Effects of natural antimicrobial compounds propolis and copaiba on periodontal ligament fibroblasts, molecular docking, and in vivo study in Galleria mellonella.

Root canal treatment addresses infectious processes that require control. Occasionally, the radicular pulp is vital and inflamed, presenting a superficial infection. To preserve pulpal remnants, conservative procedures have gained favor, employing anti-inflammatory medications. This study investigated the effects of propolis (PRO), and copaiba oil-resin (COR) associated with hydrocortisone (H) and compared their impact to that of Otosporin® concerning cytotoxic and genotoxic activity, cytokine detection, and toxicity in the Galleria mellonella model. Human periodontal ligament fibroblasts (PDLFs) were exposed to drug concentrations and evaluated by the MTT assay. Associations were tested from concentrations that did not compromise cell density. Genotoxicity was evaluated through micronucleus counting, while cytokines IL-6 and TGF-ß1 were detected in the cell supernatant using ELISA. Molecular docking simulations were conducted, considering the major compounds identified in PRO, COR, and H. Increasing concentrations of PRO and COR were assessed for acute toxicity in Galleria mellonella model. Cellular assays were analyzed using one-way ANOVA followed by Tukey tests, while larval survivals were evaluated using the Log-rank (Mantel-Cox) test (α = 0.05). PRO and COR promoted PDLFs proliferation, even in conjunction with H. No changes in cell metabolism were observed concerning cytokine levels. The tested materials induce the release of AT1R, proliferating the PDFLs through interactions. PRO and COR had low toxicity in larvae, suggesting safety at tested levels. These findings endorse the potential of PRO and COR in endodontics and present promising applications across medical domains, such as preventive strategies in inflammation, shedding light on their potential development into commercially available drugs.

Different concentrations of fetal bovine serum affect cytokine modulation in Lipopolysaccharide-activated apical papilla cells in vitro.

BACKGROUND: Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. METHODOLOGY: Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-ß1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). RESULTS: In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. CONCLUSION: FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-ß1, OPG, IL-6, and CCL2 by APCs in vitro.

Simulação Clínica Realística: relato da experiência do laboratório de simulação clínica realística da FOUSP / Simulación clínica realista: informe de la experiencia del laboratorio de simulación clínica realista de la FOUSP / Realistic Clinical Simulation: report of the experience of the realistic clinical simulation laboratory at FOUSP

Apresenta-se a experiência da simulação clínica realística (SCR) efetuada no Laboratório de Simulação Clínica Realística e de Realidade Virtual Aumentada da Faculdade de Odontologia da Universidade de São Paulo (inaugurado em junho de 2022). Essa metodologia contribui significativamente na formação do estudante de Odontologia em todas as áreas do conhecimento odontológico. Sua aplicação deve submeter-se a protocolos corretos construção do problema (briefing), a simulação e discussão posterior (debriefing). Este cenário constitui o presente e o futuro da realidade do ensino odontológico. De fato, sua estratégia possibilita a simulação de uma multiplicidade de situações clínicas. Para tal, impõe-se a estruturação de um ambiente específico (aqui denominado "laboratório"). Este trabalho resume os fundamentos dessa simulação e a avaliação interna de sua aplicabilidade e inequívoca eficácia na nossa Faculdade. De fato, a simulação é uma estratégia facilitadora imprescindível no processo ensino-aprendizagem da Odontologia, cujo papel nesse processo deve delinear-se cada vez mais importante, na medida em que outros centros formadores forem adotando esse recurso e forem progressivamente intercambiando suas experiências e avaliações (AU).

Se presenta la experiencia de simulación clínica realista (SCR) realizada en el Laboratorio de Simulación Clínica Realista y Realidad Virtual Aumentada de la Facultad de Odontología de la Universidad de São Paulo (inaugurado en junio de 2022). Esta metodología contribuye significativamente a la formación de los estudiantes de odontología en todas las áreas del conocimiento odontológico. Su aplicación debe someterse a protocolos correctos para la construcción del problema (briefing), simulación y posterior discusión (debriefing). Este escenario constituye la realidad presente y futura de la educación odontológica. De hecho, su estrategia permite simular una multiplicidad de situaciones clínicas. Para lograrlo, es necesario estructurar un entorno específico (aquí llamado "laboratorio"). Este trabajo resume los fundamentos de esta simulación y la evaluación interna de su aplicabilidad y efectividad inequívoca en nuestra Facultad. De hecho, la simulación es una estrategia facilitadora esencial en el proceso de enseñanza-aprendizaje de la Odontología, cuyo papel en este proceso debe ser cada vez más importante, a medida que otros centros de formación adopten este recurso e intercambien progresivamente sus experiencias y valoraciones (AU).

