Bacterial Endotoxin Inhibits Migration,Attachment,and Orientation of Human Gingival Fibroblasts in vitro and Delays Collagen Gel Contraction

Abstract

The purpose of this study was to assess the effect of endotoxin adsorbed to dental surfaces and to collagen type I on the migration, attachment, and orientation of human gingival fibroblasts (HGF). Transversely cut porcine tooth root slices (RS), 200 μm thick, were prepared. Half of the RS obtained were partially demineralized in EDTA. Half of the demineralized and non-demineralized RS were incubated with 400 μg/mL of endotoxin for 24 hr, whereas the other half were maintained in PBS and served as controls. Experimental and control RS were placed on confluent layers of HFG and cultured for six days.

Cell migration toward and cell attachment to the periphery of the RS and the formation of oriented cell sheets were assessed by means of photographic techniques. Additionally, six-day-old cultures were fixed and processed for SEM observation. In separate experiments, the effect of endotoxin on cell attachment to collagen type I and on contraction of three-dimensional collagen gels was assessed.

It was found that: (i) bacterial endotoxin inhibited migration and attachment of HGF to both demineralized and non-demineralized cementum and interfered with the development of oriented cellular structure ; (ii) the inhibitory effect was significantly more pronounced for non-demineralized than for demineralized cementum; (iii) the morphology of HGF attached to endotoxin-treated dental surfaces was altered compared with that of their controls; and (iv) bacterial endotoxin inhibited cell attachment to collagen type I and delayed the contraction of collagen gel.

Get full access to this article

View all access options for this article.

References

Adelson, L.J. ; Hanks, K.T. ; Ramfjord, S.P. ; and Caf-Fesse, R.G. (1980): In vitro Cytotoxicity of Periodontally Diseased Root Surfaces, J Periodontol 51:700-704.

Aleo, J.J. ; DeRenzis, F.A. ; and Farber, P.A. (1975): In vitro Attachment of Human Gingival Fibroblasts to Root Surfaces, J Periodontol 46:639-645.

Aleo, J.J. ; DeRenzis, F.A. ; Farber, P.A. ; and Varebon-Coeur, A.P. (1974): The Presence and Biologic Activity of Cementum-bound Endotoxin, J Periodontol 45:672-674.

Allison, A. ; Schorlemmer, H. ; and Bitter-Suermann, D. (1976): Activation of Complement by the Alternate Pathway as a Factor in the Pathogenesis of Periodontal Disease , Lancet 2:1001-1003.

Bellows, C.G. ; Melcher, A.H. ; and Aubin, J.E. (1981): Contraction and Organization of Collagen Gels by Cells Cultured from Periodontal Ligament, Gingiva and Bone Suggest Functional Differences Between Cell Types, J Cell Sci 50:299-314.

DeRenzis, F.A. and Chen, S.Y. (1983): Ultrastructural Study of Cultured Human Gingival Fibroblast Exposed to Endotoxin, J Periodontol 54:86-90.

Gelman R.A.; Poppke, D.C. ; and Piez, K.A. (1979): Collagen Fibril Formation in vitro, the Role of the Nonhelical Terminal Regions , J Biol Chem 254:11741-11745.

Johnson, D. ; Chen, C. ; Dombrowski, J. ; and Novotny, Y. (1976): Role of Bacterial Products in Periodontitis, J Periodont Res 11:349-359.

Karp, W. ; Sodek, J. ; Aubin, J.E. ; and Melcher, A.H. (1986): A Comparison of Fibronectin and Laminin Binding to Mineralized and Demineralized Tooth Surfaces, J Periodont Res 21:30-38.

10.

Kleinman, H.K. ; Wilkes, C.M. ; and Martin, G.R. (1981): Interaction of Fibronectin with Collagen Fibres , Biochem 30 :2325-2330.

11.

Lucas, R.M. ; Subramoniam, A. ; and Aleo, J.J. (1985): Intracellular Localization of Bacterial Lipopolysaccharide Using Avidin Biotin Complex Method at the Electron Microscopic Level, J Periodontol 56:553-557.

12.

Pitaru, S. ; Aubin, J.E. ; Gray, A. ; Metzger, Z. ; and Melcher, A.H. (1984a): Cell Migration, Attachment and Orientation in vitro are Enhanced by Partial Demineralization of Dentine and Cementum and are Inhibited by Bacterial Endotoxin, J Periodont Res 19:661-665.

13.

Pitaru, S. ; Gray, A. ; Aubin, J.E. ; and Melcher, A.H. (1984b): The Influence of the Morphological and Chemical Nature of Dental Surfaces on the Migration, Attachment and Orientation of Human Gingival Fibroblasts in vitro, J Periodont Res 19:408-418.

14.

Pitaru, S. and Melcher, A.H. (1983): Orientation of Gingival Fibroblasts and Newly synthesized Collagen Fibres in vitro: Resemblance to Transseptal and Dentogingival Fibres, J Periodont Res 18:483-500.

15.

Porvaznik, M. ; Cohen, M.E. ; Bockowski, S.W. ; Muller, E.J. ; and Wirthlin, M.R., Jr. (1982): Enhancement of Cell Attachment to Substrate Coated with Oral Bacterial Endotoxin by Plasma Fibronectin, J Periodont Res 17:154-168.

16.

Selvig, K.A. ; Hofstad, T. ; and Kristoffersen, T. (1971): Electron-microscopic Demonstration of Bacterial Lipopolysaccharides in Dental Plaque Matrix, Scand J Dent Res 79:409-421.

17.

Simon, B. ; Goldman, H. ; Ruben, M. ,; and Baker, E. (1970): The Role of Endotoxins in Periodontal Disease. II. Correlation of the Amount of Endotoxin in Human Gingival Exudate with the Clinical Degree of Inflammation, J Periodontol 41:81-86.

18.

Simon, B. ; Goldman, H. ; Ruben, M. ; and Baker, E. (1971): The Role of Endotoxins in Periodontal Disease. III. Correlation of the Amount of Endotoxin in Human Gingival Exudate With the Histologic Degree of Inflammation, J Periodontol 42:210-216.

19.

Simon, B. ; Goldman, H. ; Ruben, M. ; and Baker, E. (1972): The Role of Endotoxins in Periodontal Disease. IV. Bacteriological Analyses of Human Gingival Exudate as Related to the Quantity of Endotoxin and Clinical Degree of Inflammation, J Periodontol 43:468-175.

20.

Singer, R.E. and Dutton, W.G. (1979): A Comparison of the Effects of Endotoxin Upon Fibroblast Proliferation and Macromolecular Synthesis, J Dent Res 58:1634-1639.

21.

Snyderman, R. (1973): Humoral and Bacterial Mediators of Inflammation . In: Comparative Immunology of the Oral Cavity, S.E. Mergenhagen and H.W. Scherp , Eds., Washington, DC: United States Department of Health, Education and Welfare, pp. 183-191.

22.

Stevens, R.H. ; Sela, M.N. ; McArthur, W.P. ; and Ham-Mond, B.F. (1980): Biological and Chemical Characterization of Endotoxin from Capnocytophaga sputigena, Infect Immun 27:246-254.

23.

Wahl, L. ; Wahl, S. ; Mergenhagen, S. ; and Martin, G.R. (1974): Collagenase Production by Endotoxin Activated Macrophages, Proc Natl Acad Sci USA 71:3598-3603.