An assessment of the overexpression of BMP-2 in transfected human osteoblast cells stimulated by mineral trioxide aggregate and Biodentine.

AIM: To evaluate the effect of MTA and Biodentine on viability, osteogenic differentiation and BMP-2 expression in osteogenic cells. METHODOLOGY: Saos-2 cells were used as a model of osteoblastic cells. Overexpression of BMP-2 was induced by transfection of a CMV-driven plasmid construct including the human BMP-2 coding sequence, and stably transfected cells were selected. Cell viability was assessed by the mitochondrial dehydrogenase enzymatic (MTT) assay. The bioactivity of the materials was evaluated by the alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2 and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). RESULTS: Viability tests revealed that MTA and Biodentine were not cytotoxic at the higher dilution (1 : 8) to BMP-2-transfected cells. MTA and Biodentine exhibited the highest ALP activity when compared to the Saos-BMP-2-unexposed control group (P < 0.05). Cell exposure to Biodentine and MTA had a significant stimulatory effect on the formation of mineralized nodules (P < 0.05). The highest increase in BMP-2 gene expression was observed after 3 days of BMP-2-transfected cells exposure to MTA and Biodentine in non-osteogenic medium in relation to Saos-BMP-2-unexposed control cells (P < 0.05). Exposure of cells to MTA in osteogenic medium for 1 day increased ALP gene expression by approximately 1.3-fold in relation to Saos-BMP-2-unexposed control cells (P < 0.05). CONCLUSIONS: Both MTA and Biodentine showed biocompatibility and bioactivity in Saos-BMP-2 overexpressing cells. Biodentine had a significantly greater effect on mineralization than MTA. Both MTA and Biodentine enhanced BMP-2 mRNA expression in the transfected system. Both MTA and Biodentine are suitable materials to improve osteoblastic cell mineralization.

Human dental pulp cells response to mineral trioxide aggregate (MTA) and MTA Plus: cytotoxicity and gene expression analysis.

AIM: To investigate the cytotoxicity, osteogenic bioactivity and mRNA expression of osteogenic markers of bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP) induced by the extracts of set MTA Plus (MTA P) (Avalon Biomed Inc. Bradenton, FL, USA) in comparison with MTA (Angelus, Londrina, PR, Brazil) on human dental pulp cells (hDPCs). METHODOLOGY: Cell viability was assessed by mitochondrial dehydrogenase enzymatic (MTT) assay, and the mechanism of cell death was evaluated by flow cytometry. Bioactivity was evaluated by alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). RESULTS: MTA and MTA P were not cytotoxic and did not induce apoptosis. MTA P had significant higher ALP activity in relation to MTA and the control (P < 0.05). MTA had a significantly higher percentage of mineralized area than MTA P (P < 0.05). The expression of BMP2 and OC mRNA was significantly higher in cells exposed to MTA than MTA P after 1 day (P < 0.05). At day 3, the mRNA expression of ALP was significantly higher in MTA P compared with MTA (P < 0.05). CONCLUSIONS: MTA and MTA Plus were noncytotoxic, increased mineralization processes in vitro and induced the expression of osteogenic markers.

Bioactivity of MTA Plus, Biodentine and an experimental calcium silicate-based cement on human osteoblast-like cells.

AIM: To compare the bioactivity of Biodentine (BIO, Septodont), MTA Plus (MTA P, Avalon) and calcium silicate experimental cement (CSC) with resin (CSCR) associated with zirconium (CSCR ZrO2 ) or niobium (CSCR Nb2 O5 ) oxide as radiopacifiers. METHODOLOGY: According to the relevance of osteoblastic cell response for mineralized tissue repair, human osteoblastic cells (Saos-2) were exposed to test materials and assessed for viability (MTT), cell proliferation, gene expression of alkaline phosphatase (ALP) osteogenic marker by real-time PCR (RT-qPCR), ALP activity assay and alizarin red staining (ARS) to detect mineralization nodule deposition in osteogenic medium. Unexposed cells acted as the control group (C). Statistical analysis was carried out using ANOVA and the Bonferroni post-test (P < 0.05). RESULTS: All tested cements showed dose-dependent responses in cell viability (MTT). Exposed cells revealed good viability (80-130% compared to the control group) in the highest dilutions of all types of cement. MTA P, BIO and CSCR ZrO2 significantly increased the velocity of cell proliferation after three days of cell exposure in the wound-healing assay (P < 0.05), which corroborated MTT data. On day 3, the ALP transcript level increased, especially to CSCR Nb2 O5 (P < 0.05). All cements exhibited suitable ALP enzyme activity, highlighting the 7-day period of cell exposure. ARS, CSCR Nb2 O5 , revealed a significant potential to induce mineralization in vitro. CONCLUSIONS: All materials had suitable biocompatibility and bioactivity. The MTA P, BIO and CSCR ZrO2 groups had the highest viability rates and velocity of proliferation whilst the CSCR Nb2 O5 group produced more mineralized nodules.

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos.

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions.

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17ß-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.