Abstract: BACKGROUND: Conductive hydrogels can help endogenous nerve regeneration in the spinal cord, inhibit the formation of glial fibrosis and establish a neural bridge network, and promote the recovery of motor function in rats with hemisection injury model, but their ability to reduce inflammatory response after spinal cord injury is limited. Cell-free fat extract has immunomodulatory and tissue regeneration promoting effects. 
OBJECTIVE: To explore the role of supramolecular conductive hydrogel loaded with cell-free fat extract in the repair of spinal cord injury in rats.
METHODS: (1) Cell-free fat extract was extracted from the inguinal adipose tissue of SD rats. Agar/gelatin hydrogel and agar/gelatin/polypyrrole hydrogel were prepared respectively, and the micromorphology, rheology, swelling rate, and conductivity of the hydrogel were characterized. Agar/gelatin/polypyrrole hydrogel loaded with cell-free fat extract was prepared, and the in vitro drug release performance of the hydrogel was tested. (2) A total of 80 SD rats were used to establish a hemisection model of spinal cord injury and randomly divided into four intervention groups: the model group (n=20) received no intervention; the ordinary hydrogel group (n=20), the conductive hydrogel group (n=20), and the drug-loaded conductive hydrogel group (n=20) were implanted with agar/gelatin hydrogel, agar/gelatin/polypyrrole hydrogel, and agar/gelatin/polypyrrole hydrogel loaded with cell-free fat extract at the injured spinal cord, respectively. On day 42 after surgery, the motor function of the right hind limb of the rats was evaluated by BBB score and inclined board test. The morphology of the spinal cord injury tissue of the rats was observed by hematoxylin-eosin staining. The expression levels of CD86, CD206, microtubule-associated protein 2, tubulin βIII, chondroitin sulfate 56, neurofilament protein 200, and myelin basic protein in the spinal cord injury center were detected by immunofluorescence. The degree of axonal myelination was evaluated by fast blue staining and transmission electron microscopy. On day 14 after surgery, the levels of tumor necrosis factor α, interleukin 1β, interleukin 10, and interleukin 6 in the injured spinal cord tissue were detected by ELISA.
RESULTS AND CONCLUSION: (1) Agar/gelatin hydrogel and agar/gelatin/polypyrrole hydrogel presented interconnected porous structures with high porosity and rheological properties. The pore size and swelling rate of agar/gelatin/polypyrrole hydrogel were lower than those of agar/gelatin hydrogel (P < 0.05), and the conductivity was higher than that of agar/gelatin hydrogel (P < 0.05). After soaking in PBS, the cell-free fat extract showed slow and sustained release from the agar/gelatin/polypyrrole hydrogel, with a cumulative release rate of about 31% in 5 days. (2) Compared with the other three groups, the motor function of the hind limbs of rats in the drug-loaded conductive hydrogel group was significantly improved, the area of the cyst in the spinal cord injury area was significantly reduced, the levels of tumor necrosis factor α, interleukin 1β, and interleukin 6 in the spinal cord tissue decreased, and the level of interleukin 10 increased. The expression of CD86 and chondroitin sulfate 56 was significantly reduced, and the expression of CD206, microtubule-associated protein 2, tubulin βIII, neurofilament protein 200, and myelin basic protein was significantly increased, and myelin regeneration was obvious. The results showed that the drug-loaded conductive hydrogel reduced the activity of microglia/macrophages and the expression of proinflammatory factors in the injured spinal cord tissue of rats, promoted the differentiation of endogenous neural stem cells into neurons, and ultimately promoted axon regeneration, myelination of regenerated axons, and recovery of neural function.

Key words: spinal cord injury, conductive polymer, hydrogel, electroactive biomaterial, cell-free fat extract, neural regeneration, engineered spinal cord material