Physicochemical properties, cytotoxicity and bioactivity of a ready-to-use bioceramic repair material

Abstract The aim of this study was to evaluate the physicochemical properties, cytotoxicity and bioactivity of a ready-to-use bioceramic material, Bio-C Repair (Angelus), in comparison with White MTA (Angelus) and Biodentine (Septodont). The physicochemical properties of setting time, radiopacity, pH, solubility, dimensional and volumetric changes were evaluated. Biocompatibility and bioactivity were assessed in Saos-2 osteoblast cell cultures by the MTT assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Neutral Red (NR), Alizarin Red (ARS), and cell migration tests. Statistical analysis was performed by ANOVA, Tukey or Bonferroni tests (α = 0.05). Bio-C Repair had the longest setting time (p < 0.05), but radiopacity and solubility were accordance with the ISO 6876/2012 standards, besides linear expansion. Bio-C Repair and MTA had similar volumetric change (p > 0.05); lower than Biodentine (p < 0.05). All the materials evaluated had an alkaline pH. Bio-C Repair was cytocompatible and promoted mineralized nodule deposition in 21 days and cell migration in 3 days. In conclusion, Bio-C Repair had adequate radiopacity above 3mm Al, solubility less than 3%, dimensional expansion, and low volumetric change. In addition, Bio-C Repair promoted an alkaline pH and presented bioactivity and biocompatibility similar to MTA and Biodentine, showing potential for use as a repair material.

Resumo O objetivo deste estudo foi avaliar as propriedades físico-químicas, citotoxicidade e bioatividade de um novo material biocerâmico pronto para uso, Bio-C Repair (Angelus), em comparação com MTA Branco (Angelus) e Biodentine (Septodont). Foram avaliadas as propriedades físico-químicas de tempo de presa, radiopacidade, pH, solubilidade, alterações dimensionais e volumétricas. A biocompatibilidade, bioatividade e capacidade de reparo foram avaliadas em culturas de células de osteoblastos Saos-2 pelo ensaio MTT brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio), vermelho neutro (NR), vermelho de alizarina ( ARS) e testes de migração celular. A análise estatística foi realizada pelos testes ANOVA, Tukey ou Bonferroni (α = 0,05). Bio-C Repair apresentou o maior tempo de presa (p < 0,05), mas radiopacidade e solubilidade de acordo com as normas ISO 6876/2012, além de expansão linear. Bio-C Repair e MTA tiveram variação volumétrica semelhante (p > 0,05), menor que Biodentine (p < 0,05). Todos os materiais avaliados apresentaram pH alcalino. Bio-C Repair foi citocompatível, além de promover deposição de nódulos mineralizados em 21 dias e migração celular em 3 dias. Em conclusão, o Bio-C Repair apresentou radiopacidade adequada acima de 3mmAl, solubilidade menor que 3%, expansão dimensional e baixa perda volumétrica.. Além disso, o Bio-C Repair promoveu um pH alcalino e apresentou bioatividade e biocompatibilidade semelhantes ao MTA e Biodentine, mostrando potencial para uso como material reparador

Cytotoxicity and bioactive potential of new root repair materials for use with BMP-2 transfected human osteoblast cells

