Objective To investigate the effects of chitosan/polyvinyl alcohol (PVA) nerve conduits for repairing radial nerve defect in Macaques. Methods Twelve adult Macaques weighing 3.26-5.35 kg were made the models of radial nerve defect (2 cm in length) and were randomly divided into 3 groups according to nerve grafting, with 4 Macaques in each group. Chitosan/PVA nerve conduit, non-graft, and autografts were implanted in the defects in groups A, B, and C, respectively. And the right radial nerves were used as normal control. At 8 months postoperatively, the general observation,electrophysiological methods, and histological examination were performed. Results At 8 months postoperatively, theregenerated nerve bridged the radial nerve defect in group A, but no obvious adhesion was observed between the tube and the peripheral tissue. The regenerated nerve had not bridged the sciatic nerve defect in group B. The adhesions between the implanted nerve and the peri pheral tissue were significant in group C. Compound muscle action potentials (CMAP) were detected in group A and group C, and no CMAP in group B. Peak ampl itude showed a significantly higher value in normal control than in groups A and C (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). Nerve conduction velocity and latency were better in normal control than in groups A and C, and in group C than in group A, all showing significant differences (Plt; 0.05). The density of myl inated fibers in groups A and C was significantly lower than that in normal control (P lt; 0.05), but there was no significant difference between groups A and C (P gt; 0.05). The diameter and the myel in sheath thickness of the myl inated fibers in normal control were significantly higher than those in groups A and C, and in group C than in group A, all showing significant differences (P lt; 0.05). Conclusion The chitosan/PVA nerve conduits can promote the peripheral nerve regeneration, and may promise alternative to nerve autograft for repairing peripheral nerve defects.
Objective To investigate the promotion effect of neurotropic reinnervation with chemically extracted acellular nerve allograft. Methods The sciatic nerves of 5 healthy adult SD rats, regardless of gender and weighing 270-300 g, were collected to prepare chemically extracted acellular nerve allograft. Eighteen healthy adult Wistar rats, regardless of genderand weighing 300-320 g, were made the model of left sciatic nerve defect (10 mm) and randomly divided into 2 groups: autograft (control group, n=9) and allograft group (experimental group, n=9). The defects were bridged by acellular nerve allograft in experimental group and by autograft by turning over the proximal and distal ends of the nerve in control group. At 3 months after transplantation, dorsal root ganglion (DRG) resection operation was performed in 6 rats of 2 groups. At 3 weeks after operation, the sural nerves were harvested to calculate the misdirection rate of nerve fibers with pathological staining and compute-assisted image analysis. Cholinesterase staining and carbonic anhydrase staining were performed in the sural nerve of 3 rats that did not undergo DRG resection at 3 months. Results The results of chol inesterase staining and carbonic anhydrase staining showed that experimental group had less brown granules and more black granules than control group. After DRG resection, count of myelinated nerve fiber were 4 257 ± 285 in the experimental group and 4 494 ± 310 in the control group significant (P lt; 0.05). The misdirection rate was 2.27% ± 0.28% and 7.65% ± 0.68% in the experimental group and in the control group respectively, showing significant difference (P lt; 0.05). Conclusion Chemically extracted acellular nerve allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinnervation.
Objective To observe the revascularization process of chemically extracted acellular allogeneous nerve graft in repairing rat sciatic nerve defect. Methods Eighty adult male SD rats were selected. The sciatic nerve trunks from ischial tuberosity to the ramus of tibiofibular nerve of 16 SD rats were obtained and were prepared into acellular nerve stents by chemical reagent. Sixty-four SD rats were used to prepare the models of sciatic nerve defect (1.0 cm) and thereafter were randomized into two groups (n=32): experimental group in which acellular allogeneous nerve grafts were adopted and control group in which orthotopic transplantation of autologous nerve grafts were adopted. Postoperatively, the general conditions of all rats were observed, and the gross and ALP staining observation were conducted at 5, 7, 10, 14, 21, 28 days and 2, 3 months, respectively. Results All the incisions were healed by first intention. Trail ing status and toe’s dysfunction in extension happened to the right hindl imb of rats in two groups and were improved 6 weeks after operation. General observation showed that the grafts of two groups connected well to the nerves, with appearances similar to that of normal nerve. ALP staining demonstrated that the experimental group had no ingrowth of microvessel but the control group had ingrowth of microvessel 5 days after operation; the experimental group had ingrowth of microvessel but both groups had no microvessel 7 days after operation; few longitudinal microvessel throughout the grafts were observed in both groups 10, 14 and 21 days after operation; no obvious difference in capillary network of grafts was observed between two groups 28 days after operation; and the microvascular architecture of grafts in both groups were similar to that of normal nerve 2 and 3 months after operation. Conclusion When the chemically extracted allogeneous nerve graft is adopted to repair the peripheral nerve defect, new blood microvessels can grow into grafts timely and effectively.
