Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND/OBJECTIVES: Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll-like receptor (TLR) 2- and 4-induced response in human periodontal ligament cells (hPDLCs). MATERIAL AND METHODS: Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 µg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 µg/mL); TLR4 agonist Escherichia coli LPS (0.1 µg/mL; 1 µg/mL); TLR2 agonist Pam3CSK4 (0.1 µg/mL; 1 µg/mL). The protein expression of interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 was analyzed with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction. RESULTS: Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL-6, IL-8 and monocyte chemotactic protein-1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non-pretreated cells. CONCLUSION: The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.

Smoking influences salivary histamine levels in periodontal disease.

OBJECTIVES: Histamine, a potent vasoactive amine, is increased in saliva of periodontitis patients. The present study aimed to further investigate the diagnostic potential of histamine for periodontal disease and assessed smoking, a major risk factor of periodontitis, as a possible influencing factor. METHODS: Salivary and serum samples of 106 participants (60 periodontitis patients, 46 controls) were collected. Salivary histamine was determined by a commercially available ELISA kit, and serum C-reactive protein was measured by a routine laboratory test. Cigarettes per day and packyears were assessed as smoking exposure parameters. RESULTS: Statistically significantly increased levels of salivary histamine and serum C-reactive protein were detected between the patient and control group (P = 0.022 and P = 0.001). Salivary histamine levels were significantly higher in smoking compared with non-smoking patients (P < 0.001), and salivary histamine as well as serum C-reactive protein correlated significantly positively with smoking exposure parameters (P < 0.05). CONCLUSIONS: Smoking, an established and common risk factor of periodontitis, was assessed as a possible influencing factor for salivary histamine. Most interestingly, salivary histamine differed highly significantly between smoking and non-smoking periodontitis patients. Our results suggest a possible involvement of histamine in tobacco-exacerbated periodontal disease, but do not suggest salivary histamine as a reliable diagnostic marker for periodontitis.

Characteristic features of trabecular bone in edentulous mandibles.

BACKGROUND: The density and architecture of the alveolar trabecular bone are crucial to the stability of an endosseous implant. A significantly higher implant failure rate can be expected when implants are placed in alveolar bone with reduced density and stability. Therefore, the present study aimed to describe the trabecular bone structure of edentulous mandibles. METHOD: Two hundred and seventy-eight bone sections, including the lateral incisor, first premolar, and first molar regions, were obtained from 128 edentulous lower jaws (68 females and 60 males; mean age: 77.58 years). Ground sections were prepared for each region using the 'sawing and grinding' technique. The following standard structural histomorphometric parameters were determined using a semiautomatic image analysis: trabecular bone volume, trabecular thickness, trabecular number, trabecular separation, and the trabecular bone pattern factor, which describes the connectedness of cancellous bone structures. Also, the maximum height of the jaw section was determined, to detect any possible correlations between vertical height and histomorphometric parameters. RESULTS: All the histomorphometric parameters examined showed an unexpectedly huge range of variation. The mean trabecular bone volume ranged between 20.9% and 36.9%. The mean trabecular thickness showed values between 165.9 and 224.7 microm. The mean trabecular number ranged between 1.22 and 1.77/ mm, and the mean trabecular separation ranged between 436.7 and 720.0 microm. The mean trabecular bone pattern factor showed values between -0.05 and -3.01/ mm. The maximum height of the jaw sections showed values between 16.05 and 23.42 mm. The trabecular bone volume, thickness, number and connectivity were significantly lower in the molar region than in the incisal and premolar regions. Significant sex-specific differences were found in all the regions, female mandibles showing a smaller amount and lower connectivity of cancellous bone than male mandibles. No correlation could be found between the maximum height of the jaw and the histomorphometric parameters of the cancellous bone. CONCLUSION: A possible explanation for the difference in the density between the incisal and the molar region may be that molars are generally lost at an earlier age than anterior and premolar teeth. As a result, atrophy-related resorptive and remodeling processes commence earlier and progress further in this region than in the anterior and premolar regions. Sex-specific differences are probably due to an increased postmenopausal bone loss of the females.

Response of L-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations.

