Applications of inflammation-derived gingival stem cells for testing the biocompatibility of dental restorative biomaterials.

BACKGROUND: Normal or inflamed gingival tissues are regarded as a source of mesenchymal stem cells (MSCs) abundant and easily accessible through minimally invasive dental procedures. Due to the proximity of dental resin composites to gingival tissues and to the possible local cytotoxic effect of the eluted components, gingiva-derived MSCs could be used to investigate the biocompatibility of dental biomaterials. PURPOSE: The present research aimed to isolate (MSCs) from inflamed and normal gingiva, to fully characterize them and to observe their behavior in relation with some commercial resin composite materials and one experimental material. MATERIAL AND METHODS: Following their isolation, putative MSCs from both gingival sources were grown under the same culture conditions and characterized by immunophenotyping of cell surface antigens by flow-cytometry and transcription factors by immunocytochemical staining. Moreover, stemness gene expression was evaluated by RT-PCR analysis. Multipotent mesenchymal differentiation potential was investigated. Osteogenic and neurogenic differentiated cells were highlighted by immunocytochemical staining, chondrogenic cells by cytochemical staining, and adipocytes by cytochemical staining and spectrophotometry, respectively. Resin composite cytotoxicity was evaluated by cell membrane fluorescent labeling with PKH 26 and MTT assay. The results of PKH labeling were statistically analysed using two-way RM ANOVA with Bonferroni post-tests. For MTT assay, two-way RM ANOVA with Bonferroni post-tests and unpaired t test with Welch's correction were used. RESULTS: A similar expression pattern of surface markers was observed. The cells were positive for CD105, CD73, CD90, CD49e, CD29, CD44 and CD166 and negative for CD45, CD34, CD14, CD79, HLA-DR and CD117 indicating a mesenchymal stem cell phenotype. The qRT-PCR analysis revealed a low gene expression for NOG, BMP4 and Oct3/4 and an increased expression for Nanog in both cells lines. Immunocytochemical analysis highlighted a more intense protein expression for Nanog, Oct3/4 and Sox-2 in MSCs derived from normal gingiva than from inflamed gingiva. Multipotent differentiation capacity of MSCs isolated from both sources was highlighted. The tested materials had no hazardous effect on MSCs as the two cell lines developed well onto resin composite substrates. Cell counting revealed some significant differences in the number of PKH-labeled MSCs at some experimental moments. Also, some differences in cell viability were recorded indicating better developmental conditions offered by some of the tested biomaterials. CONCLUSIONS: The experimental resin composite behaved like the most biocompatible commercial material. Inflamed gingiva-derived MSCs retain their stem cell properties and could be used as a valuable cell line for testing dental biomaterials.

In vitro studies on the mechanisms involved in chemoprevention using Calluna vulgaris on vascular endothelial cells exposed to UVB.

The study aims to investigate the mechanisms involved in the in vitro effect of UVB on endothelial vascular cells (HUVECs) pretreated with a photochemopreventive agent, the Calluna vulgaris (Cv) extract. Two concentrations of Cv, below the limit of cytotoxicity IC50 (2.5 and 7.5 µg GAE/ml) and two doses of UVB (50 and 100 mJ/cm(2)) were used. Oxidative stress parameters were quantified at 1 h and 24 h after irradiation and apoptosis, DNA damage and the induction/activation of NF-κB were evaluated at 24 h. UVB exposure led to the formation of lipid peroxides in a dose dependent manner (p<0.001), induced apoptosis, increased the γ-H2AX levels and the activation of NF-κB. Pretreatment with 2.5 µg GAE/ml Cv improved the antioxidant defense, protected against DNA lesions and was able to decrease cellular death at low dose of irradiation. 7.5 µg GAE/ml Cv was prooxidant, favored the formation of DNA lesions, amplified the NF-κB activation UVB-induced (p<0.01) and led to high levels of cellular death. Both doses of Cv inhibited caspase-3 activation. The modulatory effect of Cv extract on endothelial cells exposed to UVB depend on the concentration of Cv used. This study provides insides into the mechanisms triggered by UVB and antioxidants on skin endothelial cells.

Evaluation of bovine embryo biopsy techniques according to their ability to preserve embryo viability.

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

Establishment of an embryonic stem cell line from blastocyst stage mouse embryos.

Embryonic stem cells have the ability to remain undifferentiated and proliferate in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. The aim of the present study was to establish mouse ES lines from blastocyst stage embryos obtained after CD1/EGFP mice superovulation. We isolated, cultured and determined the characteristics of mouse embryonic stem cells in early passages, which were first described by Evans M and Kaufman M. Therefore, we evaluated the morphological criteria for the approval of ES cells in early expansion stage. Two cell lines were isolated (CDE1 and CDE2) and analyzed. They showed similar characteristics to those reported earlier for blastocyst-derived ES cell lines.