Elucidating angiogenesis mechanism of stem cells from human exfoliated deciduous teeth incorporated with chitosan hydrogel via MAPK/ERK pathway

Abstract

Dental stem cell research is advancing regenerative medicine by exploring innovative approaches to tissue repair and regeneration. This study investigates the angiogenic potential of stem cells derived from human exfoliated deciduous teeth (SHEDs) in combination with chitosan hydrogel and examines the role of the MAPK/ERK signalling pathway. SHEDs are recognized for their high proliferation rates and multipotency, while chitosan hydrogel is valued for its biocompatibility and support for cell growth. The study aimed to optimize chitosan hydrogel formulations and their impact on the angiogenic potential of SHEDs. In Phase 1, SHEDs were isolated from deciduous teeth, and three chitosan hydrogel formulations with varying β- glycerophosphate (BGP) concentrations (40 mg, 50 mg, and 60 mg) were analyzed using Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray (EDX). The 50 mg BGP formulation exhibited the most favourable gel-like consistency, with pore diameters of 189.78 ± 59.7 μm, compared to smaller diameters of 133.64 ± 61.61 μm and bigger diameters of 325.63 ± 24.70 μm for the 40 mg and 60 mg BGP formulations, respectively. EDX analysis confirmed the presence of essential elements such as oxygen, sodium, iridium, and phosphorus. In Phase 2, SHEDs were cultured under five experimental conditions: 1) hydrogel only, 2) hydrogel with cells, 3) hydrogel with cells and VEGF, 4) hydrogel with cells and ERK inhibitors, and 5) hydrogel with cells and both VEGF and ERK inhibitors. Proliferation, assessed by BrdU assay, was significantly enhanced in conditions with VEGF and diminished with ERK inhibition. Angiogenesis, measured by branching points and CD31 expression, peaked in the VEGF-treated group and was markedly reduced with ERK inhibition. The combination of VEGF and ERK inhibitors initially promoted angiogenesis but showed a subsequent decline. This study demonstrates that the angiogenic potential of SHEDs is enhanced by the combination of chitosan hydrogel and VEGF through the involvement of the MAPK/ERK signalling pathway. The findings from this study provide crucial information for developing the optimal formulation of hydrogel combined with growth factors, which can improve clinical outcomes in tissue engineering and dental regeneration