This report on the experience of realistic clinical simulation (RCS) is linked to the Realistic Clinical Simulation and Augmented Virtual Reality Laboratory, launched in June 2022 at the Faculty of Dentistry of the University of São Paulo (São Paulo). RCS is an active methodology that can significantly contribute to the education of dental students in all areas of dental knowledge. Its application must adhere to correct protocols for problem construction (briefing), the simulation itself, and the subsequent discussion (debriefing). This scenario represents both the present and the future of dental education. Indeed, its strategy enables the simulation of a multitude of clinical situations, for which the establishment of a specific environment (herein referred to as a "laboratory") is needed. The present work summarizes the basis of this simulation and the internal evaluation of its applicability and effectiveness. It is concluded that simulation is a facilitating strategy in the dental teaching-and-learning process. In fact, its role in this process will become increasingly important as far as other educational institutions adopt this resource and progressively exchange their experiences and assessments (AU).

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells.

Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells

Abstract Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

Different concentrations of fetal bovine serum affect cytokine modulation in Lipopolysaccharide-activated apical papilla cells in vitro

Abstract Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Methodology Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). Results In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. Conclusion FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.

Exposure to lipopolysaccharide and calcium silicate-based materials affects the behavior of dental pulp cells.

This study assessed the cell viability, cytokine production, and mineralization potential of human dental pulp cells (hDPCs) after exposure to lipopolysaccharide (LPS) and application of calcium silicate-based materials (CSBM). Characterization of the CSBM was performed by infrared spectroscopy (n = 3). Extracts of Bio-C Repair, Biodentine, Cimmo HD, and MTA Repair HP were prepared and diluted (1:1, 1:4, and 1:16). Culture of hDPCs was established and treated or not with 1 µg/mL of LPS from Escherichia coli for 7 days. MTT assay was used to assess cell viability at 24, 48, and 72 h (n = 6). Alkaline phosphatase (ALP) activity was assayed on day 7 (n = 4). Il-10 and TNF-α were quantified by ELISA at 24 h (n = 6). Data were analyzed by ANOVA and Tukey's test (α = 0.05). Cell viability of LPS-activated hPDCs was higher than untreated control in 48 and 72 h (p < 0.05). Differences between non-treated and LPS-activated hPDCs were observed for Biodentine and Cimmo HP (p < 0.05). The CSBM influenced the cell viability (p < 0.05). ALP activity was higher in LPS-activated hDPCs (p < 0.05). No changes in the concentration of TNF-α were observed between groups (p > 0.05). The CSBM increased the Il-10 production (p < 0.05). LPS-activated hDPCs presented increased cell viability and ALP activity. The CSBM showed mild toxicity and was able to enhance the cell viability and mineralization potential of untreated and LPS-activated hDPCs. The CSBM also induced anti-inflammatory mechanisms without compromising pro-inflammatory ones.

Exposure to lipopolysaccharide and calcium silicate-based materials affects the behavior of dental pulp cells

Abstract This study assessed the cell viability, cytokine production, and mineralization potential of human dental pulp cells (hDPCs) after exposure to lipopolysaccharide (LPS) and application of calcium silicate-based materials (CSBM). Characterization of the CSBM was performed by infrared spectroscopy (n = 3). Extracts of Bio-C Repair, Biodentine, Cimmo HD, and MTA Repair HP were prepared and diluted (1:1, 1:4, and 1:16). Culture of hDPCs was established and treated or not with 1 µg/mL of LPS from Escherichia coli for 7 days. MTT assay was used to assess cell viability at 24, 48, and 72 h (n = 6). Alkaline phosphatase (ALP) activity was assayed on day 7 (n = 4). Il-10 and TNF-α were quantified by ELISA at 24 h (n = 6). Data were analyzed by ANOVA and Tukey's test (α = 0.05). Cell viability of LPS-activated hPDCs was higher than untreated control in 48 and 72 h (p < 0.05). Differences between non-treated and LPS-activated hPDCs were observed for Biodentine and Cimmo HP (p < 0.05). The CSBM influenced the cell viability (p < 0.05). ALP activity was higher in LPS-activated hDPCs (p < 0.05). No changes in the concentration of TNF-α were observed between groups (p > 0.05). The CSBM increased the Il-10 production (p < 0.05). LPS-activated hDPCs presented increased cell viability and ALP activity. The CSBM showed mild toxicity and was able to enhance the cell viability and mineralization potential of untreated and LPS-activated hDPCs. The CSBM also induced anti-inflammatory mechanisms without compromising pro-inflammatory ones.

