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Background: Different pulp capping materials have different origins and compositions, require different preparations, and may vary in their bioactive properties., Aim: The purpose of this study was to evaluate the antibacterial activity, biocompatibility, and mineralization-inducing potential of calcium silicate-based pulp capping materials., Design: Six contemporary calcium silicate-based cements, ProRoot MTA, MTA Angelus, Biodentine, EndoSequence, NeoMTA 2, and NeoPutty, were evaluated. The antibacterial effects of these materials against Streptococcus mutans UA159 and Enterococcus faecalis ATCC 29212 were determined by the agar diffusion assay and the direct culture test. The biocompatibility and mineralization-inducing potential of these materials in preodontoblastic 17IIA11 cells were evaluated by the MTT assay and by Alizarin Red S staining, respectively., Results and Conclusion: In agar diffusion test, only Biodentine showed distinct antibacterial effects against S. mutans. All the tested materials, however, showed antibacterial effects against S. mutans and E. faecalis in the direct culture test, with Biodentine showing the strongest growth inhibition against both S. mutans and E. faecalis. All the tested materials showed acceptable biocompatibility and mineralization-supporting potential in our experimental conditions. In summary, ProRoot MTA, MTA Angelus, Biodentine, EndoSequence, NeoMTA 2, and NeoPutty demonstrated acceptable in vitro antimicrobial, biocompatible, and mineralization-supporting properties., (© 2024 The Authors. International Journal of Paediatric Dentistry published by BSPD, IAPD and John Wiley & Sons Ltd.)

Global ornamental fish transportation ranging from hours to days can produce multiple stress factors impact fish health and cause mortality. Clownfish, particularly Amphiprion ocellaris, are among the most traded saltwater ornamental fish. Vibrio includes several pathogenic strains that affect aquatic animals. Consequently, prophylactic treatment of the water or fish is recommended. In this study, six Vibrio strains including V. alginolyticus, V. parahaemolyticus and V. harveyi isolated from sick A. ocellaris and one V. harveyi strain from a sick East Asian fourfinger threadfin (Eleutheronema rhadinum) were tested for their sensitivity to a popular disinfectant, chlorine dioxide (ClO 2 ). The results showed that 0.25 ppm ClO 2 effectively suppressed five of the seven tested Vibrio strains for 24 h; however, 0.1 ppm ClO 2 is safer for A. ocellaris. Meanwhile, ClO 2 2.5 ppm reduced the bacterial counts to below 3.3 × 10 5  CFU/mL for 24 hours. The LC 50 of ClO 2 for A. ocellaris was 0.87 ppm at 10 min and 0.72 ppm at 24 h post treatment. Mild changes in water quality, including dissolved oxygen (DO), temperature and pH, were recorded during the trial. More research is necessary to understand the sensitivity of various aquatic animal pathogens to ClO 2 and its toxicity to different aquatic animals., (© 2024 John Wiley & Sons Ltd.)

Aim: To evaluate the inflammatory reaction and the ability to induce mineralization activity of a new repair material, NeoPUTTY (NPutty; NuSmile, USA), in comparison with Bio-C Repair (BC; Angelus, Brazil) and MTA Repair HP (MTA HP; Angelus, Brazil)., Methodology: Polyethylene tubes were filled with materials or kept empty (control group, CG) and implanted in subcutaneous tissue of rats for 7, 15, 30, and 60 days (n = 6/group). Capsule thickness, number of inflammatory cells (ICs), fibroblasts, collagen content, and von Kossa analysis were performed. Unstained sections were evaluated under polarized light and by immunohistochemistry for osteocalcin (OCN). Data were submitted to two-way anova followed by Tukey's test (p ≤ .05), except for OCN. OCN data were submitted to Kruskal-Wallis and Dunn and Friedman post hoc tests followed by the Nemenyi test at a significance level of 5%., Results: At 7, 15, and 30 days, thick capsules containing numerous ICs were seen around the materials. At 60 days, a moderate inflammatory reaction was observed for NPutty, BC while MTA HP presented thin capsules with moderate inflammatory cells. In all periods, NPutty specimens contained the highest values of ICs (p < .05). From 7 to 60 days, the number of ICs reduced significantly while an increase in the number of fibroblasts and birefringent collagen content was observed. At 7 and 15 days, no significant difference was observed in the immunoexpression of OCN (p > .05). At 30 and 60 days, NPutty showed the lowest values of OCN (p < .05). At 60 days, a similar immunoexpression was observed for BC and MTA HP (p > .05). In all time intervals, capsules around NPutty, BC, and MTA HP showed von Kossa-positive and birefringent structures., Conclusions: Despite the greater inflammatory reaction promoted by NeoPutty than BC and MTA HP, the reduction in the thickness of capsules, the increase in the number of fibroblasts, and the reduction in the number of ICs indicate that this bioceramic material is biocompatible Furthermore, NeoPutty presents the ability to induce mineralization activity., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: Previous studies have evaluated the pulpal responses to calcium silicate cements (CSCs) on normal dental pulp, but investigations on the effects of CSCs on inflamed pulp are limited. This study aimed to test the inflammatory response and odontogenic differentiation of inflamed rat dental pulp after direct pulp capping with CSCs., Methodology: Wistar rat molars pulps were exposed for 48 h to induce inflammation and then capped with ProRoot MTA (Dentsply), Biodentine (Septodont), RetroMTA (Bio MTA) and Dycal (Dentsply Caulk). The degree of pulpal inflammation and hard tissue formation was evaluated by histological analysis. Immunofluorescence staining for interleukin (IL)-6, osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) was also performed., Results: After 4 weeks, complete recovery from inflammation was evident in 22%, 37.5%, 10% and none of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. Heavy hard tissue deposition as a continuous hard tissue bridge was observed in 77.8%, 75%, 70% and 60% of the ProRoot MTA, Biodentine, RetroMTA and Dycal samples, respectively. IL-6, OCN and RUNX2 were detected in all materials, mainly adjacent to areas of inflammation and reparative dentine formation. At one, two and 4 weeks, significant differences were not observed between the inflammation and hard tissue formation scores of the four material groups (p > .05)., Conclusions: In this study, pulpal inflammation was still present in most specimens at 4 weeks after pulp capping and a significant number of samples showed incomplete and discontinuous dentine bridge formation. The results of this study suggest that initial inflammatory conditions of the pulp may risk the prognosis of teeth treated with CSCs., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: This study aimed to evaluate the cytotoxicity, biocompatibility and osteoinductive profile of a mineral trioxide aggregate (MTA)-hydrogel-based material (MTA Flow) in comparison with MTA Angelus., Methodology: Cell viability was evaluated in human periodontal ligament stem cells (hPDLSCs) using the methyl-thiazol-tetrazolium (MTT) colourimetric assay. Polyethylene tubes containing the tested materials and empty polyethylene tubes (control) were implanted in the subcutaneous tissue of Wistar rats. Cellular (lymphocyte infiltration) and extracellular events (ECM; collagen fibres) were analysed in histological sections. Immunohistochemical (collagen I, osteopontin, bone sialoprotein, bone morphogenetic protein4) analyses were also performed., Results: At 24, 48 and 72 h, all tested groups showed cell viability similar to control (p > .05). Regarding biocompatibility, all groups showed similar cellular events represented by a slight inflammatory reaction characterized by hyperaemia and a mild lymphocytic inflammatory infiltrate. The analysis of lymphocytes during the time showed a decrease in these cells in the control group and a significant interaction between MTA Angelus and control (p < .001), with MTA Angelus showing a more extensive inflammatory infiltrate. Regarding fibres, an increase in content was observed in all groups during the experimental time (7, 30 and 60 days), however, no difference was detected among the experimental groups (p = .063). After 60 days, the immunoexpression of bone matrix proteins in the MTA Flow group was similar to or higher than that observed in the MTA Angelus and in the control group., Conclusions: MTA Flow showed a non-cytotoxic behaviour, biocompatibility and ability to stimulate tissue mineralization., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: Inflammatory-regenerative cell interaction is believed to mediate hard tissue formation. This study aimed to investigate the interaction between human inflammatory monocytes with human regenerative fibroblasts after exposure to different calcium silicate materials., Methodology: Human monocytes were cultured on three materials, polystyrene (PS), mineral trioxide aggregates (MTA) and biodentine (BD), in the presence or absence of lipopolysaccharide (LPS). Half of the monocyte-conditioned media (MoCM) of each group was used to analyse inflammatory cytokine secretion, namely TNF-α, IL-1β, IL-1RA and IL-6. The remaining MoCM was used to culture recipient fibroblasts, measuring the cell number (proliferation) and levels of alkaline phosphatase (differentiation) and lactic acid dehydrogenase (cytotoxicity)., Results: In absence of LPS, MTA was associated with higher secretion of TNF-α and lower secretion of IL-1β, while BD triggered higher secretions of both cytokines when both materials were compared to control (PS). When LPS was added, higher levels of all analysed cytokines were observed in the PS and BD groups, whereas for the MTA group, only TNF-α and IL-6 were increased. Fibroblasts responded differently to the MoCM from the different groups, revealing significant increases in proliferation and differentiation