Bioactivity of flavonoids on LPS-challenged human pulp cells and association with fibrous scaffolds for modulating dentinogenesis in a degenerative inflammatory environment

• Grant number: 21/13096-2
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): April 01, 2022
• Effective date (End): July 27, 2026
• Field of knowledge: Health Sciences - Dentistry - Dental Materials
• Principal Investigator: Josimeri Hebling Costa
• Grantee: Igor Paulino Mendes Soares
• Host Institution: Faculdade de Odontologia (FOAr). Universidade Estadual Paulista (UNESP). Campus de Araraquara. Araraquara , SP, Brazil
• Associated scholarship(s): 23/04465-0 - Flavonoid-laden fibrous scaffolds for modulating the inflammatory response and dentinogenesis in vital pulp therapy: physicochemical and biological analyses, BE.EP.DR

Abstract

Reverting the pro-inflammatory into a pro-regenerative environment remains a challenge in Vital Pulp Therapy (VPT). Biomaterials that mimic the native extracellular matrix have been widely explored for endodontic regeneration, and their functionalization with bioactive molecules makes them capable of modulating dentinogenesis in degenerative inflammatory environments, i.e., in adverse pulp conditions. Flavonoids (natural phenolic compounds) have several well-established biological/pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, and progenitor cell differentiation-inducing properties. All these properties make these molecules ideal candidates for application in regenerative therapies. Considering the lack of scientific evidence regarding the effects of flavonoids on human pulp cells, especially under inflammatory conditions, further investigations are needed to use these compounds for managing pulpopathies. The controlled release of a bioactive flavonoid, especially in the first days, from a scaffold capable of stimulating cell differentiation and biomineralization processes may be an interesting alternative to modulate dentinogenesis in human pulp cells under inflammatory conditions, culminating in more favorable prognoses for TPV procedures. Therefore, this study aims to evaluate the bioactivity of flavonoids on human pulp cells (HDPCs) cultured under normal or inflammatory conditions and to associate the most bioactive flavonoid with fibrous scaffolds for modulating dentinogenesis in a degenerative inflammatory environment. Firstly, the cytocompatibility and bioactivity of different concentrations of the flavonoids quercetin, hesperetin, and taxifolin will be evaluated on HDPCs isolated from third molars cultivated in the absence and presence of degenerative inflammatory stimulus with lipopolysaccharide (LPS) from E. coli. Next, functionalization strategies of PCL and PCL/nHA fibrous scaffolds with the previously selected flavonoid will be addressed and evaluated according to physicochemical properties, bioactivity, and modulation of in situ dentinogenesis using human dentin discs and HDPCs. Lastly, PCL and PCL/nHA fibrous scaffolds with controlled release of the flavonoid will be evaluated for bioactive potential in a degenerative inflammatory environment (with LPS) and using in vitro direct pulp capping model with a three-dimensional (3D) culture of HDPCs and human dentin discs. As physicochemical properties, morphology and composition (SEM/EDS and FTIR), surface free energy, flavonoid release profile (UV-Vis), mass change, calcium release (o-cresolphthalein), and medium pH modulation will be evaluated. As biological analyses, it will be performed cell viability tests (alamarBlue/LiveDead), adhesion/spreading (F-actin probe), chemotactic potential (Wound Healing), gene expression of inflammatory mediators, dentinogenic/osteogenic, and integrins (RT-qPCR), modulation of intracellular oxidative stress (Carboxy-H2DCFDA probe), production of nitric oxide (Griess), total protein synthesis (Lowry), alkaline phosphatase activity (ALP; Thymolphthalein), dentin sialophosphoprotein synthesis (DSPP; Immunofluorescence), and mineralized matrix formation (Alizarin Red). 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%. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SOARES, IGOR PAULINO MENDES; ANSELMI, CAROLINE; PIRES, MARIA LUIZA BARUCCI ARAUJO; RIBEIRO, RAFAEL ANTONIO DE OLIVEIRA; LEITE, MARIA LUISA; SOARES, DIANA GABRIELA; COSTA, CARLOS ALBERTO DE SOUZA; HEBLING, JOSIMERI. Chronic exposure to lipopolysaccharides as an in vitro model to simulate the impaired odontogenic potential of dental pulp cells under pulpitis conditions. Journal of Applied Oral Science, v. 31, p. 12-pg., 2023-01-01. (21/13096-2, 19/16473-1)