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Aim: The present study examined the leaching and cytotoxicity of bismuth from ProRoot MTA and aimed to identify whether bismuth leaching was affected by the cement base and the immersion regime used., Methodology: The leaching profile of bismuth was examined from ProRoot MTA and compared with hydroxyapatite containing 20% bismuth oxide as well as hydroxyapatite and tricalcium silicate to investigate whether bismuth release changed depending on the cement base. Bismuth leaching was determined after 30 and 180 days of ageing immersed in Dulbecco's modified Eagle's medium (DMEM) using mass spectroscopy (ICP-MS). The media were either unchanged or regularly replenished. The pH, surface microstructure and phase changes of aged materials were assessed. Wistar rat femoral bone marrow stromal cells (BMSCs) and cutaneous fibroblasts were isolated, cultured and seeded for cell counting (trypan blue live/dead) after exposure to non-aged, 30- and 180-days-aged samples in regularly replenished DMEM. Aged DMEM in contact with materials was also used to culture BMSCs to investigate the effect of material leachates on the cells. Gene expression analysis was also carried out after direct exposure of cells to non-aged materials. Differences between groups were statistically tested at a significance level of 5%., Results: All materials exhibited alterations after immersion in DMEM and this increased with longer exposure times. The bismuth leached from ProRoot MTA as detected by ICP-MS. Aged ProRoot MTA samples exhibited a black discolouration and surface calcium carbonate deposition. ProRoot MTA influenced cell counts after direct exposure and its 180-days leachates reduced BMSC viability. After direct BMSC contact with non-aged ProRoot MTA an upregulation of metallothionein (MT1 and MT2A) expression and down-regulation of collagen-1a (Col-1a) and bone sialoprotein (BSP) expression was identified., Conclusions: Bismuth leaching was observed throughout 180-days observation period from all materials containing bismuth oxide. This negatively influenced cell viability and gene expression associated with bismuth exposure. This is the first study to report that metallothionein gene expression was influenced by exposure to ProRoot MTA., (© 2024 The Author(s). International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