Abstract: Modified formulations of calcium silicate repair materials with additives have been developed to enhance handling, consistency, biocompatibility and bioactivity. Considering the relevance of osteoblastic cell response to mineralized tissue repair, human osteoblastic cells (Saos-2 cells overexpressing BMP-2) were exposed to mineral trioxide aggregate (MTA) (with calcium tungstate - CaWO4), MTA HP Repair, Bio-C Repair and Bio-C Pulpo. Cell viability was assessed by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR), and cell death, by flow cytometry. Gene expression of bone morphogenetic protein 2 (BMP-2), runt-related transcription factor 2 (RUNX-2), and alkaline phosphatase (ALP) osteogenic markers were evaluated by real-time polymerase chain reaction (RT-qPCR). ALP activity and alizarin red staining (ARS) were used to detect mineralization nodule deposition. Bioactive cements presented no cytotoxic effect, and did not induce apoptosis at the higher dilution (1:12). MTA, Bio-C Repair and Bio-C Pulpo exhibited higher ALP activity than the control group (P < 0.05) after 7 days. MTA, MTA HP and Bio-C Pulpo affected the formation of mineralized nodules (p < 0.05). Exposure to all cement extracts for 1 day increased BMP-2 gene expression. RUNX-2 mRNA was greater in MTA, MTA HP and Bio-C Repair. MTA, MTA HP and Bio-C Pulpo increased the ALP mRNA expression, compared with BMP-2 unexposed cells (P < 0.05). Calcium silicate cements showed osteogenic potential and biocompatibility in Saos-2 cells transfected BMP-2, and increased the mRNA expression of BMP-2, RUNX-2, and ALP osteogenic markers in the BMP-2 transfected system, thereby promoting a cellular response to undertake the mineralized tissue repair.

Different formulations of peracetic acid: effects on smear layer removal, dentine erosion, cytotoxicity and antibiofilm activity

Abstract Objective: To assess the effects of different peracetic acid (PAA) formulations on smear layer (SL) removal, dentine erosion, cytotoxicity, and antibiofilm activity. Methodology: SL removal and dentine erosion were assessed using 90 premolars, distributed into six groups, according to final irrigation: PAA formulations (1% Sigma, 1% Bacterend OX, 1% Arposept, and 0.09-0.15% Anioxyde), 17% ethylenediaminetetraacetic acid (EDTA), and water (control). Cytotoxicity was assessed by methyl-thiazol-tetrazolium (MTT) and neutral red assays. Antibacterial and antibiofilm effectiveness was evaluated against Enterococcus faecalis. For cytotoxicity and antibiofilm activity assessment, the 2.5% NaOCl was also included. Results: EDTA, Sigma, and Bacterend OX removed more SL than Arposept, Anioxyde, and water (p<0.05). EDTA caused more severe dentine erosion than Sigma and Bacterend OX (p<0.05). Sigma and Bacterend OX had higher cytotoxicity than the other solutions (p<0.05). NaOCl, Bacterend OX, Sigma, and Anioxyde significantly reduced E. faecalis colony-forming units (CFU) (p<0.05). The 2.5% NaOCl solution promoted greater biofilm biomass reduction (p<0.05) than the other solutions. All PAA formulations promoted greater biomass reduction than 17% EDTA (p<0.05). Conclusions: Although Sigma and Bacterend OX had higher cytotoxicity, they had a SL removal capability similar to that of EDTA, were as effective as NaOCl against E. faecalis biofilm, and promoted less dentine erosion than EDTA. Arposept and Anioxyde failed to remove the SL, had lower cytotoxicity, and showed less bacterial activity than NaOCl.

Physicochemical properties, cytocompatibility and antibiofilm activity of a new calcium silicate sealer

Abstract The aim of this study was to evaluate the physicochemical properties, cytocompatibility and antibiofilm activity of a new calcium silicate-based endodontic sealer, Sealer Plus BC (MK Life, Brazil), in comparison with TotalFill BC Sealer (FKG Dentaire SA, Switzerland) and AH Plus (Dentsply, Germany). Setting time and flow were evaluated based on ISO 6876 standard. The pH was evaluated after different periods, and radiopacity by radiographic analysis (mmAl). Solubility (% mass loss) and volumetric change (by micro-CT) were assessed after 30 days of immersion in distilled water. Cytocompatibility was assessed by methyltetrazolium (MTT) and neutral red (NR) assays, after exposure of Saos-2 cells to the sealer extract for 24 h. An additional analysis was performed by using MTT assay after 1, 3 and 7 days of exposure of Saos-2 to the sealers 1:8 dilution extracts. Antibiofilm activity against Enterococcus faecalis and/or Candida albicans was evaluated by crystal violet assay and modified direct contact test. The physicochemical properties were analyzed using ANOVA/Tukey tests; MTT and NR data were analyzed by ANOVA and Bonferroni tests; the antimicrobial tests were analyzed by Kruskal-Wallis and Dunn tests (α=0.05). Sealer Plus BC had proper setting time, radiopacity, flow and alkalization capacity. Sealer Plus BC was significantly more soluble than AH Plus (p<0.05) and presented volumetric change similar to AH Plus and TotalFill BC (p>0.05). Sealer Plus BC presented antibiofilm activity and no cytotoxic effect. In conclusion, although Sealer Plus BC had higher solubility, this sealer showed proper physicochemical properties, cytocompatibility, and antibiofilm activity.

