Faculty Bibliography

Short-chain carboxylic acids (e.g., lactic acid, propionic acid, butyric acid) are metabolic by-products of bacterial metabolism which can accumulate in the gingival crevice. It is of no small consequence, therefore, that 1- to 5-mM concentrations of these acids exhibit significant biological activity, including the ability to alter cell proliferation and gene expression in cells of importance to the periodontium. This communication reports on the in vivo concentrations of propionic and butyric acid in the gingival crevices of periodontal subjects with severe and mild disease. The results indicated that severely diseased subjects exhibited a > 10-fold increase in the mM concentration of these acids when compared with mildly diseased subjects (mean propionic acid-severe = 9.5 +/- 1.8 mM, and mild = 0.8 +/- 0.3 mM; mean butyric acid-severe = 2.6 +/- 0.4 mM, and mild = 0.2 +/- 0.04 mM). These differences (mean +/- SE) were significant (p < 0.0001). The propionic and butyric acid concentrations were below detection limits in healthy sites of mildly diseased subjects. The propionic and butyric acid concentrations also associated significantly with clinical measures of disease severity (e.g., pocket depth, attachment level) and inflammation (e.g., subgingival temperature, % of sites bleeding when probed), and with the total microbial load (all p < 0.05). Taken together, these data suggest that short-chain carboxylic acids play a mediating role in periodontal disease pathogenesis.

This communication reviews the effects of short-chain carboxylic acids on human cells of importance to the periodontium. The central hypothesis is that these acids can alter both cell function and gene expression, and thus contribute to the initiation and prolongation of gingival inflammation. Short-chain carboxylic acids [CH3-(CH2)x-COOH, x < 3] are metabolic intermediates with a broad range of apparently paradoxical biological effects. For example, lactic acid (CH3-CHOH-COOH), a 3-carbon alpha-hydroxy-substituted acid, is widely recognized for its cariogenicity. Lactic acid, however, also occurs in tropical fruits, and is the active ingredient in a variety of anti-wrinkle creams developed by dermatologists. In marked contrast, the unsubstituted 3-carbon propionic acid (CH3-CH2-COOH) is used as a food preservative and is the active principle for one class of non-steroidal anti-inflammatory agents. Interestingly, the addition of one carbon to propionic acid dramatically changes the biological effects. The unsubstituted 4-carbon butyric acid (CH3-CH2-CH2-COOH) is used by hematologists as a de-differentiating agent for the treatment of sickle cell anemia, but by oncologists as a differentiating agent for cancer chemotherapy. Finally, acting either individually or in concert, these acids can increase vascular dilation. Clearly, these acids, while metabolically derived, have a number of very divergent activities which are cell-type-specific (Fig. 1). It may be telling that periodontal bacteria produce these acids in millimolar concentrations, and that these bacteria can be characterized by their acid production profiles. It is no less interesting that these acids occur in the gingival crevices of human subjects with severe periodontal disease at millimolar levels which are > 10-fold higher than those found in mildly diseased subjects, and are undetectable in healthy subjects. Further, when applied directly to healthy human gingiva, short-chain carboxylic acids stimulate a gingival inflammatory response and inflammatory cytokine release. At the cellular level, these acids inhibit proliferation of gingival epithelial and endothelial cells, and inhibit leukocyte apoptosis and function, but can stimulate leukocyte cytokine release. At the molecular level, these acids can stimulate neutrophil gene transcription, translation, and protein expression. Thus, the likelihood is high that these acids, in addition to their cariogenic activity, can promote and prolong gingival inflammation. Our challenge will be to identify the cell or cells of the periodontium which respond to short-chain carboxylic acids, to delineate their responses and the molecular mechanism(s) of these effects, and to categorize the aspects of the inflammatory components which damage and those which protect the host. With this information, it may be possible to begin to rationally identify and test pharmaceutical agents which diminish the harmful aspects, while enhancing the beneficial components, of the inflammatory response.

