Simvastatin-enriched macro-porous chitosan-calcium-aluminate scaffold for mineralized tissue regeneration

Abstract The present study evaluated the odontogenic potential of human dental pulp cells (HDPCs) exposed to chitosan scaffolds containing calcium aluminate (CHAlCa) associated or not with low doses of simvastatin (SV). Chitosan scaffolds received a suspension of calcium aluminate (AlCa) and were then immersed into solutions containing SV. The following groups were established: chitosan-calcium-aluminate scaffolds (CHAlCa - Control), chitosan calcium-aluminate with 0.5 µM SV (CHAlCa-SV0.5), and chitosan calcium-aluminate with 1.0 µM SV (CHAlCa-SV1.0). The morphology and composition of the scaffolds were evaluated by SEM and EDS, respectively. After 14 days of HDPCs culture on scaffolds, cell viability, adhesion and spread, mineralized matrix deposition as well as gene expression of odontogenic markers were assessed. Calcium aluminate particles were incorporated into the chitosan matrix, which exhibited regular pores homogeneously distributed throughout its structure. The selected SV dosages were biocompatible with HDPCs. Chitosan-calcium-aluminate scaffolds with 1 µM SV induced the odontoblastic phenotype in the HDPCs, which showed enhanced mineralized matrix deposition and up-regulated ALP, Col1A1, and DMP-1 expression. Therefore, one can conclude that the incorporation of calcium aluminate and simvastatin in chitosan scaffolds had a synergistic effect on HDPCs, favoring odontogenic cell differentiation and mineralized matrix deposition.

Resumo O presente estudo avaliou o potencial odontogênico de células da polpa dental humana (HDPCs) em contato com scaffolds de quitosana contendo aluminato de cálcio (CHAlCa) associado ou não à baixas dosagens de sinvastatina (SV). Scaffolds de quitosana receberam uma suspensão de aluminato de cálcio e foram imersos em soluções contendo a droga. Foram estabelecidos três grupos experimentais: scaffolds de quitosana e aluminato de cálcio (CHAlCa - controle), scaffolds de quitosana-aluminato de cálcio com 0.5 µM SV (CHAlCa-SV0.5), e quitosana-aluminato de cálcio com 1.0 µM SV (CHAlCa-SV1.0). A morfologia e composição foram avaliados por MEV e EDS, respectivamente. Após 14 dias do cultivo das HDPCs sobre os scaffolds, foram avaliados a viabilidade celular, adesão e espalhamento, deposição de matriz mineralizada e expressão gênica de marcadores odontogênicos. Observou-se que as partículas de aluminato de cálcio foram incorporadas à matriz de quitosana, a qual exibiu poros regulares distribuídos por toda sua estrutura. As dosagens selecionadas de sinvastatina foram biocompatíveis com as HDPCs. A concentração de 1 µM de SV induziu intensa expressão de fenótipo odontoblástico pelas HDPCs, demonstrando aumento da deposição de matriz mineralizada e maior expressão de ALP, Col1A1 e DMP-1. Portanto, podemos concluir que a incorporação de aluminato de cálcio e sinvastatina em scaffolds de quitosana apresentou um efeito sinérgico nas HDPCs, favorecendo a diferenciação celular e deposição de matriz mineralizada.

