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.

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 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.

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.

Abstract: Despite the crucial role of osteoclasts in the physiological process of bone repair, most bone tissue engineering strategies have focused on osteoblast-biomaterial interactions. Although Biosilicate® with two crystalline phases (BioS-2P) exhibits osteogenic properties and significant bone formation, its effects on osteoclasts are unknown. This study aimed to investigate the in vitro and in vivo effects of BioS-2P on osteoclast differentiation and activity. RAW 264.7 cells were cultured in osteoclastogenic medium (OCM) or OCM conditioned with BioS-2P (OCM-BioS-2P), and the cell morphology, viability, and osteoclast differentiation were evaluated. BioS-2P scaffolds were implanted into rat calvarial defects, and the bone tissue was evaluated using tartrate-resistant acid phosphatase (TRAP) staining and RT-polymerase chain reaction (PCR) after 2 and 4 weeks to determine the gene expressions of osteoclast markers and compare them with those of the bone grown in empty defects (Control). OCM-BioS-2P favored osteoclast viability and activity, as evidenced by an increase in the TRAP-positive cells and matrix resorption. The bone tissue grown on BioS-2P scaffolds exhibited higher expression of the osteoclast marker genes (Ctsk, Mmp 9, Rank) after 2 and 4 weeks and the RankL/Opg ratio after 2 weeks. Trap gene expression was lower at 2 weeks, and a higher number of TRAP-stained areas were observed in the newly formed bone on BioS-2P scaffolds at both 2 and 4 weeks compared to the Controls. These results enhanced our understanding of the role of bioactive glass-ceramics in bone repair, and highlighted their role in the modulation of osteoclastic activities and promotion of interactions between bone tissues and biomaterials.