Abstract This study aimed to evaluate, in vitro and in vivo, the biocompatibility of experimental methacrylate-based endodontic sealers containing α-tricalcium phosphate (α-TCP) or nanostructured hydroxyapatite (HAp). Experimental methacrylate-based dual-cure sealers with the addition of α-TCP or HAp, at 10%wt were formulated and compared to AH Plus (AHP). Cell viability was assessed by 3-(4,5-dimethyl-thiazoyl)-2,5-diphenyl-tetrazolium bromide (MTT), and sulforhodamine B (SRB). Sealers were implanted in rats’ subcutaneous tissue and histologically evaluated. Bioactivity was assessed by alkaline phosphatase enzyme activity (ALP) and Alizarin Red (AR), using apical papillary cells (SCAPs), and by the bone deposition measured in surgical cavities on rats’ femur filled with AH Plus or α-TCP. In both viability assays, HAp and AHP sealers were similar, and α-TCP presented lower viability compared to the others at MTT assay (p<0.05). A gradual decrease of the inflammatory response according to the periods was observed and AHP was the only that presented giant cells (7-day period). Collagen fibers condensation increased according to the periods, with no differences among sealers. There was an increase at ALP activity and mineralized nodules deposition according to periods. HAp and α-TCP presented higher values for ALP activity at 5 days and at 5, 10, and 15 days for AR and were different from AHP (p<0.05). α-TCP presented superior values at 10 and 15 days compared to HAp and AHP for AR (p<0.05). At 90 days, α-TCP and control (empty cavity) showed high bone deposition compared to AHP (p<0.05). α-TCP and HAp, in a methacrylate-based sealer, presented biocompatibility and bioactivity, with the potential to be used as endodontic sealers in clinical practice. Further investigations are required to gain information on the physicochemical properties of these sealers formulation before its clinical implementation.
Resumo O objetivo deste estudo foi avaliar a biocompatibilidade de cimentos endodônticos experimentais à base de metacrilato contendo fosfato α-tricálcico ou hidroxiapatita nanoestruturada in vitro e in vivo. Cimentos experimentais de cura dual à base de metacrilato com a adição de fosfato de α-tricálcico (α-TCP) ou hidroxiapatita (HAp), a 10% em peso, foram formulados e comparados com AH Plus (AHP). Viabilidade celular foi avaliada por brometo de 3- (4,5-dimetil-tiazoil) -2,5-difenil-tetrazólio (MTT) e sulforodamina B (SRB). Cimentos foram implantados no tecido subcutâneo dos ratos e avaliados histologicamente. Bioatividade foi avaliada pela atividade da enzima fosfatase alcalina (ALP) e Alizarin Red (AR) utilizando células da papila apical (SCAPs) e pela deposição óssea, medida em cavidades cirúrgicas no fêmur de ratos preenchidos com AH Plus e α-TCP. Nos dois ensaios de viabilidade, HAp e AHP não apresentaram diferenças estatísticas, α-TCP apresentou menores resultados de viabilidade para o ensaio MTT (p <0,05). Resultados histológicos mostraram que houve uma diminuição do conteúdo inflamatório de acordo com os períodos, e o AHP foi o único grupo que apresentou células gigantes (período de 7 dias). A condensação das fibras colágenas aumentou conforme os períodos, sem diferenças entre os grupos. Houve aumento da atividade da ALP e deposição de nódulos mineralizados de acordo com os períodos. HAp e α-TCP apresentaram maiores valores para a atividade de ALP em 5 dias e em 5, 10 e 15 dias para AR, com diferença para o AHP (p <0,05). O α-TCP apresentou valores superiores aos 10 e 15 dias quando comparado ao HAp e AHP para AR (p <0,05). Aos 90 dias, α-TCP e controle (cavidade vazia) apresentaram maior deposição de tecido ósseo quando comparado ao AHP (p <0,05). α-TCP e HAp, presentes nos cimentos à base de metacrilato, apresentaram biocompatibilidade e potencial para serem utilizados como seladores endodônticos na prática clínica. Investigações adicionais são necessárias para obter informações sobre as propriedades físico-químicas dessas formulações de cimentos antes de sua implementação clínica.
Abstract This study aimed to evaluate the role of photobiomodulation (PBM) in apexification and apexogenesis of necrotic rat molars with an open apex. Rat molars were exposed to the oral environment for 3 weeks. Canals were rinsed with 2.5% NaOCl and 17% EDTA, filled with antibiotic paste and sealed. After 7 days, canals were rinsed and divided into six groups (n=6): mineral trioxide aggregate (MTA); blood clot (BC); human dental pulp stem cells (hDPSC); MTA+PBM; BC+PBM; and hDPSC+PBM. In hDPSC groups, a 1% agarose gel scaffold was used. Two groups were not exposed: healthy tooth+PBM (n = 6), healthy tooth (n = 3); and one was exposed throughout the experiment: necrotic tooth (n = 3). In PBM groups, irradiation was performed with aluminum gallium indium phosphide (InGaAlP) diode laser for 30 days within 24-h intervals. After that, the specimens were processed for histological and immunohistochemical analyses. Necrotic tooth showed greater neutrophil infiltrate (p < 0.05). Necrotic tooth, healthy tooth, and healthy tooth+PBM groups showed absence of a thin layer of fibrous condensation in the periapical area. All the other groups stimulated the formation of a thicker layer of fibers (p < 0.05). All groups formed more mineralized tissue than necrotic tooth (p < 0.05). PBM associated with MTA, BC, or hDPSC formed more mineralized tissue (p < 0.05). MTA+PBM induced apexification (p < 0.05). Rabbit polyclonal anti-bone sialoprotein (BSP) antibody confirmed the histological findings of mineralized tissue formation, and hDPSC groups exhibited higher percentage of BSP-positive cells. It can be concluded that PBM improved apexification and favored apexogenesis in necrotic rat molars with an open apex.
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.
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.
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 materials in vitro. MTAP may be considered a promising material for endodontic treatments.
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.