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: To investigate the effects of N-acetyl cysteine (NAC), Biodentine, ProRoot MTA and their combinations, on cell viability, mitochondrial reactive oxygen species (mtROS) production, mineralization and on the expression of genes related to inflammatory cytokine production, mitochondrial dynamics and cell apoptosis of lipopolysaccharide (LPS)-induced human dental pulp cells (hDPCs)., Methodology: Isolated hDPCs were exposed to 20 μg mL -1 of Escherichia coli (E. coli) LPS for 24 h, before the experiment, except for the control group. Eight experimental groups were assigned: (i) control (hDPCs cultured in regular medium), (ii) +LPS (hDPCs cultured in LPS medium throughout the experiment), (iii) -LPS/Media, (iv) -LPS/BD, (v) -LPS/MTA, (vi) -LPS/NAC, (vii) -LPS/BD + NAC and (viii) -LPS/MTA + NAC. Cell viability was measured using Alamar blue assay at 24 and 48 h. Production of mtROS was evaluated at 6 and 24 h by MitoSOX Red and MitoTracker Green. The expressions of IL-6, TNF-α, Bcl-2, Bax, Mfn-2 and Drp-1 genes were investigated at 6 h using reverse transcriptase-polymerase chain reaction (RT-PCR). For differentiation potential, cells were cultured in the osteogenic differentiation media and stained using Alizarin red assay at 14 and 21 days. The Kruskal-Wallis test, Mann-Whitney U test and one-way anova were performed for statistical analysis., Results: NAC was associated with significantly greater LPS-induced hDPC viability (P < 0.05). Both Biodentine and MTA extracts promoted cell survival, whereas the combination of NAC to these material extracts significantly increased the number of viable cells at 24 h (P < 0.05). Biodentine, MTA or NAC did not alter the mtROS level (P > 0.05). NAC supplementation to the MTA extract significantly reduced the level of IL-6 and TNF-α expression (P < 0.05). Regarding mitochondrial dynamics, the use of NAC alone promoted significant Mfn-2/Drp-1 expression (P < 0.05). Most of the groups exhibited a level of Bcl-2/Bax gene expression similar to that of the control group. The increases in mineralization productions were observed in most of the groups, except the LPS group (P < 0.05)., Conclusions: The antioxidant effect of NAC was not evident under the LPS-induced condition in DPC in vitro. NAC combined either with Biodentine or MTA improved LPS-induced hDPCs survival at 24 h. The combination of NAC with MTA promoted mineralization., (© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the in vitro effect of the novel adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate (4-MET), on the proliferation, mineralization and differentiation of odontoblast-like cells, comparing with 4-MET, calcium hydroxide (CH) and mineral trioxide aggregate (MTA)., Methodology: Rat odontoblast-like MDPC-23 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% foetal bovine serum. The powder of four tested materials (CMET, 4-MET, CH and MTA) was first dissolved in distilled water (dH 2 O) and then was diluted by DMEM to yield final concentrations. Solvent (dH 2 O) was used as a control. Cell viability was assessed using CCK-8 assay. Real-time RT-PCR was used to quantify the mRNA expression of odontogenic markers, cytokines and integrins. Mineralization inducing capacity was evaluated by alkaline phosphatase (ALPase) activity and alizarin red S staining. Statistical analyses were performed using one-way anova and post hoc Tukey's HSD test, with the significance level at 1%., Results: Cell viability was significantly greater in the CMET- (83 to 828 mmol L -1 ), CH- and MTA-treated (low concentrations) groups than that in the control group (P < 0.01). Higher concentrations of each material decreased the viable cells to different extents (P < 0.01). CMET treatment augmented the expression of several integrin subunits and exhibited the highest mRNA expression levels of odontogenic markers among all groups (P < 0.01). CH and MTA treatment caused significantly greater upregulation of pro-inflammatory cytokines expression than the other groups (P < 0.01). The calcific deposition of MDPC-23 cells was dose-dependently accelerated by the addition of CMET (P < 0.01); the enhancement of mineralization was also found in the fresh prepared CH and MTA treatments. Besides, CMET showed consistency in mineralization induction after 8 weeks storage. Exposure to SB202190, a specific p38 mitogen-activated protein kinases inhibitor, significantly decreased the ALPase activity as well as the mineral deposition which was enhanced by CMET treatment (P < 0.01)., Conclusions: The novel bio-active monomer had the lowest cytotoxicity among all groups and it induced the proliferation, mineralization and differentiation of odontoblast-like cells under appropriate concentrations. This adhesive monomer possesses excellent biocompatibility and hence exhibits great potential in dentine regeneration., (© 2020 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 evaluate in vitro cytotoxicity and in vivo inflammatory response to new nanostructural materials based on active calcium silicate systems (CS) and hydroxyapatite (HA-CS)., Methodology: Cytotoxicity of eluates of new nanostructural noncommercial materials CS and HA-CS, and MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil) as a control, were tested using the MTT assay on MRC-5 cells. Eluates of set materials were tested in 100% and 50% concentrations, 24 h, 7 days and 21 days post-elution. The pH values were determined for undiluted eluates of set materials. Polyethylene tubes containing the test materials (CS, HA-CS, MTA) were implanted in subcutaneous tissue of Wistar rats. Histopathological examinations were conducted at 7, 15, 30 and 60 days after the implantation. Data were statistically analyzed using three-way and one-way anova Tukey's post hoc test as well as Kruskall-Wallis test with Dunn's post hoc test at α = 0.05., Results: All materials significantly reduced cell viability; especially when undiluted eluates were used (P < 0.001). After 24 h elution, cell viability was 10 ± 1.8%, 49.5 ± 4.2% and 61 ± 7.4%, for MTA, and HA-CS, respectively. However, CS and HA-CS were significantly less toxic than the control material MTA (P < 0.05). Cytotoxicity could be at least partially attributed to pH kinetics over time. Dilution of eluates of all tested materials resulted in better cell survival. Histopathological examination indicated similar inflammatory reaction, vascular congestion and connective tissue integrity associated with CS, HA-CS and MTA at each observation period (P > 0.05). The only significant difference was found for capsule thickness, that is thicker capsule was associated with HA-CS compared to MTA at 60 days (P = 0.0039). HA-CS induced moderately thick capsules (median score 3, score range 2-3), whereas MTA resulted in thin capsule formation (median score 2, score range 1-3)., Conclusions: Evaluation of cytotoxicity and inflammatory response indicated better biocompatibility of CS and HA-CS, in comparison with MTA (White MTA, Angelus(®) Soluções Odontológicas, Londrina, Brazil)., (© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To evaluate the cytotoxicity and mineralization effects of iRoot BP in human dental pulp cells (hDPCs) and to compare them with those of white mineral trioxide aggregate (WMTA)., Methodology: hDPCs were exposed to prepared dilutions (1 : 1-1 : 10) of the test materials. Cell viability was evaluated using the XTT assay after incubation periods of 24, 48 or 72 h. The expression of mineralization-related genes (bone morphogenic protein, osteonectin, bone sialoprotein, osteopontin, dentine sialophosphoprotein and collagen type 1) and heme oxygenase 1 was measured by quantitative real-time polymerase chain reaction (qRT-PCR) at 24 and 72 h. Statistical differences between test materials were analysed with the Mann-Whitney test., Results: The 1 : 1 and 1 : 2 dilutions of iRoot BP were associated with higher cell viability after 24 h (P < 0.05). Only the 1 : 1 dilution of iRoot BP had higher cell viability after 48 h (P < 0.05), and there was no difference between iRoot BP and WMTA after 72 h (P > 0.05). Although somewhat variable, according to the gene expression results, iRoot BP had a mineralization potential similar to that of WMTA. WMTA revealed a higher heme oxygenase 1 (HO-1) mRNA level than iRoot BP (P < 0.001)., Conclusions: iRoot BP and WMTA were biocompatible and facilitated odontoblastic differentiation of hDPCs., (© 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 investigate and compare the systemic toxic effect of DiaRoot BioAggregate and grey ProRoot Mineral trioxide aggregate (MTA) on the liver and kidney after 7 and 30 days., Methodology: Forty-two white albino rats were divided into two main groups. Group (1), considered the control group (n = 18), was further divided into two subgroups. The negative control subgroup (n = 6) received no treatment. The empty tube subgroup (n = 12) received empty sterile Teflon tubes. In Group (2), considered the experimental group (n = 24), the rats were divided equally into two subgroups. One subgroup received MTA, whilst the other received BioAggregate. The materials in the Teflon tubes were implanted subcutaneously in the dorsal side of the rats. Blood samples were taken to investigate the change of kidney and liver functions on day 7 and day 30. The liver and kidney organs were subjected to histopathological examination and calculation of the number of inflammatory cells. Data analysis was performed using one-way anova with post hoc multiple comparisons with the Tukey's test. Student's t-test was used to compare the changes in liver and kidney functions amongst the groups., Results: On day 7, a significantly more severe inflammatory reaction was observed in both experimental subgroups compared with the control (P < 0.05); the severity decreased after 30 days. The kidney functions were not affected after 7 days but had subsequently increased after 30 days (P < 0.001). Liver functions increased after 7 days and had decreased in the BioAggregate subgroup after 30 days, whilst in the MTA subgroup, a continuous increase in the level of liver function was observed., Conclusions: Mineral trioxide aggregate had adverse effects on the liver and kidney that were significantly more severe than BioAggregate but with no permanent damage., (© 2013 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Aim: To characterize and investigate the genotoxic effect of a new endodontic cement based on dicalcium- and tricalcium-silicate (CS) with hydroxyapatite (HA) on human lymphocytes., Methodology: Hydrothermal treatment was applied for synthesis of CS and HA. The final mixture HA-CS, with potential to be used in endodontic practice, is composed of CS (34%) and HA (66%). Human lymphocytes were incubated with HA, HA-CS and CS for 1 h, at 37 °C and 5% CO2. Cell viability was determined using the trypan blue exclusion assay. To evaluate the level of DNA damage comet assay (single cell gel electrophoresis) was performed. For the statistical analysis anova and Duncan's Post Hoc Test were used., Results: The SEM analysis indicated that CS consisted mostly of agglomerates of several micrometers in size, built up from smaller particles, with dimensions between 117 and 477 nm. This is promising because dimensions of agglomerates are not comparable with channels inside the cell membranes, whereas their nano-elements provide evident activity, important for faster setting of these mixtures compared to MTA. Values of DNA damage obtained in the comet assay indicated low genotoxic risk of the new endodontic materials., Conclusions: The significantly improved setting characteristics and low genotoxic risk of the new material support further research., (© 2012 International Endodontic Journal.)