Resumo Este estudo avaliou a viabilidade celular, produção de citocinas e potencial de mineralização de células da polpa dentária humana (hDPCs) após exposição a lipopolissacarídeo (LPS) e aplicação de materiais à base de silicato de cálcio (CSBM). A caracterização do CSBM foi realizada por espectroscopia (n = 3). Extratos de Bio-C Repair, Biodentine, Cimmo HD e MTA Repair HP foram preparados e diluídos (1: 1, 1: 4 e 1:16). A cultura de hDPCs foi estabelecida e tratada ou não com 1 µg / mL de LPS de Escherichia coli por 7 dias. O ensaio de MTT foi usado para avaliar a viabilidade celular em 24, 48 e 72 h (n = 6). A atividade da fosfatase alcalina (ALP) foi avaliada no dia 7 (n = 4). Il-10 e TNF-α foram quantificados por ELISA em 24 h (n = 6). Os dados foram analisados ​​por ANOVA e teste de Tukey (α = 0,05). A viabilidade celular das hPDCs ativados por LPS foi maior do que o controle não tratado em 48 e 72 h (p <0,05). Diferenças entre hPDCs não tratados e ativados por LPS foram observados para Biodentine e Cimmo HP (p < 0,05). Os CSBM influenciaram na viabilidade celular (p <0,05). A atividade de ALP foi maior em hDPCs ativadas por LPS (p <0,05). Não foram observadas alterações na concentração de TNF-α entre os grupos (p> 0,05). Os CSBM aumentaram a produção de Il-10 (p < 0,05). Os hDPCs ativados por LPS apresentaram um aumento na viabilidade celular e atividade ALP. Os CSBM apresentaram toxicidade moderada e foram capazes de aumentar a viabilidade celular e o potencial de mineralização de hDPCs não tratados e ativados por LPS. Os CSBM também induziram mecanismos anti-inflamatórios sem comprometer os pró-inflamatórios.

Endocannabinoids Modulate Production of Osteoclastogenic Factors by Stem Cells of the Apical Papilla In Vitro.

INTRODUCTION: Many mediators are produced during pulp inflammation and necrosis, including endocannabinoids (ECbs), which might affect the function of stem cells of the apical papilla (SCAP), cells of paramount importance for root formation, and regenerative endodontic treatment. The aim of this study was to evaluate the production of osteoclastogenesis-related mediators by SCAP modulated by ECbs and lipopolysaccharide (LPS) in vitro. METHODS: SCAP were cultured and treated with ECb anandamide (AEA), 2-arachidonoylglycerol, or N-arachidonoylaminophenol. All groups were incubated in the presence of a vehicle or LPS and the antagonist of transient receptor potential cation channel subfamily V member 1, capsazepine. After 24 hours, the culture medium supernatants were collected for further quantification of tumor necrosis factor alpha, CCL2, macrophage colony-stimulating factor, osteoprotegerin, and receptor activator of nuclear factor kappa B ligand. RESULTS: Small amounts of tumor necrosis factor alpha and receptor activator of nuclear factor kappa B ligand were detected in SCAP supernatants, and none of the experimental conditions altered their production. A down-regulation in constitutive CCL2 production was observed in the AEA group compared with that in the LPS group. The production of macrophage colony-stimulating factor was significantly increased in all groups treated with AEA compared with the control and LPS-treated groups. Osteoprotegerin was significantly increased by AEA alone and by 2-arachidonoylglycerol and N-arachidonoylaminophenol in the presence of LPS and capsazepine. CONCLUSIONS: AEA modulates some of the osteoclastogenic factors produced by SCAP in a bone resorption protective fashion.

