Autoregulation of periodontal ligament cell phenotype and functions by transforming growth factor-beta1.

During orthodontic tooth movement, mechanical forces acting on periodontal ligament (PDL) cells induce the synthesis of mediators which alter the growth, differentiation, and secretory functions of cells of the PDL. Since the cells of the PDL represent a heterogeneous population, we examined mechanically stress-induced cytokine profiles in three separate clones of human osteoblast-like PDL cells. Of the four pro-inflammatory cytokines investigated, only IL-6 and TGF-beta1 were up-regulated in response to mechanical stress. However, the expression of other pro-inflammatory cytokines such as IL-1 beta, TNF-alpha, or IL-8 was not observed. To understand the consequences of the increase in TGF-beta1 expression following mechanical stress, we examined the effect of TGF-beta1 on PDL cell phenotype and functions. TGF-beta1 was mitogenic to PDL cells at concentrations between 0.4 and 10 ng/mL. Furthermore, TGF-beta1 down-regulated the osteoblast-like phenotype of PDL cells, i.e., alkaline phosphatase activity, calcium phosphate nodule formation, expression of osteocalcin, and TGF-beta1, in a dose-dependent manner. Although initially TGF-beta1 induced expression of type I collagen mRNA, prolonged exposure to TGF-beta1 down-regulated the ability of PDL cells to express type I collagen mRNA. Our results further show that, within 4 hrs, exogenously applied TGF-beta1 down-regulated IL-6 expression in a dose-dependent manner, and this inhibition was sustained over a six-day period. In summary, the data suggest that mechanically stress-induced TGF-beta1 expression may be a physiological mechanism to induce mitogenesis in PDL cells while down-regulating its osteoblast-like features and simultaneously reducing the IL-6-induced bone resorption.

Regulation of periodontal ligament cell functions by interleukin-1beta.

Periodontal ligament (PDL) cells maintain the attachment of the tooth to alveolar bone. These cells reside at a site in which they are challenged frequently by bacterial products and proinflammatory cytokines, such as interleukin-1beta (IL-1beta), during infections. In our initial studies we observed that IL-1beta down-regulates the osteoblast-like characteristics of PDL cells in vitro. Therefore, we examined the functional significance of the loss of the PDL cell's osteoblast-like characteristics during inflammation. In this report we show that, during inflammation, IL-1beta can modulate the phenotypic characteristics of PDL cells to a more functionally significant lipopolysaccharide (LPS)-responsive phenotype. In a healthy periodontium PDL cells exhibit an osteoblast-like phenotype and are unresponsive to gram-negative bacterial LPS. Treatment of PDL cells with IL-1beta inhibits the expression of their osteoblast-like characteristics, as assessed by the failure to express transforming growth factor beta1 (TGF-beta1) and proteins associated with mineralization, such as alkaline phosphatase and osteocalcin. As a consequence of this IL-1beta-induced phenotypic change, PDL cells become responsive to LPS and synthesize proinflammatory cytokines. The IL-1beta-induced phenotypic changes in PDL cells were transient, as removal of IL-1beta from PDL cell cultures resulted in reacquisition of their osteoblast-like characteristics and lack of LPS responsiveness. The IL-1beta-induced phenotypic changes occurred at concentrations that are frequently observed in tissue exudates during periodontal inflammation (0.05 to 5 ng/ml). The results suggest that, during inflammation in vivo, IL-1beta may modulate PDL cell functions, allowing PDL cells to participate directly in the disease process by assuming LPS responsiveness at the expense of their normal structural properties and functions.

LPS responsiveness in periodontal ligament cells is regulated by tumor necrosis factor-alpha.

Gingival fibroblasts function as accessory immune cells and are capable of synthesizing cytokines in response to lipopolysaccharides (LPS) from Gram-negative microbes. Recently, we have isolated, cloned, and characterized two cell lines which exhibit characteristics of periodontal ligament (PDL) cells. In this report, we demonstrate that PDL cells showing osteoblast-like phenotype are not LPS-responsive cells. However, treatment of PDL cells with tumor necrosis factor-alpha (TNF-alpha) inhibits the expression of their osteoblast-like characteristics. As a consequence of this TNF-alpha-induced phenotypic change, PDL cells become LPS-responsive, i.e., synthesize several pro-inflammatory cytokines in response to LPS. These phenotypic changes occur at concentrations of TNF-alpha that are frequently observed in tissue exudates during periodontal inflammation, suggesting a physiological significance for these in vitro observations. It is of interest that TNF-alpha-induced phenotypic changes in PDL cells are transient, since removal of rhTNF-alpha from the supernatants of PDL cell cultures results in re-acquisition of the osteoblast-like characteristics and lack of LPS responsiveness of PDL cells. These results suggest that TNF-alpha, by regulating the PDL cell functions, may allow these cells to participate in the disease process as accessory immune cells at the expense of their structural properties.

Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1 beta.

We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1 beta (IL-1 beta). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1 beta, IL-6 and IL-8. IL-1 beta activation of GF cells showed that IL-1 beta non only induces the expression of IL-6, IL-8 and TNF-alpha, but also acts in an autocrine manner of GF cells and induces IL-1 beta expression. Furthermore, the continuous presence of IL-1 beta in GF cell cultures did not down regulate the response of GF cells to IL-1 beta. Pretreatment of GF cells with IL-1 beta resulted in the enhanced synthesis of TNF-alpha in response to additional IL-1 beta. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.

Toxicity testing of sealants: a tissue implant study.

The toxicity of pit and fisure sealants implanted into the subcutaneous tissues of guinea pigs was tested using the protocol for toxicity testing derived from the ADA/ANSI Document No. 41, 1982. The materials tested were Delton autopolymerized and photopolymerized, and Concise White autopolymerized and photopolymerized. After two weeks, reactive fibrosis and mild to moderately severe foreign body reactions were noted. After 12 weeks, only thin fibrous walls infiltrated occasionally by small numbers of chronic inflammatory cells were observed. The results of this investigation appear to indicate that following subcutaneous implantation of a pit and fissure sealant, a foreign body reaction will most likely take place during the first two weeks, but will be resolved by 12 weeks. Furthermore, than an initial reactive fibrosis will give way to a thin fibrosis wall by 12 weeks, and the initial inflammatory response will subside. It can also be stated that in this study, Delton and Concise White sealant materials produced similar tissue reactions, and that there were few differences between materials which were autopolymerized or photopolymerized.

Use of lipids to potentiate the antibacterial activity of aminoglycosides.

Linolenyl alcohol has been shown to inhibit the in vitro growth of several species of gram-positive bacteria. Since the double bonds in linolenyl alcohol could undergo autooxidation, the antimicrobial activities of saturated primary alcohols of similar molecular sizes against Streptococcus mutans BHT were evaluated. Tridecan-1-ol was identified as the most active compound, eliciting a bacteriostatic effect at concentrations at which growth occurred in the presence of other saturated alcohols or linolenyl alcohol. Evidence is also presented that the combined use of tridecan-1-ol and gentamicin sulfate produces a synergistic effect that is bactericidal to S. mutans BHT. A similar bactericidal response was observed when tridecan-1-ol was combined with other aminoglycosides or tested alone against other strains of S. mutans.

Effect of linolenyl alcohol on the in-vitro growth of the oral bacterium Streptococcus mutans.

The effect of primary aliphatic alcohols of varying chain length and degree of unsaturation on bacterial growth was assessed, using Strep. mutans BHT as the main test organism. Unsaturated alcohols, linoleyl and linolenyl, effectively inhibited bacterial growth. Of the saturated alcohols, only lauryl and myristyl alcohols inhibited the growth of Strep. mutans BHT, but at concentrations much higher than those required for the unsaturated alcohols. All Gram-positive organisms tested were sensitive to linolenyl alcohol. Gram-negative bacteria did not exhibit the sensitivity. Linoleic and linolenic acid were inactive as antibacterial agents at the same concentration as the related alcohol. Repeated exposure of Strep. mutans BHT to linolenyl alcohol produced no change in the sensitivity of the organism to the alcohol. Significant amounts of linolenyl alcohol were found in bacteria grown in the presence of this lipid for 24 h but linolenic acid was not detected. Thus the primary polyunsaturated aliphatic alcohols, particularly linolenyl alcohol, could be effective antibacterial agents for the prevention of dental caries and periodontal disease.

Scanning electron microscopy of epithelial cells grown on enamel, glass and implant materials.

The purpose of the present study was to examine with scanning electron microscopy gingival epithelial cells grown in cell culture on tooth enamel, glass, Vitallium, titanium and vitreous carbon. SGL (Smulow-Glickman) gingival epithelial cells were grown for 5 days and processed using a critical point drying apparatus. Scanning electron microscopy carried out at X 1500 magnification revealed that the gingival epithelial cells gres equally well on all materials on either smooth or rough (sand-blasted) surfaces.