capacities, particularly when cultured in CM from monocytes exposed to MTA. The morphological evaluation revealed different patterns of fibroblast shape and spread in the different MoCM groups., Conclusion: Calcium silicate materials modulate the monocyte inflammatory response, which subsequently induce differential effects on the recipient fibroblasts. MTA appears to promote the secretion of prodifferentiation signals from the monocytes, which are received by fibroblasts, promoting their proliferation and differentiation. The model represents a promising tool to evaluate the interaction of different cells in response to different materials., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: To evaluate whether the bioceramic materials Bio-C Pulpo (Bio-C, Angelus) and mineral trioxide aggregate (MTA) Repair HP (MTA-HP, Angelus) induce fibroblast proliferation and release of interleukin-10 (IL-10), an anti-inflammatory cytokine, stimulating connective tissue remodelling. The tissue response of Bio-C and MTA-HP was compared with the White MTA (WMTA; Angelus) since studies have demonstrated that WMTA induces tissue repair., Methodology: Bio-C, MTA-HP and WMTA were inserted into polyethylene tubes and implanted in the subcutaneous tissue of Holtzman rats for 7, 15, 30 and 60 days. As a control group (CG), empty tubes were implanted subcutaneously. The number of fibroblasts (FB), Ki-67-, fibroblast growth factor-1- (FGF-1) and IL-10-immunolabelled cells and collagen content in the capsules was obtained. The data were subjected to two-way anova followed by Tukey's test (p ≤ .05)., Results: At 7 days, significant differences in the number of FB were not detected amongst Bio-C, MTA-HP and WMTA groups (p ˃ .05). The capsules of all groups exhibited a significant increase in the number of FB and content of collagen over time. From 7 to 60 days, a significant reduction in the number of FGF-1- and Ki-67-immunolabelled cells was seen in the capsules of all specimens. In all periods, no significant difference in the number of FGF-1-immunolabelled cells was detected between Bio-C and CG specimens. At 60 days, significant differences in the immunoexpression of FGF-1 were not observed amongst the groups. At 7 and 15 days, the highest immunoexpression for Ki-67 was present in Bio-C specimens whilst, after 30 and 60 days, no significant difference was observed amongst the bioceramic materials. At 7 days, few IL-10 immunolabelled cells were present in the capsules of all specimens whereas, at 60 days, a significant increase in the IL-10-immunostaining was present in all groups. At 60 days, the Bio-C, MTA-HP and WMTA groups showed a greater number of IL-10-immunolabelled cells than in the CG specimens (p < .0001)., Conclusions: Bio-C, MTA-HP and WMTA stimulate fibroblast proliferation, leading to the formation of collagen-rich capsules. FGF-1 and IL-10 may mediate the remodelling of capsules around Bio-C, MTA-HP and WMTA bioceramic materials., (© 2022 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the tissue reaction of a tricalcium silicate-based repair material associated with 30% calcium tungstate (TCS + CaWO 4 ) in comparison to Bio-C Repair (Bio-C; Angelus) and to MTA Repair HP (MTA HP; Angelus)., Methodology: Polyethylene tubes filled with one of the materials or left empty (control group, CG) were implanted into the subcutaneous tissues of rats for 7, 15, 30 and 60 days (n = 32/group). The capsule thickness, number of inflammatory cells, collagen content, interleukin-6 (IL-6), osteocalcin (OCN), von Kossa reaction and analysis under polarized light were evaluated. The data were subjected to generalized linear models for repeated measures, except the OCN. OCN data were submitted to Kruskal-Wallis and Dunn's post hoc test and Friedman followed by Nemenyi's test at significance level of 5%., Results: At all time points, significant differences in the number of inflammatory cells were not observed between TCS + CaWO 4 and Bio-C, whereas, at 15, 30 and 60 days, no significant difference was detected between TCS + CaWO 4 and MTA HP. At all periods, significant differences were not detected in the number of fibroblasts in TCS + CaWO 4 versus MTA HP, and, at 60 days, no significant difference was demonstrated between these groups and CG. Significant differences in the immunoexpression of IL-6 were not detected amongst bioceramic materials at all periods. From 7 to 60 days, significant reduction in the number of inflammatory cells, number of IL-6-immunopositive cells and in the capsule thickness was accompanied by significant increase in the collagen in all groups. OCN-immunolabelled cells, von Kossa-positive structures and amorphous calcite deposits were observed around all materials, whereas, in the CG, these structures were not seen., Conclusions: These findings indicate that the experimental material (TCS + CaWO 4 ) is biocompatible and has a bioactive potential, similar to the MTA HP and Bio-C Repair, and suggest its use as a root repair material., (© 2022 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: To evaluate in vitro whether MTA Repair HP can induce repair processes at a distance, including its effects on biofilm, cell viability, migration, production of TGF-β, phosphate and ALP, evaluated through MTA diluted extracts., Methodology: Initially, antibacterial tests were performed with the bacterium Streptococcus mutans (ATCC 25175) in the presence of MTA extracts (dilutions of 1:1, 1:2 and 1:4). Growth inhibition assay by microdilution in broth, antibiofilm plate assay of young biofilm and antibiofilm assay in confocal microscopy of mature biofilm were carried out. Then, pulp cells were stimulated in the presence of several MTA dilutions, and cell viability (MTT assay), proliferation and migration capacity (scratch assay) were evaluated. To evaluate the capacity of 1:1, 1:2 and 1:4 dilutions of MTA Repair HP to promote the production of important agents of odontogenic differentiation and mineralization, ALP activity, TGF-β secretion and phosphate quantification were measured. Statistical differences were verified using one-way and two-way anova and Tukey's post-tests., Results: The test dilutions of MTA Repair HP did not inhibit planktonic S. mutans growth but were able to reduce young and mature S. mutans biofilm (p < 0.001). In addition, none of the MTA Repair HP dilutions was cytotoxic for pulp cells. The 1:2 and 1:4 dilutions of MTA Repair HP induced migration and proliferation of pulp cells (p < 0.05). ALP activity and TGF-β secretion were independent of the tested dilution (p < 0.001). Diluted 1:4 MTA Repair HP produced less phosphate than the more concentrated 1:1 and 1:2 MTA dilutions (p < 0.001)., Conclusions: Undiluted MTA Repair HP reduced S. mutans biofilm, when compared to 1:2 and 1:4 MTA dilutions. Furthermore, none of the tested dilutions was cytotoxic to pulp cells. MTA Repair HP promoted cell migration and proliferation at a distance, assessed through the dilution of the MTA. Even from a distance, MTA Repair HP has the ability to participate in some events related to repair, such as migration, proliferation and TGF production., (© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To explore the proliferation, adhesion and differentiation response and the underlying mechanisms that occur in lipopolysaccharide (LPS)-induced inflamed dental pulp cells (DPCs) in contact with Biodentine and mineral trioxide aggregate (MTA)., Methodology: The DPCs were isolated from three healthy donors and named DPC-H1 to DPC-H3. The DPCs were pre-cultured with 2 or 5 μg mL -1 LPS for 24 h to induce inflammation. The expression of inflammation marker miR-146a was detected by q-PCR. The normal and LPS-induced DPCs were further treated with 0.14 mg mL -1 Biodentine or 0.13 mg mL -1 MTA for 24 h. MTT assay and adhesion assay were used to analyse the changes of cell phenotypes. DSPP, AKT and ERK expressions were detected by Western blotting. The data were analysed by Mann-Whitney test or two-way anova. Differences were considered statistically significant when P < 0.05., Results: In LPS-induced DPCs, Biodentine and MTA treatment neither induced nor aggravated LPS-induced inflammation, but their presence did increase the expression of the odontogenic differentiation marker DSPP. Under 2 or 5 μg mL -1 LPS-induced inflammation, Biodentine and MTA promoted the proliferation of DPC cells, and significantly in DPC-H2 (P < 0.0001 for both reagents). With the treatment of 2 μg mL -1 LPS, the cell adhesion of DPCs on the fibronectin-coated culture plates was increased significantly by Biodentine (P = 0.0413) and MTA (P < 0.0001). Biodentine and MTA regulated cell adhesion on the fibronectin-coated culture plates (P < 0.0001 for both reagents) and proliferation (P < 0.0001 for both reagents) via the AKT pathway. However, the AKT pathway was not involved in the expression of DSPP induced by Biodentine and MTA., Conclusion: Biodentine and MTA enhanced the proliferation, adhesion and differentiation of LPS-induced DPCs. The proliferation and adhesion process induced by Biodentine and MTA was via the AKT pathway. However, the cellular differentiation process might not use the same pathway, and this needs to be explored in future studies., (© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the influence of powder-to-gel ratio (0.19 g powder to 50 μL of gel, thick MTA Flow, and 0.06 g powder to 50 μL of gel, fluid MTA Flow) on biocompatibility of MTA Flow (Ultradent Products Inc., South Jordan, UT, USA, lot: 2015122901) and compare it with Biodentine (Septodont Inc., Saint-Maur-des-Fossés, France, lot: B18542A)., Methodology: The materials were manipulated and inserted into polyethylene tubes for implantation in twenty rats. After 7, 15, 30 and 60 days, the specimens were removed and embedded in paraffin. Haematoxylin and eosin sections were used to count the number of inflammatory cells (IC) and fibroblasts mm -2 (Fb). In the Masson's trichrome-stained sections, the fibrous capsule thickness was measured; picrosirius red-stained sections were used for birefringent collagen quantification. The