Response of periodontal ligament stem cells to lipopolysaccharide and calcium silicate-based materials.

This study was conducted to assess the in vitro response of human periodontal ligament stem cells (hPDLSCs) to bacterial lipopolysaccharide (LPS) activation and application of three calcium silicate-based materials (CSBM): Bio-C Sealer, MTA Fillapex and Cimmo HP. Characterization of the CSBM was performed by FTIR (n = 3). Extracts of Bio-C Sealer, MTA Fillapex and Cimmo HP were prepared and diluted (1:1, 1:4 and 1:16). Culture of hPDLSCs was established and treated or not with LPS from Escherichia coli (1 µg/mL) for 7 days. MTT assay was used to assess cell viability at 24, 48 and 72 h (n = 9). Alkaline phosphatase (ALP) activity was indirectly assayed at day 7 (n = 5). TNF-α and Il -1 0 cytokines were quantified by ELISA at 24h-cell supernatants (n = 6). Data were analyzed by ANOVA and Tukey's test (α = 0.05). The cell viability of the LPS-activated hPDLSCs were higher than untreated control (p < 0.05). The application of CSBM affected the cell viability of untreated and LPS-activated cells (p < 0.05). ALP activity was higher for Bio-C Sealer and Cimmo HP in untreated and LPS-activated cells, respectively (p < 0.05). Application of CSBM normalized the TNF-α secretion in the LPS-activated cells (p < 0.05). Only MTA Fillapex in untreated hPDLSCs presented higher values of Il -1 0 (p < 0.05). Taken collectively, the results suggests that the simulation of the inflammatory process by LPS affect the in vitro response the hPDLSCs to the application of the CSBM.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway.

PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

Response of periodontal ligament stem cells to lipopolysaccharide and calcium silicate-based materials

Abstract This study was conducted to assess the in vitro response of human periodontal ligament stem cells (hPDLSCs) to bacterial lipopolysaccharide (LPS) activation and application of three calcium silicate-based materials (CSBM): Bio-C Sealer, MTA Fillapex and Cimmo HP. Characterization of the CSBM was performed by FTIR (n = 3). Extracts of Bio-C Sealer, MTA Fillapex and Cimmo HP were prepared and diluted (1:1, 1:4 and 1:16). Culture of hPDLSCs was established and treated or not with LPS from Escherichia coli (1 µg/mL) for 7 days. MTT assay was used to assess cell viability at 24, 48 and 72 h (n = 9). Alkaline phosphatase (ALP) activity was indirectly assayed at day 7 (n = 5). TNF-α and Il -1 0 cytokines were quantified by ELISA at 24h-cell supernatants (n = 6). Data were analyzed by ANOVA and Tukey's test (α = 0.05). The cell viability of the LPS-activated hPDLSCs were higher than untreated control (p < 0.05). The application of CSBM affected the cell viability of untreated and LPS-activated cells (p < 0.05). ALP activity was higher for Bio-C Sealer and Cimmo HP in untreated and LPS-activated cells, respectively (p < 0.05). Application of CSBM normalized the TNF-α secretion in the LPS-activated cells (p < 0.05). Only MTA Fillapex in untreated hPDLSCs presented higher values of Il -1 0 (p < 0.05). Taken collectively, the results suggests that the simulation of the inflammatory process by LPS affect the in vitro response the hPDLSCs to the application of the CSBM.

