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.

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.

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.

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.

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.

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.

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.

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.

Early effect of laser irradiation in signaling pathways of diabetic rat submandibular salivary glands.

Low-power laser irradiation (LPLI) is clinically used to modulate inflammation, proliferation and apoptosis. However, its molecular mechanisms are still not fully understood. This study aimed to describe the effects of LPLI upon inflammatory, apoptotic and proliferation markers in submandibular salivary glands (SMGs) in an experimental model of chronic disorder, 24h after one time irradiation. Diabetes was induced in rats by the injection of streptozotocin. After 29 days, these animals were treated with LPLI in the SMG area, and euthanized 24h after this irradiation. Treatment with LPLI significantly decreased diabetes-induced high mobility group box 1 (HMGB1) and tumor necrosis factor alpha (TNF-α) expression, while enhancing the activation of the transcriptional factor cAMP response element binding (CREB) protein. LPLI also reduced the expression of bax, a mitochondrial apoptotic marker, favoring the cell survival. These findings suggest that LPLI can hamper the state of chronic inflammation and favor homeostasis in diabetic rats SMGs.

Molecular response of pulp fibroblasts after stimulation with pulp capping materials

Abstract This in vitro study evaluated cell viability and metabolism, nitric oxide release and production of two chemokines and one cytokine by cultured human dental pulp fibroblasts (HDPF) in contact with two glass ionomer cements (Ketac Molar-KM and Vitrebond-VB), Single Bond (SB) and calcium hydroxide (Dycal-DY). Cultures of HDPF were established by means of an explant technique. The specimens were prepared under sterile conditions and in disks measuring 5 mm x 2 mm obtained from a prefabricated mold and placed on a permeable membrane to avoid direct contact with the cells. Cytotoxicity was assessed by Trypan Blue exclusion method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide release in cell supernatant was detected by the Griess Method whereas stromal derived factor-1 alpha (SDF-1α or CXCL12), chemokine (C-X-C motif) ligand 8 [Interleukin 8 (IL-8 or CXCL8)] and interleukin-6 (IL-6) were detected by ELISA. RT-qPCR was employed for gene expression analysis. Statistical analyses were performed by One-way ANOVA followed by Tukey's post hoc test for materials independent of the time, and Two-way ANOVA followed by Bonferroni correction test for the comparisons between materials and experimental time (p<0.05). Cytotoxic tests showed significant differences only for DY. Protein levels and mRNA expression were significantly increased for IL-8 for both periods of time. IL-6 production increased when fibroblasts were stimulated by KM. SDF-1α protein production and mRNA expression were not affected by any of the materials. There was a decrease in nitrate/nitrite levels only for KM. Although DY caused intense cell death and did not stimulate the production of the inflammatory mediators evaluated in this work, it is known that this event seems to be fundamental for the process of repair of the pulp tissue and formation of mineralized barrier. KM and VB increased production of proteins related to the inflammatory process, thus favoring tissue repair. Therefore, although these glass ionomer cements did not lead to large cell death, they should be used with caution.

Resumo Este estudo avalia in vitro a viabilidade e metabolismo celular, a liberação de óxido nítrico e a produção de duas quimiocinas e uma citocina por fibroblastos de polpa dentária humana em cultura (FPDH) em contato com dois cimentos de ionômero de vidro (Ketac Molar-KM e Vitrebond-VB), Single Bond (SB) e hidróxido de cálcio (Dycal-DY). As culturas de FPDH foram estabelecidas por meio de uma técnica de explante. As amostras foram preparadas em condições estéreis e em discos de 5 mm x 2 mm, obtidas de um molde pré-fabricado e colocadas em uma membrana permeável (Maxicell 24 W 0,4 µm) para evitar o contato direto com as células. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de MTT. A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método Griess, enquanto fator 1 derivado do estroma (SDF-1α ou CXCL12), interleucina-8 (IL-8 ou CXCL8) and interleucina-6 (IL-6) foram detectados por ELISA. RT-qPCR foi empregada para análise de expressão gênica. As análises estatísticas foram realizadas por ANOVA a 1 critério, seguida pelo pós-teste de Tukey para os materiais independentes do tempo, e ANOVA a 2 critérios, seguida pelo teste de correção de Bonferroni para comparações entre materiais e tempo experimental (p<0,05). Os testes citotóxicos mostraram diferenças significativas apenas para DY. Os níveis da proteína e a expressão de RNAm para IL-8 aumentaram significativamente para ambos os tempos estudados. A produção de IL-6 aumentou quando os fibroblastos foram estimulados por KM. A produção da proteína e a expressão de RNAm para SDF-1α não foram afetadas por nenhum dos materiais. Houve uma diminuição nos níveis de nitrato/nitrito apenas para KM. Embora o DY tenha causado intensa morte celular e não tenha estimulado a produção dos mediadores inflamatórios avaliados neste trabalho, sabe-se que esse evento parece ser fundamental para o processo de reparo do tecido pulpar e formação de barreira mineralizada. Os cimentos de ionômero de vidro utilizados aumentaram a produção de proteínas relacionadas ao processo inflamatório, favorecendo a reparação tecidual e, portanto, esses materiais, embora não causem grande morte celular, devem ser utilizados com cautela.

