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.

New method of synthesis and in vitro studies of a porous biomaterial.

Biomaterials for bone reconstruction represent a widely studied area. In this paper, a new method of synthesis of a porous glass-ceramic obtained by thermal treatment is presented. The prepared biomaterial was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and induced couple plasma-optical emission spectroscopy (ICP-OES), mercury porosimetry and by the Archimedes method. In vitro evaluations in a simulated body fluid (SBF) and in contact with SaOS2 human osteoblasts were also carried out. The porous glass-ceramic is composed of a total porous network of 60% suitable for body fluid and cell infiltration, with pore sizes varying from 60 nm to 143 µm. The presence of two crystalline phases decreases the kinetic of bioactivity compared to an amorphous biomaterial (bioactive glass). A hydroxyapatite layer appears from 15 days of immersion on the surface and inside the pores, showing a biodegradation and a bioactivity in four steps. Cytotoxicity assessments present an increase of the cellular viability after 72 h proving the non-cytotoxic effect of the glass-ceramic. Thus, the results of these different studies indicate that the porous biomaterial may have a potential application for the bone regeneration. This paper also presents the novelty of this method. It is a rapid synthesis which combines simplicity and low cost. This represents an advantage for an eventual industrialization.

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.

Activation of brain estrogen receptors in mice lactating from mothers exposed to DDT.

The insecticide dichlorodiphenyltrichloroethane (DDT) interferes with physiological endocrine processes modulating estrogens receptor activity. Most of the data describing the DDT mechanism of action have been collected in vitro or in reproductive tissues in vivo. Here we use a new transgenic mouse model to investigate the DDT effects on estrogens receptor activation in vivo in non-reproductive tissues. In particular, we demonstrate that DDT is able to activate estrogen receptors in the brain and the liver of adult mice after acute administration, and it is active in lactating mice when accumulated in the mother's milk. Furthermore, we demonstrate that the acute administration of DDT activates estrogen receptors with a different kinetics with respect to 17beta-estradiol. Experiments with a breast cancer cell line engineered to express luciferase under the transcriptional control of activated estrogen receptors reveal that the microsomal metabolization of DDT is required for its full activity on estrogen receptors. Taken together these data lead to hypothesize that the delayed DDT time course on estrogen receptor activation in vivo might be due to a necessary step of metabolism of the compound.

Estrogen neuroprotection: the involvement of the Bcl-2 binding protein BNIP2.

To identify genes selectively induced by estrogens in cells of neural origin we have treated with a low concentration of 17 beta-estradiol (E2) the estrogen-receptor positive SK-ER3 neuroblastoma cells and we have isolated messages modulated by the hormonal treatment at short (1 h) and longer (17 h) times. By using the ddPCR approach we identified numerous messages which content was significantly and reproducibly altered by the hormonal treatment. Among these messages we focused our attention on bnip2, which expression was inhibited by estradiol. bnip2 was found to be a member of the BNIP family of genes of unknown physiological activity at the time. Investigations carried out in our laboratory proved a strong correlation between the increased expression of bnip2 gene and cell death induced by toxic stimuli. Furthermore, we showed that transfection of the bnip2 cDNA results in massive cell death and Bcl-2 overexpression counteracts the toxic effect of bnip2. These findings suggest that the proteins encoded by these two genes either interact or act in an opposite manner on the same mechanisms triggering the apoptotic cascade of events. Time-course experiments carried out in different cell systems and with a variety of neurotoxic agents proved a strong correlation between estrogen-induced decrease in bnip2 expression and the time required for estrogen to exert its protective effect. These observations led us to hypothesize an involvement of bnip2 in estrogen effects on cell survival. The finding that bnip2 is developmentally regulated may suggest a role of this gene in those brain areas where the differentiation is orchestrated by estradiol. Investigations in non-neural cells show that bnip2 is the mediator of the anti-apoptotic activity of estrogens in a variety of cells and thus might represent an important target for the evaluation of the activity of novel synthetic ligands for the estrogen receptor.