Influence of zirconia-coated bioactive glass on gingival fibroblast behavior

Abstract The objective of this study was the development of a bioactive glass coating on zirconia (Zr) to modulate the gingival fibroblast phenotype. For this purpose, Biosilicate® (BS) particles in a water/isopropyl alcohol (1:1) vehicle (6 mg/mL) were applied to zirconia discs followed by thermal treatment at 1100 °C for 20 min. The surface topography (SEM), chemical composition (EDX), surface roughness (Ra; confocal microscopy), surface free energy (goniometry), and color alteration (UV-vis spectrophotometry) were assessed (n=6). Thereafter, L929 fibroblasts were seeded onto Zr and Zr+BS discs, and cell proliferation (Alamar Blue; n=6), morphology (SEM; n=2), migration (wound healing; n=4), and collagen synthesis (Sirius Red; n=6) were evaluated up to 7 days. Data were analyzed by ANOVA/Tukey tests (a=5%). A homogeneous coating consisting of Si, Na, O, and Ca was detected on the Zr surface after thermal treatment with BS, which led to a significant increase in surface roughness and free energy (p<0.05). No change in color parameters was observed (p>0.05). Cells seeded on the Zr+BS surface featured increased proliferation, collagen expression, and migration capability in comparison with those cultured on plain Zr (p<0.05). SEM images revealed that cell spreading occurred faster in the presence of BS. Therefore, it was concluded that thermal treatment of the Zr surface with BS led to the deposition of a bioactive coating, which induced gingival fibroblast spread, proliferation, migration, and collagen expression in vitro.

Resumo O objetivo deste estudo foi o desenvolvimento de um recobrimento de vidro bioativo sobre a zircônia (Zr) para modular o fenótipo de fibroblastos gengivais. Para este propósito, partículas de Biosilicato® (6 mg/mL) em um veículo a base de água/álcool isopropílico (1:1) foram aplicadas sobre discos de zircônia seguido por tratamento térmico a 1100 °C por 20 min. A topografia de superfície (MEV), composição química (EDX), rugosidade de supefície (Ra; microscopia confocal), energia livre de superfície (goniômetro) e alteração de cor (Espectrofotometria UV-vis) foram avaliadas (n=6). A seguir, fibroblastos L929 foram semeados sobre discos de Zr e BS+Zr e a proliferação (Alamar Blue; n=6), morfologia (MEV; n=2), migração celular (wound healing; n=4) e a síntese de colágeno (Sirius Red; n=6) foram avaliados até 7 dias. Os dados foram analisados pelos testes ANOVA/Tukey (a=5%). Um recobrimento homogêneo consistindo de Si, Na, O e Ca foi detectado na superfície da Zr após o tratamento térmico com BS, o qual promoveu um aumento significante na rugosidade e energia livre de superfície (p<0,05). Nenhuma mudança nos parâmetros de cor foi observada (p>0,05). Células semeadas na superfície de Zr+BS apresentaram maior proliferação, expressão de colágeno e capacidade de migração em comparação com aquelas cultivadas sobre a superfície de Zr (p<0,05). Imagens de MEV revelaram que o espalhamento celular ocorreu mais rápido na presença de BS. Assim, conclui-se que o tratamento térmico da superfície da Zr com BS levou a deposição de um recobrimento bioativo, o qual induziu in vitro o espalhamento, proliferação e migração de fibroblastos gengivais e expressão de colágeno.

Biostimulatory effects of simvastatin on MDPC-23 odontoblast-like cells

Abstract: The aim of this study was to evaluate the bioactivity and cytocompatibility of simvastatin (SV) applied to MDPC-23 odontoblast-like cells. For this purpose, MDPC-23 cells were seeded in 96-well plates and submitted to treatments with 0.01 or 0.1 μM of SV for 24 h, 72 h or continuously throughout the experimental protocol. The negative control group (NC) was maintained in DMEM. Cell viability (MTT), ALP activity (thymolphthalein monophosphate), and mineralized matrix deposition (alizarin red) were analyzed at several time points. The data were submitted to ANOVA and Tukey's test (α = 0.05). Although cell viability was observed in the groups treated with SV, these groups did not differ from the NC up to 7 days. There was a reduction in cell viability for the groups treated with 0.1 μM of SV for 72 h, and submitted to continuous mode after 14 days. A significant increase in ALP activity occurred in the group treated with 0.01 μM of SV for 24 h, compared with the NC; however, only the group treated with 0.1 μM of SV in continuous mode reduced the ALP activity, in comparison with the NC. After 14 days, only continuous treatment with 0.1 μM of SV did not differ from NC, whereas the other experimental groups showed increased mineralized matrix deposition. Thus, it was concluded that low concentrations of simvastatin were bioactive and cytocompatible when applied for short periods to cultured MDPC-23 odontoblast-like cells.