Mineral-induced bubbling effect and biomineralization as strategies to create highly porous and bioactive scaffolds for dentin tissue engineering

Author(s)

Camila Correa da Silva Braga de Melo

• Document type: Master's Dissertation
• Press: Bauru.
• Institution: Universidade de São Paulo (USP). Faculdade de Odontologia de Bauru (FOB/SDB)
• Defense date: 2021-12-08

Examining board members

Diana Gabriela Soares dos Passos; Josimeri Hebling Costa; Andréa Candido dos Reis

• Advisor: Diana Gabriela Soares dos Passos

Abstract

The objective of this study was to develop porous chitosan (CH) scaffolds containing different mineral phases, based on a bubbling effect technique and subjected to a biomineralization protocol, to create biomimetic strategies to improve the odontogenic potential of human dental pulp cells (HDPCs). Suspensions containing the mineral phases calcium hydroxide (Ca), nanohydroxyapatite (nHA), and - tricalcium phosphate (TCP) were added to the CH solution at a 1:2 (v/v) ratio. The scaffolds were synthesized by a phase-separation technique and biomineralized by incubation in Simulated Body Fluid (SBF) for 5 days at 37°C and under constant stirring. The samples were characterized as to their morphology, chemical composition, degree of degradability, and calcium-release capacity. The biological behavior of the HDPCs in direct and indirect contact with the scaffolds was analyzed by cell viability (Live/Dead® and Alamar Blue) and odontogenic differentiation (ALP activity and Alizarin Red) assays. The addition of mineral phases to CH led to the appearance of larger pores and higher degrees of porosity, with complexation of calcium and phosphate ions to the chitosan structure. CH-Ca and CH-nHA pores were round, well-distributed, and with an interconnected network, whereas other formulations had more disorganized architecture. Incubation in SBF was responsible for disorganization of the porous structure, except for CH-Ca formulation. A disperse and slightly mineral-like deposition was detected on CH and CH-Ca scaffolds subjected to SBF treatment, whereas large mineral deposits were observed on CHnHA and CH-TCP. The deposition of calcium phosphate by SBF, indicative of hydroxyapatite, was confirmed chemically by FTIR. The mineral-containing scaffolds, incubated or not in SBF, featured a controlled degradation profile, whereas plain CH formulations were stable throughout 28 days of incubation in phosphate-buffered saline solution. The CH-TCP and CH-nHA showed continuous Ca2+ release, whereas CH-Ca released less Ca2+, with a peak at 7 days. A similar trend was detected for SBFtreated counterparts. The cells seeded onto the scaffolds remained viable at all timepoints. The porous and interconnected porous architecture of CH-Ca, CH-nHA, and CH-Ca-SBF allowed cells to infiltrate and spread throughout the scaffold structure, whereas in other formulations cells were dispersed or agglomerated. There was an increase in cell viability in the groups containing mineral phases and biomineralized compared with the control (CH), which was more evident for SBF-treated samples. There was also an increase in odontogenic differentiation and mineralized matrix deposition in these groups compared with the groups without biomineralization, in both experimental models. A significant increase in mineralized matrix deposition (by 8.4 to 18.9 times) was observed for CH-Ca-SBF, CH-nHA-SBF, and CH-TCP-SBF in comparison with plain CH. Therefore, biomineralization of chitosan scaffolds containing different mineral phases was responsible for increasing the capacity of mineralized matrix deposition by pulpal cells, with potential for use in dentin tissue engineering (AU)

• FAPESP's process: 19/06884-4 - Development and analysis of bioactive scaffolds for dentin regeneration
• Grantee: Camila Correa da Silva Braga de Melo
• Support Opportunities: Scholarships in Brazil - Master