Host-bacteria crosstalk at the dentogingival junction.

The dentogingival junction is of crucial importance in periodontal host defense both structurally and functionally. Oral bacteria exert a constant challenge to the host cells and tissues at the dentogingival junction. The host response is set up to eliminate the pathogens by the innate and adaptive defense mechanisms. In health, the commensal bacteria and the host defense mechanisms are in a dynamic steady state. During periodontal disease progression, the dental bacterial plaque, junctional epithelium (JE), inflammatory cells, connective tissue, and bone all go through a series of changes. The tissue homeostasis is turned into tissue destruction and progression of periodontitis. The classical study of Slots showed that in the bacterial plaque, the most remarkable change is the shift from gram-positive aerobic and facultatively anaerobic flora to a predominantly gram-negative and anaerobic flora. This has been later confirmed by several other studies. Furthermore, not only the shift of the bacterial flora to a more pathogenic one, but also bacterial growth as a biofilm on the tooth surface, allows the bacteria to communicate with each other and exert their virulence aimed at favoring their growth. This paper focuses on host-bacteria crosstalk at the dentogingival junction and the models studying it in vitro.

Epithelial cell response to challenge of bacterial lipoteichoic acids and lipopolysaccharides in vitro.

Accumulating dental plaque at the gingival margin contains lipoteichoic acids (LTAs) from the cell walls of gram-positive bacteria. In subgingival plaque associated with periodontal disease the amount of lipopolysaccharides (LPSs) from gram-negative bacteria increases. As the gingival junctional epithelium (JE) is an important structural and functional tissue, participating in the first line defence against apical advancement of dental plaque, this study examined the direct effects of LTAs (from Streptococcus mutans and S. sanguis) and LPSs (from Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Escherichia coli) on two epithelial cell lines (HaCaT and ERM) and a culture model for human JE. The cells were exposed to the LTAs or LPSs (10-50 microg/ml) for variable periods of time. None of the bacterial surface components had any effect on primary adhesion or on the epithelial attachment of the JE cultures. However, cell growth and mitotic activity were consistently reduced in all cultures treated with LTAs. In contrast, LPSs showed only slight or no effects on cell growth and mitotic activity depending on the epithelial cells used. This suggests that LPSs, despite their established role as modulators of inflammation, do not have direct harmful effects - at the concentrations found in dental plaque and gingival crevicular fluid - which would explain the mechanism of epithelial degeneration and detachment from the tooth surface. However, the LTAs appear to inhibit the renewal of epithelium and may thus contribute to degeneration of coronal JE and subgingival colonisation by periodontal pathogens.

Effect of short chain fatty acids on human gingival epithelial cell keratins in vitro.

Hemidesmosomal attachment of the junctional epithelial cells to the tooth and the ability of the attached cells to divide are essential features of the healthy dentogingival junction. Short chain fatty acids are bacterial metabolites associated with gingival inflammation and periodontal pockets. In vitro, short chain fatty acids have been shown to inhibit epithelial cell division and increase the density of their keratin filaments. This study examined these keratin changes by making use of human gingival keratinocyte cultures, gel electrophoresis and Western blot. Short chain fatty acids, butyrate and propionate, increased the relative amount of keratin proteins in the cells, most strikingly keratin K17. The distribution of K17 was further studied in a culture model for human junctional epithelium and in gingival biopsies. In butyrate-treated cultures of junctional epithelium, K17 expression was markedly increased and extended to the basal cells and to the cells mediating the attachment of the explant to the substratum. In clinically healthy gingiva, K17 was expressed predominantly in sulcular epithelium. The dividing basal cells and the cells attached to the tooth were negative. In advanced periodontitis, a strong reaction for K17 was localised to the pocket epithelium. The inhibition of epithelial cell division and the simultaneous upregulation of K17 in vitro, and the strong expression of this protein in detached pocket epithelium suggest a role for the short chain fatty acids in the degenerative process that leads to subgingival advancement of pathogens and, eventually, to periodontal pocket formation.

Lactoferrin impedes epithelial cell adhesion in vitro.

In the process of host defence against microbial challenge, neutrophils release granule contents with the potential side effect of damaging structural tissues. In the junctional epithelium such damage may contribute to the degeneration and renewal of the epithelial cells attached directly to the tooth (DAT cells), and subsequently to periodontal pocket formation. This study reports on lactoferrin, one of the substances released by neutrophils, and its effects on epithelial cell adhesion, growth, DNA synthesis and spreading of cell colonies at concentrations recorded in the crevicular fluid. We show that, in opposition to what has been reported on bacterial cells, lactoferrin has no effect on the DNA synthesis of attached epithelial cells in model systems attempting to simulate the DAT cells in vivo. However, both iron-saturated and unsaturated lactoferrin hampered cell adhesion, growth and spreading of cell colonies in a dose-dependent manner. These findings suggest that lactoferrin does not affect epithelial cell proliferation but it may have a role in delaying the repair of the DAT cell population during inflammation by interfering with cell adhesion.

Bacterial metabolites sodium butyrate and propionate inhibit epithelial cell growth in vitro.

The structural and functional barrier preventing the free advancement of microbial plaque subgingivally along the tooth surface is formed by the junctional epithelial (JE) cells directly attached to the tooth (DAT cells). The mechanism leading to degeneration of the DAT cells is not known. In the present study we examined the possible role of short chain fatty acids (SCFAs) on epithelial cells by making use of 2 epithelial cell cultures (HaCaT and ERM) and an explant culture model of human JE. The SCFAs butyrate and propionate were used in concentrations found in human plaque and gingival crevicular fluid (0.25-16.0 mM). The SCFAs had no effect on primary cell adhesion nor on the epithelial attachment apparatus (EAA). By contrast, even 0.25 mM of butyrate significantly retarded epithelial cell growth. Similar effects with propionate were first observed at concentrations higher than 1.0 mM. The retardation of epithelial cell growth was found to be due to inhibition of cell division. Furthermore, after butyrate treatment dense accumulations of intermediate filaments and cytoplasmic vacuolization were characteristically seen in cells adjacent to cells of normal appearance. This suggests that some cells of the growing epithelial cell population are more sensitive to the SCFAs than others, and agrees with previous reports on the DAT cells of periodontally-involved teeth in vivo. The results suggest that SCFAs are microbial factors that play a role in the initiation and progression of periodontal pocket formation by impairing epithelial cell function rather than having a direct effect on the EAA.