Development of oxidized hyaluronic acid based hydrogels for neuronal tissue engineering: Effects of matrix stiffness on primary neurons

This study introduces oxidized hyaluronic acid (OHA)–gelatin (GEL) hydrogels crosslinked enzymatically with microbial transglutaminase (mTG) for mimicking brain tissue stiffness in neuronal culture systems. Hydrogels were formulated in various OHA/GEL ratios (5/5, 3.75/3.75, and 2.5/2.5% w/v) and crosslinked with 10% or 20% mTG to modulate stiffness, stability, and degradation rate. Compression and rheological analyses revealed that increasing OHA and GEL concentrations raised stiffness (35–900 Pa range) and long-term structural stability. The optimal modulus (~0.5 kPa) supported neuronal survival and dendrite outgrowth of primary E18 rat neurons, while higher stiffness hindered branching. Neurons in softer hydrogels (2.5/2.5) demonstrated enhanced viability and neurite extension compared to stiffer matrices (5/5). Results underscore the critical role of hydrogel elasticity and crosslinking density in directing neuronal behavior, suggesting the OHA-GEL-mTG system as a tunable ECM analog for brain repair.