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

Due to the presence of hyaluronic acid (HA) in the human body, specifically the brain, HA-based hydrogels are promising candidates for neural tissue engineering applications. Providing the right mechanical and biological properties is essential to mimic the native tissue with the aim of achieving stimulatory effects and promoting regeneration. In this study, HA was oxidized using sodium metaperiodate (NaIO4) to produce oxidized hyaluronic acid (OHA). Hydrogels were then synthesized by crosslinking OHA with gelatin (GEL) through a Schiff base reaction, facilitated by microbial transglutaminase (mTG). The hydrogels were further modified to achieve different mechanical properties, and their long-term stability was investigated by varying the concentrations of OHA, GEL, and mTG. Compression tests as well as swelling/degradation studies confirmed an important influence of the precursor amount on the mechanical characteristics in these hydrogels. Increasing the amount of GEL and OHA at the same time led to a higher effective modulus and beneficial properties regarding long-term stability, and vice versa. Microstructural analyses proved the connection of the respective mechanical properties to the crosslinking density and mesh size. To investigate the applicability of the different hydrogel concentrations as ECM substitutes, three hydrogel compositions were selected and evaluated using E18 primary neurons. The experiments showed that the neuron survival rate as well as their development was optimal at lower ratios of the components with higher crosslinking amount and an intermediate stiffness (modulus) of ∼0.5 kPa. The results thus confirmed the versatility of the OHA-GEL system to be used as matrix in brain tissue engineering. STATEMENT OF SIGNIFICANCE: Neural damage poses a significant medical challenge, with the mechanics of native neural tissue still not fully understood. Hyaluronic acid (HA), a natural component of the brain's extracellular matrix, holds promise for neural tissue engineering. This study developed a hydrogel by oxidizing HA (OHA) and crosslinking it with gelatin (GEL) using a Schiff base reaction and microbial transglutaminase (mTG). By adjusting OHA, GEL, and mTG concentrations, the hydrogels were engineered to mimic brain tissue stiffness and maintain long-term stability. Compression and microstructural analyses linked crosslinking density and mesh size to mechanical properties. Testing with primary neurons demonstrated optimal survival and growth at intermediate stiffness, emphasizing the OHA-GEL system's potential for advancing neural repair.