Calcium silicate-based cements cause environmental stiffness and show diverse potential to induce osteogenesis in human osteoblastic cells.

Calcium silicate-based cements differ markedly in their radiopacifiers and the presence of calcium sulfate, aluminates, carbonates and other components that can affect their biological properties. This study aimed to compare the biological properties of six calcium silicate cements in human osteoblastic cell culture (Saos-2 cells): Bio-C Repair (Bio-C), PBS HP (PBS-HP), Biodentine (Biodentine), MTA Repair HP (MTA-HP), NeoMTA Plus (NeoMTA-P), and ProRoot MTA (ProRoot). After exposure to these materials, the cells were analyzed by MTT, wound healing, cell migration, and alkaline phosphatase activity (ALP) assays, real-time PCR (qPCR) analysis of the osteogenesis markers (osteocalcin or bone gamma-carboxyglutamate protein, BGLAP; alkaline phosphatase, ALPL; bone sialoprotein or secreted phosphoprotein 1, BNSP), and alizarin red staining (ARS). Curiously, the migration rates were low 24-48 h after exposure to the materials, despite the cells showing ideal rates of viability. The advanced and intermediate cell differentiation markers BGLAP and BNSP were overexpressed in the Bio-C, MTA-HP, and ProRoot groups. Only the Biodentine group showed ALPL overexpression, a marker of initial differentiation. However, the enzymatic activity was high in all groups except Biodentine. The mineralization area was significantly large in the NeoMTA-P, ProRoot, PBS-HP, MTA-HP, and Bio-C groups. The results showed that cellular environmental stiffness, which impairs cell mobility and diverse patterns of osteogenesis marker expression, is a consequence of cement exposure. Environmental stiffness indicates chemical and physical stimuli in the microenvironment; for instance, the release of cement compounds contributes to calcium phosphate matrix formation with diverse stiffnesses, which could be essential or detrimental for the migration and differentiation of osteoblastic cells. Cells exposed to Bio-C, PBS-HP, ProRoot, NeoMTA-P, and MTA-HP seemed to enter the advanced or intermediate differentiation phases early, which is indicative of the diverse potential of cements to induce osteogenesis. Cements that quickly stimulate osteoblast differentiation may be ideal for reparative and regenerative purposes since they promptly lead to dentin or bone deposition.

Influence of the Vehicle and Antibiotic Formulation on Cytotoxicity of Triple Antibiotic Paste.

INTRODUCTION: The aim of this study was to assess the influence of antibiotic formulations (tablet/capsule [TC] or United States Pharmacopeia [USP]-grade antibiotics) and vehicles (water [H2O] or macrogol + propylene glycol [MP]) on the cytotoxicity and pH of triple antibiotic pastes (TAPs). METHODS: L929 fibroblasts were exposed to TAPs prepared with TC or USP-grade antibiotics mixed with H2O or MP for 72 hours. Each isolated antibiotic with each vehicle, each isolated vehicle, and the culture medium were used as controls. Cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and neutral red assays. The pH was measured after 3 and 8 hours of immersion of the pastes in water. Data were analyzed using analysis of variance, the Bonferroni or Tukey posttests (α = 0.05), and the Pearson correlation test (α = 0.05). RESULTS: The pastes prepared with TC were less cytotoxic than pastes prepared with USP-grade antibiotics (P < .05), and pastes with the MP vehicle were less cytotoxic than pastes with H2O (P < .05). TC TAP + MP showed the lowest cytotoxicity, whereas USP-grade TAP + H2O showed the highest cytotoxicity (P < .05). All TAPs showed a pH ranging from 4.64-5.20. Irrespective of the vehicle, USP-grade TAP showed a lower pH than TC TAP (P < .05). TAPs with H2O had a lower pH than TAPs with MP (P < .05). CONCLUSIONS: The vehicle and the antibiotic formulations influenced the cytotoxicity and pH of TAP. The pastes prepared with TC and MP were less acidic and less cytotoxic than the type prepared with USP-grade antibiotics and H2O.

Cytotoxicity, genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles and farnesol as irrigating solutions.

