The Fundamental Research And Clinical Application Of A Novel Bioactive Artificial Nerve In Repairing Peripheral Nerve Defects

Ⅰ. BACKGROUNDThe treatment of peripheral nerve injury remains a clinical challenge, especially in some large-diameter, critical peripheral nerve defect or Multiple nerve injury, The regeneration is very difficult to rebuild the normal structure, also it is hard to establish normal connection with the distal nerve. Currently autologous nerve grafting is still the "gold standard" in bridging nerve defects in the clinic setting. However, harvesting a nerve requires extra operation, thus resulting in denervation distal to the donor site, and the availability of donor nerves are limited, Furthermore, scarring, neuropathic pain, neuroma formation and other symptoms have all been concerned. Therefore, promising strategies and alternatives to autologus nerve grafts need to be developed for clinical use. Synthetic substances and biogenic conduits like Neurotube (polyglycolic acid, PGA, Synovis), Neurolac (poly-DL-lactide-caprolactone, PLCL Poly-ganics BV) and NeuraGen (collagen type I, Integra NeuroSciences) have been verified experimentally and clinically as an alternative for repairing both sensory and motor peripheral nerve defects. However, with the increasing length of the nerve defect, the clinical outcomes of functional reinnervation are not satisfactory, perhaps because of lack of neurotrophic factors and viable cells, which normally help to remove myelin and cell debris and remodel the nerve regeneration environment of the graft, hindered the regenerating progress. Ideally, The tissue engineering artificial nerve should be biocompatible, timely biodegradable, with low toxicity and low cost, not only last long enough for axonal regrowth and tissue repair but also provide the growth factors essential for axonal regrowth. Numerous experiments indicated that after nerve injury, cell adhesion molecules and neurotrophic factors played very important roles in nerve regeneration and functional recovery. The current research is focusing on fabricating neurotrophic factors sustained-release nerve conduit and integrating with seed cells to promote the structural and functional recovery of injured nerve. Ⅱ. OBJECTIVE1) In the present study, we isolated and cultured rat Bone marrow derived mesenchymal stem cells(BMSCs) and treated with a mixture of glial growth factors(basic fibroblast growth factor, bFGF; epidermal growth factor, EGF) to differentiated them into NC-like BMSCs(BMSC-NCs), then took them as the seed cells combining with self-manufactured PRGD/PDLLA/β-TCP/NGF(PNGF) composite for peripheral nerve tissue engineering construction, then evaluated the nerve regeneration and functional recovery by in vivo animal experiments.2) Evaluate the efficacy and safety in treating peripheral nerve injury of PRGD/PDLLA/β-TCP composite nerve conduit by clinical application, and explore the effect, safety and indication in promoting peripheral nerve regeneration through comparing to similar products.Ⅲ. METHODS1. BMSCs were isolated and purificated from the bone marrow of Wistar rats by using the differential adherence method. Then used bFGF and EGF to induce the BMSCs differentiated into neuron like cells. The morphological change was observed by using inverted phase contrast microscope, the neuronal specific markers Neuron specific enolase(NSE), neurofilament protein(NF-M) and synaptic vesicle specific protein Synaptophysin protein(SYN) were detected by immunohistochemistry technique.2.40isogenic adult male Wister rats were randomly divided into4groups (n=10each), The left sciatic nerve was exposed to create a12-mm gap in all rats. Three groups received nerve-bridging with PNGF conduits filled with:(A) PBS;(B) BMSCs;(C) BMSC-NCs; and Animals in the group(D) were bridged with autografts. The left was the experimental side and the right was the normal,intact control. Wound healing and gait were recorded after surgery.3. The nerve conduits were removed to observe the gross morphology of the vascularization and tissue growing3months post-operation, we made rat foot print out box and performed sciatic nerve functional index(SFI) to evaluate sciatic nerve recovery. Electrophysiological analyses were performed in all experimental rats under anesthesia at3months, The difference in latency of CMAPs was computed, and the distance between the proximal and distal sites of stimulation was measured for the determination of the conduction velocity across the regenerated nerve. The rats were sacrificed, the triceps surae muscles from both intact and injured sides of rats were dissected and weighed immediately using an electronic balance to calculate the recovery rate of wet triceps surae muscle, the triceps surae muscle on each side was harvested and paraffin-embedded for hematoxylin and eosin(H&E) staining. The regenerated nerve and normal,intact nerve were used for H&E staining and methylene blue staining, then investigated by light microscopy.4. The product registration standards was developed on the basis of animal experiments. The product adopted by national medical equipment testing center in Tianjin. This study is aim at exploring the clinical efficacy of PRGD/PDLLA/β-TCP composite nerve conduit in the treatment of human large-diameter, critical peripheral nerve defect in upper extremity by sensory and motor examination, electrophysiologic study and ultrasonic examination.