The influence of hydrogel stiffness on axonal regeneration after spinal cord injury

Abstract

The core challenge in spinal cord injury(SCI) treatment is promoting axonal regeneration and forming new neural connections in damaged areas. However, mature CNS neurons have limited regenerative capacity, causing long-term dysfunction. Axonal regeneration involves elongating axons guided by growth cones, which sense and respond to external mechanical signals, integrating them into cytoskeleton reconstruction. After injury, growth cones experience altered mechanical forces due to changes in ECM stiffness. However, systematic studies on matrix stiffness's impact on axonal regeneration post-SCI remain insufficient. This study investigates the influence of hydrogel stiffness on axonal regeneration following SCI. Using gelatin methacryloyl (GelMA) hydrogels with varying stiffness levels, we cultured dorsal root ganglia (DRG) neurons in vitro and applied the hydrogels to a complete transection SCI mouse model. Results demonstrated that higher stiffness GelMA (15% w/v) significantly enhanced axonal extension and sensory functional recovery compared to lower stiffness (7.5% w/v). The study highlights the critical role of ECM stiffness in regulating axonal regeneration and suggests that optimizing hydrogel stiffness can promote neural regeneration and functional recovery after SCI. These findings provide valuable insights for developing therapeutic strategies in SCI treatment.

Fig 1. Material characterization of hydrogels.

A. Mira3-TESCAN SEM images showed the microstructures of the hydrogel. Scale bar: 100 μm. Magnification: 100X. Scale bar, 50 μm (enlarged view). B. The Young’s modulus (stiffness) of GelMA hydrogel with different concentrations, **p < 0.01. C. Quantitative schematic of dorsal root ganglion (DRG) axon length and number of branches.

Fig 2. In vivo characterization of hydrogels.

A. Representative images of HE- staining of the heart, liver, spleen, lung, and kidney tissue showed no differences between the Sham, 7.5% Hydrogel and 15% hydrogel treated mice groups. Scale bar, 100μm. Magnification: 40X.

Fig 3. In vitro evaluation of axonal growth promotion by GelMA of different concentrations in DRG.

A. Representative images of length of the axons and branches of the axons treated with 7.5% Hydrogel and 15% Hydrogel in DRG. Magnification: 40X. Scale bar 200 μm. B. Quantification of branches of the axons are shown in (A), n = 6. C. Quantification results for length of the axons are shown in (A), n = 6. The data was shown as a mean ± SD, * P < 0.05, ** P < 0.01. (One-way ANOVA between multiple groups plus Tukey’s hoc test).

Fig 4. Motor function assessment treated with hydrogels of different concentrations after SCI.

A. Schematic diagram of the modeling process for the complete transection model of SCI. B. BMS scores at different time points after SCI in the treatment groups. n = 6. The data was shown as a mean ± SD, & P < 0.05 15% Hydrogel vs. 7.5% Hydrogel, # P < 0.05 7.5% Hydrogel vs. Control, ^^ P < 0.01 15% Hydrogel vs. Control, && P < 0.01, ## P < 0.01. (Two-way ANOVA plus Turkey’S hoc test between multiple groups).

Fig 5. In vivo evaluation of axonal regeneration promotion by hydrogels of different concentrations after SCI.

A. Representative images of NF (green-Alexa Fluor® 488) immunostained neurons and GFAP (red-Alexa Fluor® 594) astrocytes in mice treated with Control, 7.5% Hydrogel and 15% hydrogel after spinal cord injury. Magnification: 10X. Scale bar, 150 μm, Scale bar, 30 μm (enlarge view). B. Quantification of labeled neurons in different groups. Each group n = 6. C. Curves showing the continuous distribution of NF positive neuronal fiber area in (A).The data was shown as a mean ± SD, * P < 0.05, ** P < 0.01. (One-way ANOVA between multiple groups plus Tukey’s hoc test).

Fig 6. Histological evaluation treated with hydrogels of different concentrations after SCI.

A. H&E staining of spine in each group at 56 days post-SCI. Scale bar, 150 μm. B. H&E staining of bladder sections from each group at 56 days post-SCI. The black line indicates the detrusor muscle. Magnification: 10X. Scale bar, 100 μm. C. Quantification results for cavity area are shown in (A), n = 6. D. Quantification of detrusor muscle thickness in (B), n = 6. The data was shown as a mean ± SD, * P < 0.05, ** P < 0.01. (One-way ANOVA between multiple groups plus Tukey’s hoc test).