Expression of factors involved in dental pulp physiopathological processes by nemotic human pulpal fibroblasts.

AIM: To investigate in human dental pulp fibroblasts (HDPF) the expression of factors involved in dental pulp physiopathological processes and in an experimental model of cell activation called nemosis, and to compare the behaviour of pulp cell activation with sound lung fibroblast MRC5, employed as a reference model for nemosis. METHODOLOGY: Nemotic response was induced in three-dimensional cultures of HDPF and lung fibroblasts. The expressions of molecules involved in physiological (alkaline phosphatase, type I collagen) and in inflammatory processes (IL-6, CXCL8, CCL20, COX-2) were studied using real-time PCR. Concentrations of IL-6 and CXCL8 were analysed during 4 days with ELISA. Nonparametric tests were used to determine statistical differences between groups. RESULTS: A significant decrease (P < 0.001) in type I collagen and alkaline phosphatase was observed in MRC5 and HDPF nemotic responses. Although the amounts of mRNA differed between these cell types, there was an increase in CCL20, CXCL8 and COX-2 expression (P < 0.001). Unlike HDPF, MRC5 spheroids displayed significant amounts of IL-6 concentrations and mRNA expression. Notably, increased concentrations of CXCL8 were recorded in all three-dimensional cultures compared with monolayers as a function of time (P < 0.05). CONCLUSION: Although the nemotic responses observed were not identical in the pulpal and lung fibroblasts, similarities occurred in the expression of chemokines and cyclooxygenase-2. Nemotic reactions and inflammatory processes in pulp diseases share similarities in terms of the expression of factors. Thus, this in vitro model could constitute a powerful tool to study intercellular relations within the dental pulp and to develop new local treatments to counteract the inflammatory reaction that occurs during pulpitis.

3D cell culture to determine in vitro biocompatibility of bioactive glass in association with chitosan.

This study reports the in vitro biocompatibility of a composite biomaterial composed of 46S6 bioactive glass in association with chitosan (CH) by using 3D osteoblast culture of SaOS2. The 46S6 and CH composite (46S6-CH) forms small hydroxyapatite crystals on its surface after only three days immersion in the simulated body fluid. For 2D osteoblast culture, a significant increase in cell proliferation was observed after three days of contact with 46S6 or 46S6-CH-immersed media. After six days, 46S6-CH led to a significant increase in cell proliferation (128%) compared with pure 46S6 (113%) and pure CH (122%). For 3D osteoblast culture, after six days of culture, there was an increase in gene expression of markers of the early osteoblastic differentiation (RUNX2, ALP, COL1A1). Geometric structures corresponding to small apatite clusters were observed by SEM on the surface of the spheroids cultivated with 46S6 or 46S6-CH-immersed media. We showed different cellular responses depending on the 2D and 3D cell culture model. The induction of osteoblast differentiation in the 3D cell culture explained the differences of cell proliferation in contact with 46S6, CH or 46S6-CH-immersed media. This study confirmed that the 3D cell culture model is a very promising tool for in vitro biological evaluation of bone substitutes' properties.

Characterization of a programmed necrosis process in 3-dimensional cultures of dental pulp fibroblasts.

AIM: To analyse and compare the expression of necrosis markers in human lung and dental pulp fibroblasts and to determine whether this process differs by the type of mesenchymal cell. METHODS: Human dental pulp fibroblasts were obtained from unerupted third molars. Sound lung and pulpal fibroblasts were cultured in vitro as spheroids to determine the expression of the necrosis hallmark cyclooxygenase-2 (COX-2) mRNA using RT-PCR and the concentrations of vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF) proteins using an ELISA test. Cell viability within spheroids was also compared with spheroid diameters over time. RESULTS: Increased expression of COX-2 and VEGF was found in all spheroids compared with corresponding monolayers. Although HGF/SF was highly expressed in MRC5 cells, dental pulp fibroblasts aggregates maintained only a basal level compared with monolayer cultures. Further, the observed progressive loss of viable cells explained the decreased diameters of spheroids over time. The results demonstrate that necrosis occurs in sound lung and pulpal fibroblasts. This cell death also displays differences between these two different cell types, as they do not produce the same growth factors quantity release. CONCLUSIONS: The necrosis process occurred in human dental pulp fibroblasts and is different between the two cell types studied. This in vitro experimental necrosis model could become an interesting inflammatory tool. More investigations are needed to compare necrosis process in dental pulp fibroblast and inflammation during pulpitis.

