Potencial bioativo de scaffolds de nanofibras de policaprolactona contendo hidróxido de cálcio e associados à fibronectina sobre células pulpares humanas
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Data
2021-03-08
Autores
Oliveira, Caroline Anselmi de
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Universidade Estadual Paulista (Unesp)
Resumo
O objetivo do presente estudo foi sintetizar e caracterizar scaffolds de nanofibras a base de policaprolactona (PCL) incorporados com hidróxido de cálcio (HC), e avaliar seu potencial tóxico e bioativo sobre células da polpa dental humana (HDPCs) quando associados à fibronectina (FN). Inicialmente, concentrações de HC (0,1%; 0,2% ou 0,4%) foram incorporadas a scaffolds de PCL confeccionados por electrospinning. Scaffolds sem HC serviram como controle. Os scaffolds foram caracterizados quanto à morfologia por MEV associada à análise por EDS (n=4). Também foram analisados quanto a solubilidade, alteração de pH do meio e liberação de cálcio (n=8). Então, HDPCs foram cultivadas sobre os scaffolds e avaliadas quanto a viabilidade (alamarBlue, n=8; ensaio de Live/Dead n=4) nos períodos de 1, 7 e 14 dias, e adesão e espalhamento (F-actina, n=4) em 1, 3 e 7 dias do cultivo celular. O aumento da concentração de HC ampliou o diâmetro das nanofibras sem interferir na porcentagem de espaços interfibrilares e aumentou a liberação de cálcio, mantendo a neutralidade do meio (pH 7,0-8,0). Em comparação ao grupo controle, aumento significativo de viabilidade celular foi observado apenas para o grupo contendo HC 0,4%, em todos os períodos. Portanto, na segunda fase, scaffolds contendo ou não 0,4% de HC foram adsorvidos com FN (20 µg/mL). HDPCs semeadas na superfície foram avaliadas quanto a viabilidade, adesão e espalhamento, migração (Trans-well; n=4), expressão gênica de marcadores de diferenciação odontogênica (RT-qPCR; n=6), atividade de fosfatase alcalina (ALP; n=8) e formação de nódulos de mineralização (Alizarin red; n=8). Os dados foram submetidos a ANOVA e pós testes de Tukey, Games-Howell ou Sidak (α=5%). Os resultados de Live/Dead e F-actina foram avaliados qualitativamente. A incorporação de HC e FN nos scaffolds, isolados ou em associação, aumentou a capacidade de migração celular, além de permitir um melhor espalhamento citoplasmático, intensificado quando houve associação. O mesmo foi visto para a viabilidade celular. A expressão de ALPL e DSPP não foi regulada pela presença de HC e/ou FN, assim como a atividade de ALP. Regulação negativa do gene COL1A1 foi observada em todos os grupos experimentais quando comparados ao controle, enquanto o gene DMP1 foi superexpresso na presença de HC. O HC favoreceu a formação de matriz mineralizada, a qual não foi influenciada pela presença de FN. Assim, foi possível concluir que a incorporação de 0,4% de HC a scaffolds de PCL proporcionou topografia de superfície e propriedades favoráveis à adesão, espalhamento, proliferação e expressão do fenótipo odontogênico por HDPCs. Entretanto, apenas viabilidade, espalhamento e migração celular foram intensificados na presença de FN.
