Pascal Pellen-Mussi

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.

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.

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.

In vitro effects of Choukroun's platelet-rich fibrin conditioned medium on 3 different cell lines implicated in dental implantology.

Objective: The aim of this work was to determine the relevance of Choukrouns platelet-rich fibrin (PRF) in dental implantology by determining the in vitro effects of soluble factors released by PRF clot. We used 3 different cell lines implicated in dental implantology: osteoblast, keratinocyte, and fibroblast. Methods: Cellular viability, cell proliferation, and gene expression were analyzed using PRF conditioned medium. Three different cells lines were used: SaOS2 (osteoblast), MRC5 (fibroblast), and KB (epithelial cell). Results: The sulforhodamine B assay showed a significant increase in cell number for the undiluted and 1:3 diluted conditioned medium after 24 and 48 hours. There was no effect for the 1:9 dilution. Cell cycle analysis by flow cytometry confirmed the viability test results. After 48 hours, PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin, late osteogenic markers, was observed using reverse transcriptase-polymerase chain reaction (RT-PCR). Conclusions: This study establishes a model to evaluate, in vitro, the effects of soluble growth factors released by PRF clot. Our work confirmed PRF is useful in stimulating tissue healing and bone regeneration. This work should recommend Choukrouns PRF in numerous implantology clinical applications.

Development of a three-dimensional model for rapid evaluation of bone substitutes in vitro: effect of the 45S5 bioglass.

The evaluation of innovative bone substitutes requires the development of an optimal model close to physiological conditions. An interesting alternative is the use of an immortalized cell line to construct multicellular spheroids, that is, three-dimensional (3D) cultures. In this study, a modified hanging drops method has resulted in the generation of spheroids with a well-established human fetal osteoblasts line (hFOB 1.19), and tests have been focused on the effect of 45S5 bioglass ionic dissolution products in comparison with two-dimensional (2D) cultures. Depending on cell culture type, quantitative analysis (cell proliferation, viability/cytotoxicity, and cellular cycle) and qualitative analysis (electron microscopy and genes expression) showed a differential effect. Cell proliferation was enhanced in 2D-conditioned cultures in accordance with literature data, but decreased in 3D cultures submitted to the same conditions, without change of gene expression patterns. The decrease of cell proliferation, observed in conditioned spheroids, appears to be in agreement with clinical observations showing the insufficiency of commercially available bioglasses for bone repairing within nonbearing sites, such as periodontal defects or small bone filling, in general. Therefore, we suggest that this model could be adapted to the screening of innovative bioactive materials by laboratory techniques already available and extended monitoring of their bioactivity.

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.

Chitosan effects on glass matrices evaluated by biomaterial. MAS-NMR and biological investigations

Bioactive glass 46S6 and biodegradable therapeutic polymer (Chitosan: CH) have been elaborated to form 46S6-CH composite by freeze-drying process. The kinetics of chemical reactivity and bioactivity at the surface were investigated by using physicochemical techniques, particularly solid-state MAS-NMR. Immortalized cell line used to construct multicellular spheroids was employed as three-dimensional (3D) cell cultures for in vitro studies. Obtained results showed a novel structure of the composite; the chemical treatment (ultrasound, magnetic stirring, freeze drying process and lyophilization) led the bioactive glass particles to be loaded in the chitosan-based materials. 29Si and 31P MAS-NMR results showed the emergence of two new species, QSi3(OH) and QSi4, which are characteristic of the vitreous network dissolution in simulated body fluid (SBF). MAS-NMR also confirmed the formation of amorphous calcium phosphate (ACP) at the surface of the initial 46S6-CH. Three-dimensional (3D) cell cultures highlighted the effect of chitosan, where the cell viability reached up to 78% in 46S6-CH composite and up to 67% in 46S6. The association of (CH) and bioactive glass (BG) matrix promotes a highly significant bioactivity, demonstrating surface bone formation and satisfactory behavior in biological environment.

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.

Evaluation of functional SiO2 nanoparticles toxicity by a 3D culture model

CONTEXT as a kind of non-metal oxide SiO2 NPs have been extensively used in biomedicine, pharmaceuticals and other industrial manufacturing fields, such as DNA delivery, cancer therapy… Our group had developed a method based on microemulsion process to prepare SiO2 NPs incorporating photonic or magnetic nanocrystals and luminescent nanosized inorganic metal atom clusters. However, the toxicity of nanoparticles is known to be closely related to their physico-chemical characteristics and chemical composition. OBJECT it is therefore of interest to investigate the toxicity of these novel SiO2 NPs to the cells that may come in contact. MATERIALS AND METHODS the potential toxic effect of the functional @SiO2 NPs containing Mo6 clusters with or without gold nanoparticles was investigated, at concentrations 1 μg/mL, 10 μg/mL and 100 μg/mL each, on three different cell lines. Cell viability was measured by the MTT test in monolayers culture whereas the cytotoxicity in spheroid model was examined by the APH assay. In a second time, oxidative-stress-induced cytotoxicity was investigated through glutathione levels dosages. RESULTS the results indicated that both A549 and L929 cell lines did not exhibit susceptibility to functional @SiO2 NPs-induced oxidative stress unlike KB cells. DISCUSSION SiO2 NPs containing CMB may become toxic to cultured cells but only at a very high dosage level. Therefore, this toxicity depends on cell lines and more, on the model of cell cultures. The selection of appropriate cell line remains a critical component in nanotoxicology. CONCLUSION these results are relevant to future applications of SiO2 gold-cluster NPs in controlled release applications.

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.