. Bioactive potential of calcium hidroxyde-containing polycaprolactone nanofiber scaffolds loaded with fibronectin on human pulp cells

Author(s)

Caroline Anselmi de Oliveira

• Document type: Master's Dissertation
• Press: Araraquara. 2021-04-08.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Defense date: 2021-03-08
• Advisor: Josimeri Hebling

Abstract

The aim of this study was to synthesize and characterize polycaprolactone-based nanofiber scaffolds (PCL) incorporated with calcium hydroxide (CH), as well as to evaluate their potential toxicity and bioactivity on human dental pulp cells (HDPCs) when loaded with fibronectin (FN). Initially, different concentrations of CH (0.1%; 0.2% or 0.4%) were incorporated in PCL scaffolds made using electrospinning. Scaffolds without CH served as control. The morphology and composition of the scaffolds were characterized using SEM/EDS (n=4). They were also tested for solubility, pH change of the medium and calcium release (n=8). Then, HDPCs were seeded on the surface of the scaffolds and evaluated regarding their viability (alamarBlue, n=8; Live/Dead assay, n=4) in the time intervals of 1, 7 and 14 days, and adhesion and spreading (Factin, n=4) in 1, 3 and 7days from cell culture. The increase in CH concentration increased the diameter of the nanofibers without interfering with the percentage of interfibrillar spaces, and increased the release of calcium, maintaining the neutrality of the medium (pH 7.0-8.0). In comparison to the control group, a significant increase in cell viability was seen only for the group containing 0.4% CH, in all time intervals. Therefore, in the next experiment, scaffolds containing or not 0.4% CH were loaded with FN (20 µg/mL). HDPCs were seeded on the scaffolds and evaluated for viability, adhesion and spreading, migration (Trans-well; n=4), gene expression of odontogenic differentiation markers (RT-qPCR; n=6), alkaline phosphatase activity (ALP; n=8) and mineralization nodules (Alizarin red; n=8). Data were submitted to ANOVA and Tukey, Games-Howell or Sidak post-hoc tests (α=5%). The results of live/dead and f-actin were evaluated qualitatively. The incorporation of HC and FN into the scaffolds increased cellular migration and spread, both intensified when CH and FN were combined. The same was seen for the viability of DHPCs. ALPL and DSPP expression, and ALP activity were not affected by HC and FN. COL1A1 was downregulated in all groups compared to the control, while DMP1 was upregulated in the presence of CH. The CH increased the formation of mineralized matrix which was not influenced by FN. In conclusion, the incorporation of 0.4% CH enabled PCL scaffolds with surface topography and properties that enhanced the viability, spread, proliferation and expression of odontoblast phenotype by HDPCs. However, only viability, spread and migration were improved by FN. The PCL+0.4%CH formulation may be a useful strategy for use in dentin tissue engineering. (AU)

• FAPESP's process: 19/11192-4 - Bioactive potential of calcium-containing nanofiber scaffolds enriched with fibronectin on human pulp cells
• Grantee: Caroline Anselmi de Oliveira
• Support Opportunities: Scholarships in Brazil - Master