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.

Penetration of sodium hypochlorite into root canal dentine: effect of surfactants, gel form and passive ultrasonic irrigation.

AIM: To assess the penetration of sodium hypochlorite (NaOCl) gel or NaOCl solutions with surfactants, and the effect of passive ultrasonic irrigation (PUI) on penetration into dentinal tubules. METHODOLOGY: Bovine incisor root canals were instrumented, the roots sectioned and the dentine blocks obtained were stained with crystal violet. Dentine blocks (n = 10 per group) were exposed to 3% NaOCl gel or 3% NaOCl solution for 10 and 20 min. Other dentine blocks (n = 10 per group) were exposed to Chlor-Extra (6% NaOCl + surfactant), 6% NaOCl, 2.5% NaOCl with 0.2% cetrimide and 2.5% NaOCl for 10 and 20 min. The penetration depth of irrigants into dentinal tubules was measured in micrometres by viewing the bleached crystal violet under a stereomicroscope. Additionally, bovine incisor root canals, instrumented and stained with crystal violet, were distributed into two groups (n = 10) and irrigated with 2.5% NaOCl with PUI or conventional syringe irrigation (CSI). The penetration depth of irrigants into dentinal tubules was assessed 3 and 7 mm from the apex. Statistical analysis was performed by ANOVA and Tukey tests (α = 0.05). RESULTS: There was significantly greater penetration of 3% NaOCl solution into dentinal tubules compared with the gel form (P < 0.05). There was no difference (P > 0.05) between 6% NaOCl and Chlor-Extra, and between 2.5% NaOCl and 2.5% NaOCl + cetrimide. PUI significantly increased the penetration depth of NaOCl into dentinal tubules when compared with CSI (P < 0.05). CONCLUSIONS: In extracted bovine incisors, NaOCl gel penetrated less into dentinal tubules than NaOCl solution. The addition of surfactants did not increase the penetration depth. The use of PUI significantly increased NaOCl penetration into dentinal tubules.

Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

AIM: To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). METHODOLOGY: L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10 min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α = 0.05). RESULTS: The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P < 0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. CONCLUSIONS: Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl.

Biocompatibility and mineralized nodule formation of Neo MTA Plus and an experimental tricalcium silicate cement containing tantalum oxide.

AIM: To evaluate the biocompatibility and mineralized nodule formation of an experimental tricalcium silicate cement with tantalum oxide (TSC/Ta2 O5 ) as radiopacifier, Neo MTA Plus (Avalon Biomed Inc., Bradenton, FL, USA) and MTA (Angelus, Londrina, PR, Brazil) on human osteoblast-like cells (Saos-2). METHODOLOGY: Biocompatibility was evaluated by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT) and neutral red (NR) assays, after exposure of Saos-2 to cement extracts at 1 : 1, 1 : 2, 1 : 4 and 1 : 8 dilutions for 24 h. Bioactivity was evaluated by alkaline phosphatase (ALP) activity, and calcium deposits were detected with alizarin red staining (ARS). Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05). RESULTS: The MTT assay revealed lower cytotoxicity for NEO and MTA (P < 0.05), and higher for TSC/Ta2 O5 at 1 : 1 and 1 : 2 dilutions when compared to serum-free medium - control (P > 0.05). At 1 : 4 dilution, the TSC/Ta2 O5 cytotoxicity was similar to the control (P > 0.05). At 1 : 8 dilution, cell viability was significantly greater than the control (P < 0.05). Saos-2 cell viability performed using the NR assay at all dilutions revealed no cytotoxic effect of MTA, NEO and TSC/Ta2 O5 . ALP activity at 1 and 3 days was similar to the control (P > 0.05). TSC/Ta2 O5 had significantly greater ALP activity at 7 days when compared with the control (P < 0.05). All materials induced the production of mineralized nodules, and NEO produced significantly more mineralized nodules than MTA and TSC/Ta2 O5 (P < 0.05). CONCLUSIONS: Neo MTA Plus and TSC/Ta2 O5 were biocompatible and induced ALP activity in Saos-2 cells. Both materials induced mineralized nodule formation by Saos-2 with Neo MTA Plus producing significantly more.