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.

Heparin is biocompatible and can induce differentiation of human dental pulp cells.

AIM: To investigate the biocompatibility, osteogenic bioactivity and mRNA expression of the osteo/odontogenic markers bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP), induced by heparin in human dental pulp cells (hDPCs). METHODOLOGY: hDPCs were exposed to the heparin, and cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT), and cell death was evaluated by flow cytometry. Osteogenic bioactivity was evaluated by the alkaline phosphatase (ALP) assay, and the detection of calcium deposits by alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with ANOVA and Bonferroni or Tukey post-test and t-test (α = 0.05). RESULTS: Heparin had no cytotoxic effect and did not induce apoptosis. After 3 days, heparin had significantly higher ALP activity in comparison with the control (P < 0.05). Heparin had a significant (P < 0.05) stimulatory effect on the formation of mineralized nodules. BMP-2 and OC mRNA expressions were significantly higher in cells exposed to heparin than control group after 1 day (P < 0.05). CONCLUSIONS: Heparin was biocompatible in hDPCs, induced osteogenic bioactivity and enhanced mRNA expression of osteo/odontogenic markers BMP-2 and OC. These results suggest that heparin has potential to induce osteo/odontogenic cell differentiation of hDPCs.

Functionality and opposite roles of two interleukin 4 haplotypes in immune cells.

Cytokines expression can be influenced by polymorphisms in their respective coding genes. We associated the CTI/TTD haplotype (Hap-1) and TCI/CCI haplotype (Hap-2) in the IL4 gene formed by the -590, +33 and variable number of tandem repeat polymorphisms with the severity of chronic periodontitis in humans. The functionality of these IL4 haplotypes in the response of immune cells to phorbol 12-myristate 13-acetate (PMA) with Ionomycin and IL-1ß (as inflammatory stimuli) was evaluated. Gene expression (quantitative real-time PCR), profile of secreted cytokines (multiplex) and phenotypic polarization of T cells (flow cytometry) were the outcomes assessed. Green fluorescent protein reporter plasmid constructs containing specific IL4 haplotype were transiently transfected into JM cells to assess the influence of the individual haplotypes on promoter activity. In response to inflammatory stimuli the immune cells from Hap-1 haplotype had increased expression of anti-inflammatory IL4; conversely, the Hap-2 haplotype showed higher levels of pro-inflammatory cytokines. The haplotype CTI proved to be the most important for the regulation of IL4 promoter, regardless of the nature of the inflammatory stimulation; whereas the polymorphism in the promoter region had the least functional effect. In conclusion, IL4 haplotypes studied are functional and trigger opposite immune responses: anti-inflammatory (Hap-1) and pro-inflammatory (Hap-2). In addition, we identified the CTI haplotype as the main responsible for the regulation of IL4 transcriptional activity.

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.

Modulation of immune response by RAGE and TLR4 signalling in PBMCs of diabetic and non-diabetic patients.

Diabetes is associated with increased glucose levels and accumulation of glycated products. It is also associated with impairment in the immune response, such as increased susceptibility to infections. In this study, we assessed the possible interactions between TLR4 and RAGE signalling on apoptosis and on the expression of inflammatory cytokines in PBMC from individuals with and without diabetes. PBMCs were isolated from seven diabetic patients and six individuals without diabetes and stimulated in vitro with bacterial LPS (1 µg/ml) associated or not with BSA-AGE (200 µg/ml). This stimulation was performed for 6 h, both in the presence and in the absence of inhibitors of TLR4 (R. sphaeroides LPS, 20 µg/ml) and RAGE (blocking monoclonal antibody). Apoptosis at early and late stages was assessed by the annexin-V/PI staining using flow cytometry. Regulation of TNF-α and IL-10 gene expression was determined by RT-qPCR. PBMCs from diabetes patients tended to be more resistant apoptosis. There were no synergistic or antagonistic effects with the simultaneous activation of TLR4 and RAGE in PBMCs from either diabetes or non-diabetes group. Activation of TLR4 is more potent for the induction of TNF-α and IL-10; RAGE signalling had a negative regulatory effect on TNF-α expression induced by LPS. TLR and RAGE do not have relevant roles in apoptosis of PBMCs. The activation of TLR has greater role than RAGE in regulating the gene expression of IL-10 and TNF-α.

Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit the production of IL-1ß, IL-6 and IL-10 on human macrophages.

OBJECTIVE: Tea tree oil (TTO) is an essential oil with anti-inflammatory properties, steam distilled from the plant Melaleuca alternifolia. We investigated the immunomodulatory properties of TTO and its components (terpinen-4-ol and alpha-terpineol) using lipopolysaccharide (LPS)-stimulated macrophages. METHODS: The ability of TTO, terpinen-4-ol and alpha-terpineol to modulate the macrophage response to bacterial LPS stimulation was assessed by ELISA for tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and IL-10 cytokine production and by western blotting for the activation of nuclear factor kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling, which are associated with the expression of pro-inflammatory cytokines. We used a human monocytic cell line (U937) differentiated into macrophages. RESULTS: LPS induced the production of all cytokines, and TTO and its components significantly reduced the production of IL-1ß, IL-6 and IL-10. The production of TNF-α was not affected by either TTO or its major components. The modulation of cytokine production was not mediated by changes in NF-κB or p38 MAPK activation. CONCLUSION: TTO, terpinen-4-ol and alpha-terpineol can suppress the production of inflammatory mediators in LPS-stimulated human macrophages; this inhibition was mediated by interfering with the NF-kB, p38 or ERK MAPK pathways.