Objective To investigate the appropriate concentration of tripterygium wilfordii and immunological rejection of rats’ sciatic nerve allograft with the tripterygium wilfordii’s pretreatment so as to explore tripterygium wilfordii’ s suppression. Methods Sixty SD rats (male, weighing 270-290 g), as sciatic nerve allograft acceptor were randomized into5 groups (groups A, B, C, D and E, n=12). To repair the sciatic nerve defect of SD rats, the Wistar rats’ sciatic nerve allografts about 15 mm long were used with 24 hours’ soak of different concentrations of tripterygium wilfordii (group A: 200 mg/L, group B: 400 mg/L, group C: 800 mg/L). The control groups (group D: the fresh sciatic nerve allograft from donors; group E: the fresh sciatic nerve allograft from themselves) were establ ished. At different time points after operation, the morphological examinations (the observation of histology, l ight microscope, electron microscope), the detection of myelin basic protein’s (MBP) content and the analyses of CD4+ and CD8+ T cells on the allografts in the acute phase were performed Results There was no significant difference in morphology among groups A, B and C, the adhesions between allografts and connective tissue were milder than that of group D, and the allografts’ morphous and the inflammatory cell infiltration were better than that of group D. The degeneration of myel in sheath was observed at different levels and there was no significant difference between group B and group E (P gt; 0.05). There was a significant difference in immunological rejection between groups A, B, C and group D (P lt; 0.05). Conclusion Tripterygium wilfordii can effectively suppress the acute immunological rejection in the early stage after operation, and protect the myel in sheath to a certain extent.
Objective To study the outcomes of nerve defect repair with the tissue engineered nerve, which is composed of the complex of SCs, 30% ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeable poly (D, L-lacitic acid) (PDLLA) catheters. Methods SCs were cultured and purified from the sciatic nerves of 1-day-old neonatal SD rats. The 1st passage cells were compounded with bFGF-PLGA sustained release microspheres andECM gel, and then were injected into permeable PDLLA catheters with PLGA microfilaments inside. In this way, the tissueengineered nerve was constructed. Sixty SD rats were included. The model of 15-mm sciatic nerve defects was made, and then the rats were randomly divided into 5 groups, with 12 rats in each. In group A, autograft was adopted. In group B, the blank PDLLA catheters with PBS inside were used. In group C, PDLLA catheters, with PLGA microfilaments and 30% ECM gel inside, were used. In group D, PDLLA catheters, with PLGA microfilaments, SCs and 30% ECM gel inside, were used. In group E, the tissue engineered nerve was appl ied. After the operation, observation was made for general conditions of the rats. The sciatic function index (SFI) analysis was performed at 12, 16, 20 and 24 weeks after the operation, respectively. Eelectrophysiological detection and histological observation were performed at 12 and 24 weeks after the operation, respectively. Results All rats survived to the end of the experiment. At 12 and 16 weeks after the operation, group E was significantly different from group B in SFI (P lt; 0.05). At 20 and 24 weeks after the operation, group E was significantly different from groups B and C in SFI (P lt; 0.05). At 12 weeks after the operation, electrophysiological detection showed nerve conduct velocity (NCV) of group E was bigger than that of groups B and C (P lt; 0.05), and compound ampl itude (AMP) as well as action potential area (AREA） of group E were bigger than those of groups B, C and D (P lt; 0.05). At 24 weeks after the operation, NCV, AMP and AREA of group E were bigger than those of groups B and C (Plt; 0.05). At 12 weeks after the operation, histological observation showed the area of regenerated nerves and the number of myel inated fibers in group E were significantly differents from those in groups A, B and C (Plt; 0.05). The density and diameter of myel inated fibers in group E were smaller than those in group A (Plt; 0.05), but bigger than those in groups B, C and D (P lt; 0.05). At 24 weeks after the operation, the area of regenerative nerves in group E is bigger than those in group B (P lt; 0.05); the number of myel inated fibers in group E was significantly different from those in groups A, B, C (P lt; 0.05); and the density and diameter of myel inated fibers in group E were bigger than those in groups B and C (Plt; 0.05). Conclusion The tissue engineered nerve with the complex of SCs, ECM gel, bFGF-PLGA sustained release microspheres, PLGA microfilaments and permeables PDLLA catheters promote nerve regeneration and has similar effect to autograft in repair of nerve defects.