Otosporin reduz reação pulpar inflamatória após clareação dentária de molares de ratos / Otosporin reduces pulp inflammatory reactions after dental bleaching of rat molars

Pacientes submetidos à clareação dentária relatam sensibilidade pós-operatória relacionada ao peróxido de hidrogênio (H2 O2 ) que penetra no tecido pulpar. Objetivo: Avaliar o efeito anti-inflamatório do ibuprofeno, Otosporin® e gel de curcumina na polpa dentária de ratos após procedimento clareador. Métodos: Cinquenta ratos foram divididos em GC ­ controle (gel placebo); CLA ­ clareação (H2 O2 35%, 30 minutos); CLA-I ­ clareação e administração oral de ibuprofeno (duas vezes a cada 12 horas, 2 dias sucessivos); CLA-O ­ clareação seguida da aplicação de Otosporin® nas superfícies dos molares (10 minutos); e CLA-C ­ sessão clareadora seguida do gel de curcumina (10 minutos). Após dois dias, os ratos foram mortos para análise histológica e testes estatísticos foram realizados(p<0,05). Resultados: CLA, CLA-I e CLA-C apresentaram inflamação severa ou necrose no terço oclusal da polpa coronária (p>0,05); CLA-O apresentou inflamação leve e foi semelhante ao GC (p>0,05) e dife- rente dos outros grupos (p<0,05). No terço médio, o grupo CLA-O apresentou menor infiltrado inflamatório e permaneceu diferente do grupo CLA (p<0,05); CLA, CLA-I e CLA-C foram semelhantes (p>0,05). No terço cervical, CLA, CLA-I e CLA-C tiveram redução da inflamação, sem diferença entre os grupos clareados (p>0,05). Conclusões: O Otosporin® pode reduzir a inflamação na polpa após clareação dentária; esse resultado não foi observado utilizando ibuprofeno ou gel de curcumina. Portanto, esse estudo mostra uma nova possibilidade de pós-tratamento em dentes clareados por meio do uso de Otosporin®, que minimiza a inflamação gerada ao tecido pulpar pelo gel clareador. Consequentemente, poderá haver redução da sensibilidade pós-operatória (AU).

Introduction: Patients undergoing dental bleaching relate to postoperative sensitivity, that is linked to hydrogen peroxide (H2 O2 ) penetrating on the dental pulp. This study evaluated the anti-inflammatory effect of ibuprofen, Otosporin®, and curcumin gel on the pulp of the rats' teeth after bleaching. Methods: Fifty rats were divided into CG: controlplacebo gel; BLE: bleached (35% H2O2, 30 minutes); BLE-I: bleached and ibuprofen oral administration (twice every 12 hours in 2 successive days); BLE-O: bleached followed by Otosporin® application in the molar surfaces (10 minutes); and BLE-C: bleaching session followed curcumin gel (10 minutes). After two days, the rats were killed for histological analysis. Statistical tests were performed (P<.05). Results: BLE, BLE-I, and BLE-C had severe inflammation or necrosis in the occlusal third of coronal pulp (P>.05); BLE-O had mild inflammation and was similar from CG (P>.05) and different from other groups (P<.05). In the middle third, BLE-O group had lower inflammatory infiltration and remained different from BLE group (P<.05); BLE, BLE-I, and BLE-C were similar (P>.05). In the cervical third, BLE, BLE-I, and BLE-C had a reduction of inflammation, without difference between bleached groups (P>.05). Conclusions: Otosporin® can reduce the inflammation in the pulp after dental bleaching; this result was not observed using ibuprofen or curcumin gel. Therefore, this study shows a new teeth bleaching posttreatment possibility using Otosporin®, which minimizes the inflammation generated to the pulp tissue by the bleaching gel. This could consequently minimize the postoperative sensitivity (AU).

