Sugarcane cystatin CaneCPI-1 promotes osteogenic differentiation in human dental pulp cells: a new insight into cysteine proteases inhibitors.

AIM: To investigate the biocompatibility, type of cell death, osteogenic bioactivity and mRNA expression of the osteogenic markers, induced by CaneCPI-1 in human dental pulp cells (hDPCs). METHODOLOGY: hDPCs exposed to CaneCPI-1 and not exposed (control) were evaluated for cell viability by the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay; apoptosis by flow cytometry; alkaline phosphatase (ALP) activity by calculation of thymolphthalein release; gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX2), ALP, osteocalcin (OC), bone sialoprotein (BSP) by qPCR; and mineralized nodules production by using alizarin red staining. The data were analysed by one-way analysis of variance (anova) and Turkey's post-test, two-way anova and Bonferroni post-test or t-test (P < 0.05). RESULTS: CaneCPI-1 induced no apoptosis and had no cytotoxic effect, except in the concentration of 33.20 µm, in which cell viability was significantly lower than the control (α-MEM nonosteogenic medium serum-free) (P < 0.05). There was significantly greater ALP activity, greater expression of the BMP-2, RUNX2, ALP, OC and BSP genes and greater mineralized nodules production in the CaneCPI-1 group in comparison with the control or osteogenic α-MEM control (α-MEM osteogenic medium - L-ascorbic acid and ß-glycerophosphate) (P < 0.05). CONCLUSIONS: CaneCPI-1 was cytocompatible and also induced the differentiation of hDPCs in osteogenic phenotype in vitro. CaneCPI-1 is a promising molecule to induce pulp repair.

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.

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.