Ⅳ. RESULTS1. BMSCs were isolated and purificated from the bone marrow of Wistar rats successfully. Cultivation of spindle fibroblast like cells with oval nucleus and abundant cytoplasm were formed after10days, cells were interconnected like meshwork. BMSCs had been induced to differentiate into neuron like cells directionally by using bFGF and EGF. After immunohistochemical staining, NSE, NF-M and SYP positive cells were detected, meaning that these cells acquired morphological and molecular level change.2. results of animal experiment2.1General observation:The site of incision healed without swelling and foreign body reaction. All animals survival to the end of the experiment. The left hindlimbs showed denervation performance like mopping the floor, limping, foot skin redness and heel ulcers after two weeks post-operation, then Gradually restored. The triceps surae muscles in all animals became atrophied compared with the normal, but the Group D was less serious than the other three groups.2.2SFI:At three months post-operation, the SFI of Group A,B,C,D were-75.15±4.54、-66.99±4.76、-65.75±5.20、-63.33±3.82, respectively. Group B,C,D was significantly higher than Group A(p<0.001),there was no significant difference among Group B,C and D.2.3At3months, The difference in latency of CMAPs was computed, and the distance between the proximal and distal sites of stimulation was measured, the conduction velocity across the regenerated nerve were acquired in four groups. The recovery rate of nerve conduction velocity in Group B was superior to that of Group A, the difference was statistically significant(p<0.05), Group C and Group D were significantly higher than Group A(p<0.001), Group D was significantly higher than Group B(p<0.05), There was no significant difference between Group C and Group D(p>0.05).2.4The recovery rate of wet triceps surae muscle:The triceps surae muscles in all animals became atrophied compared with the normal, intact side. The recovery rate of wet triceps surae muscle in Group B,C,D were superior to that of Group A, the difference was statistically significant(p<0.001);Group D was significantly higher than Group B(p<0.05); There was no significant difference between Group B and Group C(p>0.05), between Group C and Group D(p>0.05).2.5Histological observation2.5.1H&E staining of the triceps surae muscles:Normal muscle fiber of triceps appeared polygon, uniform shape and regulation alignment. the nuclei close to the muscle membrane. The muscle fibers in Group D were similar to normal muscle. The muscle in Group C is smaller than Group D, the muscle fibers were arranged in dense, a small amount of loose connective tissue between bundles was found. Most of the muscle fibers in Group B showed a round or oval shape, more fibrous tissue between bundles compared with Group C. The morphology of muscle fibers in Group A in varied forms, showing different size with some tiny muscle fibers scattered, has more nuclei, staining reaction increasing, also there are a large number of loose connective tissue between bundles. The recovery rate of rat triceps surae muscle cell cross sectional area in Group B,C,D were superior to that of Group A, the difference was statistically significant(p<0.001);Group D was significantly higher than Group B(p <0.01); There was no significant difference between Group B and Group C(p>0.05), between Group C and Group D(p>0.05).2.5.2General observation of regenerative nerves:No obvious adhesion between conduit and surrounding tissue, the conduit wall became thinner three months post-operation. Continuous regenerated nerve tissue located in the conduit, the surface of regenerated nerve can be seen creeping growth of nutrient vessels, all regenerated nerves were thinner than normal, intact nerves. No exudate, abscess and other untoward effects were found.2.5.2H&E staining of regenerative nerves showed that nerve fibers appeared good continuity and regulation alignment with clear myelin, and some unmyelinated wrapped node of Ranvier(Ranvier’s node) were visible in Group C and Group D. Sparse nerve fiber were arranged in order in Group A and B. Methylene blue staining: The neural membrane in Group D were complete, a great many of mature myelinated nerve fibers were found, set close and neatly arranged. The nerve fibers in Group C were similar to Group D. In Group B, the regenerated nerve fibers were sparse with thin myelin sheath. The regenerated nerve fibers in Group A were disorder with thin myelin, a great deal of vessels and fibrous connective tissue were visible. The number of myelinated nerve fibers, diameters of fibres and mean thickness of myelin sheath in Group B,C,D were superior to that of Group A, the difference was statistically significant(p<0.001), there was no significant difference among Group B,C and D. The diameters of fibres and mean thickness of myelin sheath in Group D was significantly higher than Group B(p<0.01), there was no significant difference between Group B and Group C(p>0.05), between Group C and Group D(p>0.05).3. Outcome of clinical trialsThe mean follow-up time was13.1months(ranging6-23months),after surgery, all patients with wound I healing, without infection, exudates and other untoward effects, sensory and motor function recovered in11patients with the efficient rate of78.9%(11/14),equal to the clinical outcome of similar products. Excellent in5patients, satisfactory in4patients, tolerable in2patients and with no improvement in 4patients were obtained according to the peripheral nerve function assessment standard made by The Hand Surgery Institute of Chinese Medical Association. Electromyogram examination identified that11patients with nerve conduction function recovery post-operation; high-frequency ultrasound confirmed that no adhesion between the implantation and the surrounding tissue was found.Ⅴ. CONCLUSION1. Rat BMSCs can be induced to differentiate into neuron like cells directionally by using bFGF and EGF, but whether these cells have the conduction function of mature neurons still need further study.2. Bone marrow derived mesenchymal stem cells derived Neural like cells seeded into PRGD/PDLLA/β-TCP/NGF composite nerve conduit can successfully repair12mm sciatic nerve defect of Wistar rats and the effect is similar to the autograft.3. The clinical curative effect of using PRGD/PDLLA/β-TCP composite nerve conduit in repairing human peripheral nerve defect is equivalent to the performance of similar products, can effectively promote the regeneration of peripheral nerve without adverse reactions. We considered it as an alternative for repairing peripheral nerve injury in the clinic setting.