Prostaglandin E2 Affects Interleukin 6 and Monocyte Chemoattractant Protein 1/CCL2 Production by Cultured Stem Cells of Apical Papilla.

INTRODUCTION: Root canal treatment of immature necrotic teeth is a major challenge in current endodontics. The effect of inflammatory mediators, such as prostaglandin, on the modulation of stem cells of the apical papilla (SCAP) is not completely understood. The aim of this study was to investigate the role of prostaglandin E2 (PGE2) on SCAP activation by Escherichia coli lipopolysaccharide (LPS) in vitro. METHODS: SCAP cultures were established and characterized. Increasing concentrations of lipopolysaccharide (0.1-10 µg/mL) were used to investigate cyclooxygenase-2 (COX-2/PTGS2) and PGE2 receptors (EP1-4) gene expression. Then, SCAP were treated with a COX-2 inhibitor (indomethacin) before treatment with different concentrations of LPS. The levels of the chemokine CCL2/monocyte chemoattractant protein 1 and interleukin (IL)-6 were detected in cell supernatants (24 hours) by enzyme-linked immunosorbent assay. Data analysis was performed using analysis of variance followed by the Tukey post test. RESULTS: The expression of COX-2 was up-regulated in the group treated with LPS at 1µg/mL compared with that in the control group. EP1-4 were detected in all experimental conditions at similar levels. SCAP treated with indomethacin presented a down-regulation in the production of LPS-induced CCL2 and the secretion of IL-6. CONCLUSIONS: SCAP showed increased COX-2 (PTGS2) gene expression induced by LPS and a PGE2-dependent production of IL-6 and CCL2.

Salivary Glands, Saliva and Oral Findings in COVID-19 Infection

Abstract The world is under the threat of the novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite several efforts to contain the disease spread, it still constitutes a public health emergency of international concern. Several published reports in the scientific literature called attention of the oral cavity as the potential route of infection, the implications for dental practice and the use of saliva in the diagnose of the COVID-19. The aim of this article is to provide an overview of the literature on the salivary glands and saliva in the context of SARS-CoV-2 infection. A brief discussion of taste disturbances and oral findings in COVID-19 patients is also presented. The literature shows that SARS-CoV-2 could infect the salivary glands. It is not possible, however, to make speculations regarding them as reservoirs for the SARS-CoV-2. In addition, patients with COVID-19 presented several oral repercussions, including hyposalivation and taste disturbances. A few reports showed oral ulcers and blisters associated with SARS-CoV-2 infection. However, it remains not fully understood and might lead to erroneous assumptions. Overall, further studies are necessary to understand the real role of salivary glands and saliva in the context of SARS-CoV-2 infection.

Root canal dressings for revascularization influence in vitro mineralization of apical papilla cells

Abstract Endodontic revascularization is based on cell recruitment into the necrotic root canal of immature teeth after chemical disinfection. The clinical outcome depends on the ability of surviving cells from the apical tissue to differentiate and promote hard tissue deposition inside the dentinal walls. Objective To investigate the effect of calcium hydroxide (CH) and modified triple antibiotic paste (mTAP - ciprofloxacin, metronidazole and cefaclor) on the viability and mineralization potential of apical papilla cells (APC) in vitro . Material and Methods APC cultures were kept in contact with CH or mTAP (250-1000 µg/mL) for 5 days, after which cell viability was assessed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Next, APCs were subjected to CH or mTAP at 250 µg/mL for 5 days before inducing the differentiation assay. After 14 and 21 days, calcium deposition was assessed by the Alizarin Red S staining method, followed by elution and quantification using spectrophotometry. Data were analyzed using ANOVA followed by Tukey post hoc test. Results CH induced cell proliferation, whereas mTAP showed significant cytotoxicity at all concentrations tested. APC treated with CH demonstrated improved mineralization capacity at 14 days, while, for mTAP, significant reduction on the mineralization rate was observed for both experimental periods (14 and 21 days). Conclusion Our findings showed that CH induces cell proliferation and improves early mineralization, whereas mTAP was found cytotoxic and reduced the mineralization potential in vitro of APCs.