Aim: To investigate the effects of root repair materials on the cytotoxicity and gelatinolytic activity of matrix metalloproteinases (MMPs) in 3T3 fibroblasts., Methodology: Fibroblasts (3T3, 3 × 10(5) cells per well) were incubated with elutes of calcium hydroxide (Biodinâmica, Ibiporã, PR, Brazil), EndoBinder (Binderware, São Carlos, SP, Brazil) and mineral trioxide aggregate (MTA) (Angelus, Londrina, PR, Brazil) for 24 h. The cytotoxicity of all root repair materials was determined using the MTT assay. Supernatants of cell cultures incubated with materials were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity by gelatin zymography. Data were analysed using anova and Tukey's test., Results: Cells secreted MMP-2 after 24 h with calcium hydroxide inducing significantly greater MMP-2 expression in relation to the control and the other root repair materials (P < 0.05). The cytotoxicity results revealed that there was no significant difference in the cell viability of MTA, EndoBinder and the control group. However, there was a significantly reduced cell viability of 3T3 fibroblasts in association with calcium hydroxide (P < 0.05)., Conclusions: Calcium hydroxide was associated with significantly less cell viability when compared with EndoBinder and MTA. All materials had gelatinolytic activity for MMP-2 with calcium hydroxide being associated with the greatest activity., (© 2012 International Endodontic Journal.)

