[Effect of composite graphene-protein hydrogels on neural regeneration after spinal cord injury in rats].

OBJECTIVE: To investigate the role of composite graphene-protein hydrogels in repairing spinal cord injury (SCI) and promoting neural regeneration in rats.

METHODS: A composite graphene-protein hydrogel was prepared using the radical copolymerization method. Its physical properties, including adhesion, were evaluated through shear testing, and cytotoxicity was assessed using the MTT assay. Twenty-four adult female Sprague-Dawley rats were randomly divided into four groups: sham surgery, injury, hydrogel, and hydrogel+graphene groups (6 rats per group). The sham surgery group only exposed the T 10 spinal cord tissue. The other three groups underwent laminectomy combined with spinal cord tissue block resection to establish a T 10 SCI model. Post-modeling, the hydrogel group and the hydrogel+graphene group received implants of the protein hydrogel and composite graphene-protein hydrogels, respectively, at the injury defect site. The injury group received no additional implant treatment. Postoperative survival rates were monitored across groups. Hindlimb motor function recovery was assessed weekly via Basso-Beattie-Bresnahan (BBB) scores during the 12-week postoperative period. At 12 weeks, motor-evoked potentials were measured to assess neurophysiological function. T 10 spinal cord tissue was harvested for histopathological examination via HE staining, followed by immunofluorescence staining for glial fibrillary acidic protein (GFAP), Laminin, and 5-hydroxytryptamine (5-HT) immunofluorescence staining to observe glial scar formation and axonal regeneration at the injury site.

RESULTS: Shear testing and MTT assays demonstrated that the composite graphene-protein hydrogels exhibited excellent underwater adhesion and biocompatibility. All rats in each group survived until the end of the experiment. During 12-week postoperative period, the BBB scores in the hydrogel and hydrogel+graphene groups showed a sustained upward trend over time ( P<0.05). At 12 weeks after operation, BBB scores were significantly higher in the hydrogel and hydrogel+graphene groups than in the injury group, in the hydrogel+graphene group than in the hydrogel group, showing significant differences between groups ( P<0.05). Neurophysiological testing revealed that the motor evoked potential amplitude in the hydrogel+graphene group was significantly higher than that in the injury group and the hydrogel group ( P<0.05), with no significant difference compared to the sham surgery group ( P>0.05). HE staining revealed that the hydrogel+graphene group exhibited spinal cord morphology most similar to the sham surgery group, with significantly restored tissue structural integrity and minimal vacuolation and inflammatory cell infiltration. Quantitative immunofluorescence analysis revealed that the relative fluorescence intensity of GFAP and Laminin in the injury group was significantly higher than that in the other groups ( P<0.05). The relative fluorescence intensity of GFAP and Laminin in the hydrogel+graphene group was significantly lower than that in the hydrogel group ( P<0.05). The relative fluorescence intensity of 5-HT in the injury group was significantly lower than that in the other groups ( P<0.05). The relative fluorescence intensity of 5-HT in the hydrogel+graphene group was significantly higher than that in the hydrogel group ( P<0.05), with no significant difference compared to the sham surgery group ( P>0.05).

CONCLUSION: The composite graphene-protein hydrogels effectively repairs SCI in rats by significantly inhibiting glial scar formation at the injury site, promoting 5-HT-positive axonal regeneration, and improving post-injury neurophysiological function and hindlimb motor recovery. It represents a spinal cord repair material with potential clinical application value.