The aim of this study was to synthesize and characterize polycaprolactone-based nanofiber scaffolds (PCL) incorporated with calcium hydroxide (CH), as well as to evaluate their potential toxicity and bioactivity on human dental pulp cells (HDPCs) when loaded with fibronectin (FN). Initially, different concentrations of CH (0.1%; 0.2% or 0.4%) were incorporated in PCL scaffolds made using electrospinning. Scaffolds without CH served as control. The morphology and composition of the scaffolds were characterized using SEM/EDS (n=4). They were also tested for solubility, pH change of the medium and calcium release (n=8). Then, HDPCs were seeded on the surface of the scaffolds and evaluated regarding their viability (alamarBlue, n=8; Live/Dead assay, n=4) in the time intervals of 1, 7 and 14 days, and adhesion and spreading (Factin, n=4) in 1, 3 and 7days from cell culture. The increase in CH concentration increased the diameter of the nanofibers without interfering with the percentage of interfibrillar spaces, and increased the release of calcium, maintaining the neutrality of the medium (pH 7.0-8.0). In comparison to the control group, a significant increase in cell viability was seen only for the group containing 0.4% CH, in all time intervals. Therefore, in the next experiment, scaffolds containing or not 0.4% CH were loaded with FN (20 µg/mL). HDPCs were seeded on the scaffolds and evaluated for viability, adhesion and spreading, migration (Trans-well; n=4), gene expression of odontogenic differentiation markers (RT-qPCR; n=6), alkaline phosphatase activity (ALP; n=8) and mineralization nodules (Alizarin red; n=8). Data were submitted to ANOVA and Tukey, Games-Howell or Sidak post-hoc tests (α=5%). The results of live/dead and f-actin were evaluated qualitatively. The incorporation of HC and FN into the scaffolds increased cellular migration and spread, both intensified when CH and FN were combined. The same was seen for the viability of DHPCs. ALPL and DSPP expression, and ALP activity were not affected by HC and FN. COL1A1 was downregulated in all groups compared to the control, while DMP1 was upregulated in the presence of CH. The CH increased the formation of mineralized matrix which was not influenced by FN. In conclusion, the incorporation of 0.4% CH enabled PCL scaffolds with surface topography and properties that enhanced the viability, spread, proliferation and expression of odontoblast phenotype by HDPCs. However, only viability, spread and migration were improved by FN. The PCL+0.4%CH formulation may be a useful strategy for use in dentin tissue engineering.
The aim of this study was to synthesize and characterize polycaprolactone-based nanofiber scaffolds (PCL) incorporated with calcium hydroxide (CH), as well as to evaluate their potential toxicity and bioactivity on human dental pulp cells (HDPCs) when loaded with fibronectin (FN). Initially, different concentrations of CH (0.1%; 0.2% or 0.4%) were incorporated in PCL scaffolds made using electrospinning. Scaffolds without CH served as control. The morphology and composition of the scaffolds were characterized using SEM/EDS (n=4). They were also tested for solubility, pH change of the medium and calcium release (n=8). Then, HDPCs were seeded on the surface of the scaffolds and evaluated regarding their viability (alamarBlue, n=8; Live/Dead assay, n=4) in the time intervals of 1, 7 and 14 days, and adhesion and spreading (Factin, n=4) in 1, 3 and 7days from cell culture. The increase in CH concentration increased the diameter of the nanofibers without interfering with the percentage of interfibrillar spaces, and increased the release of calcium, maintaining the neutrality of the medium (pH 7.0-8.0). In comparison to the control group, a significant increase in cell viability was seen only for the group containing 0.4% CH, in all time intervals. Therefore, in the next experiment, scaffolds containing or not 0.4% CH were loaded with FN (20 µg/mL). HDPCs were seeded on the scaffolds and evaluated for viability, adhesion and spreading, migration (Trans-well; n=4), gene expression of odontogenic differentiation markers (RT-qPCR; n=6), alkaline phosphatase activity (ALP; n=8) and mineralization nodules (Alizarin red; n=8). Data were submitted to ANOVA and Tukey, Games-Howell or Sidak post-hoc tests (α=5%). The results of live/dead and f-actin were evaluated qualitatively. The incorporation of HC and FN into the scaffolds increased cellular migration and spread, both intensified when CH and FN were combined. The same was seen for the viability of DHPCs. ALPL and DSPP expression, and ALP activity were not affected by HC and FN. COL1A1 was downregulated in all groups compared to the control, while DMP1 was upregulated in the presence of CH. The CH increased the formation of mineralized matrix which was not influenced by FN. In conclusion, the incorporation of 0.4% CH enabled PCL scaffolds with surface topography and properties that enhanced the viability, spread, proliferation and expression of odontoblast phenotype by HDPCs. However, only viability, spread and migration were improved by FN. The PCL+0.4%CH formulation may be a useful strategy for use in dentin tissue engineering.
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Palavras-chave
Polpa dentária, Nanofibras, Hidróxido de cálcio, Fibronectinas, Expressão gênica, Dental pulp, Nanofibers, Calcium, Fibronectins, Gene expression