Aim:   To compare the cytotoxicity of four endodontic sealers (Sealapex, Pulp Canal Sealer EWT, Real Seal and MTA Fillapex) either 1 or 7 days after mixing, when assessed through a multiparametric analysis employing human primary cells closely related to periapical tissues., Methodology:   Extracts of each sealer were prepared following 24-h exposure to culture media, at either 24 h or 7 days after mixing. Primary human osteoblasts were exposed to extracts for 24 h, at 37 °C with 5% CO(2) , and cell viability was evaluated by a multiparametric assay assessing sequentially, on the same cells, mitochondrial activity (XTT), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). Results from each test and experimental time were compared by 2-way analysis of variance (anova)., Results:   All endodontic sealers had strong cytotoxicity 24 h after mixing, according to all parameters evaluated. At a longer setting period (7 days), viability for Sealapex was significantly increased (P < 0.05) and Pulp Canal Sealer achieved levels of cytocompatibility similar to the control group. The anova indicated a general correlation between the cytotoxicity of the materials and the time after mixing, with some level of dependence on the cell viability assay employed., Conclusions:   All materials had high cytotoxic levels for human primary cells, mostly on a time-dependent basis, as shown by three different cell viability tests., (© 2011 International Endodontic Journal.)

Aim: To compare the subcutaneous tissue response to grey mineral trioxide aggregate (GMTA), white mineral trioxide aggregate (WMTA) and a new experimental cement (calcium enriched cement, CEM)., Methodology: Thirty-six Wistar male albino rats each received three implants, containing one of the tested materials, and an empty tube as a control. Seven, 30 and 60 days after implantation, the animals were sacrificed. After histological preparation and H&E staining, the specimens were evaluated for capsule thickness, necrosis, and for the type, the severity, and the extent of inflammation. Kruskal Wallis and Chi-square tests were used for data analysis., Results: After 1 week, CEM produced no necrosis compared to both types of WMTA and GMTA (P = 0.007). After 30 days, GMTA specimens had significantly less inflammation compared with WMTA and CEM (P = 0.011). After 60 days, less inflammation was associated with CEM specimens (P = 0.0001) compared to the other materials. Dystrophic calcifications in the connective tissue adjacent to all experimental material were detected., Conclusion: Histological observation illustrated that all materials were well tolerated by the subcutaneous tissues., (© 2010 International Endodontic Journal.)

Aim: To evaluate the cytotoxicity of two forms of the novel root-end filling materials, polymer nanocomposite (PNC) resins [C-18 Amine montmorillonate (MMT) and VODAC MMT] both containing Chlorhexidine Diacetate Salt Hydrate 2%, and to compare it to that of two widely accepted commercially available materials, ProRoot® MTA and Geristore®., Methodology: Elutes of experimental materials extracted after 24 h, 1, 2 and 3 weeks were interacted with the mouse fibroblasts L-929 using a colorimetric cell viability assay (MTS) based on mitochondrial dehydrogenases activity. Using 100% and 50% concentrations of the extracted elutes of the experimental materials the effect of different concentrations of elutes on the cells was analysed. In the positive control group Hygrogold® was added to the cell culture to arrest cells bioactivity. In the negative control group, fresh Dulbecco's Eagle's minimum essential medium supplemented with 10% foetal bovine serum was used to enhance cell bioactivity. Differences in mean bioactivity values were assessed using a t-test and one-way anova (P<0.05)., Results: No significant difference was found in cytotoxicity between ProRoot® MTA, Geristore® and PNC resin C-18 Amine MMT on 24 h, 1, 2 and 3 weeks samples. Sample elutes of PNC resin VODAC MMT, however, revealed cytotoxic activity during most of these experiments., Conclusion: Cytotoxicity of the elutes of PNC resin C-18 Amine MMT was not significantly different from that of ProRoot® and Geristore®. PNC resin VODAC MMT, revealed significantly more cytotoxicity compared to the other tested materials., (© 2010 International Endodontic Journal.)