data were submitted to two-way ANOVA and Tukey test (P ≤ 0.05)., Results: A significantly lower number of IC and consequently higher number of Fb were observed in the capsules adjacent to thick MTA Flow at all periods, in comparison with other materials (P ≤ 0.05). At 60 days, the quantity of birefringent collagen was significantly greater in the tissue in contact with thick MTA Flow, when compared with fluid MTA Flow and Biodentine., Conclusions: Although thick MTA Flow induced a less intense inflammatory response, all evaluated materials are biocompatible because they allowed regression of this process after 60 days., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To compare the formation of fibrous capsules around Biodentine and MTA Angelus implants as well as the participation of fibroblast growth factor-1 (FGF-1) and mast cells in the tissue response to these endodontic materials., Methodology: Sixty polyethylene tubes filled with Biodentine or MTA, and empty tubes (control group) were implanted into the dorsal subcutaneous tissues of male rats. After 7, 15, 30 and 60 days, the specimens were embedded in paraffin and the number of fibroblasts and mast cells was quantified in the sections stained with Masson's trichrome or Alcian Blue, respectively. FGF-1 and Ki-67 were detected by immunohistochemistry, and the number of immunolabelled cells was computed. The collagen content was estimated in the picrosirius red-stained sections. The data were subjected to two-way ANOVA followed by Tukey's test (P ≤ 0.05)., Results: The capsules were associated with a significant increase (P < 0.0001) in the number of fibroblasts and mast cells, and in the collagen content over time. A significant decrease (P < 0.0001) in the immunoexpression of FGF-1 and Ki-67 was observed in all groups from the 7th-60th day. At 60 days, the number of fibroblasts (P = 0.0226) and the collagen content (P < 0.0001) were significantly greater in MTA than Biodentine specimens, while the greatest number of mast cells and FGF-1-immunolabelled cells was observed in Biodentine specimens (P < 0.0001). A significant difference in Ki-67 immunoexpression was not detected between specimens of Biodentine and MTA., Conclusions: The collagen-rich capsule formed slowly around Biodentine in comparison with MTA. FGF-1 and mast cells participated in capsule remodelling, stimulating fibroblast proliferation and subsequent collagen production, in response to subcutaneous implants., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue., Methodology: A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test., Results: In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days., Conclusions: The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the expression of TNF-α, IL-6, IFN-γ, TGF-β, IL-4, IL-10, RANKL, RANK and OPG on mouse calvarial bone treated with MTA, Geristore ® and Emdogain ® ., Methodology: Bone wounds were made on the heads of C57BL/6 mice, breaking the periosteum and the cortical surface of the calvaria. Each repair agent was inserted into sectioned Eppendorf microtubes and placed on the bone wound, and soft tissues were sutured. At 14 and 21 days, animals were sacrificed and the treated region was dissected. The calvaria bone was removed, and RNA was extracted. mRNA expression of the aforementioned cytokines was assessed using real-time PCR. Data were analysed by nonparametric methods, including the Mann-Whitney and Kruskal-Wallis tests (P < 0.05)., Results: Following treatment with Emdogain ® and MTA, mRNA expression of RANKL, RANK and OPG increased significantly (P < 0.05) between days 14 to 21. Geristore ® did not alter the basal expression of these mediators during the same period of evaluation. Whilst treatment with Emdogain ® did cause a significant increase in TNF-α mRNA expression between days 14 and 21 (P < 0.05), treatment with MTA did not alter the basal expression of this cytokine at either experimental time point. However, TNF-α mRNA expression was down-regulated significantly at day 21 (P < 0.05) when Geristore ® was applied. A significant increase in the mRNA expression of IL-6, TGF-β, IL-10, IL-4 and IFN-γ was observed with Emdogain ® and MTA treatment between days 14 to 21, whereas Geristore ® reduced significantly the expression of IL-6, TGF-β and IL-4 (P < 0.05)., Conclusion: The clinical indication of these repair agents depends on the root resorption diagnosis. Whilst MTA and Emdogain ® induce a pro- and anti-inflammatory response early and late, respectively, Geristore ® was not associated with an inflammatory reaction when compared with both repair agents., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To investigate whether a combination of mineral trioxide aggregate (MTA) and fluoride compounds affects bone cells., Methodology: Mineral trioxide aggregate (MTA) discs (ProRoot ® , Dentsply Sirona, Ballaigues, Switzerland) with and without the addition of 0.1%, 0.25% and 0.5% sodium fluoride were characterized for their surface roughness by laser scanning microscopy and for the adhesion of human alveolar osteoblasts by scanning electron microscopy. Using eluates from fluoride-enriched MTA discs, the cell proliferation was measured by monitoring the DNA incorporation of 5-bromo-2'-deoxyuridine. Further, gene expression was evaluated by qPCR arrays, extracellular matrix mineralization was quantified by absorption measurement of Alizarin red stains, and effects were calculated with repeated measures analysis and post hoc P-value adjustment., Results: Irrespective of fluoride addition, cell adhesion was similar on MTA discs, of which the surface roughness was comparable. Control osteoblasts had a curvilinear proliferation pattern peaking at d5, which was levelled out by incubation with MTA. The addition of fluoride partly restored the MTA-related reduction in the cellular proliferation rate in a dose-dependent manner. At the mRNA level, both fluoride and MTA modulated a number of genes involved in osteogenesis, bone mineral metabolism and extracellular matrix formation. Although MTA significantly impaired extracellular matrix mineralization, the addition of fluoride supported the formation of mineralized nodules in a dose-dependent manner., Conclusion: The addition of fluoride modulated the biocompatibility of MTA in terms of supporting bone cell proliferation and hard tissue formation. Hence, fluoride enrichment is a trend-setting advancement for MTA-based endodontic therapies., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

The aim of this systematic review was to address the question: Do different irrigating protocols have an impact on the dislocation resistance of mineral trioxide aggregate (MTA)-based materials? The review was performed using a well-defined search strategy in three databases (PubMed, Scopus, Web of Science) to include laboratory studies performed between January 1995 and May 2017, in accordance with PRISMA guidelines. Two reviewers analysed the papers, assessed the risk of bias and extracted data on teeth used, sample size, size of root canal preparation, type of MTA-based material, irrigants, canal filling method, storage method and duration, region of roots and the parameters of push-out testing (slice thickness, plunger dimensions and plunger loading direction), the main results and dislocation resistance values (in MPa). From 255 studies, 27 were included for full-text analysis. Eight papers that met the inclusion criteria were included in this review. There was a wide variation in dislocation resistance due to differences in irrigation sequence, time and concentration of irrigants, storage method and duration, and the parameters of push-out bond strength testing. A meta-analysis was not done but qualitative synthesis of the included studies was performed. No definitive conclusion could be drawn to evaluate the effect of irrigation protocols on dislocation resistance of MTA-based materials. Recommendations have been provided for standardized testing methods and reporting of future studies, so as to obtain clinically relevant information and to understand the effects of irrigating protocols on root canal sealers and their interactions with the dentine walls of root canals., (© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To investigate the effect of accelerated-set mineral trioxide aggregate (MTA) on the proliferation and odontoblastic differentiation of human dental pulp cell niches (DPSC)., Methodology: ProRoot White MTA (WMTA; Dentsply Tulsa Dental, Johnson City, TN, USA) was mixed with various additives, which included distilled water, 2.5% disodium hydrogen phosphate (Na 2 HPO 4 ; Merck, Darmstadt, Germany) and 5% calcium chloride (CaCl 2 ; Merck). DPSC niches extracted from third molars were cultured directly on MTA in the culture medium. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulphophenyl)-2H-tetrazolium (MTS) assay. Cell growth and expression of odontoblastic differentiation markers (dentine sialophosphoprotein (DSPP) and collagen type 1 (COL1)) were determined using Real-Time Polymerase Chain Reaction analysis. Osteo-/odontogenic differentiation of DPSC niches was evaluated by measurement of alkaline phosphatase activity (ALP). Calcium deposition was assessed using von Kossa staining. The results were analysed statistically using Mann-Whitney tests and Kruskal-Wallis tests., Results: MTA mixed with 5% CaCl 2 and 2.5% Na 2 HPO 4 exhibited optimal cell viability (P < 0.05) compared to MTA mixed with distilled water. MTA mixed with 5% CaCl 2 and 2.5% Na 2 HPO 4 significantly increased ALP activity (P < 0.05), significantly promoted mineralization nodule formation (P < 0.05) and significantly enhanced the mRNA expression level of the osteogenic/odontogenic markers (P < 0.05; DSPP and COL1) compared with MTA mixed with distilled water., Conclusions: MTA mixed with 5% CaCl 2 and 2.5% Na 2 HPO 4 was biocompatible with dental pulp stem cell niches. Accelerated-set MTA promoted better differentiation in DPSC niches than conventional MTA. The accelerators could provide an alternative to MTA mixed with distilled water., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To investigate whether hypertension affects mineralization associated with white and grey mineral trioxide aggregate (MTA Angelus ® ) implanted subcutaneously into rats by assaying osteoblastic biomarkers., Methodology: Polyethylene tubes containing grey MTA Angelus ® , white MTA Angelus ® , intermediate restorative material (IRM; positive control) or an empty tube (negative control) were implanted into the dorsal connective tissue of spontaneous hypertensive (n = 12) and Wistar (normotensive; n = 10) rats. Half of the rats in each group were killed after 7 days, and the remaining after 30 days. Tubes with surrounding tissue were removed, and immunostaining was performed to detect RUNX-2, OPN and OCN proteins. The normality of data was analysed using the Shapiro-Wilk test. Comparison of two independent groups was performed using the Mann-Whitney U-test, to detect a significant difference. A post hoc test accounting for multiple comparisons was performed following Tukey's test (P < 0.05)., Results: Under hypertensive conditions after 30 days, both MTA materials were associated with immunolabelling for RUNX-2 from low to moderate, which was less than that observed at normal blood pressure and the 7-day groups (P < 0.05). The expression of OPN and OCN proteins under both MTA conditions was considered low after both 7 and 30 days for the hypertensive condition, and was less than that in animals with normal blood pressure after 30 days (P < 0.05). No immunostaining for any biomarkers in the control and IRM groups was observed (P < 0.05)., Conclusion: Hypertension decreased the immunostaining of RUNX-2, OPN and OCN biomarkers in response to MTA. Thus, hypertension can jeopardize the mineralization ability of MTA and may have a negative impact on endodontic treatment outcomes., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To examine the effect of reduction in MTA particle size on dento-alveolar and osseous healing in dogs., Methodology: Root canals of 24 mandibular premolars in four 2-year-old beagles were prepared and filled with gutta-percha and sealer. Two to four weeks later, during periapical surgery, the root-end cavity preparations in these teeth were filled with either grey ProRoot MTA or modified (reduced particle sizes with faster setting time) MTA. The animals were sacrificed 4 months later. Degrees of inflammation, type of inflammatory cells, fibrous connective tissue adjacent to the root-end filling materials, cementum formation over the resected roots and root-end filling materials and bone healing were examined. Data were analysed using the McNemar test., Results: No significant differences in healing of periapical tissues were found when comparing ProRoot MTA to a modified MTA containing reduced particle sizes., Conclusion: Reducing the particle sizes of MTA did not impact its biological properties., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the abilities of three calcium silicate-based pulp-capping materials (ProRoot MTA, TheraCal LC and a prototype tricalcium silicate cement) to produce apatite-like precipitates after being subcutaneously implanted into rats., Methodology: Polytetrafluoroethylene tubes containing each material were subcutaneously implanted into the backs of Wistar rats. At 7, 14 and 28 days post-implantation, the implants were removed together with the surrounding connective tissue, and fixed in 2.5% glutaraldehyde in cacodylate buffer. The chemical compositions of the surface precipitates formed on the implants were analysed with scanning electron microscopy-electron probe microanalysis (SEM-EPMA). The distributions of calcium (Ca) and phosphorus (P) at the material-tissue interface were also analysed with SEM-EPMA. Comparisons of the thicknesses of the Ca- and P-rich areas were performed using the Friedman test followed by Scheffe's test at a significant level of 5%., Results: All three materials produced apatite-like surface precipitates containing Ca and P. For each material, elemental mapping detected a region of connective tissue in which the concentrations of Ca and P were higher than those in the surrounding connective tissue. The thickness of this Ca- and P-rich region exhibited the following pattern: ProRoot MTA > prototype tricalcium silicate cement ≥ TheraCal LC. ProRoot MTA had a significantly thicker layer of Ca and P than the other materials at all time-points (P < 0.05), and a significant difference was detected between the prototype cement and TheraCal LC at 28 days (P < 0.05)., Conclusion: After being subcutaneously implanted, all of the materials produced Ca- and P-containing surface precipitates and a Ca- and P-rich layer within the surrounding tissue. The thickness of the Ca- and P-rich layer of ProRoot MTA was significantly thicker than that of the other materials., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the biological effects in vitro of MTA-Angelus (MTA-Ang; Angelus, Londrina, PR, Brazil), MTA Repair HP (MTA-HP; Angelus) and NeoMTA Plus (NeoMTA-P; Avalon Biomed Inc, Bradenton, FL, USA) on human dental pulp stem cells (hDPSCs)., Methodology: Cell viability and cell migration assays were performed using eluates of each material. To evaluate cell morphology and cell attachment to the different materials, hDPSCs were directly seeded onto the material surfaces and analysed by immunocytofluorescence and scanning electron microscopy, respectively. The chemical composition of the materials was determined by energy-dispersive X-ray (EDX), and eluates were analysed by inductively coupled plasma-mass spectrometry (ICP-MS). Statistical analysis was performed with the analysis of variance and Bonferroni or Tukey post-test (α < 0.05)., Results: Undiluted MTA-Ang, MTA-HP and NeoMTA-P displayed a significant increase in cell viability greater than that obtained using complete medium alone (control) (*P < 0.05; **P < 0.01; ***P < 0.001). Moreover, a cell migration assay revealed cell migration rates after incubation with extracts of MTA-Ang, MTA-HP and NeoMTA-P that were similar to levels obtained in the control group. In addition, stretched cytoskeletal F-actin fibres were detected in the cells treated with the three material extracts. SEM studies revealed a high degree of cell proliferation and attachment on all three materials. EDX analysis demonstrated similar weight percentages of C, O and Ca in all three materials, whilst other elements such as Al, Si and S were also found., Conclusions: MTA-Ang, MTA-HP and NeoMTA-P were associated with biological effects on hDPSCs in terms of cell proliferation, morphology, migration and attachment., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the biocompatibility and mineralized nodule formation of an experimental tricalcium silicate cement with tantalum oxide (TSC/Ta 2 O 5 ) 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/Ta 2 O 5 at 1 : 1 and 1 : 2 dilutions when compared to serum-free medium - control (P > 0.05). At 1 : 4 dilution, the TSC/Ta 2 O 5 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/Ta 2 O 5 . ALP activity at 1 and 3 days was similar to the control (P > 0.05). TSC/Ta 2 O 5 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/Ta 2 O 5 (P < 0.05)., Conclusions: Neo MTA Plus and TSC/Ta 2 O 5 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., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the influence of the addition of microparticulate (micro) and nanoparticulate (nano) zirconium oxide (ZrO 2 ) and niobium pentoxide (Nb 2 O 5 ) to a calcium silicate-based cement (CS) on the subcutaneous healing process in rats compared with MTA Angelus™., Methodology: In each rat, two polyethylene tubes filled with the following materials: (i) MTA; (ii) CS + ZrO 2 micro; (iii) CS + ZrO 2 nano; (iv) CS + Nb 2 O 5 micro or (v) CS + Nb 2 O 5 nano were implanted subcutaneously; empty polyethylene tubes were used in the Control group. After 7, 15, 30 and 60 days, the specimens (n = 5 per group in each period) were fixed and embedded in paraffin. Masson's trichrome sections were used to obtain the volume density of the inflammatory cells (VvIC) and fibroblasts (VvFb). The sections were also stained with Picrosirius-red to calculate the birefringent collagen content. Fibroblast growth factor-1 (FGF-1) was detected by immunohistochemistry, and the number of immunolabelled cells was obtained. The data were subjected to two-way anova followed by Tukey's test (P ≤ 0.05)., Results: At all periods, the VvIC was significantly lower (P < 0.001) in all the CS and Control groups than in the MTA group. At all periods, the VvFb was reduced significantly (P = 0.023) in the MTA group in comparison with the other groups. In addition, the number of immunolabelled cells in the capsules of the CS groups was significantly higher (P < 0.001) than in the MTA group at all time-points., Conclusions: The experimental materials (CS + ZrO 2 and CS + Nb 2 O 5 ) induced fibroblast proliferation and accelerated the regression of the inflammatory reaction. However, the addition of nanoparticulate radiopacifiers did not improve the biological properties of a calcium silicate-based cement when compared to microparticulate agents., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aims: To investigate the cytotoxicity and bioactivity of several pulpotomy materials: Biodentine (Septodont, Saint-Maur-des-Fosses, France) MTA (Angelus, Londrina, PR, Brazil), Theracal LC (Bisco Inc., Schamburg, IL, USA) and IRM (Dentsply DeTrey GmbH, Konstanz, Germany), after contact with stem cells isolated from human exfoliated primary teeth (SHEDs)., Methodology: SHEDs were cultured in the presence of the eluates of various pulpotomy materials for 24, 48 and 72 h. Cell viability was determined by mitochondrial dehydrogenase enzymatic (MTT) assay. Apoptosis and changes in cell phenotype were evaluated by flow cytometry. Also, an in vitro scratch wound-healing assay was used to determine their effects on cell migration. To assess cell morphology and attachment to the different pulpotomy materials, SHEDs were directly seeded onto the material surfaces and analysed by scanning electron microscopy (SEM). Finally, the deposition of a calcified matrix in presence of these materials was verified by Alizarin Red staining. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α = 0.05)., Results: Cell viability in the presence of Biodentine eluates was significantly higher to that obtained using complete medium alone (control; P < 0.01) and was also significantly higher than using MTA Angelus from 48 h of incubation (P < 0.01). However, Theracal LC and IRM were associated with low rates of cell viability (P < 0.001). Similar results were obtained in an apoptosis assay. In addition, SHEDs maintained their mesenchymal phenotype in all conditions although their capacity to migrate was higher in the presence of Biodentine. SEM studies revealed a suitable proliferation rate, cell spreading and attachment, especially when using Biodentine and MTA Angelus discs. Finally, Biodentine eluates significantly induced calcified matrix deposition from 7 days of culture (P < 0.01)., Conclusions: Biodentine exhibited better cytocompatibility and bioactivity than MTA Angelus, Theracal LC and IRM., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To determine the roles of autophagy and Notch signalling in mineral trioxide aggregate (MTA)-mediated proliferation and differentiation of human dental pulp cells (hDPCs)., Methodology: hDPCs were separated from human healthy molar teeth using collagenase I/dispase II digestion and then cultured in α-MEM medium with 15% foetal bovine serum. hDPCs were seeded in 96-well plates, and cell counting kit assays were carried out to test their viability. Real-time quantitative polymerase chain reaction (qPCR) was used to evaluate ALP, Runx2, Notch1, Hes1 and Hey1 mRNA levels. Notch1, hes1, LC3 and p62 protein levels were quantified by Western blotting. Colocalization of LC3 and Notch1 was measured by immunofluorescence. Two-tailed Student's t-tests were used for statistical analysis., Results: Autophagic flux was significantly (P < 0.05) inhibited by MTA extracts, causing Notch degradation arrest. This resulted in the promotion of cell proliferation and inhibition of differentiation during the logarithmic phase of cell growth., Conclusion: MTA extract promoted the proliferation of hDPCs in part by activating Notch signalling through inhibition of autophagic flux during the early stage and, thus, might potentially induce rapid restoration of injured pulps., (© 2017 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

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., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the effect of a calcium aluminate-based cement (CAC+) on the development of the osteogenic phenotype in vitro., Methodology: Rat calvaria-derived cells were grown on Thermanox ® coverslips for 24 h and then exposed to either samples (4-h set) of CAC+ or mineral trioxide aggregate (MTA) placed on Transwell ® inserts for periods of up to 14 days. Nonexposed cultures were used as the controls. The comparisons were made using the nonparametric Kruskal-Wallis test, followed by the Student-Newman-Keuls post hoc test when appropriate., Results: The results showed that proximity to MTA or CAC+ samples inhibited cell growth, whereas at a distance, viable and proliferative cells adhered to and spread on the Thermanox ® , expressing osteoblast differentiation markers prior to mineralization of the extracellular matrix. Compared with MTA, the osteogenic cell cultures exposed to CAC+ exhibited significantly greater cell viability, alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2, osterix, ALP, bone sialoprotein and osteocalcin (P < 0.05 for all). For the osteogenic cell cultures exposed to CAC+, the quantification of matrix mineralization was not altered (P > 0.05)., Conclusions: CAC+ supported the acquisition of the osteogenic cell phenotype in vitro, rendering this novel material a potential alternative to MTA in endodontic procedures. Further in vivo studies are needed to verify if the beneficial in vitro effects of CAC+ on osteoblastic cells correspond to an increase and/or acceleration of bone repair in the periapical region., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To identify whether Krüppel-like factor 5 (KLF5) was involved in odontoblastic differentiation during reparative dentine formation., Methodology: Human Dental pulp cells (DPCs) were isolated from healthy human dental pulp tissue and induced for odontoblastic differentiation. Alizarin Red staining, alkaline phosphatase (ALPase) activity, quantitative real-time PCR and Western Blot were performed to evaluate in vitro odontoblastic differentiation. The expression profile of KLF5 during the in vitro odontoblastic differentiation was determined by quantitative real-time PCR and Western Blot. Knock-down of KLF5 by lentivirus-mediated shRNA was performed to determine the function of KLF5 in odontoblastic differentiation. After direct pulp capping with MTA, the maxillary first molar segments dissected from male Wistar rats were prepared for histology analysis and immunohistochemistry staining., Results: Odontoblastic differentiation was confirmed by significantly increased alkaline phosphatase (ALP; P = 0.004) activity and upregulated odontoblastic differentiation-related genes including dentine sialophosphoprotein (DSPP; P = 0.004) and dentine matrix protein-1 (DMP-1; P = <0.001). The expression of KLF5 was significantly upregulated during odontoblastic differentiation of in vitro cultured DPCs (P = 0.0002). KLF5 knock-down impaired odontoblastic differentiation. After direct pulp capping, dentine bridge-like calcified tissues were formed under the perforation sites. KLF5 was expressed in odontoblast-like cells and DPCs beneath the perforation sites during reparative dentine formation., Conclusions: KLF5 might be involved in the process of odontoblastic differentiation during reparative dentine formation., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To assess the biological effects, including odontoblastic differentiation of a novel light-curable material (TheraCal), on human dental pulp cells (hDPCs)., Methodology: The hDPCs were isolated from freshly extracted, caries-free third molars. Ten discs of TheraCal and MTA (8 mm in diameter and 3 mm in height) were incubated in α-minimum essential medium (α-MEM) and the supernatant collected. Viability of hDPCs in response to TheraCal and MTA was measured using the WST-1 assay. RT-PCR and real-time PCR were used to detect the gene expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1). ALP staining and Alizarin red S staining were used to evaluate the expression of alkaline phosphatase (ALP) and mineralization behaviour. One-way analysis of variance and Tukey's post hoc test were used to determine the statistically significant differences as a result of the variation in test materials (P < 0.05)., Results: The effects of TheraCal and MTA on cell viability were similar except at the highest concentration. The mRNA level of DSPP increased significantly in the MTA group relative to the control at day 1 and 3 (P < 0.05). Also, the mRNA level of DSPP increased significantly in the TheraCal group relative to the control at day 3 (P < 0.05). The increased mRNA level of DMP-1 was 2.5-fold and 2.3-fold each in the MTA and TheraCal groups relative to the control (P < 0.05). Cells exposed to MTA exhibited a 1.4-fold increase of ALP staining relative to control (P < 0.05). In the mineralization assay, increased calcium nodule formation was twofold and 1.3-fold each in the MTA and TheraCal groups compared to the control (P < 0.05)., Conclusions: TheraCal and MTA had the ability to induce odontoblastic differentiation and mineralization of hDPCs., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

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 ZrO 2 ) or niobium (CSCR Nb 2 O 5 ) 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 ZrO 2 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 Nb 2 O 5 (P < 0.05). All cements exhibited suitable ALP enzyme activity, highlighting the 7-day period of cell exposure. ARS, CSCR Nb 2 O 5 , revealed a significant potential to induce mineralization in vitro., Conclusions: All materials had suitable biocompatibility and bioactivity. The MTA P, BIO and CSCR ZrO 2 groups had the highest viability rates and velocity of proliferation whilst the CSCR Nb 2 O 5 group produced more mineralized nodules., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the cytotoxic effects of Biodentine, using a three-dimensional (3D) cell culture associated with an in situ root-end filling experimental model. White mineral trioxide aggregate (MTA) and zinc oxide cement were used as reference for comparison. IL-1α and TNF-α cytokine production were also evaluated., Methodology: The root canals of 24 human maxillary incisor teeth were prepared using a single-file reciprocating technique. After root filling, a 3-mm root-end resection was performed and 3 mm of gutta-percha was removed from the canal. The teeth were randomly distributed to receive one of the following root-end filling materials: Biodentine, white MTA or zinc oxide cement (positive control group). In the negative control group, the root canal was not retro-filled. The cytocompatibility of the materials was evaluated using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay in an in situ root-end filling experimental model. Balb/c 3T3 fibroblasts, cultured in rat tail collagen type I 3D scaffold, were exposed to the root apex for 24 h, and cell viability was measured by means of reduction MTT salt. IL-1α and TNF-α production were analysed using enzyme-linked immunosorbent assay. One-way analysis of variance was performed and, when the F-ratios were significant, data were compared by Duncan's multiple-range test. The alpha-type error was set at 0.05., Results: Biodentine and MTA groups had similar cell activity to the negative control group (P > 0.05), indicating low cytotoxicity for both materials. The stronger cytotoxicity effect was identified on the zinc oxide cement (P < 0.05). Zinc