The Sonicare sonic toothbrush and a traditional manual toothbrush were compared for efficacy in improving periodontal health in young orthodontic patients with existing gingival inflammation. A 4-week, single-blind clinical trial was employed. Twenty-four subjects, ages 11-17 years, who were fully bonded and banded with fixed orthodontic appliances were selected. Subjects were randomly assigned to use either the manual or the Sonicare toothbrush, instructed in its use, and asked to brush each morning and evening for 2 minutes. Plaque index, gingival index, percentage of sites which bled on probing, pocket depth, and total gram-negative bacteria in a subgingival plaque sample were assessed at baseline and 4 weeks around the banded teeth. The results demonstrate that the Sonicare brush was significantly more effective than the manual brush in all clinical parameters. Sonicare was statistically superior to the manual brush in supragingival plaque reduction (57% vs. 10%, respectively; p < 0.001). Gingival Index scores fell by 29 percent in the Sonicare group, but only 3 percent in the manual group. Reduction of bleeding on probing was significantly greater in the Sonicare group than in the manual group (p < 0.001). The Sonicare group decreased from 78% bleeding sites at baseline to 24.5% after 1 month. In the manual group there was only a slight reduction in bleeding on probing (70% of sites at baseline and 64.6% sites after 1 month). Mean pocket depths were significantly reduced compared to baseline values in both the Sonicare and the manual groups (p < 0.001). Pocket depth reduction in the Sonicare group was, however, significantly greater than in the manual group (28% vs. 6%, respectively: p < 0.001). Total gram-negative bacteria in subgingival plaque samples from banded test teeth of a subset of patients were reduced in the Sonicare group (p < or = 0.05), but increased in the manual group. These results clearly demonstrate that the Sonicare sonic toothbrush is superior to a manual toothbrush in improving periodontal health in adolescent orthodontic patients with existing gingivitis.

Short-chain carboxylic acids (SCCA; C < or = 5; e.g., lactic acid, propionic acid, butyric acid) are metabolic by-products of bacterial metabolism which accumulate in the gingival crevice, and exhibit significant biological activity, including the ability to alter gene expression. It has been hypothesized that among the activities of SCCAs are their ability to contribute to gingival inflammation. This concept complements the notion that specific periodontal pathogens are the causative agents of gingival inflammation. To begin testing these 2 hypotheses, we examined the relationship between SCCA concentrations, specific putative periodontal pathogens, and gingival inflammation in medically healthy periodontally diseased subjects. We reasoned that if SCCAs and/or specific periodontal pathogens were causative gingival inflammatory agents, gingival inflammation should increase with increasing concentration of the inflammatory mediator. We also recognized that other clinical variables needed to be controlled for, and an objective quantitative assessment of gingival inflammation used. To accomplish these tasks, sites within subjects were stratified by location and pocket depth, and the following quantified: bacterial presence; SCCA concentration; and gingival inflammation. The results indicated that gingival inflammation directly and significantly correlated with SCCA concentrations in the maxillary and mandibular molars, incisors and canines (all r > or = 0.47; all p < or = 0.015; too few bicuspids were available for complete analysis). The relationship between gingival inflammation and SCCA concentration was best described by a natural log relationship. Gingival inflammation did not, however, correlate positively with either the total number of specific putative periodontal pathogens, or the sum of subsets of these pathogens (-0.31 < or = r < or = 0.39; 0.08 < or = p < or = 0.75) for any of the locations. Finally, the SCCA concentration did not correlate with the level of individual or groups of pathogens. These data, together with historical work and other preliminary data, support the hypothesis that SCCA, rather than specific putative periodontal pathogens, may be a causative agent in gingival inflammation. This work may, in part, begin to explain the apparent lack of a direct relationship between current gingival inflammation and the prediction of bacterially mediated periodontal attachment loss.

The cadherins are a family of cell membrane proteins that mediate calcium-dependent cell-cell adhesion. E-cadherin is required for the formation, differentiation, polarization and stratification of epithelia; P-cadherin is also expressed on many epithelia. We report here the first study of cadherin expression in immortalized human gingival epithelial cells (IHGK) and examine the role of cadherins in growth regulation of these cells. We found that the IHGK cells are similar to normal gingival epithelial cells in their cadherin expression and density-dependent inhibition of growth. The IHGK cells proliferate more rapidly at low calcium concentration (0.15 mM) than at physiological concentrations of calcium (1.8 mM) and magnesium (0.65 mM; Ca/Mg medium) suggesting that calcium is required for density-dependent regulation of proliferation. To evaluate the possibility that cadherin function is required for contact inhibition in these cells, we grew them in Ca/Mg medium in the presence of adhesion-blocking anti-cadherin monoclonal antibodies. At anti-E-cadherin concentrations sufficient to disrupt cell-cell adhesion, the proliferation of the IHGK cells was similar to that observed in medium containing 0.2 mM EDTA. Anti-P-cadherin had a much weaker effect on cell proliferation than anti-E-cadherin, and cells grown in medium containing both antibodies grew at intermediate rates. The increased proliferation of the IHGK cells in either low calcium medium or Ca/Mg medium containing adhesion-blocking anti-cadherin antibodies suggests that cadherin-mediated adhesion is required for density-dependent regulation of growth of these cells.