Cytotoxicity of intracanal dressings on apical papilla cells differ upon activation with E. faecalis LTA

Abstract Objective The aim of this study was to investigate the cytotoxic effects of modified triple antibiotic paste and an experimental composition using calcium hydroxide on lipoteichoic acid (LTA)-primed apical papilla cells (APC). Material and Methods Human APC were tested for in vitro cytotoxicity of modified Triple Antibiotic Paste (mTAP - Ciprofloxacin, Metronidazole and Cefaclor at 1:1:1) and of a paste of Ciprofloxacin, Metronidazole and Calcium hydroxide (CMC - 1:1:2) and modified CMC (mCMC - 2:2:1) by using MTT assay. The substances were reconstituted in DMEM at 1,000 µg/mL and » serially diluted before being kept in contact with cells for 1, 3, 5 and 7 days. Further, cells were primed with 1 µg/mL of Enterococcus faecalis LTA for 7 days prior to the viability test with 1,000 µg/mL of each substance. Statistical analysis was performed using one-way analysis of variance (ANOVA) and two-way ANOVA respectively followed by Tukey's post-test. Significance levels were set at p<0.05. Results In the first assay, the higher cytotoxic rates were reached by mTAP for all experimental periods. CMC was found toxic for APC at 5 and 7 days, whereas mCMC did not affect the cell viability. Only CMC and mCMC were able to induce some cellular proliferation. In the second assay, when considering the condition with medium only, LTA-primed cells significantly proliferated in comparison to LTA-untreated ones. At this context, mTAP and CMC showed similar cytotoxicity than the observed for LTA-untreated cells, while mCMC was shown cytotoxic at 7 days only for LTA-primed APC. Comparing the medications, mTAP was more cytotoxic than CMC and mCMC. Conclusion mTAP showed higher cytotoxicity than CMC and mCMC and the effect of topic antimicrobials might differ when tested against apical papilla cells under physiological or activated conditions.

Dental pulp fibroblasts response after stimulation with hema and adhesive system

Abstract This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Resumo Este estudo avaliou in vitro a viabilidade e metabolismo celular, liberação de óxido nítrico e produção de quimiocinas em cultura de fibroblastos de polpa dental humana (DPF) em contato com HEMA e Single Bond. Culturas de DPF foram estabelecidas por meio de uma técnica de explante. Uma vez plaqueadas, as células foram mantidas em contato com concentrações crescentes de HEMA (10, 100 e 1000 nM) ou Single Bond (SB) [10 vezes diluídas em série em meio de cultura (10-4, 10-3 e 10-2 v/v)] e também com SB polimerizado. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo (MTT). A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método de Griess, enquanto as quimiocinas (CXCL12 e CXCL8) foram detectadas por ELISA. RT-qPCR foi empregada para análise de expressão gênica de quimiocinas. Testes citotóxicos mostraram diferenças significativas para SB 10-2. Nenhum dos materiais testados alterou significativamente os níveis de NO. Os níveis de proteína de CXCL12 foram significativamente diminuídos apenas pelo HEMA. Por outro lado, enquanto o RNAm de CXCL12 permaneceu inalterado, a expressão gênica de CXCL8 teve redução significativa com todos os materiais, com exceção do SB polimerizado. Em conclusão, Single Bond e HEMA, em várias concentrações, diminuíram a expressão e produção de moléculas envolvidas em processos inflamatórios e, portanto, o uso de sistemas adesivos, como o material protetor da polpa, deve ser visto com cautela devido ao seu grande efeito citotóxico quando em contato com a polpa.