Synthesis and evaluation of biomaterials associated with extracellular matrix proteins for pulp tissue regeneration

Author(s)

Maria Luísa de Alencar e Silva Leite

• Document type: Doctoral Thesis
• Press: Araraquara. 2021-07-06.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2021-06-30
• Advisor: Carlos Alberto de Souza Costa; Diana Gabriela Soares dos Passos

Abstract

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. (AU)

• FAPESP's process: 17/14210-8 - Synthesis and evaluation of biomaterials associated with extracellular matrix proteins for the pulp tissue regeneration
• Grantee: Maria Luísa de Alencar e Silva Leite
• Support Opportunities: Scholarships in Brazil - Doctorate