OBJECTIVE: To evaluate the cytotoxicity, genotoxicity and antibacterial activity of poly(vinyl alcohol)-coated silver nanoparticles (AgNPs-PVA) and farnesol (FAR). DESIGN: The cytotoxicity (% of cell viability) was evaluated by MTT assay and the genotoxicity (% of DNA in the tail) was evaluated by Comet assay. Root canal disinfection with different irrigating protocols was evaluated ex vivo in human teeth contaminated with Enterococcus faecalis for 21days. Three microbiological samples were collected: initial (after contamination); post-irrigation (after irrigation); and final (after 7days). After each sample, the number of log 10 CFU mL-1 was determined. Statistical analyses was performed using two-way ANOVA and Bonferroni post-hoc tests for MTT assay, Kruskal-Wallis and Dunn post-hoc tests for Cometa and antibacterial assays (α=0.05). RESULTS: The MTT assay showed that AgNPs and FAR were less cytotoxic that sodium hypochlorite (NaOCl) and showed a lower% of DNA in the tail, in comparison with H2O2 (positive control - C+). In the post-irrigation microbiological sample, all the irrigating protocols were more effective than C+ (without irrigation). NaOCl/saline, NaOCl/saline/AgNPs-PVA and NaOCl/saline/FAR led to complete bacterial elimination (p >0.05). In comparison with the initial sample, both the post-irrigation and the final samples showed microbial reduction (p < 0.05). CONCLUSIONS: AgNPs-PVA and FAR showed low cytotoxicity and genotoxicity, and exhibit potential for use as a final endodontic irrigation protocols.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines.

The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

Cytotoxicity and Bioactivity of Calcium Silicate Cements Combined with Niobium Oxide in Different Cell Lines

Abstract The aim of this study was to evaluate the cytotoxicity and bioactivity of calcium silicate-based cements combined with niobium oxide (Nb2O5) micro and nanoparticles, comparing the response in different cell lines. This evaluation used four cell lines: two primary cultures (human dental pulp cells - hDPCs and human dental follicle cells - hDFCs) and two immortalized cultures (human osteoblast-like cells - Saos-2 and mouse periodontal ligament cells - mPDL). The tested materials were: White Portland Cement (PC), mineral trioxide aggregate (MTA), white Portland cement combined with microparticles (PC/Nb2O5µ) or nanoparticles (PC/Nb2O5n) of niobium oxide (Nb2O5). Cytotoxicity was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue exclusion assays and bioactivity by alkaline phosphatase (ALP) enzyme activity. Results were analyzed by ANOVA and Tukey test (a=0.05). PC/Nb2O5n presented similar or higher cell viability than PC/Nb2O5µ in all cell lines. Moreover, the materials presented similar or higher cell viability than MTA. Saos-2 exhibited high ALP activity, highlighting PC/Nb2O5µ material at 7 days of exposure. In conclusion, calcium silicate cements combined with micro and nanoparticles of Nb2O5 presented cytocompatibility and bioactivity, demonstrating the potential of Nb2O5 as an alternative radiopacifier agent for these cements. The different cell lines had similar response to cytotoxicity evaluation of calcium silicate cements. However, bioactivity was more accurately detected in human osteoblast-like cell line, Saos-2.