Aim: To investigate the cytotoxicity of bioaggregate (BA; Innovative Bioceramix, Vancouver, BC, Canada) to human periodontal ligament (PDL) fibroblasts and its effect on differentiation of human PDL fibroblasts and to compare its performance to that of mineral trioxide aggregate (MTA; Dentsply, Tulsa, OK, USA)., Methodology: Cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on days 1, 2, and 3 after incubation with BA or MTA. The influence of BA and MTA on differentiation of human PDL fibroblasts on days 3, 5, and 7 was evaluated by gene expression of alkaline phosphatase (ALP) and collagen type I (COLI) via quantitative real-time polymerase chain reaction (PCR). The data were analysed by one-way analysis of variance followed by Tukey's test., Results: Cell numbers in the BA group were similar to that of the control group throughout the culture period, whereas MTA suppressed the proliferation of fibroblasts. ALP expression was significantly increased in the BA group on day 7, whilst it was enhanced by MTA on day 3. Gene expression of COLI was induced by both BA and MTA compared to the control group., Conclusions: Bioaggregate was nontoxic to human PDL fibroblasts and appeared to induce the differentiation of human PDL fibroblasts., (© 2010 International Endodontic Journal.)

Aims: To evaluate whether white mineral trioxide aggregate (MTA) or white Portland cement with 15% bismuth oxide were able to induce genetic damage and cellular death ex vivo., Methodology: Aliquots of 1 × 10(4) murine fibroblasts were incubated at 37 °C for 3 h with MTA (white) or white Portland cement with 15% bismuth oxide, at final concentrations ranging from 10 to 1000 μg mL(-1) individually. Data of three independent repeats from the comet assay and the trypan blue exclusion test were assessed by the one-way anova followed by Tukey's test., Results: Mineral trioxide aggregate or Portland cement containing bismuth oxide did not produce genotoxic effects with respect to the single-cell gel (comet) assay data for all concentrations evaluated. Furthermore, no cytotoxicity was observed for MTA or Portland cement., Conclusion: White MTA or white Portland cement containing 15% bismuth oxide were not genotoxic and cytotoxic., (© 2010 International Endodontic Journal.)

Aim: To evaluate the biocompatibility of iRoot SP root canal filling material to L929 mouse fibroblasts., Methodology: Specimens of iRoot SP, AH Plus and ProRoot mineral trioxide aggregate (MTA) were prepared for direct contact using the Millipore filter diffusion test, as well as extracts in MTT assay. Mouse fibroblasts (L929) were used as toxicity targets. Differences in cytotoxicity between fresh and set specimens and between root canal material extracts were determined by t-test. A P-value <0.05 was considered statistically significant., Results: In the filter diffusion test, iRoot SP was slightly cytotoxic when tested as a fresh mixture and not cytotoxic in a set condition tested after 24 h. AH Plus was rated moderately cytotoxic when tested as a fresh mixture and slightly cytotoxic in a set (24 h) condition. MTA was noncytotoxic in both a fresh or set state. AH Plus was significantly more toxic than iRoot SP and MTA (P < 0.05). In addition, iRoot SP was significantly more toxic than MTA (P < 0.05). In the MTT assay with set specimens, extracts of iRoot SP and MTA were noncytotoxic, whilst extracts of AH Plus were rated as slightly cytotoxic. AH Plus was significantly more toxic than iRoot SP and MTA (P < 0.05)., Conclusion: AH Plus root canal sealer was significantly more toxic to L-929 cells than MTA and iRoot SP. iRoot SP had intermediate toxicity.

Aim: The aim of this study was to evaluate the cytotoxicity and genotoxicity of the new castor oil bean cement (COB) material in comparison to commonly used pulp capping materials., Methodology: Specimens of COB, calcium hydroxide (Hydro C), and mineral trioxide aggregate (white and gray MTA) were extracted in culture medium (91.6 mm(2) sample surface mL(-1)). Transfected human pulp cells (tHPCs) were exposed to dilutions of the extracts for 1 h, and the generation of reactive oxygen species (ROS) was determined by flow cytometry (FACS) using H(2)DCF-DA as a dye. Survival of tHPCs was measured photometrically using a crystal violet assay after a 24-h exposure period. Genotoxicity as indicated by the formation of micronuclei in V79 cells, and the modification of the normal cell cycle by extracts of the materials was analysed by FACS., Results: Clear cytotoxic effects were detected only with extracts of Hydro C under the current experimental conditions. The two MTA preparations induced an insignificant reduction in the number of cells. In contrast, the extracts of COB slightly induced cell proliferation. Extracts of Hydro C caused a twofold increase in ROS production, whilst the other tested materials were ineffective. An increase in the number of micronuclei was not detected with any material tested; Hydro C slightly increased the number of cells in G1 and G2., Conclusions: The COB and the two MTA preparations did not negatively influence cell survival or ROS production and may thus be further considered for pulp capping studies.

