Multilayer gradient chitosan fiber scaffolds for skin tissue regeneration with enhanced mechanical strength and cellular infiltration

Abstract

Using the solution blow spinning technique, a trilayer structure scaffold was developed for skin tissue engineering. The scaffold, composed of chitosan/polyethylene oxide (CHI/PEO) fibers, mimicking the skin's natural structure, featuring a fiber diameter gradient ranging from 300 to 1200 nm and controlled porosity. Neutralization with potassium carbonate solution stabilized the chitosan-based fibers and enhanced their mechanical properties. The subsequent removal of PEO during neutralization created a porosity gradient (80–50 %) across the scaffold layers, resulting in interconnected pores essential for cell infiltration and nutrient transport. The neutralized scaffolds enhanced mechanical performance with an ultimate tensile strength of 29.4 ± 4.8 MPa and Young's modulus of 6.5 ± 1.1 MPa. The toughness was found to be 2.3 ± 1.0 MJ/m³.

The gradient design closely mimics aspects of the skin's extracellular matrix. It substantially enhances the scaffold's mechanical integrity—doubling tensile strength, Young's modulus, and toughness compared to the as-spun structure—while preserving the essential flexibility required for dynamic wound environments.

In vitro evaluations using co-cultures of normal human dermal fibroblasts (NHDF) and human epidermal keratinocytes (HEKa) demonstrated suitable cell viability, adhesion, and proliferation on the scaffold. Moreover, confocal imaging confirmed HEKa cellular infiltration throughout the more porous scaffold parts and dense NHDF layers on the less porous fiber layer, effectively mimicking the spatial organization of native skin layers.

Our results underscore the significant potential of this gradient chitosan fiber scaffold as a scalable and versatile platform for skin tissue engineering. This innovative scaffold offers a promising new strategy for accelerating wound healing and achieving durable tissue regeneration by seamlessly integrating superior mechanical performance with enhanced cellular dynamics.