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 cytocompatibility of three endodontic materials.

The goal of this in vitro study was to evaluate the relative cytocompatibility of three endodontic materials: calcium hydroxide, a calcium oxide-based compound, and a zinc oxide-eugenol-based sealer. The evaluation was conducted 24, 72, and 168 h after contact with the compounds and involved three complementary techniques: a colorimetric cytotoxicity test, scanning electron microscopy, and flow cytometry. The results we obtained confirmed the initial cytotoxicity of the zinc oxide-eugenol-based sealer and showed that the calcium oxide-based compound had the same relative cytocompatibility as calcium hydroxide.

Divided vertical banded gastroplasty either for correction or as a first-choice operation.

BACKGROUND: Staple-line disruption may occur after vertical banded gastroplasty (VBG). METHODS: Since May 1996, the authors have performed the gastric restrictive procedure divided VBG, as described by MacLean, as a first-choice operation and not only as correction for staple-line breakdown. Divided VBG was done in 111 patients, 32 as correction after staple-line disruption and 79 as a first choice, 29 of them by hand-assisted laparoscopy with the dexterity pneumosleeve. RESULTS: Weight loss and nutritional status have been very satisfactory. Operating time for the standardized operation has been no longer than 60 minutes. CONCLUSION: Divided VBG, especially if done by hand-assisted laparoscopy using the dexterity pneumosleeve, is a valid restrictive procedure.

Evaluation of the in vitro biocompatibility of various elastomers.

A previous study highlighted the superior shock absorption of silicone rubbers compared to other elastomers. We evaluated and compared the in vitro biocompatibility of silicone-based rubbers and propose them as an alternative to conventional products. We used the MTT colorimetric test to assess cell viability and flow cytometry to evaluate cell proliferation. Tests were conducted at 24 and 72 h. Changes in cell morphology were evaluated by scanning electron microscopy. Positive (polyurethane) and negative (polystyrene) toxicity controls were included. The number of viable cells was significantly higher on polystyrene than on polyurethane. A decrease in the total number of cells from 24 to 72 h compared to the negative control was correlated with a lower percentage of S-phase cells. The differences in cell viability noted between the samples and the polystyrene control mainly resulted from an initial lack of adhesion, which was confirmed by scanning electron microscopy. The biocompatibility of the three silicone rubbers was comparable to the best of the three products currently being used. These results, combined with those of the previous study, indicate that silicone rubber could be considered for the manufacture of mouth guards.

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.

Evaluation of the biocompatibility of titanium-tantalum alloy versus titanium.

To evaluate the biocompatibility of a new titanium-tantalum alloy, with qualities superior to titanium alone, for use in oral implantology, fibroblast and epithelial cell lines were grown on plastic, titanium, copper, and titanium-tantalum supports. Studies using scanning electron microscopy, flow cytometry, and cytotoxicity assays were conducted to compare the different supports. Scanning electron microscopic observations showed high densities of fibroblasts and epithelial cells with well-developed attachment systems in the form of cytoplasmic projections. Cell densities were lower on titanium and titanium-tantalum surfaces than on plastic. Cell numbers, as determined by cytotoxicity assays, were significantly higher on plastic than on titanium or titanium-tantalum surfaces while fibroblasts proliferated better than epithelial cells on both metal surfaces. Flow cytometric analyses of cell cycles did not reveal any significant variations in the distribution of cells among the cycle phases on the three materials. We found no differences with regard to the parameters studied between titanium and the titanium-tantalum alloy.

Fimbriae and the hemagglutinating adhesin HA-Ag2 mediate adhesion of Porphyromonas gingivalis to epithelial cells.