This paper reviews the literature on the constituents and biocompatibility of mineral trioxide aggregate (MTA). A Medline search was conducted. The first publication on the material was in November 1993. The Medline search identified 206 papers published from November 1993 to August 2005. Specific searches on constituents and biocompatibility of mineral trioxide aggregate, however, yielded few publications. Initially all abstracts were read to identify which fitted one of the two categories required for this review, constituents or biocompatibility. Based on this assessment and a review of the papers, 13 were included in the constituent category and 53 in the biocompatibility category. Relatively few articles addressed the constituents of MTA, whilst cytological evaluation was the most widely used biocompatibility test.

Aim: To compare the cytotoxicity of materials used to repair perforations using permanent V79 fibroblasts and murine granulocyte-macrophage progenitor cells (CFU-GM)., Methodology: Set specimens from amalgam, glass-ionomer, SuperEBA, N-Rickert, MTA and gutta-percha were eluted with culture medium for 72 h and their cytotoxicities were assessed by incubating the extracts with V79 and bone marrow-derived progenitors for 24 h and 7 days, respectively. Cytotoxicity on V79 cells was judged using the total nucleic acid content (NAC), neutral red uptake (NRU) and reduction of the tetrazolium salt (MTT). The number of bone marrow CFU-GM colonies determined in clonal cultures stimulated with recombinant murine granulocyte-macrophage colony-stimulating factor was used to assess cytotoxicity to progenitor cells. Statistical analyses were conducted using the one-way analysis of variance and Tukey's test where appropriate., Results: All materials were cytotoxic in both cell systems; however, CFU-GM was more sensitive to the extracts than V79 cells. A similar rank order of toxicity was observed in V79 cells using the NAC and the MTT assays: glass-ionomer > N-Rickert congruent with SuperEBA > gutta-percha > amalgam congruent with MTA (P < 0.05). In contrast, the NRU test exhibited a lower sensitivity to MTA, gutta-percha and amalgam extracts. In the clonal culture assay, the toxicity was less pronounced in the presence of gutta-percha, SuperEBA and MTA. Similar cellular responses were found by placing the set specimens directly in the clonal culture dishes., Conclusions: The sensitivity of toxicity depended on the choice of the endpoint and the cell-culture system. Nevertheless, MTA was ranked as the least cytotoxic cement in both cell systems.

Aim: To examine the genotoxicity and cytotoxicity of regular and white mineral trioxide aggregate (MTA) ex vivo by the single-cell gel (comet) assay and trypan blue exclusion test, respectively., Methodology: Aliquots of 1 x 10(4) Chinese hamster ovary cells were incubated at 37 degrees C for 3 h with grey and white forms of MTA at final concentrations ranging from 1 to 1,000 microg mL(-1). The negative control group was treated with vehicle control phosphate buffer solution for 3 h at 37 degrees C and the positive control group was treated with methyl metasulfonate (at 1 microg mL(-1)) for 1 h at 37 degrees C. After incubation, the cells were centrifuged at 180 g for 5 min and washed twice with fresh medium and resuspended with fresh medium. Each individual treatment was repeated three times consecutively to ensure reproducibility. Parameters from single-cell gel (comet) and cytotoxicity assays were assessed by the Kruskal-Wallis nonparametric test., Results: Neither compounds produced genotoxic effects with respect to the single-cell gel (comet) assay in all concentrations evaluated. In the same way, the dose-response relationships of all compounds tested at concentrations ranging from 1 to 1,000 microg mL(-1) on cell viability assessed by the trypan blue assay displayed no statistically significant differences (P > 0.05) for either endodontic material., Conclusions: Regular (grey) and white MTA are not genotoxins and do not induce cellular death.

Aim: To evaluate the biocompatibility of mineral trioxide aggregate and accelerated Portland cement and their eluants by assessing cell metabolic function and proliferation., Methodology: The chemical constitution of grey and white Portland cement, grey and white mineral trioxide aggregate (MTA) and accelerated Portland cement produced by excluding gypsum from the manufacturing process (Aalborg White) was determined using both energy dispersive analysis with X-ray and X-ray diffraction analysis. Biocompatibility of the materials was assessed using a direct test method where cell proliferation was measured quantitatively using Alamar Blue dye and an indirect test method where cells were grown on material elutions and cell proliferation was assessed using methyltetrazolium assay as recommended by the International standard guidelines, ISO 10993-Part 5 for in vitro testing., Results: The chemical constitution of all the materials tested was similar. Indirect studies of the eluants showed an increase in cell activity after 24 h compared with the control in culture medium (P<0.05). Direct cell contact with the cements resulted in a fall in cell viability for all time points studied (P<0.001)., Conclusions: Biocompatibility testing of the cement eluants showed the presence of no toxic leachables from the grey or white MTA, and that the addition of bismuth oxide to the accelerated Portland cement did not interfere with biocompatibility. The new accelerated Portland cement showed similar results. Cell growth was poor when seeded in direct contact with the test cements. However, the elution made up of calcium hydroxide produced during the hydration reaction was shown to induce cell proliferation.