Objective To make a comparison between the effects of the small intestinal submucosa (SIS) graft and the insideout vein graft on repairing the peripheral nerve defects. Methods SIS was harvested from the fresh jejunum of the quarantined pig by curetting the musoca, the tunica serosa, and the myometrium; then, SIS was sterilized, dried and frozen before use. Thirty-six male SD rats were divided into 3 groups randomly, with 12 rats in each group. Firstly, the 10mm defects in the right sciatic nerves were madein the rats and were respectively repaired with the SIS graft (Group A), the insideout autologous vein graft (Group B), and the autonerve graft (Group C). At 6 weeks and 12 weeks after the operations, the right sciatic nerves were taken out, and the comparative evaluation was made on the repairing effects by the histological examination, the neural electrophysiological examination, the computerized imaging analysis, and the Trueblue retrograde fluorescence trace. Results The histological examination showed that the regenerated nerve fibers were seen across the defects in the three groups at 6 weeks after the operations. The nerve fibers were denser, the formed nerve myelin was more regular, and the fibrous tissue was less in Group A than in Group B; the nerve regeneration was more similar between Group A and Group C. At 12 weeks after the operations, the neural electrophysiological examination showed that the neural conductive rate was significantly lower in Group B than in Groups A and C (Plt;0.05),but no statistically significant difference was found between Group A and GroupC (Pgt;0.05); the component potential wave amplitude was not statistically different between Group A and Group B; however, the amplitude was significantly lower in Groups A and B than in Group C (Plt;0.05). At 6 weeks and 12 weeks after the operations, the computerized imaging analyses showed that the axiscylinder quantity per area and the nerve-tissue percentage were significantly greaterin Group A than in Group B (Plt;0.05); the average diameter of the regenerated axis cylinder, the axiscylinder quantity per area, and the nerve-tissue percentage were significantly lesser in Group B than in Group C (Plt;0.05). At 12 weeks after the operations, the Trueblue retrograde fluorescence trace revealed that the positivelylabeled neurons were found in the lumbar 3-6 dorsal root ganglion sections in the three groups. Conclusion The small intestinal submucosa graft is superior to the autologous inside-out vein graft in repairing the peripheral nerve defects and it is close to the autonerve graft in bridging the peripheral nerve defects. Therefore, the small intestinal submucosa is a promising biological material used to replace the autonerve graft.
