| Ⅰ. BACKGROUND:The treatment of peripheral nerve injury and defect is one of the problems of microsurgery. The repairment of long gap nerve defects is still a troublesome thing for the clinicians and lack of effectively treatment.Autologous nerve grafts have been the ’gold standard’ for treatment of peripheral nerve defect and have been widely applied in clinical practice. However, availability of donor nerves and donor site morbidity are concerns. We need a safer and more effective clinical treatment strategy. To overcome these problems, a considerable amount of research has been devoted to the development of tissue-engineered artificial nerve. Therefore, many scholars continue to explore other material for repairmen of nerve defects. The current research is focusing on the simulation of nerve regeneration microenvironment, the choice of appropriate scaffolds, playing the role of neurotrophic factors and seed cell chemotaxis and building a composite nerve conduit to support long-distance nerve defect.Ⅱ. OBJECTIVE: This research was according to the bionic point of view and based on analyzing structure of nerve basement membrane, a novel RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}/poly(d,1-lactic acid)/β-tricalcium phosphate/nerve growth factor(PRGD/PDLLA/β-TCP/NGF) composite nerve conduit was prepared.The structure and properties of the composite materials were characterized by animal experiments in vitro. Evaluate the efficacy of large-diameter nerve repairs by preliminary clinical application.III. METHODS:1. In this paper, according to the bionic point of view and based on analyzing structure of nerve basement membrane. A new polymer RGD peptide modification of poly{(lactic acid)-co-[(glycolic acid)-alt-(L-lysine)]}(PRGD) was synthesized. Ethyl acetate was used as the solvent.The solution was then mixed with proper quantities of PDLLA,NGF and β-TCP on a magnetic stirrer till the mixture was homogeneously. The composite nerve conduit(PRGD/PDLLA/β-TCP/NGF) was then prepared by solvent pouring method.2. RSC96cells were co-cultured with PRGD/PDLLA/β-TCP/NGF nerve conduit to evaluate their cell compatibility and potential to be used as nerve conduit material. Using the SEM photographs to observe cell adhesion and spreading of the material surface. Invitro,the NGF release was quantified using enzyme-linked imrnunosorbent assay method(ELISA)and recorded NGF release curve.3. Twelve healthy hybrid dogs were used as the animal model. The conduits were employed to bridge the30mm defects in the peroneal nerve.The PRGD/PDLLA/β-TCP/NGF nerve conduit was implanted into12dogs in the left peroneal nerve. The right peroneal nerve was reconstructed with he autograft harvested from the left side, as a control. After the surgery, Wound healing and gait were recorded. 4. The dogs were anesthetized respectively at3,6and9months, The target muscle harvested and paraffin-embedded for hematoxylin and eosin (H&E) staining. Electrophysio-logical recordings were carried out under anesthesia at9months. The peak latency and peak amplitude of the muscle action potentials were measured from the chart record ings, as was the conduction velocity through the regenerated nerve.At9months after surgery, The dogs were sacrificed. Regenerated nerve and target muscle were investigated by histological observations (by light microscopy and transmission electron microscopy).At9months after surgery, nerve conduit material removed to observe the gross morphology of the vascularization and tissue growthing, Micro-structural changes analysised by scanning electron microscopy.5. 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 in order to prove the feasibility of the artificial nerve conduit for repairing large diameter nerve defects of human with regular follow-up at1month,3months and6months.IV. RESULTS:1. The prepared PRGD/PDLLA/β-TCP/NGF nerve conduit has milky white appearance, odorless, tasteless, smooth surface, no cracks, no glitches, soft, bending and easy to suture. In vitro the composites has good cell affinity with RSC96cells, and could sustained release NGF30days.2. General observation of animal experiments postoperatively. The dorsalis pedis varying degrees of redness After2weeks postoperatively, swelling, ulcers, and then the wound healed spontaneously one month later. We could see apparent dorsiflexion of the limb postoperatively and leg tibialis anterior muscles gradually atrophy. The site of incision healed without swelling, exudates and foreign body reaction. The atrophy of tibialis anterior muscles recovered slowly after the operation at3months.3. Electrophysiological assessment:At9months after surgery, electrophysiologic tests were performed on all animals. Single electrical pulses were delivered via bipolar electrodes placed in turn at the proximal and distal trunk of the grafted nerve.The latency and amplitude were recorded via an electrode inserted into the anterior tibial muscle. The difference conduction velocity was computed. CMAPs were detected in groups PRGD/PDLLA/β-TCP/NGF and autograft. The difference in CMAP amplitude was not statistically significant between groups PRGD/PDLLA/β-TCP/NGF and autograft.(P>0.05)4. Histological observation:The tibialis anterior muscles of groups PRGD/PDLLA/β-TCP/NGF were observed by HE staining at3months,6months,9months. The muscle fiber diameter was significantly smaller, has more nuclei, staining reaction increasing. There were more connective tissue between muscle bundles. The atrophy of tibialis anterior muscles recovered slowly after the operation at3months.At9months continuous regenerated nerve tissue located in the conduit. The surface of regenerated nerve can be seen creeping growth of nutrient vessels. Methylene blue staining:Nerve fibers evenly distributed in the proximal of regenerated nerve. The myelin sheath thicker than before.There are many varying sizes of nerve bundles in the regenerated nerve. Between the nerve bundles there are rich in connective tissue.The myelin sheath is thinner than the proximal of regenerated nerve. The nerve fibers were regional and dispersed in the distal of regenerated nerve. The morphological is regular. The myelin sheath is thinner. Quantitative image analysis:the conduit group and the autologous group two sets of axon diameter of myelinated nerve fibers, myelin thickness compared with no significant difference (P>0.05) differences were not statistically significant. Ultrastructure under transmission electron microscope:Mature nerve fibers can be seen in the proximal and center of the nerve conduit and the distal nerve stump.The regenerated nerve is characteristics of clear myelin lamellar, rich microfilaments microtubules. It can be found that the Schwann cells surrounding the axon. There are part of the myelinated nerve fibers are scattering distribution.5. Follow-up results of clinical trials:After operation, all patients with wounds I/A healing, without local inflammation, without allergy symptoms (local swelling, rash, itching).All patients with vital signs stable, there are no clinical significance of routine blood test, liver and renal function, erythrocyte sedimentation rate, C-reactive protein, etc. And without related adverse events occurred.V. CONCLUSION:1. PRGD/PDLLA/β-TCP/NGF nerve conduit was successfully prepared through the bionic basement membrane components of natural nerve. In vitro experiments showed that the material has good biocompatibility and cell affinity, and can be sustained release of NGF up to30days.2. PRGD/PDLLA/β-TCP/NGF composite nerve conduit can successful repair long segment of peripheral nerve defect in dogs and the effect is similar to the autograft.3. Three cases of patients with peripheral nerve defects treated by PRGD/PDLLA/β-TCP/NGF nerve conduit as preliminary clinical trial research. Recent follow-up observation with good results,and long-term effect remains to be seen. |
PDF Full Text Request