Femtosecond Laser Assisted Fabrication of Functionalized Micro/Nanostructured Chitosan Patterns for ECM Free Cellular Adhesion and Alignment of Human Mesenchymal Stem Cells

The extracellular matrix (ECM) and tissues comprises of micro/nanoscale topographical features that critically influence cell behavior. Mimicking these features in vitro has emerged as a promising strategy in biomaterials engineering, offering the potential to guide cellular responses. However, conventional patterning techniques use multistep, mask-based fabrication and synthetic polymers that lack biocompatibility, and require additional surface modifications for cell culture applications. This emphasizes the need for a functionalized micro/nanostructured platform that better recapitulate the native microenvironment. In this study, high-resolution, biocompatible, functionalized substrates with anisotropic/isotropic patterns are fabricated on chitosan using single-step femtosecond (fs) laser lithography and physiochemically characterized. The anisotropic patterned substrates are checked for cytocompatibility and supported the direct adhesion and growth of human mesenchymal stem cells (hMSCs), eliminating the need for any surface modifications. Surface characterization of these micro/nanostructured patterns confirmed the presence of functional carbonaceous surface groups, suitable for ECM-free cell attachment. hMSCs cultured on these substrates showed directional growth and alignment along the grooves, with notable changes in focal adhesion orientation, actin remodelling and nuclear elongation indicating effective mechanotransduction. This study demonstrates the potential of fs laser-fabricated biopolymeric patterns for controlled cell adhesion and alignment, offering promising applications in mechanobiology, tissue engineering, and regenerative medicine.