Resumo O objetivo deste estudo foi avaliar a citotoxicidade e bioatividade de cimentos à base de silicato de cálcio associados com óxido de nióbio (Nb2O5) micro e nanoparticulados, e comparar a resposta em diferentes linhagens celulares. Foram utilizadas quatro linhagens celulares: duas culturas primárias (células da polpa dentária humana - hDPCs e células do folículo dentário humano - hDFCs) e duas culturas imortalizadas (células osteoblásticas humanas - Saos-2 e células do ligamento periodontal de ratos - mPDL). Os materiais analisados foram: Cimento Portland branco (PC); Agregado trióxido mineral (MTA); PC associado com micropartículas (PC/Nb2O5µ) ou nanopartículas (PC/Nb2O5n) de óxido de nióbio (Nb2O5). A citotoxicidade foi avaliada pelos ensaios de brometo de metil-tiazolil-difeniltetrazólio (MTT) e azul de tripan, e a bioatividade pela atividade da enzima fosfatase alcalina (ALP). Os resultados foram analisados por ANOVA e teste de Tukey (a=0,05). O grupo do PC/Nb2O5n apresentou viabilidade celular semelhante ou maior do que o grupo do PC/Nb2O5μ em todas as linhagens celulares. Além disso, ambos os grupos apresentaram viabilidade celular semelhante ou maior do que o MTA. Saos-2 apresentaram maior atividade de ALP, com destaque para o material PC/Nb2O5μ aos 7 dias de exposição. Concluiu-se que cimentos de silicato de cálcio associados com Nb2O5 micro ou nanoparticulado apresentaram citocompatibilidade e bioatividade, demonstrando potencial do Nb2O5 como agente radiopacificador alternativo para estes cimentos. As linhagens celulares estudadas apresentaram resposta semelhante na avaliação da citotoxicidade de cimentos de silicato de cálcio. No entanto, a bioatividade é melhor detectada na linhagem de células osteoblásticas humanas, Saos-2.

Cytotoxicity and genotoxicity of calcium silicate-based cements on an osteoblast lineage.

Several calcium silicate-based biomaterials have been developed in recent years, in addition to Mineral Trioxide Aggregate (MTA). The aim of this study was to evaluate the cytotoxicity, genotoxicity and apoptosis/necrosis in human osteoblast cells (SAOS-2) of pure calcium silicate-based cements (CSC) and modified formulations: modified calcium silicate-based cements (CSCM) and three resin-based calcium silicate cements (CSCR1) (CSCR 2) (CSCR3). The following tests were performed after 24 hours of cement extract exposure: methyl-thiazolyl tetrazolium (MTT), apoptosis/necrosis assay and comet assay. The negative control (CT-) was performed with untreated cells, and the positive control (CT+) used hydrogen peroxide. The data for MTT and apoptosis were submitted to analysis of variance and Bonferroni's posttest (p < 0.05), and the data for the comet assay analysis, to the Kruskal-Wallis and Dunn tests (p < 0.05). The MTT test showed no significant difference among the materials in 2 mg/mL and 10 mg/mL concentrations. CSCR3 showed lower cell viability at 10 mg/mL. Only CSC showed lower cell viability at 50 mg/mL. CSCR1, CSCR2 and CSCR3 showed a higher percentage of initial apoptosis than the control in the apoptosis test, after 24 hours exposure. The same cements showed no genotoxicity in the concentration of 2 mg/mL, with the comet assay. CSC and CSCR2 were also not genotoxic at 10 mg/mL. All experimental materials showed viability with MTT. CSC and CSCR2 presented a better response to apoptosis and genotoxicity evaluation in the 10 mg/mL concentration, and demonstrated a considerable potential for use as reparative materials.

Cytotoxicity and genotoxicity of calcium silicate-based cements on an osteoblast lineage

Abstract Several calcium silicate-based biomaterials have been developed in recent years, in addition to Mineral Trioxide Aggregate (MTA). The aim of this study was to evaluate the cytotoxicity, genotoxicity and apoptosis/necrosis in human osteoblast cells (SAOS-2) of pure calcium silicate-based cements (CSC) and modified formulations: modified calcium silicate-based cements (CSCM) and three resin-based calcium silicate cements (CSCR1) (CSCR 2) (CSCR3). The following tests were performed after 24 hours of cement extract exposure: methyl-thiazolyl tetrazolium (MTT), apoptosis/necrosis assay and comet assay. The negative control (CT-) was performed with untreated cells, and the positive control (CT+) used hydrogen peroxide. The data for MTT and apoptosis were submitted to analysis of variance and Bonferroni's posttest (p < 0.05), and the data for the comet assay analysis, to the Kruskal-Wallis and Dunn tests (p < 0.05). The MTT test showed no significant difference among the materials in 2 mg/mL and 10 mg/mL concentrations. CSCR3 showed lower cell viability at 10 mg/mL. Only CSC showed lower cell viability at 50 mg/mL. CSCR1, CSCR2 and CSCR3 showed a higher percentage of initial apoptosis than the control in the apoptosis test, after 24 hours exposure. The same cements showed no genotoxicity in the concentration of 2 mg/mL, with the comet assay. CSC and CSCR2 were also not genotoxic at 10 mg/mL. All experimental materials showed viability with MTT. CSC and CSCR2 presented a better response to apoptosis and genotoxicity evaluation in the 10 mg/mL concentration, and demonstrated a considerable potential for use as reparative materials.

Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells.

UNLABELLED: Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus. OBJECTIVE: The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs). MATERIAL AND METHODS: The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP); MTA Fillapex (MTAF) and FillCanal (FC). Biocompatibility was evaluated with MTT and Neutral Red (NR) assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4). Unexposed cells were the positive control (CT). Bioactivity was assessed by alkaline phosphatase (ALP) enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution). All data were analyzed by ANOVA and Tukey post-test (p≤0.05%). RESULTS: MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%). Cells exposed to MTAF and FC (1:2 and 1:4 dilutions) showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure. CONCLUSIONS: The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

Biocompatibility and bioactivity of calcium silicate-based endodontic sealers in human dental pulp cells

Mineral Trioxide Aggregate (MTA) is a calcium silicate-based material. New sealers have been developed based on calcium silicate as MTA Fillapex and MTA Plus.Objective The aim of this study was to evaluate biocompatibility and bioactivity of these two calcium silicate-based sealers in culture of human dental pulp cells (hDPCs).Material and Methods The cells were isolated from third molars extracted from a 16-year-old patient. Pulp tissue was sectioned into fragments with approximately 1 mm3 and kept in supplemented medium to obtain hDPCs adherent cultures. Cell characterization assays were performed to prove the osteogenic potential. The evaluated materials were: MTA Plus (MTAP); MTA Fillapex (MTAF) and FillCanal (FC). Biocompatibility was evaluated with MTT and Neutral Red (NR) assays, after hDPCs exposure for 24 h to different dilutions of each sealer extract (1:2, 1:3 and 1:4). Unexposed cells were the positive control (CT). Bioactivity was assessed by alkaline phosphatase (ALP) enzymatic assay in cells exposed for one and three days to sealer extracts (1:4 dilution). All data were analyzed by ANOVA and Tukey post-test (p≤0.05%).Results MTT and NR results showed suitable cell viability rates for MTAP at all dilutions (90-135%). Cells exposed to MTAF and FC (1:2 and 1:4 dilutions) showed significant low viability rate when compared to CT in MTT. The NR results demonstrated cell viability for all materials tested. In MTAP group, the cells ALP activity was similar to CT in one and three days of exposure to the material. MTAF and FC groups demonstrated a decrease in ALP activity when compared to CT at both periods of cell exposure.Conclusions The hDPCs were suitable for the evaluation of new endodontic materialsin vitro. MTAP may be considered a promising material for endodontic treatments.

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles

Objective Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: 1) PC; 2) White MTA; 3) PC+30% Nbµ; 4) PC+30% Nbη. Material and Methods For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay. Results The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA. Conclusions It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA. .

Radiopacity and cytotoxicity of Portland cement associated with niobium oxide micro and nanoparticles.

OBJECTIVE: Mineral Trioxide Aggregate (MTA) is composed of Portland Cement (PC) and bismuth oxide (BO). Replacing BO for niobium oxide (NbO) microparticles (Nbµ) or nanoparticles (Nbη) may improve radiopacity and bioactivity. The aim of this study was to evaluate the radiopacity and cytotoxicity of the materials: (1) PC; (2) White MTA; (3) PC+30% Nbµ; (4) PC+30% Nbη. MATERIAL AND METHODS: For the radiopacity test, specimens of the different materials were radiographed along an aluminum step-wedge. For cell culture assays, Saos-2 osteoblastic-cells (ATCC HTB-85) were used. Cell viability was evaluated through MTT assay, and bioactivity was assessed by alkaline phosphatase activity assay. RESULTS: The results demonstrated higher radiopacity for MTA, followed by Nbµ and Nbη, which had similar values. Cell culture analysis showed that PC and PC+NbO associations promoted greater cell viability than MTA. CONCLUSIONS: It was concluded that the combination of PC+NbO is a potential alternative for composition of MTA.

