Influence of photobiomodulation therapy on root development of rat molars with open apex and pulp necrosis

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.

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.

Avaliação de três técnicas de instrumentação com diferentes diâmetros apicais na formação de smear layer / Evaluation of three instrumentation techniques with different apical diameters on smear layer formation

Objetivo: avaliar os efeitos de diferentes instrumentações na formação de smear layer no terço apical de 90 raízes mesiobucais de molares superiores por microscopia eletrônica de varredura (MEV). Materiais e método: três grupos foram formados baseados nas técnicas utilizadas: instrumentação manual, sistema rotatório K3 e sistema reciprocante NSK. Os grupos foram subdivididos em três, de acordo com o diâmetro apical: #30, #35 ou #40. Após o preparo, as raízes foram seccionadas no sentido transversal, separando os terços apicais do restante das raízes; esses terços foram divididos em duas metades e preparados para MEV. A formação de smear layer na superfície do canal radicular e os túbulos den-tinários foram avaliados por escores num aumento de 1.000×. Os dados foram analisados pelo teste Kruskal-Wallis complementado pelo teste de Dunn (p < 0.05). Resultados: o aumento no diâmetro do preparo apical não influenciou na formação de smear layer nas paredes dentinárias. Conclusão: apesar das técnicas e dos diâmetros apicais utilizados durante o preparo, nenhuma parede livre de smear layer foi observada.

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. .

Estudo das células primárias dentais, viabilidade celular e bioatividade de materiais á base de silicato de cálcio / Cell viability and bioactivity of Portland cement-based materials in human dental follicle and pulp stem cells, fibroblasts and osteoblasts

Os objetivos deste estudo foram o isolamento e diferenciação de células da polpa (hDPCs) e do folículo (hDFCs) dentário de terceiros molares humanos e utilização para avaliação da citotoxicidade e bioatividade de novos cimentos endodônticos. (Capítulo 1) - Após a obtenção das culturas primárias foram realizados os ensaios vermelho de Alizarina, atividade da enzima fosfatase alcalina (ALP). A expressão gênica de proteína morfogenética óssea (bmp7), sialofosfoproteína dentinária (dspp) e alp foi avaliada. Os ensaios de bioatividade demonstraram maior atividade para hDPCs, além de maior expressão de dspp e alp; a expressão de bmp7 foi semelhante nas duas linhagens. Conclui-se que hDPCs demonstram maior capacidade de diferenciação em células com potencial de mineralização, quando comparadas com hDFCs. (Capítulo 2) – Foram utilizadas linhagens primárias (hDPCs e hDFCs), em comparação com linhagens imortalizadas (células osteoblásticas humanas Saos-2, e do ligamento periodontal de ratos mPDL) para análise dos materiais: 1) Cimento Portland branco (CP); 2) MTA; 3) CP/ óxido de nióbio microparticulado (Nb2O5?); 4) CP/ óxido de nióbio nanoparticulado (Nb2O5n). A citotoxicidade foi avaliada pelos ensaios MTT e Azul de Trypan e a bioatividade pela atividade da ALP. Viabilidade celular foi observada em todas as linhagens; e a atividade de ALP apresentou maior resposta na linhagem Saos-2. Conclui-se que as diferentes linhagens celulares são similares para avaliação da citotoxicidade; entretanto, para avaliação da bioatividade o uso de uma linhagem celular osteoblástica é mais indicado. Além disso, observa-se que materiais à base de Nb2O5? e Nb2O5n mostram potencial como radiopacificador alternativo no MTA

The aims of this study were to isolate and differentiate pulp (hDPCs) and follicle (hDFCs) cells obtained from human third molars and to use them in cytotoxicity and bioactivity assays of new dental materials. (Chapter 1) - After obtaining the stem cultures, Alizarin Red, alkaline phosphatase (ALP) activity and gene expression by Real Time PCR of bone morphogenetic protein (BMP7), dentin sialophosphoprotein (DSPP) and ALP were performed. The bioactivity asssays showed greater activity for hDPCs, and increased expression of dspp and alp; the expression of bmp7 was similar in both cell lines. We conclude that hDPCs demonstrate greater ability to differentiate into cells with mineralization potential when compared with hDFCs. (Chapter 2) - stem lineages (hDPCs and hDFCs) were used, compared to immortalized lineages (human osteoblastic cells Saos-2, and periodontal ligament of mice cells mPDL) for analysis of the following materials: 1) white Portland cement (PC); 2) MTA; 3) C/ microparticulated of niobium oxide (Nb2O5µ); 4) CP/nanoparticulated niobium oxide (Nb2O5n). Cytotoxicity was performed by MTT and Trypan Blue assays and bioactivity by ALP activity assay. Cell viability was observed in all cell lines, and ALP activity showed greater response in Saos-2. We conclude that the different cell lines are similar in cytotoxicity, however, to evaluate the bioactivity, using an osteoblastic cell line is more appropriate. Moreover, it is observed that materials based on Nb2O5µ and Nb2O5n show potential for alternative use to MTA