Recent data suggest that under certain conditions, various metal cations are released from dental alloys. These ions may produce adverse effects in various cell types in vivo. In this study, the cytopathogenic effects of 13 metal cations on murine L-929 fibroblasts, human gingival fibroblasts, and human tissue mast cells were analyzed in vitro. Several metal cations (dose range, from 0.0033 to 1.0 mmol/L) were found to induce dose-dependent inhibition of 3H-thymidine incorporation into cultured fibroblasts. The rank order of potency (lowest observed effect level, LOEL) for L-929 fibroblasts was: Ag+ > Pt4+ > Co2+ > In3+ > Ga3+ > Au3+ > Cu2+ > Ni2+ > Zn2+ > Pd2+ > Mo5+ > Sn2+ > Cr2+. A similar rank order of potency was obtained for primary human gingival fibroblasts: Pt4+ > Ag+ > Au3+ > In3+ > Ga3+ > Ni2+ > Co2+ > Zn2+ > Cu2+ > Cr2+ > Pd2+ > Mo5+ > Sn2+. In primary human mast cells, Ag+ and Au3+ caused dose-dependent toxic histamine release, whereas the other metal cations were ineffective over the dose range tested. To investigate the mechanism of metal cation-induced effects, we performed DNA as well as electron microscopic analyses on cultured fibroblasts. Both the DNA pattern and the ultrastructure of L-929 cells and gingival fibroblasts after exposure to cytopathogenic metal cations revealed signs of necrosis but no signs of apoptosis. Together, our data provide evidence that various metal cations produce dose-dependent cytopathogenic effects in distinct cell types, including human gingival fibroblasts and human tissue mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic effects of dental composites, adhesive substances, compomers and cements.

OBJECTIVES: Although research interest in biocompatibility of dental materials has been increasing, findings are frequently controversial and non-harmonized experimental approaches often lead to the production of contradictory results. The aim of this study was to compare the cytotoxic effects of six different light-cured dental composites, one compomer, one advanced glass-ionomer, two glass-ionomer cements, two zinc phosphate cements, one calcium hydroxide liner, one composite cement and one carboxylate cement with the same standardized cell-culture system. Two composites, one compomer and one advanced glass-ionomer were also tested in combination with the appropriate bonding substances and surface primers. METHODS: Specimens were added to the cultures immediately after production or after preincubation for 1, 2 or 7 days or 6 weeks under cell-culture conditions. Specimens were incubated with L-929 fibroblasts for 72 h and cell numbers determined by flow cytometry. RESULTS: All freshly prepared composite materials were cytotoxic. These effects diminished with increased preincubation times and were not significant after 7 days. Combinations of composites and bonding substances were still cytotoxic after preincubation for 7 days, but not after 6 weeks. Combinations of compomers and bonding substances demonstrated stronger toxicity than composites, although these effects were reduced earlier during preincubation. Glass-ionomer and phosphate cements showed similar effects to the composites with the exception of carboxylate cement, which demonstrated severe and persistent effects even after 6 weeks' preincubation. Together, our data provide evidence that all dental materials tested are cytotoxic immediately after production and that these effects are reduced after different preincubation periods in most cases. SIGNIFICANCE: Tested with a standardized cell-culture system, differences in toxicological potency between various commonly used dental materials were observed. Cytotoxicity data from standardized protocols should form the basis of screening the cytotoxic effects of new materials.

Potential of the Tannerella forsythia S-layer to delay the immune response.

UNLABELLED: The periodontal pathogen Tannerella forsythia possesses a glycosylated S-layer as an outermost cell decoration. While the S-layer provides a selection advantage to the bacterium in the natural habitat, its virulence potential remains to be investigated. In the present study, the immune responses of human macrophages and gingival fibroblasts upon stimulation with wild-type T. forsythia and an S-layer-deficient mutant were investigated. The mRNA expression levels of the pro-inflammatory mediators IL-1ß, TNF-α, and IL-8 were analyzed by qPCR, and the production of the corresponding cytokines was investigated by ELISA. The S-layer-deficient T. forsythia mutant induced significantly higher levels of pro-inflammatory mediators compared with wild-type T. forsythia, especially at the early phase of response. Analysis of these data suggests that the S-layer of T. forsythia is an important virulence factor that attenuates the host immune response to this pathogen by evading the bacterium's recognition by the innate immune system. ABBREVIATIONS: DMSO, dimethylsulfoxide; FBS, fetal bovine serum; GAPDH, glycerinaldehyde-3-phosphate-dehydrogenase; HGFs, human gingival fibroblasts; LPS, lipopolysaccharide; MEM, minimal essential medium; MTT, 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide; OD, optical density; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain-reaction; SD, standard deviation; Tannerella forsythia ATCC 43037, Tf wt; Tannerella forsythia ATCC 43037 S-layer mutant, Tf ΔtfsAB.

Influence of dental amalgam and heavy metal cations on in vitro interleukin-1beta production by human peripheral blood mononuclear cells.

