Influence of electrospinning parameters on polycaprolactone fiber alignment for the differentiation of embryonic stem cells into neuronal lineage � A systematic study

Electrospun fiber alignment plays a significant role in the proliferation and differentiation of embryonic stem cells (ESCs) into mature neurons. Although previous studies have reported the impact of aligned fibers on increasing neurite outgrowth, there is still a knowledge gap in the influence of synthesis parameters on fiber formation, alignment, and stem cell differentiation. To bridge this gap, we aimed to utilize polycaprolactone (PCL) electrospun fibers as a scaffold model to test synthesis parameters to facilitate the differentiation of ESCs into mature neurons with aligned neurites. This study is divided into two distinct phases. Phase 1 focuses on optimizing the synthesis parameters, including solution viscosity, applied voltage, and the rotational speed of the mandrel to produce aligned fibers. Phase 2 investigates the physicochemical properties of these aligned fibers in comparison to PCL random fibers and two-dimensional (2D) flat membranes. Our results demonstrated that the fiber group exhibited enhanced hydrophilicity, greater resistance to degradation, and superior tensile strength when compared to the 2D flat membranes. Immunocytochemistry analysis of neural markers, including Nestin, Sox2, GFAP, and NEFL, revealed that both aligned and random fiber groups showed higher expression levels of these markers compared to the flat membrane group. Notably, the aligned fiber group displayed NEFL expressions along the axis of the fibers, in contrast to the random fibers, suggesting the importance of fiber alignment in supporting aligned nerve regeneration. The outcomes of phase 1 address the critical factors and key areas that must be considered when developing an electrospun fiber-based scaffold, where phase 2 provides valuable insights into how the geometry and topography of electrospun fibrous scaffolds influence the mechanotransduction of stem cells during differentiation into mature neurons. This systematic analysis of PCL based electrospun fibers, starting from parameter optimisation towards physicochemical characterization, followed by biological validation for nerve regeneration, fills the gap in existing literature. © 2025 Elsevier B.V., All rights reserved.