Resumo Este estudo objetivou avaliar a resposta in vitro de células-tronco do ligamento periodontal humano (hPDLSCs) à ativação por lipopolissacarídeo bacteriano (LPS) e aplicação de três materiais à base de silicato de cálcio (CSBM): Bio-C Sealer, MTA Fillapex e Cimmo HP. A caracterização dos CSBM foi realizada por FTIR (n = 3). Extratos de Bio-C Sealer, MTA Fillapex e Cimmo HP foram preparados e diluídos (1:1, 1: 4 e 1:16). A cultura de hPDLSCs foi estabelecida e tratada ou não com 1 µg / mL de LPS de Escherichia coli por 7 dias. O ensaio de MTT foi usado para avaliar a viabilidade celular em 24, 48 e 72 h (n = 9). A atividade de ALP foi avaliada indiretamente no dia 7 (n = 5). As citocinas TNF-α e Il-10 foram quantificadas por ELISA em sobrenadantes de células em 24h (n = 6). Os dados foram analisados por ANOVA e teste de Tukey (α = 0,05). A viabilidade celular das hPDLSCs ativados por LPS foi maior do que o controle (p <0,05). A aplicação dos CSBM afetou a viabilidade celular de células ativadas ou não por LPS (p <0,05). A atividade de ALP foi maior para Bio-C Sealer e Cimmo HP em células não ativadas e ativadas por LPS, respectivamente (p <0,05). A aplicação dos CSBM normalizou a secreção de TNF-α nas células ativadas por LPS (p <0,05). Apenas o MTA Fillapex em hPDLSCs não ativadas apresentou valores mais elevados de Il-10 (p <0,05). Em conclusão, os resultados sugerem que a simulação do processo inflamatório por LPS afetou a resposta in vitro de células-tronco do ligamento periodontal e de materiais à base de silicato de cálcio.

Protease-activated receptor type 1 (PAR1) increases CEMP1 gene expression through MAPK/ERK pathway

Abstract: PAR1 is a G-coupled protein receptor that regulates several cellular metabolism processes, including differentiation and proliferation of osteogenic and cementogenic related cells and our group previously demonstrated the regenerative potential of PAR1 in human periodontal ligament stem cells (hPDLSCs). In this study, we hypothesized that PAR1 regulates the cementogenic differentiation of hPDLSCs. Our goal was to identify the intracellular signaling pathway underlying PAR1 activation in hPDSLC differentiation. hPDLSCs were isolated using the explant technique. Cells were cultured in an osteogenic medium (OST) (α-MEM, 15% fetal bovine serum, L-glutamine, penicillin, streptomycin, amphotericin B, dexamethasone, and beta-glycerophosphate). The hPDLSCs were treated with a specific activator of PAR1 (PAR1 agonist) and blockers of the MAPK/ERK and PI3K pathways for 2 and 7 days. The gene expression of CEMP1 was assessed by RT-qPCR. The activation of PAR1 by its agonist peptide led to an increase in CEMP1 gene expression when compared with OST control. MAPK/ERK blockage abrogated the upregulation of CEMP1 gene expression induced by PAR1 agonist (p < 0.05). PI3K blockage did not affect the gene expression of CEMP1 at any experimental time (p > 0.05). We concluded that CEMP1 gene expression increased by PAR1 activation is MAPK/ERK-dependent and PI3K independent, suggesting that PAR1 may regulate cementogenetic differentiation of hPDLSCs.

Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

Cytotoxicity and cytokine production by calcium silicate-based materials on periodontal ligament stem cells.

This study investigated the effect of three commercial calcium silicate-based materials (CSBM) on cytotoxicity and pro-and anti-inflammatory cytokines production in cultured human periodontal ligament stem cells (hPDLSCs). Culture of hPDLSCs was established and characterized. Extracts of Bio-C Sealer (Angelus, Londrina, PR, Brazil), MTA Fillapex (Angelus, Londrina, PR, Brazil) and PBS Cimmo HP (Cimmo Soluções em Saúde, Pouso Alegre, MG, Brazil) were prepared by placing cement specimens (5 x 3 mm) in culture medium. Then, the extracts were serially two-fold diluted (1, 1:2, 1:4, 1:8, 1:16) and inserted into the cell-seeded wells for 24, 48 and 72 h for MTT assays. TNF-α and IL-10 cytokines were quantified by ELISA at 24h-cell supernatants. Data were analyzed by ANOVA and Tukey's test (α = 0.05). All CSBM exhibited some cytotoxicity that varied according to extract concentration and time of evaluation. MTA Fillapex presented the highest cytotoxic effects with significant reduction of metabolic activity/cell viability when compared to Bio-C Sealer and Cimmo HP®. TNF-α was significantly upregulated by the three tested cements (p < 0.05) while only MTA Fillapex significantly upregulated IL-10 in comparison to control. Taken collectively, the results showed that PBS Cimmo HP®, Bio-C Sealer and MTA Fillapex present mild and transient cytotoxicity and slightly induced TNF-α production. MTA Fillapex upregulated IL-10 release by hPDLSCs.

