Fabricating a biomimetic artificial nerve with polylactic acid glycolic acid copolymer composite ordered multi tunnel collagen scaffold to repair the sciatic nerve defects in rats

Abstract

Objective To investigate the potential of polylactic acid glycolic acid copolymer(PLGA) composite ordered multi tunnel collagen scaffold in fabricating a biomimetic artificial nerve graft to repair the sciatic nerve defects in rats. Methods The ordered multi tunnel collagen scaffold was prepared by vacuum freeze-drying and directional drawing method to simulate the epineurium; the outer conduit was prepared by PLGA to simulate the epineurium; and then, the ordered multi tunnel collagen scaffolds were loaded in the PLGA conduit (5∶1) under a stereomicroscope to develop a novel biomimetic artificial nerve. Sixty-four rats were randomly divided into four groups: artificial nerve group, PLGA group, peripheral nerve group, and non-graft control group (n=16); rats in the artificial nerve group, PLGA group, and peripheral nerve group were repaired with artificial nerve graft, hollow PLGA conduit and allogeneic sciatic nerve to bridge the sciatic nerve defect, while the sciatic nerve with the gap in rats of the control group was without any grafting. After 11 weeks of operation, the hind limbs of rats in each group were detected by behavioral test, electrophysiological examination and Fluoro-Gold retrograde tracing method. The changes of muscle tissues (gastrocnemius) were observed by hematoxylin staining and TMR-α-BTX staining, and the regenerated axons were observed by immunohistochemical staining with NF200 and the regenerative spinal anterior horn motor neurons were observed by Nissl fluorescence staining 12 weeks after operation. Results After 11 weeks of operation, the recoveries of the motor functions (the distance between the injured hindlimb and forelimb, the rotation angle of the injured foot) in the peripheral nerve group, artificial nerve group, PLGA group and control group were significantly deteriorated in turn, and the differences were statistically significant (P<0.05). Electrophysiological examination showed that the recovery effect of peripheral nerve group was the best in both latency and amplitude of the compound muscle action potential, followed by artificial nerve group. The latency of PLGA group was the longest and the amplitude of compound action potential was the smallest; significant differences were noted between each two groups (P<0.05). At 12 weeks after operation, the wet weight ratio of muscle fibers, area of muscle fibers and neuromuscular junction area were significantly different between each two groups (P<0.05); the degree of gastrocnemius atrophy in the artificial nerve group was significantly improved than that in the PLGA group, but not yet reached the level of peripheral nerve group. NF200 immunohistochemical staining showed that a large number of NF200-positive axons were seen in the grafts of the artificial nerve group, but the number was slightly smaller than that of the peripheral nerve group; the number of regenerated axons in the PLGA group was the smaller and mainly distributed near the proximal side. In the PLGA group, only (19.33±6.73)% regenerated spinal anterior horn motor neurons were labeled with Fluoro-Gold, while the positive rates of Fluoro-Gold in the artificial nerve group and peripheral nerve group were (42.67±7.45)% and (50.13±4.33)%; the differences between each two groups were statistically significant (P<0.05). Conclusion The biomimetic artificial nerve made of PLGA conduit and ordered multi tunnel collagen scaffold can efficiently reconstruct the defected peripheral nerve with guiding axonal regeneration and promoting functional restoration in rats; however, its effect is poor than peripheral nerve grafting. Key words: Peripheral nerve injury; Artificial nerve; Polylactic acid glycolic acid copolymer; Collagen scaffold; Nerve regeneration