The influence of dental amalgam and heavy metal cations on interleukin-1beta (IL-1beta) expression by peripheral blood mononuclear cells from healthy donors was studied. A marked decrease in the production of IL-1beta was caused by freshly prepared amalgam or amalgam-conditioned culture medium, but not by amalgam aged for 6 weeks. When metal cations were added as salts, Cu(2+), Hg(2+), and Ag(+) at high concentrations (33.3 and 333.3 microM) were highly inhibitory. Among other heavy metal cations, Au(3+), Pt(4+), Ni(2+), Pd(2+), but not Ga(3+) or Sn(2+), inhibited IL-1beta production in a concentration-dependent manner. Flow cytometry studies indicated that Hg(2+) and Ag(+) strongly reduced the percentage of CD14(+) cells containing IL-1beta intracellularly. As shown by Northern blot analysis, Hg(2+) inhibited the level of IL-1beta-specific mRNA by 28% at 3.3 microM and completely at 33.3 microM. Only slight inhibitory effects were induced by Cu(2+) at 33.3 microM. Interestingly, Ag(+) at a concentration of 3.3 microM increased twofold the amount of IL-1beta-specific mRNA. Our data show that IL-1beta production is altered at protein and mRNA levels by components released from fresh amalgam and by other heavy metal cations, suggesting a role of these cations in changes in the cell phenotype and IL-1-mediated cell functions.

Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro.

The effects of dental amalgam on cytokine production by human peripheral blood mononuclear cells (PBMC) from healthy donors were analyzed. To induce cytokine production, PBMC were stimulated with lipopolysaccharide, phytohemagglutinin, or staphylococcal enterotoxin A and cultured for 48 h in the presence of either freshly prepared amalgam, aged amalgam, or amalgam-conditioned culture medium (ACCM). The concentrations of several cytokines were measured in PBMC supernatants by enzyme-amplified sensitivity immunoassays (EASIAs). Freshly prepared amalgam as well as ACCM induced a decrease in the production of interferon-gamma (IFN-gamma) and interleukin-10 (IL-10), and an increase in the concentrations of tumor necrosis factor-alpha (TNF-alpha). Both fresh amalgam and ACCM showed no effects on IL-2, IL-6, or granulocyte-macrophage colony-stimulating factor levels. Amalgam aged for 6 weeks did not affect the concentration of any of the above cytokines. To investigate which heavy metal cations released from amalgam caused the observed immunomodulatory effects, Cu2+, Hg2+, and Sn2+, which were detected in amalgam supernatants by inductively coupled plasma atomic spectrophotometry, were added as salts to the cultures. Cu2+ and Hg2+ induced a decrease in IFN-gamma and IL-10 levels, and Hg2+ an increase in TNF-alpha concentrations. Cytokine production was not significantly modulated by Sn2+. Under these experimental conditions, release of Ag+ into culture medium was not detectable. However, Ag+ markedly suppressed the production of IFN-gamma, IL-10, and TNF-alpha. In summary, our results show that fresh amalgam, but not amalgam aged for 6 weeks, causes changes in the cytokine pattern of PBMC in vitro, and that these effects are due to the release of Cu2+ and Hg2+.

Metal ion-induced toxic histamine release from human basophils and mast cells.

Recent data suggest that distinct metal ions can be released from dental alloys or other biomaterials, and may cause toxic effects on various cells. In this study, the effects of 14 metal ions on histamine release from human blood basophils (n = 4), isolated tissue mast cells (lung n = 8, uterus n = 2, skin n = 1, gingiva n = 1), the basophil cell line KU-812, and the mast cell line HMC-1 were analyzed. Of the 14 metal ions, Ag+ (0.33 mM) and Hg2+ (0.33 mM) were found to induce release of histamine in blood basophils, KU-812, mast cells, and HMC-1. The effects of Ag+ and Hg2+ were dose dependent and were observed within 60 min of incubation. In primary mast cells and basophils, AU3+ (0.33 mM) also induced histamine release, whereas no effects of Au3+ on HMC-1 or KU-812 cells were seen. The other metal ions showed no effects on primary or immortal cells within 60 min. However, Pt4+ (0.33 mM) induced histamine liberation in HMC-1 and lung mast cells after 12 h. The Ag+- and Hg2+-induced rapid release of histamine from HMC-1 was associated with ultrastructural signs of necrosis, but not apoptosis. In contrast, prolonged exposure to Pt4+ (0.33 mM, 14 h) induced apoptotic cell death in HMC-1 cells, as assessed by electron microscopy and DNA analysis. Together, certain metal ions induce distinct cytopathogenic effects in mast cells and basophils. Whereas Ag+, Hg2+, and Au3+ cause direct toxicity, Pt4 causes cell death through induction of apoptosis. Whether such effects contribute to local adverse reactions to metal-containing biomaterials in vivo remains to be determined.