Aim: To evaluate the cytotoxic effects of two brands of mineral trioxide aggregate (MTA) (Pro-Root MTA and MTA Angelus) and Portland cement (PC) on the human ECV 304 endothelial cell line., Methodology: Endothelial ECV 304 cells were incubated at 37 degrees C in an atmosphere of 95% air, 5% carbon dioxide and 100% humidity for 7 days and grown in F12 medium supplemented with 10% fetal bovine serum with 50 microg mL(-1) of gentamicin sulphate. Effects of the materials on mitochondrial functions were measured by a colorimetric assay. At each experimental time interval (24, 48 and 72 h), a dimethyl-thiazol-diphenyl tetrazolium bromid assay was conducted to measure cell viability. All assays were repeated three times to ensure reproducibility. Results were expressed as average absorbance (A(570/nm)) +/- SD and the data were analysed statistically by one-way analysis of variance and the Bonferroni post-test. A P-value < 0.05 was considered statistically significant., Results: No statistically significant difference was shown between any of the experimental materials (P > 0.05)., Conclusions: The two brands of MTA analysed, as well as the PC, initially showed a similar elevated cytotoxic effect that decreased gradually with time allowing the cell culture to become reestablished.

Aim: To investigate the in vitro behaviour of rat bone marrow cells (RBM) on mineral trioxide aggregate (MTA) (ProRoot, MTA Root Canal Repair Material; Dentsply Tulsa, Tulsa, OK, USA) compared with intermediate restorative materials (IRM) (Dentsply Caulk, Milford, DE, USA)., Methodology: RBM were obtained from rat femur and were primary cultured and then subcultured. Cells were then seeded on three dishes of each material, and cultured for 3 days, after which they were evaluated morphologically using scanning (SEM) and transmission (TEM) electron microscopy. Furthermore, the calcium released from hydrated material, the cell proliferation ratio and alkaline phosphatase (ALP) activity were analysed, and the expression of type I collagen and bone-related protein mRNAs were evaluated. The data were averaged and analysed via one-way analysis of variance (anova) and were then compared by the Scheffe's test., Results: SEM showed that RBM attached to MTA and had a flattened appearance without nuclear protrusions and microspikes. TEM showed that the cells attached in the same manner as the control group, but gaps larger than 2 microm were frequently seen. The calcium released from hydrated MTA was about 130 ppm after 3 days of immersion in saline. The ALP activity was similar to the control group. Cell proliferation and expression of type I collagen mRNA was significantly lower, while the expression of osteopontin mRNA was significantly higher than the control group at the third day of culture. In IRM groups, a few rounded cells were observed on the material but no living cells were seen., Conclusions: MTA is a material of low toxicity which does not inhibit cell growth, but does suppress the differentiation of osteoblast-like cells.

Aim: To compare the percentage of apoptotic cells and the cell cycle profile of fibroblasts and macrophages exposed to either ProRoot mineral trioxide aggregate (MTA) mixed with chlorhexidine (CHX), or exposed to ProRoot MTA mixed with sterile water., Methodology: Mouse gingival fibroblasts or mouse macrophages were seeded in six-well plates and allowed to attach overnight. Freshly mixed or set (allowed to dry for 24 h) specimens of tooth-coloured (white) ProRoot MTA were prepared with 0.12% CHX gluconate (MTA/CHX) or with sterile water (MTA/H2O). The cells were exposed for 24 h to the MTA specimens, which were placed over permeable membrane inserts to avoid direct contact with the cells. Untreated cells served as controls. Propidium iodide staining followed by flow cytometry was used to evaluate the effects of ProRoot MTA on cell apoptosis and cell cycle. Statistical analyses were performed by one-way anova followed by post-hoc tests with the use of the SigmaStat 2.0 software, and significance was determined at P < or = 0.05., Results: MTA specimens containing CHX induced apoptosis of macrophages and fibroblasts (P < 0.05). In contrast, no change in the proportion of apoptotic cells was observed when sterile water was used to prepare the specimens (P > 0.05). Cell cycle analysis showed that exposure to MTA/CHX decreased the percentage of fibroblasts and macrophages in S phase (DNA synthesis) as compared with exposure to MTA/H2O (P < 0.05)., Conclusion: This in vitro study demonstrated that the substitution of CHX for sterile water in MTA increases its cytotoxicity. This suggests that the potentially beneficial antimicrobial effect of CHX may be accompanied by an increase in the cytotoxicity of the resulting MTA-based material.

