Scaffolds nanofibrilares de policaprolactona/nano-hidroxiapatita para regeneração do complexo dentino-pulpar: síntese, caracterização e avaliação em células pulpares humanas
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Data
2021-06-08
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Universidade Estadual Paulista (Unesp)
Resumo
Visando uma terapia citocompatível e bioativa para aplicação sobre o tecido pulpar exposto, neste estudo foram desenvolvidos e caracterizados scaffolds nanofibrilares de policaprolactona (PCL) incorporados com nano-hidroxiapatita (nHA) e foi avaliada a resposta de células da polpa dental humana (HDPCs) semeadas sobre esses scaffolds. Soluções de PCL (10% m/v em clorofórmio/dimetilformamida) foram incorporadas ou não com nHA (0,5; 1,0; ou 2,0% m/v) e eletrofiadas em scaffolds. Os scaffolds foram caracterizados quanto à morfologia (MEV), composição (EDS), solubilidade (alteração de massa), liberação de cálcio/fosfato (espectrofotometria) e modulação do pH do meio. HDPCs foram cultivadas na superfície dos scaffolds e avaliadas quanto à viabilidade (Live/Dead e alamarBlue) e adesão/espalhamento celular (F-actina) ao longo do tempo. A expressão dos genes COL1A1, ALPL, DSPP e DMP1 (RT-qPCR), dosagem de proteínas totais (PT; Lowry) e a atividade de fosfatase alcalina (ALP; timolftaleína) foram investigadas aos 14 e 21 dias. A formação de matriz mineralizada (Alizarin Red) foi avaliada em 21 dias. Os dados foram analisados com ANOVA a um ou dois fatores, complementada por Tukey, Games-Howell ou Sidak (α=0,05). Todas as formulações originaram fibras variando entre 600 a 900 nm de diâmetro, com disposição aleatória. A identificação de cálcio e fósforo nas fibras foi concentração-dependente, confirmando a incorporação de nHA ao polímero. Concentrações mais altas de nHA tornaram as superfícies das nanofibras irregulares. PCL+0,5% nHA ampliou o diâmetro da nanofibra, enquanto PCL+2%nHA aumentou os espaços interfibrilares. PCL+1%nHA ou PCL+2%nHA promoveram significativa liberação de cálcio e fosfato, porém a modulação de pH não excedeu o valor 8,0. A viabilidade de HDPCs não foi afetada pela adição de nHA, enquanto adesão/espalhamento celular foram favorecidas. A expressão de DSPP e DMP1 foi regulada positivamente em 14 dias, e COL1A1, ALPL e DMP1 em 21 dias pelas formulações PCL+1%nHA e PCL+2%nHA. Maior quantidade de proteínas foi identificada em scaffolds contendo nHA no período mais tardio e a presença de nHA aumentou a atividade de ALP em 14 e 21 dias. A formação de matriz mineralizada foi dependente da concentração de nHA, cerca de 9× maior para PCL+2%nHA em comparação ao controle. Em conclusão, scaffolds nanofibrilares de PCL incorporados com nHA foram citocompatíveis, estimularam a adesão, espalhamento, proliferação e o potencial odontogênico de HDPCs. A formulação PCL+2%nHA é uma estratégia bioativa de engenharia tecidual para terapia pulpar vital.
