Bioactivity of retinoic acid and ascorbic acid and loading into thermosensitive synthetic hydrogel for endodontic regeneration

• Grant number: 22/07140-1
• Support Opportunities: Scholarships in Brazil - Master
• Start date: November 01, 2022
• End date: February 29, 2024
• Field of knowledge: Health Sciences - Dentistry - Dental Materials
• Principal Investigator: Josimeri Hebling Costa
• Grantee: Lídia de Oliveira Fernandes
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil

Abstract

Immature teeth, especially those with incomplete root formation, have a high regenerative capacity due to a direct blood supply route and a rich source of progenitor cells from the apical papilla. Thence, regenerative endodontic procedures have been investigated to preserve and stimulate these cells to develop a new dentin-pulp complex. Researchers have been working on strategies that, after disinfection of the root canal, allows the continuity of the root formation process with apical closure, reestablishing homeostasis, innervation, and revascularization, aiming to promote the regeneration of lost tissue. Moreover, the tooth stability and mechanical strength depend on the length of the root and the thickness of the root dentin walls. For this purpose, hydrogels of natural origin (e.g., collagen-based) associated with growth factors have been proposed as scaffolds to facilitate regenerative processes, demonstrating promising results. However, immunogenic and commercial-related questions are raised about the use of natural polymers and growth factors for this purpose. Therefore, it is relevant to investigate bioactive synthetic molecules that, associated with synthetic hydrogels, may signal the migration and shelter progenitor cells of the apical papilla to promote events compatible with endodontic regeneration. Within the reasoned context, this project aims to investigate the bioactive effect of retinoic acid (RA) and ascorbic acid (AA) and formulate a synthetic thermosensitive hydrogel loaded with bioactive dosages of these molecules for endodontic regeneration. To accomplish that, three sequential and interconnected studies were designed. In Study 1, the bioactive dose-response effect of RA and AA will be investigated on human apical papilla cells (hAPCs). Also, the same dosages will be tested in human endothelial cells (HUVECs) to assess the angiogenic potential in vitro. In Study 2, synthetic thermosensitive hydrogels cytocompatible with hAPCs will be formulated. Finally, in Study 3, the molecule presenting the most bioactive effects will be incorporated into the pre-established hydrogel formulation, and the biomaterials will be applied to root segments of bovine incisors, where hAPCs and HUVECs will be co-cultured to evaluate the in-situ pulp regeneration. As biological analyses, it will be performed cell viability/proliferation assays (alamarBlue and Live/Dead), cell adhesion/spreading (actin immunofluorescence stain), active cell migration (transwell/invasion assay), gene expression regulation (RT-qPCR), and protein synthesis (immunofluorescence) of interest markers for endodontic regeneration. The number of replicates (n) will vary according to data obtained on each experimental occasion and adjusted for each experimental protocol. Data distribution (Shapiro-Wilk) and homoscedasticity (Levene) will be evaluated to guide the selection of the statistical methods. The statistical inferences will be made considering a level of significance of 5%.