Aim: To evaluate the biological properties of a variety of materials that could be used in apical surgery., Methodology: The intraosseous implant technique recommended by the FDI (1980) and ADA (1982) was used to test the following materials: zinc oxide-eugenol (ZOE), mineral trioxide aggregate (MTA), and Z-100 light-cured composite resin. Thirty guinea-pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks, received one implant on each side of the lower jaw symphysis. The connective tissue response alongside the lateral wall outside the cup served as a negative control for the technique. At the end of the observation periods, the animals were killed and the specimens prepared for routine histological examination to evaluate their biocompatibility., Results: The reaction of the tissue to the materials diminished with time. The ZOE cement was highly toxic during the 4-week experimental period, but this profile changed significantly after 12 weeks, when it showed biocompatible characteristics. MTA and Z-100 showed biocompatibility in this test model at both time periods., Conclusions: MTA and Z-100 composite were biocompatible at 4 and 12 weeks in this experimental model.

Aim: To examine the biocompatibility of two commercial forms of mineral trioxide aggregate (MTA), by evaluating the morphology of an established cell line., Methodology: The two cements were cast on glass cover slips and cured for 1 or 28 days. Saos-2 osteosarcoma cells were trypsinized and seeded at a density of 1 x 10(5) cells and were then placed in medium over the material-coated coverslips for 1, 5 and 7 days. After these time intervals the media were discarded and the cells fixed. Cell morphological investigation was performed by scanning electron microscopy at various magnifications ranging from x 250 to x 500. The biocompatibility of cement constituents, alusilicate flux and bismuth oxide was also investigated., Results: All cement samples cured for 1 day showed a confluent cell monolayer after 5 and 7 days. The response to both materials was similar. Materials cured for 28 days showed incomplete cell confluence after 1 and 5 days. Alusilicate flux and bismuth oxide did not demonstrate biocompatibility., Conclusions: The 1-day cured samples of two commercial forms of MTA showed good biocompatibility. However, the 28-day cured samples were less biocompatible after 1 and 5 days.

Aim: The aim of the present study was to evaluate the short-term response of periradicular tissues to MTA when used as a root-end filling material in ideal tissue conditions., Methodology: The experimental procedures were performed on the healthy teeth of dogs. Pulps were removed and root canals prepared and filled with gutta-percha and sealer. At the same session, buccal mucoperiosteal flaps were reflected and the root ends resected. MTA or IRM were used as root-end filling materials. The periradicular tissue reactions were evaluated histologically from 1 to 5 weeks. Hard tissue formed on the MTA surface was further examined by scanning electron microscopy., Results: The most characteristic tissue reaction to MTA was the presence of connective tissue after the first postoperative week. Inflammation was seen occasionally. Early tissue healing events after MTA root-end filling were characterized by hard tissue formation, activated progressively from the peripheral root walls along the MTA-soft tissue interface. In contrast, hard tissue was not seen over the IRM root-end filling., Conclusions: MTA is a biocompatible material that stimulates periradicular tissue repair at the root-end situation; however, the nature of the newly formed tissues requires further elucidation.

Aim: This work was designed to investigate the potential cytotoxicity of two of the newer dental restorative materials. Spectrum composite resin and Dyract AP compomer., Methodology: Cultured human endothelial cells (ECV-304) were exposed to each of the restorative materials through a 70-microm dentine barrier to simulate the in vivo clinical situation. Cell viability was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay. The effects of different extents of light-curing were also examined by microscopic examination of stained human promyelocytic leukemia cells (HL-60). Caspase-3 activation was determined as a measure of apoptotic cell death., Results: Assessment of cellular viability indicated that both materials cause cell death, with Spectrum being the more toxic. The cytotoxicity was considerably increased in the absence of the dentine barrier. Direct exposure to Spectrum for 12 h resulted in the death of 69% of the cells after full light-curing (78% of total death was by apoptosis) and 96% after partial light-curing (73% of total death was by necrosis). Assessment of caspase activation, in the absence of the dentine barrier, showed that longer curing-times resulted in an increase in the proportion of the cells dying through apoptosis, rather than necrosis, for both materials tested., Conclusions: These results indicate the restorative materials to be potentially toxic, particularly if the degree of light-cure is inadequate.