Tissue Engineering Artificial Nerves For Long Segment Peripheral Nerve Injury: An Experimental Study

【Background】Peripheral nerve injury (PNI) is a frequently encountered trauma in clinic of orthopedic department. The incidence of PNI has been increase annually with the developing of modern architecture and transportation industry and the frequent local war. Compared with orther tissues, the prognosis of peripheral nerve injury is ugly because of limitied regenerative ability of mature neurons. Defuctionalization of muscle and sensory may occur without prompt remedy, and even permanent disability can happen to the patients, which brings about tremendous loss and burden. So the treatment of PNI has been the century challenge of medicine.Bridging the nerve gap, promoting regeneration of axon and overcoming regeneration barrier have been the main treatment of PNI. The advantage of nerve conduit in nerve injury treatment has been recognized and accepted since specificity of neurotropism was confirmed by Lundborg in 90s. Another progress has been made with the flying develop of tissue engineering technique, whose fundamental mode is "seed cell + cytokine + bioscaffold", and certain curative effect has been obtained with neural stem cell carried on tissue engineered artificial nerve which has outstanding histocompatibility and bioactivity.【Objectives】1,Bone marrow mesenchymal stem cells (BMSCs) is cultured in vitro and induced to differentiate into bone marrow-derived neural stem cells to obtain neural stem cells of higher proliferative capacity in a short period of time.2,Using PLGA as raw materials to produce a new type of three-dimensional structure biodegradable nerve conduit for tissue engineering.3,Using IKVAV self-assembled peptide to constructed tissue engineered artificial nerves.4,Tissue engineered artificial nerves constructed of biodegradable nerve conduit, IKVAV self-assembled peptide, bone marrow-derived neural stem cells and NGF were grafted to sciatic nerve defects to observe nerve regeneration and functional recovery, and to explore the feasibility of peripheral nerve repair using new type tissue-engineered artificial nerve. 【Method】1,We introduced a whole bone marrow culture method to culture primary BMSCs. Bone marrow(2~3ml) was obtained from 3-month-old rabbits under the tibial plateau, and was centrifugated in Percoll solution. The material was cultured after being re-floated in DMEM (10% FBS, 1% antibiotic). The culture fluid was removed after sub-fusion of BMSCs. Then induce fluid (DMEM, all-trans retinoic acid, bFGF) was added in to the material to induce the BMSCs differentiate toward Schwann cell-like.2,Degradable 3-D nerve conduit was structured by electrospun technique as PLGA being raw material, and biocompatibility was tested in vivo and in vitro.3,IKVAV self-assembling peptide was triggered into polypeptide-gel in certain condition, and compatibility of nerve stem cell was primarily tested.4,Nerve-repair experiment was performed by graft of tissue engineered artificial nerves. Animal models (New Zealand rabbits) were randomly divided into 3 groups: A: Auto-nerve transplatation group, B: Nerve conduct + NSC + NGF group, C: Nerve conduct + IKVAV + NSC + NGF group. The survival of NSCs and function recovery of PN was observed by means of EMG, muscle wet weight, HE stain, IMF stain and TEM at the time of 3, 6, 9, 12 weeks after operation.【Results】1,Flow cytometer indentification shows that BMSCs were isolated and cultured successfully. After the induction, BMSCs displayed morphologies of Schwann cell and its purity reached 87% by S-100 immunocytochemistry staining.2,The new type nerve conduit was produced by way of electrospun technique. We prepared new type of conduits whose outer diameter was 3mm, inner diameter was 2.5mm and the tube wall fiber diameters were 18μm. The fibers of the tube wall showed a spiral increasing arrangement in axial sections. The porosity of nerve conduit was 85.4%, and the 3D scaffold possessed features with good biocompatibi1ity, its degradation time was 3 months.3,We successfully trigger IKVAV peptides self-assemble into gels under laboratory conditions. The diameter of gels fibers was 10-30 nm and the length can reach to hundreds of nanometers. Nanofibers intertwined into three-dimensional mesh structure. Adhesion rate and biological activity determination showed that NSCs have good biocompatibi1ity.4,Different degree plantar ulcer were observed in all three groups (Severity: Group A> C> B) after reparation of long segment of sciatic nerve by artificial nerve. The result of Group C is significantly better than that of Group B (p<0.05) while it has no significant difference with that of Group A (p<0.05) in EMG test and determination of wet weight of triceps surae. More fasciculate tissue, thicker nerve fiber and neural sheath were observed in Group A but degradation of PLGA fiber in group B and C by HE stain after 12 weeks reservation. The nerve fiber were tangle and tiny with lots of collagen tissue in Group B, but raritas and neat with little collagen tissue in Group A, while much wave-shaped fascicularis tissue can be observed in Group C. TEM illustrated large quantity of neat medullated nerve fibers in Group A. Group B showed less quantity of irregular-shaped medullated nerve fibers and the medullary sheath of nerve fiber mild swelling under TEM. More regenerate medullated nerve fiber can be found in group C and more newborn capillary formation compared to group B under TEM. Fluorescence microscope illustrated that GFP fluorescence was expressed after 12 weeks, which showed that 12 weeks after transplantation, the NSCs were still alive over that period.【Conclusions】1,Using whole bone marrow culture method can get a large number of high-purity BMSCs and can be used as seed cells for tissue engineering technique for PNI repair after induction into Schwann cell-like.2,The PLGA biodegradable nerve conduits were suitable for PNI repair by tissue engineering technique with good biocompatibi1ity and biodegradability.3,IKVAV peptides can self-assembled into gels, which intertwined by nanofibers like extracellular matrix. The gels with good biocompatibility and biological activity and can be used as tissue engineering scaffold.4,Tissue-engineered artificial nerves can gain good effect for sciatic nerve injury repair. NSCs maintained a good biological activity living for 3 months. Newly born axon can be founded in the sciatic nerve defect. The function of sacistic nerve got partially restored.