Epithelial cell surface sites involved in the polyvalent adherence of Porphyromonas gingivalis: a convincing role for neuraminic acid and glucuronic acid.

We investigated the target structures of the epithelial cells responsible for the attachment of Porphyromonas gingivalis by immunocytofluorimetry, enzyme-linked immunosorbent assay, and confocal microscopy. Integrins (beta1, beta3, and alphaV) and E-cadherin played no significant role. Carbohydrates (such as alpha-D-methylglucoside, L-fucose, D- and L-mannose, N-acetylglucosamine, and N-acetylgalactosamine) had little inhibitory effect on bacterial binding. Enzymatic treatments of the epithelial membranes and sugar competition studies showed that N-acetylneuraminic acid and glucuronic acid were involved in binding.

Assessment of internalization and viability of Porphyromonas gingivalis in KB epithelial cells by confocal microscopy.

Porphyromonas gingivalis (P. gingivalis) is considered to be one of the main periodontal pathogens. The goal of this work was to confirm the ability of P. gingivalis to invade host cells. We detected P. gingivalis inside KB cells by confocal microscopy and analyzed the various aspects of the adherence and internalization process. Lysates of P. gingivalis-infected KB cells were also examined using anaerobic growth techniques. The results showed the viability and ability to replicate, inside the host cells, of the internalized pathogen. The production of vesicles was also tracked for the first time. Confocal microscopy revealed P. gingivalis in a perinuclear position.

Evaluation of the capacity of the SCGE assay to assess the genotoxicity of biomaterials.

The comet test or SCGE assay, which is already widely used in other areas, has never been used to evaluate the mutagenic potential of medical biomaterials in the final form. The purpose of our study was thus to assess the comet test as a means of assessing the genotoxic potential of finished medical biomaterials. We used silicone elastomers with increasing concentrations of 4-nitroquinoline oxide, a genotoxic agent. Hydrogen peroxide was used as the positive control, and tissue culture polystyrene as the negative control. In our study, the comet test did not detect a significant difference in genotoxicity between the pure elastomer and the same elastomer containing 0.01 mg/ml 4-nitroquinoline oxide, but did detect a significant difference between two elastomers containing 0.01 and 0.3 mg/ml of 4-nitroquinoline oxide, respectively. Since, the surface properties of the samples were identical, only the chemical composition may have caused significant differences in mutagenicity. Whatever the cause of the genotoxicity detected by the SCGE assay, testing finished biomaterials using the comet assay makes it possible to evaluate interactions between biomaterials and living tissues that are much closer to actual application conditions.

In vitro evaluation of the retention of three species of pathogenic microorganisms by three different types of toothbrush.

The retention and survival of microorganisms on toothbrushes pose a threat of recontamination for certain patients at risk. In order to measure the influence of brush design and optimize the choice of toothbrush model for complementary studies, the in vitro retention of three microbial species (Porphyromonas gingivalis ATCC 33277, Streptococcus mutans ATCC 25175 and Candida albicans ATCC 26555) was evaluated for three types of toothbrush. Two series of standardized experiments were carried out for each brush and microorganism. The first series tested the retention of the microorganisms on the head portion of the brush, while the second measured retention on the head of the brush and the part of the handle inserted in the mouth during brushing. For each series, the microorganisms were counted at T0 and T24 (after storage of the brushes at room temperature for 24 h). Depending on the microorganism studied, from 0.2% to 2% of the initial inoculum was retained on the brush. The number detected increased with the size of the exposed area. After 24 h, P. gingivalis and S. mutans were found on only one type of brush. C. albicans survived on all three. These results confirm that microorganisms can quickly colonize toothbrushes.

Adherence of Porphyromonas gingivalis to gingival epithelial cells: modulation of bacterial protein expression.

