Síntese e avaliação de biomateriais associados a proteínas da matriz extracelular para a regeneração do tecido pulpar
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2021-06-30
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Universidade Estadual Paulista (Unesp)
Resumo
A regeneração tecidual guiada tem sido considerada uma estratégia promissora para substituir terapias endodônticas convencionais em casos de dentes com rizogênese incompleta. Portanto, no presente estudo, proteínas da matriz extracelular (fibronectina, laminina e colágeno tipo I) foram avaliadas a fim de selecionar um potente agente de sinalização sobre células da papila apical humana (hAPCs). Em seguida, com o objetivo de desenvolver biomateriais inovadores para regeneração pulpar de dentes com rizogênese incompleta, um scaffold tubular de nanofibras e um hidrogel de preenchimento foram sintetizados e associados à proteína da matriz extracelular com maior potencial bioativo. Assim, foi possível avaliar a influência dos biomateriais sobre a migração, adesão, proliferação e síntese de colágeno pelas hAPCs. No Artigo 1, diferentes concentrações (1, 5 e 10 µg/mL) de fibronectina (FN), laminina (LM) e colágeno tipo I (COL) foram aplicadas no fundo de compartimentos não tratados de placas de 96 compartimentos esterilizadas. Como controles negativo (CN) e positivo (CP), compartimentos não tratados e pré-tratados, respectivamente, foram usados. Após semear as hAPCs nos diferentes substratos, os seguintes parâmetros de bioatividade foram avaliados: adesão, viabilidade, espalhamento, síntese de colágeno total/colágeno tipo I e expressão gênica (ITGA5, ITGAV, COL1A1, COL3A1). Maior potencial de adesão foi observado para todas as concentrações de FN, sendo o efeito dose-dependente. Os melhores resultados de viabilidade e espalhamento celular, bem como síntese de colágeno ocorreram quando 5 e 10 µg/mL de FN foi utilizado. FN na concentração de 10 µg/mL aumentou a expressão de ITGA5, ITGAV e COL1A1 em comparação ao CP. Embora maiores valores de bioatividade tenham sido demostrados pela LM (5 e 10 µg/mL) em comparação ao CN, essas concentrações de LM exibiram menores efeitos bioativos em relação ao CP. As concentrações de COL avaliadas não mostraram bioatividade sobre as células em cultura. Portanto, concluiu-se que FN nas concentrações de 5 e 10 µg/mL exerceu efeitos bioativos mais intensos sobre as hAPCs. Então, no Artigo 2, a técnica de electrospinning foi empregada para obter, por meio do uso de poli-caprolactona (PCL), scaffolds de nanofibras aleatórias (NR) e alinhadas (NA), os quais tiveram suas propriedades biológicas avaliadas. As melhores formulações de NR e NA foram associadas a 0, 5 ou 10 µg/mL de FN sendo a bioatividade determinada. Finalmente, scaffolds tubulares de NR e NA revestidos com FN foram desenvolvidos e seu potencial quimiotático foi analisado usando um modelo in vitro para simular a regeneração pulpar de dentes com rizogênese incompleta. Foi demonstrado que todos os scaffolds testados são citocompatíveis. No entanto, NR e NA à base de 10% de PCL promoveram maior proliferação, adesão e espalhamento de hAPCs. Células poligonais e alongadas foram observadas em NR e NA, respectivamente. Quanto maior a concentração de FN nos scaffolds, maior a migração, viabilidade, proliferação, adesão e espalhamento celular, bem como a síntese de colágeno e expressão gênica (ITGA5, ITGAV, COL1A1, COL3A1). Além disso, os scaffolds tubulares de NA associados com FN (10 µg/mL) mostraram maior potencial quimiotático sobre as APCs. Então, concluiu-se que os scaffolds tubulares de NA revestidos com FN são biomateriais promissores, os quais talvez possam ser usados, no futuro, para promover regeneração pulpar mediada por hAPCs em casos de dentes com rizogênese incompleta. No Artigo 3, hidrogéis com variada proporção de colágeno e gelatina (Col/Gel; v/v) foram inicialmente preparados e avaliados de acordo com os seguintes grupos: Colágeno (controle positivo); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. A viabilidade, adesão/espalhamento das hAPCs semeadas nos hidrogéis foi determinada. Diferentes concentrações de FN (0, 5 ou 10 µg / mL) foram incorporadas à melhor formulação do hidrogel de colágeno/gelatina selecionada. Em seguida, as hAPCs semeadas nos biomateriais foram analisadas quanto a migração, viabilidade, adesão/espalhamento e expressão gênica de ITGA5, ITGAV, COL1A1 e COL3A1. As células do grupo Col/Gel 8:2 exibiram a maior viabilidade e adesão/espalhamento, semelhante ao observado no grupo controle. Ainda, as hDPCs exibiram maior migração, viabilidade, adesão/espalhamento e expressão gênica de marcadores envolvidos na regeneração pulpar quanto maior a concentração de FN incorporada ao hidrogel de colágeno/gelatina. Assim, concluiu-se que um hidrogel de colágeno/gelatina com 10 µg/mL de FN apresentou potente efeito bioativo e quimiotático sobre hAPCs em cultura.