Calcium silicate-based cements affect the cell viability and the release of TGF-β1 from apical papilla cells

Abstract This study investigated the cytotoxicity and release of Transforming Growth Factor Beta 1 (TGF-β1) from cultured human apical papilla cells (APCs) after application of four bioactive materials. Culture of APCs was established and used for cytotoxic and quantitative assays. Extracts of Biodentine, Bio-C Repair, MTA Repair and White MTA were prepared and diluted (1, 1:4 and 1:16) and used for MTT assays up to 72 h. Total TGF-β1 was quantified by ELISA. Data were analyzed by ANOVA and Tukey's test (α = 0.05). For Biodentine, at 24 h and 48 h, cell viability was lower than control (p < 0.05). At 72 h, only undiluted extract of Biodentine were cytotoxic (p < 0.05). At 24 h, a cytotoxic effect was found for undiluted and 1:4 dilution of Bio-C Repair (p < 0.05). At 48 h, however, Bio-C Repair at 1:4 and 1:8 dilution showed higher cell viability (p < 0.05). At 24 and 48 h, the cell viability for undiluted MTA Repair were higher than control (p < 0.05). For White MTA, at 24 and 48 h, all dilutions were cytotoxic (p < 0.05). All cements led to reduced release of total TGF-β1 from the APCs (p < 0.05). In conclusion, cell viability varied depending on the material and dilution. Only Bio-C repair and MTA repair led to higher cell viability of APCs. All materials induced a decrease in the release of total TGF-β1 from the APCs.

Resumo Este estudo investigou a citotoxicidade e liberação do Fator de Crescimento Transformador Beta 1 (TGF-β1) em células da papila apical humana (APCs) cultivadas após a aplicação de quatro materiais bioativos. A cultura de APCs foi estabelecida e usada para ensaios citotóxicos e quantitativos. Extratos de Biodentine, Bio-C Repair, MTA Repair e White MTA foram preparados e diluídos (1, 1: 4 e 1:16) e usados para ensaios de MTT por até 72 h. O TGF-β1 total foi quantificado por ELISA. Os dados foram analisados por ANOVA e teste de Tukey (α = 0,05). Para o Biodentine, em 24 h e 48 h, efeito citotóxico foi observado (p <0,05). Em 72 h, apenas o extrato não diluído de Biodentine teve efeito citotóxico (p <0,05). Em 24 h, valores mais baixos de viabilidade celular foram encontrados para o extrato não diluído e diluidi 1:4 de Bio-C Repair (p <0,05). Em 48 h, no entanto, Bio-C Repair na diluição 1:4 e 1:8 mostrou maior viabilidade celular (p <0,05). A viabilidade celular para MTA Repair não diluído em 24 e 48 h foi maior que o controle (p <0,05). Para White MTA, às 24 e 48 h, a viabilidade celular em todas as diluições foram citotóxicas (p <0,05). Todos os cimentos levaram à redução da liberação de TGF-β1 total das APCs (p <0,05). Em conclusão, a viabilidade celular variou dependendo do material e da diluição. Biodentine, Bio-C Repair e MTA Repair levaram a uma maior viabilidade celular de APCs. Todos os materiais induziram uma diminuição na liberação de TGF-β1 total das APCs.

Cytotoxicity and cytokine production by calcium silicate-based materials on periodontal ligament stem cells