The protein profiles of Porphyromonas gingivalis (ATCC 33277 and W83) bound to KB gingival epithelial cells were analyzed by SDS-PAGE and immunoblotting. We found that a 51-kDa component was formed in bacteria that adhered to the KB cells, whereas 26- to 29-kDa bands were less intensive, in contrast to the protein profile of free bacteria. P. gingivalis ATCC 33277 incubated with protease-treated KB cells retained the profile of free bacteria. These results demonstrate the specificity of bacterial recognition of eukaryotic membrane components.

Adherence of Porphyromonas gingivalis to epithelial cells: analysis by flow cytometry.

Porphyromonas gingivalis, implicated in the pathogenesis of periodontitis, can adhere to epithelial cells and gingival fibroblasts. This study employed flow cytometry to evaluate the adherence of P. gingivalis to epithelial cells under various conditions. The cell lines SK-MES and KB were used in the first experiments. The P. gingivalis strains employed were ATCC 33277, ATCC 49417 and W83. Different adherence conditions were tested (contact time, bacteria/cell ratio, contact temperature). In later experiments, adherence of P. gingivalis to human gingival epithelial cells (GEC) obtained by explant was studied under various conditions. Results showed that P. gingivalis had a high affinity for buccal keratinocytes compared with SK-MES. Adherence showed a level of saturation. The number of receptors may be limited for each epithelial cell line, and there may be more receptors for gingival keratinocytes. Depending on contact time, P. gingivalis showed a higher affinity for GEC, compared with the other two lines. P. gingivalis thus showed specific adherence for a host cell type from a site associated with periodontal disease.

Oral complications during treatment of malignant diseases in childhood: effects of tooth brushing.

During anticancer treatment, oral lesions considerably aggravate the child's clinical condition and increase the risk of infection. This prospective study evaluated the incidence, nature and chronology of oral complications arising during the first 6 weeks of chemotherapy. 131 children were included in this study, and their oral and dental health evaluated on enrolment. Each child was observed once a week, for 6 weeks. Fifty-two per cent (68/131) of the children presented with at least one oral lesion. Two oral healthcare regimens (with or without tooth brushing) were evaluated. Tooth brushing significantly reduced the number of children affected. Standardised multicentre studies should permit the definition of oral care regimens which would eliminate pain and reduce the risk of infection in children hospitalised for cancer.

Changes in extracellular matrix macromolecules in human gingiva after treatment with drugs inducing gingival overgrowth.

It is generally agreed that gingival overgrowth results from an increase in the levels of gingival extracellular macromolecules infiltrated with various numbers of inflammatory cells. The relative amounts of extracellular matrix macromolecules observed in 12 cases of gingival hyperplasia associated with the use of cyclosporin, hydantoin or nifedipine were compared with those obtained in a control group on the basis of histological and immunohistochemical investigations. From tissue sections, the quantification was by computerized morphometric analysis on a BFM 186 microcomputer to which were implemented the transformations of mathematical morphology. The area fractions (AA%) occupied by total collagen, type III and type IV collagen, vessels, fibroblasts, fibronectin and elastic fibres were estimated and compared. The overall histological aspects of drug-induced gingival overgrowth were similar, but quantification of different extracellular matrix components showed differences. In the nifedipine and cyclosporin groups, the area occupied by fibroblasts were not significantly greater than in healthy gingiva and chronic gingivitis. The area occupied by collagen was significantly greater in the nifedipine group than in the other pathological groups. Fibronectin was also strongly expressed in the nifedipine group, and the elastic fibre network was preserved in this group.

Gingival biopsy in the diagnosis of giant axonal neuropathy.

We report the case of a 4-year-old child presenting with a diagnosis of giant axonal neuropathy. This rare condition was diagnosed when the child was 15 months old and was confirmed using a gingival sample removed during a dental examination. Structural and ultrastructural analyses demonstrated the presence of numerous unmyelinated fibres with distended axons and the accumulation of intermediate filaments in Schwann cells, in fibroblasts and in endothelial cells. Cytological examination of connective tissue revealed the presence of mitochondria-like particles. Scanning electron microscopic examination of hair revealed the presence of a longitudinal groove along the shaft. These characteristic features of giant axonal neuropathy demonstrate that gingival tissue can be used as an aid in its diagnosis.