Resumo O objetivo deste estudo foi avaliar as propriedades físico-químicas, a citocompatibilidade e a atividade antibiofilme de um novo cimento endodôntico à base de silicato de cálcio, Sealer Plus BC (MK Life, Brasil), em comparação com TotalFill BC Sealer (FKG Dentaire SA, Suíça) e AH Plus (Dentsply, Alemanha). O tempo de presa e o escoamento foram avaliados com base nas normas ISO 6876. O pH foi avaliado após diferentes períodos, e a radiopacidade por análise radiográfica (mmAl). A solubilidade (% de perda de massa) e alteração volumétrica (por micro-CT) foram avaliadas após 30 dias de imersão em água destilada. Citocompatibilidade foi avaliada pelos ensaios metiltetrazólio (MTT) e vermelho neutro (NR), após exposição das células Saos-2 ao extrato de cimento por 24 horas. Análise adicional foi realizada através do ensaio MTT após 1, 3 e 7 dias de exposição das células Saos-2 aos extratos dos cimentos na diluição de 1:8. Atividade antibiofilme contra Enterococcus faecalis e/ou Candida albicans foi avaliada pelos ensaios cristal violeta e contato direto modificado. As propriedades físico-químicas foram analisadas utilizando os testes ANOVA e Tukey; MTT e NR foram analisados pelos testes ANOVA e Bonferroni; os ensaios antimicrobianos foram analisados pelos testes Kruskal-Wallis e Dunn (α=0.05). Sealer Plus BC apresentou tempo de presa, radiopacidade e escoamento adequados, além de capacidade de alcalinização. Sealer Plus BC foi significantemente mais solúvel que AH Plus (p<0.05) e apresentou alteração volumétrica similar à de AH Plus e TotalFill BC (p>0.05). Sealer Plus BC apresentou atividade antibiofilme, sem efeito citotóxico. Como conclusão, embora Sealer Plus BC apresente maior solubilidade, este cimento apresentou propriedades físico-químicas adequadas, citocompatibilidade e atividade antibiofilme.

Physicochemical properties and effect of bioceramic root canal filling for primary teeth on osteoblast biology

Abstract Bio-C Pulpecto (Bio-CP) was recently developed as the first bioceramic root filling material for primary teeth. Objective To evaluate the physicochemical properties of radiopacity, setting time, pH, cytocompatibility and potential of Bio-CP to induce mineralisation, compared with (1) Calen thickened with zinc oxide (Calen-ZO), and (2) zinc oxide and eugenol (ZOE). Methodology Physicochemical properties were evaluated according to ISO 6876. Saos-2 (human osteoblast-like cell line) exposed to extracts of the materials were subjected to assays of methyl thiazolyl tetrazolium, neutral red, alkaline phosphatase (ALP) activity and mineralised nodule production. The results were analysed using one-way or two-way ANOVA and Tukey's or Bonferroni's post-tests (α=0.05). Results All the materials showed radiopacity higher than 3 mm Al. Bio-CP had lower pH than Calen-ZO, but higher pH than ZOE. Calen-ZO and Bio-CP did not set. The setting time for ZOE was 110 min. The cytocompatibility order was Calen-ZO > Bio-CP > ZOE (1:2, 1:4 dilutions) and Calen-ZO > Bio-CP = ZOE (1:12, 1:24 dilutions) and Calen-ZO = Bio-CP > ZOE (1:32 dilution). Bio-CP induced greater ALP activity at 7 days, and greater mineralised nodule production, compared to Calen-ZO (p<0.05). Conclusions Bio-CP showed adequate physicochemical properties, cytocompatibility and potential to induce mineralisation.