Abstract This study investigated the effect of three commercial calcium silicate-based materials (CSBM) on cytotoxicity and pro-and anti-inflammatory cytokines production in cultured human periodontal ligament stem cells (hPDLSCs). Culture of hPDLSCs was established and characterized. Extracts of Bio-C Sealer (Angelus, Londrina, PR, Brazil), MTA Fillapex (Angelus, Londrina, PR, Brazil) and PBS Cimmo HP (Cimmo Soluções em Saúde, Pouso Alegre, MG, Brazil) were prepared by placing cement specimens (5 x 3 mm) in culture medium. Then, the extracts were serially two-fold diluted (1, 1:2, 1:4, 1:8, 1:16) and inserted into the cell-seeded wells for 24, 48 and 72 h for MTT assays. TNF-α and IL-10 cytokines were quantified by ELISA at 24h-cell supernatants. Data were analyzed by ANOVA and Tukey's test (α = 0.05). All CSBM exhibited some cytotoxicity that varied according to extract concentration and time of evaluation. MTA Fillapex presented the highest cytotoxic effects with significant reduction of metabolic activity/cell viability when compared to Bio-C Sealer and Cimmo HP®. TNF-α was significantly upregulated by the three tested cements (p < 0.05) while only MTA Fillapex significantly upregulated IL-10 in comparison to control. Taken collectively, the results showed that PBS Cimmo HP®, Bio-C Sealer and MTA Fillapex present mild and transient cytotoxicity and slightly induced TNF-α production. MTA Fillapex upregulated IL-10 release by hPDLSCs.

Resumo Este estudo investigou o efeito de três materiais comerciais à base de silicato de cálcio (CSBM) na citotoxicidade e na produção de citocinas pró e antiinflamatórias em células-tronco do ligamento periodontal humano (hPDLSCs). Cultura de hPDLSCs foi estabelecida e caracterizada. Extratos de Bio-C Sealer (Angelus, Londrina, PR, Brasil), MTA Fillapex (Angelus, Londrina, PR, Brasil) e PBS Cimmo HP® (Cimmo Soluções em Saúde, Pouso Alegre, MG, Brasil) foram preparados com a colocação de espécimes dos cimentos (5 x 3 mm) em meio de cultura. Em seguida, os extratos foram diluídos (1, 1: 2, 1: 4, 1: 8, 1:16) e inseridos nos poços semeados de células para ensaio de citotoxicidade por meio de MTT por 24, 48 e 72 h. As citocinas TNF-α e IL-10 foram quantificadas por ELISA em sobrenadantes de células de 24 h. Os dados foram analisados por ANOVA e teste de Tukey (α = 0,05). Todos os CSBM exibiram alguma citotoxicidade que variou de acordo com a concentração do extrato e o tempo de avaliação. O MTA Fillapex apresentou os maiores efeitos citotóxicos com redução significativa da atividade metabólica / viabilidade celular quando comparado ao Bio-C Sealer e Cimmo HP®. O TNF-α foi regulado positivamente pelos três cimentos testados (p <0,05), enquanto apenas o MTA Fillapex regulou positivamente a liberação de IL-10 em comparação com o controle. Tomados em conjunto, os resultados mostraram que PBS Cimmo HP®, Bio-C Sealer e MTA Fillapex apresentam citotoxicidade leve e transitória e induziram a produção de TNF-α. O MTA Fillapex regulou positivamente a liberação de IL-10 por hPDLSCs.

Calcium silicate-based cements affect the cell viability and the release of TGF-ß1 from apical papilla cells.

This study investigated the cytotoxicity and release of Transforming Growth Factor Beta 1 (TGF-ß1) from cultured human apical papilla cells (APCs) after application of four bioactive materials. Culture of APCs was established and used for cytotoxic and quantitative assays. Extracts of Biodentine, Bio-C Repair, MTA Repair and White MTA were prepared and diluted (1, 1:4 and 1:16) and used for MTT assays up to 72 h. Total TGF-ß1 was quantified by ELISA. Data were analyzed by ANOVA and Tukey's test (α = 0.05). For Biodentine, at 24 h and 48 h, cell viability was lower than control (p < 0.05). At 72 h, only undiluted extract of Biodentine were cytotoxic (p < 0.05). At 24 h, a cytotoxic effect was found for undiluted and 1:4 dilution of Bio-C Repair (p < 0.05). At 48 h, however, Bio-C Repair at 1:4 and 1:8 dilution showed higher cell viability (p < 0.05). At 24 and 48 h, the cell viability for undiluted MTA Repair were higher than control (p < 0.05). For White MTA, at 24 and 48 h, all dilutions were cytotoxic (p < 0.05). All cements led to reduced release of total TGF-ß1 from the APCs (p < 0.05). In conclusion, cell viability varied depending on the material and dilution. Only Bio-C repair and MTA repair led to higher cell viability of APCs. All materials induced a decrease in the release of total TGF-ß1 from the APCs.