Experiment Study Of PDLLA/NGF Compound Conduit Bridging Peripheral Nerve Defect

The incidence rate of road traffic accident and various kinds of trauma gradually increased with the development of national economy. It brought about more and more peripheral nerve defect clinically. When the gap distance is short, the defect can be repaired by nerve anastomosis through local dissociation or drag-extension of nerve stumps. While the defect is long, the autologous nerve graft is the first method to choose for most neurological doctors at present. It was commonly considered as the "gold standard" to repair nerve defect according to its good effect. But the use of this method is limited because of its limited donor nerve source and the quality of donor grafts may be insufficient to guide and support axonal regeneration. Furthermore, the nerve autografting involves sacrificing one or more functioning nerves, and the sequelae include loss of sensation, scarring, and possible neuroma formation. To avoid these problems, more attention has been focused on the development of substitutes for conventional nerve autograft. The research about tissue autograft, nerve allograft, synthetic material and nerve tissue engineering attained considerable progress with the development of guided tissue regeneration, but no one technique can achieve same effect as that of nerve autograft.Thus investigating some new ideal nerve conduits to bridge nerve defect and guide the axon regeneration become a hot spot. Nerve regeneration need suitable microenvironment and proper biological factors which can promote nerve regeneration. Guidance channels help direct axons sprouting from the regenerating nerve end provide a conduit for diffusion of neurotropic and neurotrophic factors secreted by the damaged nerve stumps, and minimize infiltration of fibrous tissue. It should have certain characteristics for successful nerve regeneration: biocompatible with the surrounding nerve tissues, causing minimal tissue inflammatory reaction, stimulating axonal regeneration through its entire length; and biodegradable as the nerve regenerates. In addition to its biochemical role, the nerve conduit would also benefit from certain mechanical features: manufactured easily, readily available, flexible, and manipulated easily in the surgical setting. On these grounds, a new PDLLA/NGF compound nerve conduit was manufactured. It was used to bridge 10mm defect of the sciatic nerve of rat and the efficacy to nerve regeneration was evaluated using morphometry combined with neurophisiological technique. Then we analyzed quantitatively the effect of the compound conduit with different dose of NGF on nerve regeneration when bridging the sciatic nerve gap. The biodegradation characters and precaution role to denervated skeletal muscle atrophy of the conduit were also explored. The purpose of this study was to provide a feasible selection for bridging peripheral nerve defect. There are four parts of work as follows.1. Qualitative investigation of bridging peripheral nerve defect with biodegradable PDLLA/NGF compound conduit.Objective: To establish an animal model for repairing sciatic nerve defect with biodegradable PDLLA/NGF compound conduit in rats and to observe the effect of the conduit on improving sciatic nerve regeneration.Methods: 60 SD rats were randomly assigned into four groups: autograft(group A, n=15), PDLLA conduit(group B, n=15), PDLLA with NGF injected into conduit(group C, n=15), and PDLLA/NGF compound conduit(group D, n=15). Each rat underwent right sciatic nerve transection and kept a 10mm nerve defect, and then repaired according to the above four experimental groups. The process of peripheral nerve regeneration was evaluated at 1, 2, 3 months following operation by using gross observation, resumption rates of triceps weight, electrophysiological, histological and axon morphologic assessment of the regenerated nerve in each rat. 5 rats were checked and measured in every groups at 1,2, 3 months after operation respectively.Results: All rats survived well after operation. There were no necrosis and ulceration in hindlimb of the experimental lateral. Group A was the best in resumption rates of triceps weight at every time site(P＜0.05), then was group D, group C, and group B in turn. There were no significant differences between group A and group D(P＞0.05)in motor nerve conduct velocity(MNCV) and their MNCV were faster than that of group C and group B(P＜0.05). Regenerated nerve fibers of the PDLLA/NGF compound conduit group was comparable with that seen in autograft and significantly superior to that of other two groups in histological and axon morphologic index. There were no apparent inflammations around conduits.Conclusions: The biodegradable PDLLA/NGF compound conduit had favorable compatibility with rat tissue and it could effectively promote nerve regeneration after bridging sciatic nerve defect of rats. The effect was as good as that of autograft. PDLLA/NGF compound conduit was a potential ideal conduit. We hope it will be applied clinically with farther study to relieve pains of patients.2. Research on the suitable dose of NGF in PDLLA/NGF compound conduit to nerve regeneration.Objective: To evaluate the dose effect of PDLLA/NGF compound conduit on restoring the rat sciatic nerve defect and the suitable dose of NGF in PDLLA/NGF compound conduit.Methods: 40 male SD rats were randomly assigned into five groups: PDLLA/NGF compound conduit with NGF 100U(group A, n=8), 250U(group