oxide cement caused a significant up-regulation in IL-1α and TNF-α (P < 0.05). No significant differences amongst MTA, Biodentine and the negative control group were observed for TNF-α (P > 0.05); however, both MTA and Biodentine were associated with overproduction of IL-1α when compared to the control group (P < 0.05)., Conclusions: Biodentine and MTA had similar cytocompatibility in a 3D cell culture model associated with an in situ root-end filling model. The methodology could be used as an alternative to assess the cytocompatibility of endodontic cements because it is more closely related to the in vivo situation., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To compare dental pulp responses to capping with iRoot BP Plus and mineral trioxide aggregate (MTA) in dogs., Methodology: Pulps in 36 incisors of three 8-month-old beagle dogs were mechanically exposed and assigned to two experimental groups (iRoot BP Plus group and MTA group, n = 15 per group) and one control group (n = 6). Direct pulp capping was performed using either iRoot BP Plus or MTA. The animals were sacrificed 3 months later. Histological sections were stained with haematoxylin and eosin and categorized using a histologic scoring system. Statistical analysis was performed using the Mann-Whitney U-test, with the significance set at 0.05., Results: The majority of specimens in both experimental groups were associated with complete calcified bridge formation and the absence of pulpal inflammation. There was no significant difference in pulp response to iRoot BP Plus or MTA after 3 months (P > 0.05)., Conclusion: iRoot BP Plus and MTA had similar favourable results when used as pulp-capping agents., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the inflammatory process induced by Biodentine and mineral trioxide aggregate (MTA) in rat subcutaneous tissues., Methodology: A polyethylene tube filled with Biodentine (n = 20) or MTA (n = 20) was placed into the dorsal subcutaneous of forty male rats; in the control group (CG; n = 20), empty tubes were implanted. After 7, 15, 30 and 60 days, the polyethylene tubes surrounded by connective tissue were fixed and embedded in paraffin. The number of inflammatory cells was estimated in HE-stained sections; numerical density of interleukin-6 (IL-6)-immunolabelled cells was also performed. The differences amongst the groups were analysed statistically by Tukey's test (P ≤ 0.05)., Results: A high number of inflammatory cells and IL-6-positive cells were observed at 7 days, in all groups; however, in the Biodentine group, the number of inflammatory cells and IL-6-immunolabelled cells was significantly higher (P ≤ 0.05) in comparison with the other groups at 7 and 15 days. In the capsules of animals from all groups, a gradual and significant reduction (P ≤ 0.05) of these parameters was seen over time. At 60 days, the capsules exhibited numerous fibroblasts and bundles of collagen fibres; in addition, the number of IL-6-positive cells was not significantly different amongst Biodentine, MTA and control groups., Conclusions: There was a significant regression in the inflammatory reaction in the capsules indicating, therefore, that Biodentine is a biocompatible material., (© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Acute promyelocytic leukaemia (APL), the M3 subtype of acute myeloid leukaemia, was once a lethal disease, yet nowadays the majority of patients with APL can be successfully cured by molecularly targeted therapy. This dramatic improvement in the survival rate is an example of the advantage of modern medicine. APL is characterized by a balanced reciprocal chromosomal translocation fusing the promyelocytic leukaemia (PML) gene on chromosome 15 with the retinoic acid receptor α (RARα) gene on chromosome 17. It has been found that all-trans-retinoic acid (ATRA) or arsenic trioxide (ATO) alone exerts therapeutic effect on APL patients with the PML-RARα fusion gene, and the combination of both drugs can act synergistically to further enhance the cure rate of the patients. Here, we provide an insight into the pathogenesis of APL and the mechanisms underlying the respective roles of ATRA and ATO. In addition, treatments that lead to more effective differentiation and apoptosis of APL cells, including leukaemia-initiating cells, and more thorough eradication of the disease will be discussed. Moreover, as a model of translational research, the development of a cure for APL has followed a bidirectional approach of 'bench to bedside' and 'bedside to bench', which can serve as a valuable example for the diagnosis and treatment of other malignancies., (© 2015 The Association for the Publication of the Journal of Internal Medicine.)

Aim: To investigate the effect of BioAggregate (Innovative Bioceramix, Vancouver, BC, Canada) on lipopolysaccharide (LPS)-induced bone destruction in vivo and to compare its performance with that of mineral trioxide aggregate (MTA; Dentsply Tulsa Dental, Tulsa, OK, USA)., Methodology: Mouse bone marrow macrophages (BMMs) were primary cultured and treated with several concentrations of BioAggregate and MTA extracts. Cell viability was measured with a Cell Counting Kit-8 assay, whilst in vitro osteoclast differentiation was evaluated with tartrate-resistant acid phosphatase (TRAP) staining. LPS-induced mouse calvarial bone destruction model was established to assess the effect of BioAggregate and MTA extracts in vivo. Mice were killed on day 7, and calvarial bones were prepared for microcomputed tomography scanning, histologic analysis and double-immunofluorescence staining. Statistical tests used were one-way anova followed by Student-Newman-Keuls test., Results: BioAggregate extracts displayed no obvious cytotoxicity to BMMs and significantly inhibited (P < 0.01) the differentiation of RANKL-stimulated BMMs. Comparable effects were induced by MTA. BioAggregate and MTA extracts markedly reduced (P < 0.01) osteoclast numbers and attenuated (P < 0.05) bone resorption in LPS-challenged mouse calvaria. The expression levels of osteoclastogenic cathepsin K and its upstream regulator nuclear factor of activated T-cell cytoplasmic 1 and c-Fos were also decreased by BioAggregate and MTA extracts., Conclusions: BioAggregate and MTA showed comparable inhibitory effect on osteoclast differentiation and inflammatory bone resorption in vivo., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To investigate the influence of mineral trioxide aggregate (MTA) on angiogenesis of primary human dental pulp cells (hDPCs) via the MAPK pathway, in particular p38., Methodology: Human dental pulp cells were cultured with MTA to angiogenesis, after which cell viability, ion concentration, osmolality, NO secretion, the von Willebrand factor (vWF) and angiopoietin-1 (Ang-1) protein expression were examined. PrestoBlue(®) was used for evaluating the proliferation of hDPCs. An enzyme-linked immunosorbent assay was employed to determine vWF and Ang-1 protein secretion in hDPCs cultured on MTA and the control. Cells cultured on the tissue culture plate without the cement were used as the control. The t-test was used to evaluate the significance of the differences between the mean values., Results: Mineral trioxide aggregate elicited a significant (P < 0.05) increased viability compared with the control (15%, 16% and 13% on days 1, 3 and 5 of cell seeding, respectively). MTA consumed calcium and phosphate ions, and released more Si ions in the medium. MTA significantly (P < 0.05) increased the osmolality of the medium to 313, 328 and 341 mOsm kg(-1) after 1, 3 and 5 days, respectively. P38 was activated through phosphorylation, and the phosphorylation kinase was investigated in the cell system after being cultured with MTA. Expression levels for Ang-1 and vWF in hDPCs on MTA were higher than those of the MTA + p38 inhibitor (SB203580) group (P < 0.05) at all of the time-points., Conclusions: Mineral trioxide aggregate was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si increased the osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signalling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion decreased. These findings verify that the p38 pathway plays a key role in regulating the angiogenic behaviour of hDPCs cultured on MTA., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To comparatively examine the cell attachment, cytotoxicity, and antibacterial activity of radiopaque dicalcium silicate cement (RDSC) and ProRoot white-coloured mineral trioxide aggregate (WMTA)., Methodology: AlamarBlue was used for real-time and repeated monitoring of MG63 cell attachment on freshly mixed and set cements. The pH changes in the growth medium at different time-points were also measured. Cytotoxicity evaluation was performed according to ISO 10993-5 specifications. The antibacterial activity of the cement specimens was evaluated using Enterococcus faecalis., Results: There were no significant differences (P > 0.05) between the two cements for cell attachment either in the fresh groups or in the set groups at all culture times. Neither freshly mixed group nor set groups had significant pH differences. In the case of cytotoxicity, RDSC was significantly (P < 0.05) superior to WMTA at 12 and 24 h of incubation. RDSC and WMTA possessed similar antimicrobial activity, substantiated by the formation of growth inhibition zones and bacteriostasis ratio in E. faecalis strains., Conclusions: The cell attachment, cytotoxicity and antibacterial efficacy of RDSC were comparable to those reported for ProRoot WMTA. The results of the current study suggest that this RDSC could be used as a root-end filling material and root sealer., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To compare the mineralization inductive capacity of Biodentine and Bioaggregate with Mineral trioxide aggregate (MTA) and to investigate possible signaling pathways of mineralization in human dental pulp cells (HDPCs)., Methodology: Viability of HDPCs in response to Biodentine, Bioaggregate, and MTA was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. To investigate their potential to