Poly-L-lysine integration in PVA hydrogels enables stable, bioactive matrices for motor neuron differentiation and neural network formation.

The regeneration of motor neurons remains a major challenge in neural tissue engineering due to the complex cellular architecture and dynamic microenvironment of neural systems. While three-dimensional (3D) hydrogels offer a promising biomimetic platform, the stable integration of bioactive molecules such as laminin is hindered by high cost and impact on structural stability. Poly-L-lysine (PLL), commonly employed in 2D systems to enhance cell adhesion and laminin retention, faces critical limitations in 3D applications, including undefined optimal dosing, cytotoxicity at high concentrations, and detrimental effects on hydrogel properties. In this study, we developed a stable, biocompatible poly (vinyl alcohol) (PVA)-based hydrogel incorporating PLL via either physical entrapment (blended PVA-PLL) or covalent methacrylation (covalent PVA-PLL). Systematic optimization identified a minimal effective PLL concentration (0.002 wt%) that enhanced laminin retention and motor neuron progenitors (MNPs) adhesion without inducing cytotoxicity or compromising hydrogel stability. Notably, covalent PLL-MA incorporation provided no significant advantage over physical adsorption at this low concentration, streamlining hydrogel fabrication. A cluster-based morphometric analysis further confirmed superior cell adhesion, neurite outgrowth, and early network formation on PLL-integrated hydrogels relative to 2D controls. This work established PLL as a simple, scalable, and effective modulator for improving cell adhesion and bioactivity in 3D neural scaffolds, offering translational potential for motor neuron regeneration in spinal cord injury and neurodegenerative disease models.