Targeting a cytocompatible and bioactive therapy for the application on the exposed pulp tissue, this study developed and characterized polycaprolactone (PCL) nanofibrous scaffolds incorporated with nano-hydroxyapatite (nHA) and evaluated the response of human dental pulp cells (HDPCs) seeded on their surface. PCL-based solutions (10% w/v in chloroform/dimethylformamide) were incorporated or not with nHA (0.5; 1.0; or 2.0 % w/v) and electrospun into nanofibrous scaffolds. The scaffolds were characterized for morphology (SEM), composition (EDS), solubility (mass change), release of calcium/phosphate (spectrophotometry), and pH medium modulation. HDPCs were cultured on the surface of the scaffolds and evaluated for cell viability (Live/Dead and alamarBlue) and adhesion/spreading (F-actin) over time. The expression of COL1A1, ALPL, DSPP, and DMP1 genes (RT-qPCR), total protein synthesis (TP; Lowry), and alkaline phosphatase activity (ALP; thymolphthalein assay) were investigated at 14 and 21 days. The formation of a mineralized matrix (Alizarin Red) was assessed at 21 days. Data were analyzed with one- or two-way ANOVA complemented with Tukey, Games-Howell, or Sidak post-hocs (α=0.05). All formulations generated fibers ranging from 600 to 900 nm in diameter, with random arrangement. The incorporation of nHA into the nanofibers was dose-dependent. Higher nHA concentrations roughened nanofibers surfaces. PCL+0.5%nHA enlarged fiber diameter whereas PCL+2%nHA increased interfibrillar spaces. PCL+1%nHA or PCL+2%nHA promoted greater release of calcium and phosphate, but the medium pH was maintained below 8.0. HDPCs viability was not affected by the addition of nHA, while adhesion/spreading were favored. The expression of DSPP and DMP1 was upregulated in 14 days, and COL1A1, ALPL, and DMP1 in 21 days by the PCL+1%nHA and PCL+2%nHA formulations. The incorporation of nHA promoted higher protein synthesis and increased ALP activity. The formation of a mineralized matrix was concentration-dependent, about 9× higher for the PCL+2%nHA in comparison to the control. In conclusion, nanofibrous PCL scaffolds incorporated with nHA were cytocompatible, stimulated adhesion, spreading, proliferation, and the odontogenic potential of HDPCs. The PCL+2%nHA formulation is a bioactive tissue engineering strategy for vital pulp therapy.
Targeting a cytocompatible and bioactive therapy for the application on the exposed pulp tissue, this study developed and characterized polycaprolactone (PCL) nanofibrous scaffolds incorporated with nano-hydroxyapatite (nHA) and evaluated the response of human dental pulp cells (HDPCs) seeded on their surface. PCL-based solutions (10% w/v in chloroform/dimethylformamide) were incorporated or not with nHA (0.5; 1.0; or 2.0 % w/v) and electrospun into nanofibrous scaffolds. The scaffolds were characterized for morphology (SEM), composition (EDS), solubility (mass change), release of calcium/phosphate (spectrophotometry), and pH medium modulation. HDPCs were cultured on the surface of the scaffolds and evaluated for cell viability (Live/Dead and alamarBlue) and adhesion/spreading (F-actin) over time. The expression of COL1A1, ALPL, DSPP, and DMP1 genes (RT-qPCR), total protein synthesis (TP; Lowry), and alkaline phosphatase activity (ALP; thymolphthalein assay) were investigated at 14 and 21 days. The formation of a mineralized matrix (Alizarin Red) was assessed at 21 days. Data were analyzed with one- or two-way ANOVA complemented with Tukey, Games-Howell, or Sidak post-hocs (α=0.05). All formulations generated fibers ranging from 600 to 900 nm in diameter, with random arrangement. The incorporation of nHA into the nanofibers was dose-dependent. Higher nHA concentrations roughened nanofibers surfaces. PCL+0.5%nHA enlarged fiber diameter whereas PCL+2%nHA increased interfibrillar spaces. PCL+1%nHA or PCL+2%nHA promoted greater release of calcium and phosphate, but the medium pH was maintained below 8.0. HDPCs viability was not affected by the addition of nHA, while adhesion/spreading were favored. The expression of DSPP and DMP1 was upregulated in 14 days, and COL1A1, ALPL, and DMP1 in 21 days by the PCL+1%nHA and PCL+2%nHA formulations. The incorporation of nHA promoted higher protein synthesis and increased ALP activity. The formation of a mineralized matrix was concentration-dependent, about 9× higher for the PCL+2%nHA in comparison to the control. In conclusion, nanofibrous PCL scaffolds incorporated with nHA were cytocompatible, stimulated adhesion, spreading, proliferation, and the odontogenic potential of HDPCs. The PCL+2%nHA formulation is a bioactive tissue engineering strategy for vital pulp therapy.
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Palavras-chave
Nanofibras, Engenharia tecidual, Biomineralização, Capeamento da polpa dentária, Expressão gênica, Nanofibers, Tissue engineering, Biomineralization, Dental pulp capping, Gene expression