Guided tissue regeneration has been considered a promising strategy to replace conventional endodontic therapy of teeth with incomplete root formation. Therefore, in the present study extracellular matrix proteins (fibronectin, laminin and type I collagen) were assessed to select a potent signaling agent on human apical papilla cells (hAPCs). Next, to develop interesting biomaterials for pulp regeneration of teeth with incomplete root formation, a tubular nanofiber scaffold and a filling hydrogel were synthesized and associated with the most bioactive extracellular matrix protein previously selected. The biomaterials were evaluated concerning their influence on migration, adhesion, proliferation and collagen synthesis by hAPCs. Then, in Paper 1, different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96- well plates. As negative (NC) and positive (PC) controls, non-treated and pre-treated wells, respectively, were used. After seeding the hAPCs on the different substrates, the following bioactivity parameters were assessed: adhesion, viability, spreading, total collagen/type I collagen synthesis and gene expression of ITGA5, ITGAV, COL1A1, and COL3A1. The greatest cells attachment occurred for all concentrations of FN, and in a dose-dependent way. The highest cell viability, spreading and collagen synthesis was observed when 5 and 10 µg/mL of FN were used. FN at 10 µg/mL concentration increased the ITGA5, ITGAV, and COL1A1 expression in comparison with PC. Although the higher bioactivity values shown by LM at 5 and 10 µg/mL in comparison with NC, those concentrations of LM exhibited lower bioactive effects than PC. All concentrations of COL showed no bioactivity on cultured cells. Therefore, it was concluded that 5 and 10 µg/mL FN exerted the most intense bioactive effects on hAPCs. In Paper 2, random (NR) and aligned (NA) nanofiber scaffolds of poly-caprolactone (PCL) were obtained by electrospinning technique and their biological properties assessed. The best formulations of NR and NA were loaded with 0, 5 or 10 µg/mL of FN and their bioactivity evaluated. Finally, FN-loaded NR and NA tubular scaffolds were prepared, and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, hDPCs seeded on 10% PCL-based NR and NA scaffolds presented the highest proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA scaffolds, respectively. The higher the concentration of FN added to the scaffolds, greater the migration, viability, proliferation, adhesion/spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) by hDPCs. In addition, tubular scaffolds with NA loaded with FN (10 µg/mL) showed the highest chemotactic potential on hAPCs. Then, it was concluded that FN-loaded NA scaffold is an interesting biomaterial that may be used in a near future to promote hAPCsmediated pulp regeneration of endodontically compromised teeth with incomplete root formation. In Paper 3, diverse concentrations of collagen and gelatin (Col/Gel; v/v) were used to prepare hydrogels, which were assessed according to the established groups: Collagen (positive control); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. The viability, adhesion and spreading of cells seeded on the hydrogels were evaluated. Concentrations of 0, 5 or 10 µg/mL FN were incorporated into the best formulation of the collagen/gelatin hydrogel selected. Then, the hAPCs seeded on the biomaterials were assessed concerning their migration, viability, adhesion/spreading, and gene expression of ITGA5, ITGAV, COL1A1 and COL3A1. In Col/Gel 8:2 group, cells exhibited greater viability, adhesion and spreading in comparison with control. Higher values of hAPCs migration, viability, adhesion/spreading and gene expression of pulp regeneration markers were found, the higher the concentration was of FN incorporated into the collagen/gelatin hydrogel. Thus, one can conclude that the collagen/gelatin hydrogel with 10 µg/mL of FN had potent bioactive and chemiotactic effects on cultured hAPCs.