induce odontoblast differentiation, expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein1 (DMP1) mRNA level was evaluated by RT-PCR. For the mineralized nodule assay, Alizarin red staining was performed. To determine the role of MAPK signaling in the odontoblastic differentiation of HDPCs, activated MAPKs were investigated by Western blot and the effect of MAPK inhibitor was examined by Alizarin red S staining. The results were statistically analysed using one-way anova and the Bonferroni test., Results: The effects of MTA, Biodentine, and Bioaggregate on cell viability were similar. Biodentine and Bioaggregate enhanced DSPP and DMP1 mRNA expression compared to the control group, but to the same extent as MTA (P < 0.05). MTA, Biodentine, and Bioaggregate increased the area of calcified nodules compared to the control (P < 0.01). MTA, Biodentine, and Bioaggregate increased phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). MAPK inhibitors attenuated mineralized nodule formation, which was increased by MTA, Biodentine, and Bioaggregate, respectively (P < 0.01)., Conclusion: Biodentine and Bioaggregate stimulated odontoblastic differentiation and mineralization nodule formation by activating the MAPK pathway as did MTA. This suggests that the new materials could be useful for regenerative endodontic procedures., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To examine the effects of mineral trioxide aggregate (MTA)/fibroblast growth factor-2 (FGF-2) on material properties and in vitro human dental pulp cell (hDPCs) behaviour., Methodology: The setting time and diametral tensile strength (DTS) of MTA and MTA/FGF-2 were measured. The structure of specimens before and after soaking in DMEM was examined under a scanning electron microscope. Alamar Blue was used for evaluating hDPCs proliferation. An enzyme-linked immunosorbent assay was employed to determine ALP and osteocalcin (OC) expression in hDPCs cultured on cements. The effect of small interfering RNA (siRNA) transfection targeting fibroblast growth factor receptor (FGFR) was also evaluated. One-way analysis of variance was used to evaluate the significance of the differences between the mean values., Results: Setting time and DTS data were not found to be significant (P > 0.05) between MTA with and without FGF-2. Cell proliferation and differentiation increased significantly (P < 0.05) with FGF-2 mixed MTA. After siRNA transfection with FGFR, the proliferation and differentiation behaviour of the hDPCs appreciably decreased when cultured on an MTA/FGF-2 composite. In contrast, no significant amounts (P > 0.05) of ALP and OC were secreted by hDPCs seeded on MTA., Conclusions: Mineral trioxide aggregate with FGF-2 content enhanced the higher expression of hDPCs proliferation and osteogenic differentiation as compared to pure MTA cement., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To compare white ProRoot MTA (WMTA), EndoSequence BC sealer (BC sealer) and Biodentine with regard to their ability to produce apatites and cause Ca and Si incorporation in adjacent human root canal dentine after immersion in phosphate-buffered saline (PBS)., Methodology: Root sections of human single-rooted teeth were filled with one of the materials and immersed in PBS for 1, 7, 30 or 90 days (n = 5 each). Morphology and elemental composition of surface precipitates and interfacial dentine were analysed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with image observation function. Ca- and Si-incorporation depths in the interfacial dentine were measured. In addition, the amount of Ca ions released from the test materials was measured by EDTA titration., Results: All materials produced surface precipitates of acicular or lath-like morphology with Ca/P ratio of 1.6 : 2.0. Within dentinal tubules, the three materials formed tag-like structures that were frequently composed of Ca- and P-rich and Si-poor materials, suggesting intratubular precipitation. Ca- and Si-incorporation depths were in the order of Biodentine > WMTA > BC sealer, with a significant difference between BC sealer and the others at several time-points (P < 0.05, anova and Tukey's honestly significant difference test). The concentration of released Ca ions was in the order of Biodentine > WMTA > BC sealer with significant differences between the materials (P < 0.05)., Conclusions: Compared with Biodentine and WMTA, BC sealer showed less Ca ion release and did not show Ca and Si incorporation as deeply in human root canal dentine when immersed in PBS for up to 90 days., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate antibiofilm activity against Enterococcus faecalis, pH and solubility of AH Plus, Sealer 26, Epiphany SE, Sealapex, Activ GP, MTA Fillapex (MTA-F) and an experimental MTA-based Sealer (MTA-S)., Methodology: Sealer samples were manipulated and stored for 2 or 7 days. Prepared sealers were evaluated by a modified direct contact test (DCT) for 5 h, 10 h or 15 h with biofilm previously induced on bovine dentine for 14 days. In the control group, the biofilm was not exposed to the sealers. The number of colony-forming units (CFU mL(-1) ) in the remaining biofilm was determined. Sealer solubility was assessed by the percentage of mass loss after 15 h of immersion in distilled water. Sealer pH was measured at 5 h, 10 h and 15 h. Statistical analysis was performed using Kruskal-Wallis and Dunn or anova and Tamhane's T2 tests, at 5% significance., Results: At 2 days post-manipulation, the DCT showed that Sealapex and MTA-F were associated with a reduction in the number of bacteria in all 3 contact periods evaluated, compared with the control group (P < 0.05). At 7 days, Sealapex had the greatest antibiofilm action at 10 h and 15 h. Sealapex had the highest pH values 2 and 7 days post-manipulation. Regarding the solubility, at 2 days the highest values were observed for MTA-F, MTA-S, Sealapex and Activ GP (P < 0.05). At 7 days, MTA-S and MTA-F had greater solubility than the other materials (P < 0.05). AH Plus had the lowest solubility for both post-manipulation periods (P < 0.05)., Conclusion: Sealapex and MTA-F were associated with a reduction in the number of bacteria in biofilms and had greater solubility. The high solubility and pH may be related to the antibacterial activity of these materials., (© 2012 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To investigate cell viability and gene expression of cementoblasts (OCCM.30) exposed to extractable components released by resin-based sealers with different chemical composition Hybrid Root Seal (HRS), SimpliSeal (SS), Real Seal (RS) and AH Plus (AH) and by a MTA-based sealers Tech Biosealer Endo (TBE)., Methodology: Discs of all materials were prepared and allowed to set in humid conditions at 37° for 48 h. The discs were then incubated for 72 h at 37 °C to obtain material extracts (1/1) in DMEM. The extracts containing the components released by the sealers were filtered and other dilutions (1/2, 1/4) were prepared from the original solution (1/1). Original and diluted solutions were tested on the cementoblasts. Impedance-based real-time cell analysis (RTCA) was used to evaluate cell viability, quantitative real-time polymerase chain reaction (QRT-PCR) was used to examine the expression of mineralization-related genes (osteocalcin; OCN, Runt-related transcription factor-2; Runx2, collagen type 1; COL I, alkaline phosphatase; ALP). For statistical analysis, one-way analysis of variance (anova) and Tukey's honestly significant difference (HSD) tests were used., Results: TBE (1/2), RS (1/2, 1/4), and HRS (1/2, 1/4) significantly decreased cell viability (P < 0.001). AH (1/2, 1/4) and SS (1/2, 1/4) had similar cell viability to the control at 30 h. All tested materials significantly decreased cell viability when compared to the control group except AH (1/2, 1/4) and SS (1/4) at 90 h. All of the tested sealers reduced COL I mRNA expressions when compared to the control. SS was associated with significant increases in OCN and Runx2 mRNA expressions when compared to the control (P < 0.001). Whereas all of the dilutions of TBE, RS and HRS significantly decreased BSP mRNA expressions (P < 0,001), 1/2 and 1/4 dilutions of SS increased BSP mRNA expression (P < 0,001). Except the 1/4 dilutions of AH and SS, all the sealer dilutions significantly reduced ALP mRNA expression in cementoblasts (P < 0,001)., Conclusions: SimpliSeal and AH Plus resulted in more favourable response to cementoblasts because of their regulation potential on the mineralized tissue-associated protein's mRNA expressions., (© 2012 International Endodontic Journal.)

Aim: To verify the in vitro cytocompatibility of iRoot BP Plus (iRoot) and to compare it with White ProRoot MTA (MTA)., Methodology: Thirty-six human maxillary incisor root canals were prepared using a step-back flaring technique. The apical 3 mm was resected perpendicular to the long axis at the roots, and root-end cavities were prepared with the aid of an ultrasonic device plus a diamond retrotip with continuous irrigation using water, producing standardized preparations. After that, the root-end cavities were filled with iRoot or MTA, and each root was exposed to cell culture media for 24 or 48 h. Human osteoblast cells were exposed to the extracts thus obtained, and a multiparametric cell viability assay was performed, evaluating mitochondrial activity, membrane integrity and cell density. The results were analysed by one-way analysis of variance, complemented with the Duncan post-test (P < 0.05)., Results: Cells exposed to MTA revealed a cytocompatibility pattern similar to the untreated cells (negative control), at both experimental times (P > 0.05). iRoot, however, promoted a significantly poorer viability than MTA and the control, after 48 h of exposure (P < 0.001). Nevertheless, iRoot did not induce critical cytotoxic effects because cell viability remained higher than 70% of the control group in most tests performed., Conclusion: iRoot and MTA were biocompatible and did not induce critical cytotoxic effects., (© 2012 International Endodontic Journal.)

