Thermoresponsive injectable hydrogel and gelatin methacryloyl as delivery platforms for antimicrobials and retinoic acid, respectively, to human apical papilla cells for regenerative endodontic treatment

• Grant number: 24/17247-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: March 01, 2025
• End date: February 29, 2028
• 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

Regenerative Endodontic Procedures (REPs) aim to restore the viability and function of young teeth with pulp necrosis and incomplete rhizogenesis. Although the regenerative capacity of these teeth is promising, the current protocols have limitations, such as the cytotoxicity of the antimicrobials used and the unpredictability of tissue regeneration. Thus, the present project will investigate the performance of hydrogels as a platform to deliver antimicrobials and a signaling molecule to human apical papilla cells for endodontic regeneration (hAPSCs). Four studies were designed. The first study will evaluate the potential of Pluronic F-127 as a vehicle for controlled intracanal medication delivery aiming to reduce the cytotoxicity while preserving the antimicrobial activity of the drugs currently used. The hydrogel will be evaluated for hAPSCs viability (alamarBlue and Live/Dead), change of medium pH, solubility, capacity to inhibit bacteria-formed biofilm (CFU, CLSM, and SEM), and dental enamel color change. In the second study, gelatin methacryloyl (GelMA) scaffolds laden with retinoic acid (AR) will be developed and characterized to release a bioactive dose of 0.1 µM of AR. In the first phase of this study, the GelMA hydrogels will be evaluated for physicochemical properties and morphology (SEM), composition (FTIR), AR release (UV-Vis), swelling, and degradation rate. In the second phase, the effects of the hydrogels on hAPCs will be evaluated by performing the following assays: cell viability (alamarBlue and Live/Dead), collagen synthesis (Sirius red), alkaline phosphatase activity (Thymolphthalein monophosphate) and total protein (Lowry), immunofluorescence (COL1, DSPP, and VEGF), gene expression, and angiogenesis. In the last study, the effects of the best hydrogel found in the previous study will be evaluated on the coculture of hAPCs and human umbilical vein endothelial cells (HUVECS) after the coculture was submitted for different periods to the intracanal medication investigated in the first study. The hydrogels will be applied inside radicular segments obtained from bovine incisors that will be placed on the cell coculture prepared in a collagenous matrix, simulating the apical papilla. The in-situ pulp regeneration will be determined using cell viability and proliferation assays (alamarBlue and Live/Dead), cell adhesion and spread (F-actin), gene expression (RT-qPCR), and synthesis of proteins of interest for endodontic regeneration (immunofluorescence; COL1, DSPP, and VEGF). The number of biological repetitions for each protocol will initially be based on the current literature. Further, the number of biological repetitions will be calculated with the data obtained in the first experimental moment. Data will be subjected to specific statistical tests after checking for data distribution and homogeneity of variances. Inferences will be made considering a 5% level of significance.