Mineral trioxide aggregate-based endodontic sealer stimulates hydroxyapatite nucleation in human osteoblast-like cell culture.

INTRODUCTION: The main purpose of this study was to evaluate the biocompatibility and bioactivity of a new mineral trioxide aggregate (MTA)-based endodontic sealer, MTA Fillapex (MTA-F; Angelus, Londrina, Brazil), in human cell culture. METHODS: Human osteoblast-like cells (Saos-2) were exposed for 1, 2, 3, and 7 days to MTA-F, Epiphany SE (EP-SE; SybronEndo, Orange, CA), and zinc oxide-eugenol sealer (ZOE). Unexposed cultures were the control group (CT). The viability of the cells was assessed by MTT assay and the morphology by scanning electron microscopy (SEM). The bioactivity of MTA-F was evaluated by alkaline phosphatase activity (ALP) and the detection of calcium deposits in the culture with alizarin red stain (ARS). Energy-dispersive X-ray spectroscopy (EDS) was used to chemically characterize the hydroxyapatite crystallites (HAP). Saos-2 cells were cultured for 21 days for ARS and SEM/EDS. ARS results were expressed as the number of stained nodules per area. Statistical analysis was performed with analysis of variance and Bonferroni tests (P < .01). RESULTS: MTA-F exposure for 1, 2, and 3 days resulted in increased cytotoxicity. In contrast, viability increased after 7 days of exposure to MTA-F. Exposure to EP-SE and ZOE was cytotoxic at all time points. At day 7, ALP activity increase was significant in the MTA-F group. MTA-F presented the highest percentage of ARS-stained nodules (MTA-F > CT > EP-SE > ZOE). SEM/EDS analysis showed hydroxyapatite crystals only in the MTA-F and CT groups. In the MTA-F group, crystallite morphology and chemical composition were different from CT. CONCLUSIONS: After setting, the cytotoxicity of MTA-F decreases and the sealer presents suitable bioactivity to stimulate HAP crystal nucleation.

Cytotoxicity of Portland cement with different radiopacifying agents: a cell death study.

INTRODUCTION: The aim of this study was to investigate the cytotoxicity of white Portland cement (PC) alone or associated with bismuth oxide (PCBi), zirconium oxide (PCZir), and calcium tungstate (PCCa) in 2 cell lineages. METHODS: Murine periodontal ligament cells (mPDL) and rat osteosarcoma cells (ROS 17/2.8) were exposed for 24 hours to specific concentrations of fresh PC and PC associations with radiopacifiers. Zinc oxide-eugenol cement and hydrogen peroxide treatment were applied as cytotoxic positive controls. Cell viability after incubation with the cements was assessed by mitochondrial dehydrogenase enzymatic assay. Cell morphology was microscopically analyzed by cresyl violet staining, and the mechanism of cell death was determined by acridine orange/ethidium bromide methodology. All data were analyzed statistically by analysis of variance and Tukey post hoc test (P < .05). The correlation among cell death by apoptosis or necrosis and pH values was established by Pearson linear coefficient. RESULTS: The mitochondrial dehydrogenase enzymatic assay only revealed significant cell death rate at high concentrations of cement elutes. PC alone was not cytotoxic, even at 100 mg/mL. Microscopic images showed that none of the PC formulations caused damage to any cell lines. Statistical analysis of apoptosis/necrosis data demonstrated that PC and PC plus radiopacifying agents promoted significant necrosis cell death only at 100 mg/mL. CONCLUSIONS: The mPDL cells were more sensitive than ROS17/2.8. The results showed that PC associated with bismuth oxide, zirconium oxide, or calcium tungstate is not cytotoxic to mPDL or ROS17/2.8. Zirconium oxide and calcium tungstate might be good alternatives as radiopacifying agents.