Aim: To assess the ability of a recently developed tricalcium silicate-based cement (Biodentine™) to induce reparative dentine synthesis and to investigate its capacity to modulate pulp cells TGF-β1 secretion., Methodology: Biodentine™ was directly applied onto the dental pulp in an entire human tooth culture model. After various culture periods, the interaction of the material with dental pulp tissue was analysed on tissue sections. The effect of increasing surface area of this material on TGF-β1 secretion was investigated on pulp cell cultures and compared with that of MTA, calcium hydroxide and Xeno(®) III adhesive resin. After performing artificial injuries on pulp cell cultures, the materials eluates were added for 24 h and then TGF-β1 secretion was quantified by ELISA. Controls were performed by incubating intact cells with the culture medium, while injured cells TGF-β1 level was used as the baseline value., Results: Biodentine™ induced mineralized foci formation early after its application. The mineralization appeared under the form of osteodentine and expressed markers of odontoblasts. Biodentine™ significantly increased TGF-β1 secretion from pulp cells (P < 0.03) independently of the contact surface increase. This increase was also observed with calcium hydroxide and MTA, but not with the resinous Xeno(®) III. The statistical analysis showed statistically significant differences between capping materials and the resinous Xeno(®) III (P < 0.001)., Conclusions: When Biodentine™ was applied directly onto the pulp, it induced an early form of reparative dentine synthesis, probably due to a modulation of pulp cell TGF-β1 secretion., (© 2011 International Endodontic Journal.)

Aim: To evaluate the pH and antibacterial activity of Endo CPM Sealer and MTA Fillapex by two different methods, using white MTA and Endofill as references for comparison., Methodology: Antibacterial activity was evaluated against Enterococcus faecalis (ATCC 29212). The agar diffusion test (ADT) was performed to evaluate the effect before setting. The materials were placed in four equidistant wells made in ten agar plates. After incubation at 37 °C for 48 h, the inhibition zones were measured using a digital paquimeter. The direct contact test (DCT) was performed to assess the antibacterial effect after setting. Suspensions of crushed materials were prepared and mixed with E. faecalis. After different periods of time (1, 6, 15 and 60 min), the survival of bacteria was assessed by using 10-fold serial dilution and cultivated on agar plates in triplicate. Colony-forming units (CFU) mL(-1) were calculated after incubation. pH values were also measured in triplicate. Comparison between sealers in the ADT and DCT was performed by the Kruskal-Wallis test., Results: In the ADT, inhibition zones were found with MTA Fillapex and Endofill. They were similar to each other and greater than the other sealers (P < 0.05). None of the tested sealers demonstrated antibacterial activity in the DCT, and thus, all sealers had similar bacterial counts compared with the negative control group (P > 0.05). White MTA and Endo CPM Sealer suspensions had pH values >11, whilst MTA Fillapex and Endofill had lower values., Conclusions: MTA Fillapex and Endofill had an antibacterial effect against E. faecalis before setting, but none of the sealers maintained antibacterial activity after setting, despite the high pH of the MTA-based materials., (© 2011 International Endodontic Journal.)

Aim: To compare the histological pulp reaction to various water/powder ratios of white mineral trioxide aggregate (MTA) as a pulp-capping material in healthy human teeth., Methodology: Twenty-nine disease-free maxillary and mandibular third molar teeth, scheduled for extraction, were exposed mechanically and then capped with 0.28, 0.33 and 0.40 water/powder ratios of white MTA (ProRoot; Dentsply Maillefer, Ballaigues, Switzerland) and restored with glass ionomer. After 30 days, the teeth were extracted, resected apically and immersed in 10% formalin. For histological processing, the teeth were sectioned buccolingually in 5-μm-thick slices, stained with Haematoxylin and Eosin and evaluated by a light microscope. Samples were evaluated for intensity and type of inflammation, presence of necrosis, as well as continuity, morphology and thickness of calcified bridges. The data were analysed by Kruskal-Wallis and Mann-Whitney tests., Results: There were no significant differences in the diameter, morphology and continuity of the calcified bridges, intensity and type of inflammation or presence of necrosis (P > 0.05) in the pulps covered by MTA with various water/powder ratios. Two teeth failed to display a calcified bridge, and one had a pulp necrosis., Conclusion: Water-to-powder ratios of MTA had no significant influence on the histological outcome of direct pulp capping on healthy pulps., (© 2011 International Endodontic Journal.)

Aim: To evaluate human dental pulp cell responses to radiopaque dicalcium silicate cement and white-coloured mineral trioxide aggregate (WMTA)., Methodology: Flow cytometry was employed to quantify the phase percentage of pulp cell cycle. Alamar Blue was used for real-time and repeated monitoring of cell proliferation. Reverse transcription-polymerase chain reaction was performed to determine gene expression in pulp cells cultured on the cements., Results: The cells cultured on the radiopaque dicalcium silicate cement had similar S and G2 phases in the cell cycle and proliferation to WMTA at all culture times. In addition, the two materials presented the same evolution with similar values in interleukin-1, inducible nitric oxide synthase, alkaline phosphatase, osteocalcin and bone sialoprotein gene expression at all culture times., Conclusions: The dental pulp cell responses to radiopaque dicalcium silicate cement were similar to those reported for WMTA in terms of cell cycle, proliferation, immunocompatibility and osteogenic differentiation., (© 2011 International Endodontic Journal.)

Aim: To evaluate the biological response of the periodontium adjacent to furcation perforations in rat molars filled with Endo-CPM-Sealer (CPM), MTA-Angelus (MTA) or zinc oxide-eugenol cement (ZOE)., Methodology: The pulp chamber floors of maxillary right first molar teeth were perforated and sealed with CPM, mineral trioxide aggregate (MTA) or ZOE; the left first molars, without any treatment, were used as controls (CG). After 7, 15, 30 and 60 days, fragments of maxilla were fixed, decalcified and embedded in paraffin. Sections were stained with H&E, Masson's trichrome and submitted to tartrate-resistant acid phosphatase (TRAP) reaction, used as an osteoclast marker. The width of the periodontal space, the numerical density of inflammatory cells and the number of TRAP-positive osteoclasts in the bone surface were measured, and statistical analyses were performed using analysis of variance and Tukey test (P ≤ 0.05)., Results: In all experimental groups, the greatest number of inflammatory cells was observed at 7 days, especially in the ZOE group. In this group, the intense inflammatory process was related to a significant increase (P ≤ 0.05) in the number of osteoclasts and, thereby, in an increase in the width of the periodontal space. At 60 days, no significant differences in osteoclast numbers amongst CPM, MTA and CG groups occurred; the periodontal space was also significantly reduced in the experimental groups in comparison with the initial periods. However, in the ZOE group, the periodontal space was significantly larger (P ≤ 0.05) in comparison with MTA-based materials., Conclusions: The periodontium adjacent to perforations filled with MTA and CPM exhibited clear evidence of re-establishment and thus better biocompatibility than ZOE., (© 2010 International Endodontic Journal.)

Background and Aim: More and more microRNA (miRNA) are found to be involved in tumor genesis and progress. Arsenic trioxide has been an effective chemotherapeutic drug in cancer therapy for many years. In this study, we aimed to find the miRNA involved in the mechanisms of arsenic trioxide treatment in cancer therapy., Methods: We detected the expression profile of miRNA by miRNA microarray and quantitative real-time polymerase chain reaction. Cell viability assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis and luciferase reporter assay were carried out to determine the role of one selected miRNA, namely mir-29a, in affecting the biological behaviors of HepG-2 cells., Results: Among the 677 human miRNA in the microarray, five miRNA were upregulated and four were downregulated in HepG-2 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, four miRNA were identified, namely miR-24, miR-29a, miR-30a and miR-210, which were all upregulated. Among them, miR-29a showed a positive therapeutic effect in liver cancer cells by inhibiting cell growth and inducing cell apoptosis, and PPM1D was confirmed to be the target gene of miR-29a. Furthermore, a synergy effect was detected between miR-29a and arsenic trioxide., Conclusions: Arsenic trioxide altered miRNA expression profile in HepG-2 cells. Among the altered miRNA, miR-29a seemed to take a role in the mechanism of arsenic trioxide in liver cancer therapy. The synergy effect between miR-29a and arsenic trioxide may offer this drug a new chance in cancer therapy by decreasing its dose and toxic side-effects., (© 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)