Ytterbium oxide as radiopacifier of calcium silicate-based cements. physicochemical and biological properties

Abstract This study evaluated physicochemical properties, cytotoxicity and bioactivity of MTA Angelus (MTA), calcium silicate-based cement (CSC) and CSC with 30% Ytterbium oxide (CSC/Yb2O3). Setting time was evaluated using Gilmore needles. Compressive strength was evaluated in a mechanical machine. Radiopacity was evaluated using radiographs of materials and an aluminum scale. Solubility was evaluated after immersion in water. Cell viability was evaluated by means of MTT assay and neutral red staining, and the mineralization activity by using alkaline phosphatase activity and Alizarin Red staining. The data were submitted to ANOVA, Tukey and Bonferroni tests (5% significance). The bioactive potential was evaluated by scanning electron microscopy. The materials presented similar setting time. MTA showed the lowest compressive strength. MTA and CSC/Yb2O3 presented similar radiopacity. CSC/Yb2O3 showed low solubility. Saos-2 cell viability tests showed no cytotoxic effect, except to 1:1 dilution in NR assay which had lower cell viability when compared to the control. ALP at 1 and 7 days was similar to the control. MTA and CSC had greater ALP activity at 3 days when compared to control. All the materials present higher mineralized nodules when compared with the control. SEM analysis showed structures suggesting the presence of calcium phosphate on the surface of materials demonstrating bioactivity. Ytterbium oxide proved to be a properly radiopacifying agent for calcium silicate-based cement since it did not affected the physicochemical and biological properties besides preserving the bioactive potential of this material.

Resumo Este estudo avaliou as propriedades físico-químicas, citotoxicidade e bioatividade do MTA Angelus (MTA), cimento à base de silicato de cálcio (CSC) e CSC com 30% de óxido de itérbio (CSC/Yb2O3). O tempo de presa foi avaliado usando agulhas Gilmore. A resistência à compressão foi avaliada em uma máquina mecânica. A radiopacidade foi avaliada utilizando radiografias dos materiais e uma escala de alumínio. A solubilidade foi avaliada após imersão em água. A viabilidade celular foi avaliada por meio do ensaio MTT e coloração de vermelho neutro (NR), e a atividade de mineralização por meio da atividade da fosfatase alcalina e a coloração com Vermelho de Alizarina. Os dados foram submetidos aos testes ANOVA, Tukey e Bonferroni (5% de significância). O potencial bioativo foi avaliado por microscopia eletrônica de varredura. Os materiais apresentaram tempo de presa semelhante. O MTA mostrou menor resistência à compressão. MTA e CSC/Yb2O3 apresentaram radiopacidade semelhante. CSC/Yb2O3 apresentou menores valores de solubilidade. A viabilidade celular realizada pelos ensaios de MTT e NR não revelaram efeitos ctotóxicos em todas as diluições, exceto na diluição 1:1 no NR, o qual mostrou baixa viabilidade celular (p<0.05) em todos materiais testados quando comparado ao controle. A atividade de ALP em 1 e 7 dias foi similar ao controle (p>0.05). MTA e CSC tiveram significante aumento na atividade de ALP aos 3 dias quando comarados ao controle (p>0.05). Todos os materiais apresentaram grande produção de nódulos mineralizados quando comparados ao controle (P<0.05). A análise da SEM mostrou estruturas que sugerem a presença de depósitos de fosfato de cálcio na superfície dos materiais demonstrando bioatividade. O Yb2O3 mostrou ser um agente radiopacificador adequado em cimentos à base de silicato de cálcio uma vez que não afetou as propriedades físico-químicas e biológicas e ainda preservou o potencial bioativo desse material.

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

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

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.