Physicochemical characteristics, effects on enamel and cytotoxicity of experimental bleaching agents formulated with a chitosan hydrogel and nanohydroxyapatite: in vitro and in vivo study

• Grant number: 25/12411-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: July 01, 2026
• End date: October 31, 2028
• Field of knowledge: Health Sciences - Dentistry - Dental Clinics
• Principal Investigator: Vanessa Cavalli Gobbo
• Grantee: Marcos Roberto de Lima Benati
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil

Abstract

The formulation of a novel bleaching agent composed of a chitosan (CHI) hydrogel and nanohydroxyapatite (nHAp) aims to preserve the efficacy of dental bleaching while minimizing deleterious effects on enamel, cytotoxicity, and, consequently, the hypersensitivity associated with the treatment. In addition to functioning as a thickening agent, chitosan forms a bioadhesive matrix on the enamel surface that regulates the diffusion of reactive oxygen species (ROS) into deeper dental structures, thereby contributing to reduced cytotoxicity. Its potential as an ion-release system is further enhanced by the incorporation of nHAp, which promotes enamel remineralization and facilitates structural repair. This strategy represents a more biocompatible alternative to conventional bleaching gels. Furthermore, new measurement instruments have been suggested as more effective and suitable for clinical analysis, such as the digital spectrophotometer Rayplicker Cobra (Borea Dental), which can be applied in in vivo studies. Therefore, beyond the need to clinically evaluate the performance of these experimental hydrogels, it is crucial to validate the effectiveness of this new digital spectrophotometer for clinical color assessment. Accordingly, this study aimed to characterize and evaluate the effects on enamel, cytotoxicity, and clinical efficacy of experimental bleaching agents formulated with CHI or carbomer 940 (CAR) hydrogels containing nHAp and a high concentration of hydrogen peroxide (HP 35%). The project was divided into three studies. In Study 1, sound bovine enamel specimens were assigned to the following groups: negative control (no treatment), commercial HP 35% (positive control), CHI hydrogel + HP 35%, CHI hydrogel + HP 35% + nHAp, CAR hydrogel + HP 35%, and CAR hydrogel + HP 35% + nHAp. Bleaching was performed in three sessions at 7-day intervals. The following parameters were assessed: (1) characterization of the experimental hydrogels [retention capacity of the gel on the dental surface, pH, viscosity, transmission electron microscopy (TEM), preliminary and accelerated stability, and degradation of bleaching agents]; (2) color evaluation [spectrophotometry: L*, a*, b* parameters, color change (¿E¿¿), and whiteness index (WID)]; (3) mineral content [surface microhardness (KHN) and Fourier-transform infrared spectroscopy (FTIR)]; and (4) surface morphology [surface roughness (Ra) and scanning electron microscopy (SEM)]. Study 2 evaluated the biocompatibility of the experimental bleaching agents in human dental pulp cells (HDPCs), analyzing cellular metabolism via MTT assay, HP concentration in the cell extract, and cell morphology following exposure to the same hydrogels tested in Study 1. Study 3 consisted of a randomized, blinded clinical trial designed according to CONSORT 2010 guidelines. Patients were selected based on predefined inclusion and exclusion criteria and received one of the following treatments: commercial HP 35% gel, CHI hydrogel + HP 35%, or CAR hydrogel + HP 35%, with or without nHAp. Assessments included tooth color change, dental sensitivity using a visual analog scale (VAS) and Psychosocial Impact of Dental Aesthetics Questionnaire (PIDAQ). Data were analyzed using appropriate statistical methods, adopting a significance level of 5% across all studies. (AU)