil and fibroblast-type interstitial collagenases. Infect Immun 1992;60:4491-4495
  18. Fravalo P, Menard C, Bonnaure-Mallet M. Effect of Poyphyromonas gingivalis on epithelial cells MMP-9 typeIV collagenase production. Infect Immun 1996;64:4940-4945
  19. DeCarlo AA, Windsor LJ, Bodden MK, Birkedal-Hansen B, Birekdal-Hansen H. Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis. J Dent Res 1997;76:1260-1270 https://doi.org/10.1177/00220345970760060501
  20. Chang YC, Yang SF, Lai CC, Liu JY, HsiehYS. Regulation of matrix metalloproteinase production by cytokines, pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures. J Periodont Res 2002;37:196-203 https://doi.org/10.1034/j.1600-0765.2002.00663.x
  21. Pattamapun K, Tiranathanagul S, Yongchaitrakul T, Kuwatanasuchat J, Pavasant P. Activation of MMP-2 by Porphyromonas gingivalis in human periodontal ligament cells. J Periodont Res 2003;38:115-121 https://doi.org/10.1034/j.1600-0765.2003.01650.x
  22. Baron R. Molecular mechanisms of bone resorption by the osteoclast. Anat Rec 1989;224:317-324 https://doi.org/10.1002/ar.1092240220
  23. Delaiss JM, Engsig MT, Everts V, et al. Proteinases in bone resorption: obvious and less obvious roles. Clin Chim Acta 2000;291:223-234 https://doi.org/10.1016/S0009-8981(99)00230-2
  24. Birkedal-Hansen H. Role of matrix metalloproteinases in human periodontal diseases. J Periodontol 1993;64:474-484
  25. Reynolds JJ, Meikle MC. Mechanisms of connective tissue matrix destruction in periodontitis. Periodontol 2000 1997:144-157
  26. Heath JK, Atkinson SJ, Meikle MC, Reynolds JJ. Mouse osteoblasts synthesize collagenase in response to bone resorbing agents. Biochim Biophys Acta 1984;802:151-154 https://doi.org/10.1016/0304-4165(84)90046-1
  27. Meikle MC, Bord S, Hembry RM, et al. Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines. J Cell Sci 1992;103:1093-1099
  28. Hill PA. Docherty AJP, Bottomley KM, et al. Inhibition of bone resorption in vitro by selective inhibitors of gelatinase and collagenase. Biochem J 1995;308:167-175 https://doi.org/10.1042/bj3080167
  29. Kusano K, Miyaura C, Inada M et al. Regulation of matrix metalloproteinases (MMP-2, -3, -9, and -13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption. Endocrinology 1998;139:1338-1345 https://doi.org/10.1210/en.139.3.1338
  30. Mizutani A, Sugiyama I, Kuno E, Matsunaga S, Tsukagoshi N. Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells. J Bone Miner Res 2001;16:2043-2049 https://doi.org/10.1359/jbmr.2001.16.11.2043
  31. Ohm K, Albers H-K, Lisboa BP. Measurement of eight prostaglandins in human gingival and periodontal disease using high pressure liquid chromatography and radioimmunoassay. J Periodont Res 1984;19:501-511 https://doi.org/10.1111/j.1600-0765.1984.tb01305.x
  32. Klein DC, Raisz LG. Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 1970;86:1436-1440 https://doi.org/10.1210/endo-86-6-1436
  33. Suda T, Jimi E, Nakamura I, Takahashi N. Role of 1,25-dihydoxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 1997;282:223-235 https://doi.org/10.1016/S0076-6879(97)82110-6
  34. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63 https://doi.org/10.1016/0022-1759(83)90303-4
  35. Mallet F, Oriol G, Mary C, Verrier B, Mandrand B. Continuous RT-PCR using AMV-RT and Taq DNA polymerase: characterization and comparison to uncoupled procedures. BioTechniques 1995;18:678-687
  36. Uchida M, Shima M, Shimoaka T, et al. Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells. J Cell Physiol 2000;185:207-214 https://doi.org/10.1002/1097-4652(200011)185:2<207::AID-JCP5>3.0.CO;2-J
  37. Bellows CG, Aubin JE, Heersche JN. Initiation and progression of mineralization of bone nodules formed in vitro: the role of alkaline phosphatase and organic phosphatase. Bone and Mineral 1991;14:27-40 https://doi.org/10.1016/0169-6009(91)90100-E
  38. Woldarski KH, Reddi AH. Alkaline phosphatase as a marker of osteoinductive cells. Calcif Tissue Int 1986;39:382-385 https://doi.org/10.1007/BF02555175
  39. Matsuda N, Takemura A, Taniguchi S, Amano A, Shizukuish S. Porphyromonas gingivalis reduces mitogenic and chemotactic responses of human periodontal ligament cells to platelet-derived growth factor in vitro. J Periodontol 1996;67:1335-1341 https://doi.org/10.1902/jop.1996.67.12.1335
  40. Millar SJ, Goldstein EG, Levine MJ, Hausmann E. Modulation of bone metabolism by two chemically distict lipopolysaccharide fractions from Bacteroides gingivalis. Infect Immun 1986;51:302-306
  41. Sismey-Durrant HJ, Atkinson SJ, Hopps RM, Heath JK. The effect of lipopolysaccharide from bacteroides gingivalis and muramyl dipeptide on osteoblast collagenase release. Calcif Tissue Int 1989;44:361-363 https://doi.org/10.1007/BF02556318
  42. Goodson JM, McClatchy K, Revell C. Prostaglandin-induced resorption of the adult rat calvaria. J Dent Res 1974;53:670-677 https://doi.org/10.1177/00220345740530032601
  43. Raisz LG, Koolemans-Beynen AR. Inhibition of bone collagen synthesis by prostaglandin Ea in organ culture. Prostaglandins 1974;8:377-385 https://doi.org/10.1016/0090-6980(74)90113-0
  44. Igarashi K, Hirafuji M, Adachi H, Shinoda H, Mitani H. Role of endogenous PGE2 in osteoblastic functions of a clonal osteoblast-like cell, MC3T3-E1. Prostaglandins Leukot Essent Fatty Acids. 1994;50:169-172 https://doi.org/10.1016/0952-3278(94)90140-6