B, n=8), 400U(group C, n=8), 500U(group D, n=8), 800U(group E, n=8). Right sciatic nerve was exposed and 6mm nerve stem was excised in each rat. Then the sciatic nerves were bridged with 5 different conduits according to homologous groups and kept an 10mm nerve defect. Functional recovery of the sciatic nerve regeneration was evaluated at 6 months after operation through gross observation, triceps weight, electrophysiology, histology and axon morphologic assessment.Results: There were no necrosis and ulceration in right hindlimb of rat. Nerve regeneration of group C and D was significantly superior to that of group A, B and E in MNCV, recovery rate of triceps weight and axon morphological index(P＜0.05). There were no significant differences between group C and group D in all these indexes(P＞0.05). In MNCV, there were no significant differences among group A, B, E also(P＞0.05). The recovery rate of triceps weight between group A and group B had no statistical difference(P=0.055), but superior to that of group E. Histological and image analysis index indicated that the regenerated nerve fiber of group C and D maturated well. Among the all 5 groups, group C and D were the best in the nerve fiber number, fiber diameter, axon diameter and myelin thickness indexes, then group B, group A and E.Conclusions: There was a dose-dependent relationship between dose of NGF in PDLLA/NGF compound conduit and the quality of nerve regeneration when the dose was low. Suitable dosage of NGF could promote nerve regeneration, but too high dosage may inhibit it. The suitable dose of NGF in PDLLA/NGF compound conduit is about 400-500U.3. Biodegradation of PDLLA/NGF compound conduit in vivo.Objective: To provide basis for PDLLA/NGF compound conduit bridging peripheral nerve defect through evaluating the degradation characters of the pure PDLLA conduit and PDLLA/NGF compound conduit in vivo.Methods: PDLLA and PDLLA/NGF compound conduits were manufactured. Each compound conduit contained NGF 400U. 30 SD male rats were randomly assigned into group A and group B. The right sciatic nerve defect(10mm) models of were performed and repaired with PDLLA conduit(group A) and PDLLA/NGF compound conduit(group B) respectively. Gross observation, weight reduction and ultrastructure of the conduits were evaluated in 1 month, 2 months and 3 months after operation respectively.Results: There were loose connective tissue between the conduit and the surrounding muscle. The two kinds of conduits presented ivory white and gradually biodegraded in rat after operations. The size of micropores inside conduit wall became large and uneven at 2 months postoperatively through scanning electron microscope observation. At 3 months after operation, the boardline of micropore broke to pieces and the construction destroyed. The change is more apparent in the PDLLA/NGF compound conduit than in PDLLA conduit. The weight change of the conduits demonstrated that the weight reduction of compound conduit was faster than that of PDLLA conduit at 1, 2 and 3 months postoperatively and the weight loss rate between them had significant difference(P＜0.05). The biodegradation velocity of conduit endothecium is faster than that of exothecium. The conduit retained intact shape and the regeneration nerve passed through lumina without compression and scarring at 3 months postoperatively.Conclusions: The PDLLA/NGF compound conduit could gradually degraded in vivo and its biodegraded velocity was faster than the PDLLA conduit. The degradation time of PDLLA/NGF compound conduit exceeded 3 months in rats. The conduit could provide enough time for nerve regeneration.4. Precaution efficacy of PDLLA/NGF compound conduit to denervated muscle atrophy.Objective: To determine whether the compound conduit could prevent effectively denervated muscle atrophy and promote functional recovery of target muscle at earlier period after bridging the sciatic nerve defect in rat through observing the triceps muscle of calf.Methods: PDLLA and PDLLA/NGF compound conduits (NGF 400U) were manufactured. 32 male rats were randomly assigned into two groups on average: group A(PDLLA) and group B(PDLLA/NGF conduit). The middle part of right sciatic nerve was excised 6mm and bridged with corresponding conduit. 10mm nerve defect was retained in lumina of conduit. The precaution effect was evaluated according to the gross observation, triceps weight and histological indexes at 5, 10, 15, 20d postoperatively. Myocyte diameter and section area of triceps muscle of calf was quantitatively analyzed and contrasted between two groups at each stage.Results: The right hindlimbs limped and showed thinningz in all rats after the operation. The recovery rate of triceps weight decreased gradually following the observation period lasted. There were no significant difference between group A and B at every stage(P＞0.05). Normal muscle fiber of triceps appeared uniform shape and regulation alignment. But when it came to the 5th day after operation, the muscle fiber showed smaller diameter. General atrophy was apparent at 20th day, various appearances, hclewglnouo vaccine sizes and fibroplasias around the muscle bundle. Image analysis showed that the myocyte diameter and section area of triceps reduced postoperatively, without significant difference between the two groups(P＞0.05).Conclusions: The PDLLA/NGF compound conduit had no significant depressant effect on denervated muscle atrophy at earlier period after bridging the sciatic nerve defect of rat.