The mechanisms by which Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is pathogenic for the periodontium remain largely hypothetical. Invasion of host tissues by P. gingivalis is believed to require adhesion of the bacterium to host cells. The aim of this study was to use monoclonal antibodies (MAbs) to characterize the bacterial cell surface component(s) acting as a ligand binding to a receptor on epithelial cells. Surface antigens of P. gingivalis ATCC 33277 were obtained as a glass bead-EDTA extract (GBE), and antiserum against the GBE was produced in rabbits. Epithelial cell membrane proteins (ECMP) were prepared from a homogenate of the SK-MES-1 cell line with Triton X-100. The antigen/ligand profile of GBE was resolved by crossed immunoaffinity electrophoresis by using ECMP in the first-dimension gel. The migration of one immunoprecipitate (IP) was retarded, indicating a ligand-receptor interaction between a surface antigen of P. gingivalis and a complementary binding site on the epithelial cell membrane. The corresponding IP in the GBE/anti-GBE immunoelectrophoresis profile was excised from replicate gels to immunize mice for production of MAbs specific for the bacterial ligand. Five MAbs were obtained and tested for reactivity with GBE in immunoblots and for inhibition of the interaction between GBE and ECMP. Immunoblots revealed polypeptides at 28, 42, 43, and 49 kDa. Inhibition tests were positive for all five MAbs. These results are conclusive evidence that the MAbs recognize functional epitopes involved in the adherence of P. gingivalis to epithelial cells and that the adhesins are likely associated with fimbriae and the hemagglutinating adhesin HA-Ag2.

Evaluation of cellular proliferation on collagenous membranes.

Collagenous membranes are used in guided tissue regeneration designed to repair damage done to the periodontium by disease. The cells primarily responsible for the process are desmodontal fibroblasts. We have studied the behavior and proliferation of fibroblasts grown on collagenous membrane. Fibroblasts grown on plastic served as control. Cellular proliferation was evaluated using a colorimetric test for cytotoxicity and by flow cytometry. Scanning electron microscopy was used to observe cellular morphology. Results showed a reduction in cell number, together with a modification in cell cycle, but good preservation of cellular morphology for cultures grown on collagenous membrane. These results support the in vivo use of collagenous membrane in guided tissue regeneration.

Biliopancreatic diversion with transitory gastroplasty preserving duodenal bulb: 3 years experience.

BACKGROUND: The authors have performed 521 bariatric surgery operations (319 restrictive procedures and 202 malabsorptive procedures). METHODS: During the last few years we have introduced an evolution of biliopancreatic diversion (BPD): BPD with transitory gastroplasty, preserving the duodenal bulb (53 cases). From a technical point of view, the operation consists of a BPD, coupled with a gastroplasty which is transitory due to the use of a polydioxanone (PDS) band. In the last few cases, instead of a VBG (with PDS band) in order to make the operation completely reversible without any suture on the stomach, we made a gastric pouch by banding with PDS calibrated with the same tube as for the Lap-band (20 cc). We maintained completely the duodenal bulb (5 cm from the pylorus), making an end-to-side duodeno-ileal isoperistaltic anastomosis. RESULTS: With this anastomosis, only 2% of patients developed an anastomotic ulcer. With this new procedure, results have been good in terms of weight loss (similar to that of BPD-AHS) and in nutritional complications. No patient has had hypoalbuminemia, diarrhea or halitosis. CONCLUSION: BPD with temporary gastric restriction has provided satisfactory results.

Conservation of fimbriae and the hemagglutinating adhesin HA-Ag2 among Porphyromonas gingivalis strains and other anaerobic bacteria studied by epitope mapping analysis.

Monoclonal antibodies characterized as antifimbria and anti-HA-Ag2 were used in immunoblotting to examine the antigenic distribution of fimbriae and HA-Ag2 among a collection of human and animal Porphyromonas strains and human Prevotella and Bacteroides strains. The results showed that fimbrial and HA-Ag2 antigenic structures are peculiar to the species Porphyromonas gingivalis.