Objective To know the possibility of nerveregeneration after artery sleeve anastomosis and end-to-side suture Methods Seventy-five SD rats were divided into 5 groups. First, the distal end ofsevered peroneal nerve was sutured end-to -side with artery sleeve anastomosis withnormal nerve tibial trunk in groups A, B, C and D. Second, the tibial epineurium at the suture site was not removed in group A; the epineurium at the suturesite was removed(windowing) in group B; the distal end of pre-injured peroneal nerve was sutured after 14 days and windowing was done in group C; and the neural growth factor was injected into artery sleeve and windowing was done in group D. While the distal end of severed peroneal nerve was sutured end to side directly with normal nerve tibial trunk and windowing was done in group E. The histological observation was made and the number of nerve fibers was recorded after 4, 8 and 12 weeks of operation.Results After 4 weeks, there existed the regeneration of axons and myeline sheaths in groups C, D, E, and no nerve fiber regeneration was seen in group A. After 8 weeks, the regenerating nerve fibers were significantly more in groups C, D and E than in group B and ingroup E than groups C and D(Plt;0.05). After 12 weeks, the regenerating nervefibers were significantly more in groups C，D and E than in group B(Plt;0.05).Conclusion End-to-side coaptation with artery sleeve anastomosis is a new valuable method in repair of peripheral nerve injuries.
OBJECTIVE: To explore the possibility to bridge peripheral nerve defects by xenogeneic acellular nerve basal lamina scaffolds. METHODS: Thirty SD rats were randomly divided into 5 groups; in each group, the left sciatic nerves were bridged respectively by predegenerated or fresh xenogeneic acellular nerve basal lamina scaffolds, autogenous nerve grafting, fresh xenogeneic nerve grafting or without bridging. Two kinds of acellular nerve basal lamina scaffolds, extracted by 3% Triton X-100 and 4% deoxycholate sodium from either fresh rabbit tibial nerves or predegenerated ones for 2 weeks, were transplanted to bridge 15 mm rat sciatic nerve gaps. Six months after the grafting, the recovery of function was evaluated by gait analysis, pinch test, morphological and morphometric analysis. RESULTS: The sciatic nerve function indexes (SFI) were -30.7% +/- 6.8% in rats treated with xenogeneic acellular nerve, -36.2% +/- 9.7% with xenogeneic predegenerated acellular nerve, and -33.9% +/- 11.3% with autograft respectively (P gt; 0.05). The number of regenerative myelinated axons, diameter of myelinated fibers and thickness of myelin sheath in acellular xenograft were satisfactory when compared with that in autograft. Regenerated microfascicles distributed in the center of degenerated and acellular nerve group. The regenerated nerve fibers had normal morphological and structural characters under transmission electron microscope. The number and diameter of myelinated fibers in degenerated accellular nerve group was similar to that of autograft group (P gt; 0.05). Whereas the thickness of myelin sheath in degenerated accellular nerve group was significantly less than that of autograft group (P lt; 0.05). CONCLUSION: The above results indicate that xenogeneic acellular nerve basal lamina scaffolds extracted by chemical procedure can be successfully used to repair nerve defects without any immunosuppressants.
OBJECTIVE To explore the effect of basic fibroblast growth factor (bFGF) combined with autogenous vein graft conduit on peripheral nerve regeneration. METHODS Fifty four New Zealand rabbits were divided into three groups. The main trunk of sciatic nerve of rabbit in one side was severed and bridged by autogenous vein. 0.2 ml bFGF solution (4,000 U/ml) was intravenously injected to the vein graft conduit as group A, the same amount of saline solution as group B, and no solution injection as group C. Microscopic examination, axon video analysis and nerve conduct velocity were performed at the 10th, 30th, and 100th day after operation. RESULTS The nerve fibers were grown into vein graft conduit in all groups at 30th after operation, they were more and regular in group A than that of group B and C, and the axon regeneration rate in group A was more than that of group B and C. CONCLUSION bFGF combined with autogenous vein graft conduit can markedly promote nerve regeneration.
A 0.6cm segment of right common peroneal nerve was resected in 60 SpragueDawley rats. The nerve defects were bridged by adhering the epineurium with autogenous nerve, vein, skeletal muscle, tendon and silastic tube. According to the kinds of the grafts used, the rats were divided into 5 groups. In 6 and 12 weeks after operation, the effect was assessed by motor nerve conduction velocity, weight of the anterior tibial muscle, number of distal axons and histological examination. It was demonstrated that the result from autogenous nerve graft was superior to other grafts in all aspects and that of the vein graft was better thanthe other three. The characteristics of the nerve regeneration and the process of maturation in different types of the grafts were discussed. The related microenvironment which caused the difference was also discussed.