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Description
Introduction
External forces, notably in traumatic brain injury, can cause tissue-level damage [1, 2]. Meanwhile, mechanical cues on a smaller scale are pivotal in shaping the development, behavior, and function of individual neural cells [3]. Advancing our understanding of injury and disease mechanisms in the central nervous system depends on investigating how cellular forces and tissue mechanics affect both healthy and pathological states [4]. Due to the presence of hyaluronic acid (HA) in the human body and the extracellular matrix (ECM) mimicking properties of hydrogels, HA based hydrogels are a promising candidate for tissue engineering applications, in particular approaches designed to create scaffolds for repairing or regenerating neurological defects and diseases [5]. In this study, we developed stable oxidized hyaluronic acid (OHA) based hydrogels with tunable mechanical properties with ECM mimicking character for neuronal cells.
Experiment and Methods
HA was oxidized using sodium metaperiodate (NaIO₄) and lyophilized. Hydrogels were formed by combining various OHA and gelatin (GEL) ratios, with microbial transglutaminase (mTG) as an enzymatic crosslinker. Crosslinking occurred at room temperature for 30 minutes. Mechanical properties were evaluated via parallel plate compression testing. Swelling, degradation, and mechanical stability were monitored over 7 or 28 days.
Three hydrogel compositions were chosen for biological testing. Cell encapsulation and viability were first assessed using WST-8 assays and live/dead imaging with NIH-3T3 cells. Primary E18 rat cortical neurons were then encapsulated to evaluate suitability for neuronal tissue engineering. Neuronal development was assessed via immunostaining, confocal microscopy, and Scholl analysis.
Results and Discussion
Mechanical testing showed that higher OHA and GEL concentrations increased stiffness and long-term stability, while lower concentrations produced softer gels. mTG levels significantly influenced stiffness; higher concentrations improved mechanical integrity, particularly with low OHA/GEL ratios. NIH-3T3 cells showed high viability in all formulations.
Neuronal experiments revealed that hydrogels with intermediate stiffness (~0.5 kPa) supported optimal neuron survival and outgrowth. These conditions, achieved with lower OHA/GEL ratios and high crosslinking, promoted network formation and neurite extension.
Conclusion
OHA/GEL hydrogels with diverse material properties were synthesized. The addition of mTG enhanced long-term stability, stiffness, and temperature stability. These hydrogels could be customized to mimic neuronal extracellular matrix (ECM) properties, making them suitable for 3D neuronal cell cultivation.
References:
[1] J. D. Lai et al., “A model of traumatic brain injury using human ipsc-derived cortical
brain organoids,” bioRxiv, 2020.
[2] S. Budday, “Exploring human brain mechanics by combining experiments, modeling,
and simulation,” Brain Multiphysics, vol. 5, 2023.
[3] K. Franze, “Integrating chemistry and mechanics: The forces driving axon growth,”
Annual Review of Cell and Developmental Biology, vol. 36, pp. 61–83, 2020.
[4] S. Budday et al., “Towards microstructure-informed material models for human
brain tissue,” Acta Biomaterialia, vol. 104, pp. 53–65, 2020.
[5] S. Kuth et. al., “Oxidized hyaluronic acid-gelatin based hydrogels for tissue engineering and soft tissue mimicking” Tissue Engineering Part C: Methods, vol. 28, pp. 301-313, 2022
Acknowledgment: We acknowledge the support by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) project number 460333672 - CRC 1540 Exploring Brain Mechanics (subproject X03 and C02)
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