Guided tissue regeneration has been considered a promising strategy to replace conventional endodontic therapy of teeth with incomplete root formation. Therefore, in the present study extracellular matrix proteins (fibronectin, laminin and type I collagen) were assessed to select a potent signaling agent on human apical papilla cells (hAPCs). Next, to develop interesting biomaterials for pulp regeneration of teeth with incomplete root formation, a tubular nanofiber scaffold and a filling hydrogel were synthesized and associated with the most bioactive extracellular matrix protein previously selected. The biomaterials were evaluated concerning their influence on migration, adhesion, proliferation and collagen synthesis by hAPCs. Then, in Paper 1, different concentrations (1, 5, and 10 µg/mL) of fibronectin (FN), laminin (LM), and type I collagen (COL) were applied to the bottom of non-treated wells of sterilized 96- well plates. As negative (NC) and positive (PC) controls, non-treated and pre-treated wells, respectively, were used. After seeding the hAPCs on the different substrates, the following bioactivity parameters were assessed: adhesion, viability, spreading, total collagen/type I collagen synthesis and gene expression of ITGA5, ITGAV, COL1A1, and COL3A1. The greatest cells attachment occurred for all concentrations of FN, and in a dose-dependent way. The highest cell viability, spreading and collagen synthesis was observed when 5 and 10 µg/mL of FN were used. FN at 10 µg/mL concentration increased the ITGA5, ITGAV, and COL1A1 expression in comparison with PC. Although the higher bioactivity values shown by LM at 5 and 10 µg/mL in comparison with NC, those concentrations of LM exhibited lower bioactive effects than PC. All concentrations of COL showed no bioactivity on cultured cells. Therefore, it was concluded that 5 and 10 µg/mL FN exerted the most intense bioactive effects on hAPCs. In Paper 2, random (NR) and aligned (NA) nanofiber scaffolds of poly-caprolactone (PCL) were obtained by electrospinning technique and their biological properties assessed. The best formulations of NR and NA were loaded with 0, 5 or 10 µg/mL of FN and their bioactivity evaluated. Finally, FN-loaded NR and NA tubular scaffolds were prepared, and their chemotactic potential was analyzed using an in vitro model to mimic the pulp regeneration of teeth with incomplete root formation. All scaffolds tested were cytocompatible. However, hDPCs seeded on 10% PCL-based NR and NA scaffolds presented the highest proliferation, adhesion and spreading. Polygonal and elongated cells were observed on NR and NA scaffolds, respectively. The higher the concentration of FN added to the scaffolds, greater the migration, viability, proliferation, adhesion/spreading, as well as collagen synthesis and gene expression (ITGA5, ITGAV, COL1A1, COL3A1) by hDPCs. In addition, tubular scaffolds with NA loaded with FN (10 µg/mL) showed the highest chemotactic potential on hAPCs. Then, it was concluded that FN-loaded NA scaffold is an interesting biomaterial that may be used in a near future to promote hAPCsmediated pulp regeneration of endodontically compromised teeth with incomplete root formation. In Paper 3, diverse concentrations of collagen and gelatin (Col/Gel; v/v) were used to prepare hydrogels, which were assessed according to the established groups: Collagen (positive control); Col/Gel 4:6; Col/Gel 6:4; Col/Gel 8:2. The viability, adhesion and spreading of cells seeded on the hydrogels were evaluated. Concentrations of 0, 5 or 10 µg/mL FN were incorporated into the best formulation of the collagen/gelatin hydrogel selected. Then, the hAPCs seeded on the biomaterials were assessed concerning their migration, viability, adhesion/spreading, and gene expression of ITGA5, ITGAV, COL1A1 and COL3A1. In Col/Gel 8:2 group, cells exhibited greater viability, adhesion and spreading in comparison with control. Higher values of hAPCs migration, viability, adhesion/spreading and gene expression of pulp regeneration markers were found, the higher the concentration was of FN incorporated into the collagen/gelatin hydrogel. Thus, one can conclude that the collagen/gelatin hydrogel with 10 µg/mL of FN had potent bioactive and chemiotactic effects on cultured hAPCs.
Descrição
Palavras-chave
Proteínas da matriz extracelular, Nanofibras, Hidrogéis, Extracellular matrix proteins, Nanofibers, Hydrogels