Fabricate Novel Artificial Nerve Conduits For Reconstructing The Sciatic Nerve Defects

Peripheral nerve injury is one of the most common clinical diseases, affecting 2.8% of all trauma patients. Direct repair is just limited to short-distance gaps where the disconnected nerve stumps can be sutured end by end. Extensive nerve gaps must be reconstructed with structures creating a permissive environment for the neural regeneration and eventually reestablishing the nerve function. Current available clinical treatments for the nerve defect typically rely on donor tissues which include nerve graft, vein graft, or arterial graft, etc. However, these methods are inevitably limited by multiple disadvantages, such as; shortage of graft material, additional surgery trauma which results in functional loss at the donor sites, and the size and structural differences between donor and recipient. A promising alternative to conventional grafting is the use of artificial nerve grafts. Ideal biomaterials for neural tissue engineering should meet the critical requirements including excellent biocompatibility, biodegradability and neural integrity.In this article, we constructed three kinds of artificial conduits for repair of peripheral nerve injury. PLGA conduit filled with self-assembling nanofiber scaffold (SAPNS), SAPNS mixed schwann cells or aligned collagenscaffold respectively, and then used them to bridge the rat sciatic nerve defects.Materials and methodsPreparation of the PLGA conduit.5%PLGA solutions in chloroform were mixed with sodium chloride particulates (96-75 μm), then putting the mixture into a polytetrafluoroethylene mold make the tube material. Double distilled water was dissolved sodium chloride particles completely, and porous scaffold was prepared by freeze-drying finally. The PLGA was cut to 12mm in length., and prepared PLGA conduits were sterilized and stored in 70% alcohol for further use.PLGA tube wash three times in DMEM/F12 medium,1% of the SAPNS pure or add 2.5 x 105 SCs cells per tube filled the length of the tube to constitute the graft. Then immerse grafts in DMEM/F12 medium 30 minutes for SAPNS polymerization to gel. Empty PLGA tube and linear orderly collagen filling in PLGA also DMEM/F12 wash three times in DMEM/F12 medium and immerse in 30 minutes.Animal subjects and experimental groups.Adult Sprague-Dawley(SD) female rats were used to prepare the model of sciatic nerve defect The rats were then randomly divided into all groups.Surgical procedures.All designed rats were anaesthetised by intraperitoneal injection of 1% pentobarbital sodium. After routine skin preparation and disinfection, the right sciatic nerve was exposed and a transection was performed at the middle portion of the nerve trunk to prepare a 10 mm nerve defect, the transplant was applied to bridge the gap. Both proximal and distal nerve stumps were inserted into the conduit as far as lmm and the connections were secured with 11-0 suture. For a positive control a segment of 10 mm allogeneic fresh sciatic nerve obtained from donor rats was used to bridge the defected gap (PN group). In NG group, sciatic nerve with 10mm defect was left without any graft to serve as negative control. Antibiotics and analgesic reagents were routinely administrated for post-operative health care. Guidelines for animal care and use of Southern Medical University were strictly followed. The animals were allowed standard access to food and water ad lib throughout the study.Behavioral testing.The motor functional recovery of injured hindlimbs were assessed with a modified SFI (Sciatic Function Index) approach at 15w post-treatment as in our previous report. Briefly, the palms of forelimbs and hindlimbs were colored with different nontoxic dyes. The animals were then trained to walk on a paper-covered narrow runway (lm long,7cm wide and 8cm high). Six adequate foot prints of each rat were selected to measure the Rotation Angle of Foot injured (RAFi), which is the angle made by a stride line and the connect line of the third toe to the center of the paw pad, and the Injured Hindlimb Synergia (IHS), which is the distance between the center pads of the injured hindlimb and ipsilateral forelimb. Next, we classified the foot prints into 3 patterns and the percentage of each pattern was calculated. In second and third part, puts rat free to walk on the glass plate. Under the glass plate taken with a digital camera, and collect data through video analysis.Electrophysiology assessment. Animal subjects were anesthetized with tribromoethanol. The stimulating electrode was placed on the proximal nerve trunk 5 mm to host-conduit junction site with recording electrode placed on the ipsilateral foot. The sciatic nerve is stimulated under the same stimulation intensity and frequency. The latency and amplitude were measured in each group.Retrograde tracing.Fifteen weeks after the surgery of sciatic nerve injury and transplantation,2μl of 1% FluoroGold (FG) was microinjected at the site on nerve trunk 5mm distal to the host-implant sutured site using a Hamilton syringe with 30-gauge needle. Animals were allowed to survive for another 7 days for tracer transportation before they were sacrificed.Tissue collecting.Animals were euthanized with overdose of anesthesia and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). Bilateral gastrocnemius muscles were explanted immediately and the wet muscle weight was recorded on a laboratory scale. The weight ratio of injury side to intact side was calculated as the recovery index of gastrocnemius muscle. Involved sciatic nerve segments containing conduit implants and the 5th lumbar (L5) segment of spinal cord were also harvested after perfusion. Three nerve samples of each group were post-fixed with with 2.5% glutaraldehyde plus 2% paraformaldehyde for further transmission electron microscopy (TEM). The reamining samples were post-fixed with 4% paraformaldehyde for cryosectioning and immunofluorescence staining.Transmission electron microscopy.From the selected nerve samples, lmm segment in the middle of the graft, or the corresponding site of sham control sciatic nerve, was dissected to perform TEM as previouslydescribed. After being fixed with 2.5% glutaraldehyde plus 2% paraformaldehyde for 24 h, the samples were post-fixed in 1% OsO4 for 2h at 4 ℃ and dehydrated in graded acetone series. The stained ultrathin sections were observed with transmission electron microscope. The measurements were obtained using Image-Pro Plus software.Immunofluorescence staining.The samples designed for immunofluorescence staining were embedded in OCT Compound.after post fixed in 4% paraformaldehyde for 24 h, then immersed in 30% sucrose (w/v) until the samples were sunk in the sucrose solution. All sections were cut in 10μm thickness and mounted onto poly-1-lysine coated slides, then kept in-20℃ until used. Every tenth section of each sample was double immunostained with NF-200/MBP to detect the axon and myelin. The slides were cover slipped with mounting medium containing DAPI to counter-stain the nuclei. Digital images were captured with a fluorescence microscope.Quantification of the survived and regenerated neurons.The 5th lumbar (L5) segment of spinal cord was cryosectioned transversally in 10 μm thickness and then stained with fluorescent Nissl staining kit following the vendor’s instruction. Every eighth section of each subject was selected to count the survived and regenerated motor neurons in the spinal cord ventral horn. The motor neurons were identified by their large size, location, and dark Nissl staining. The survival rate of the motor neurons was indicated as the ratio of the neurons in the injured side divided by the number of neurons in the intact side. The regeneration rate was calculated as the number of FG labeled neuron divided by the number of Nissl neurons in the injured side.Histomorphometry of gastrocnemius muscle morphology and neuromuscular junction.The mid-belly part of gastrocnemius muscles was trimed to perform cryo sections after being post-fixed with 4% PFA. Two sets of sections were prepared from each subject:one set was transversally sectioned for routine Hematoxylin staining to count the area of muscular fibers The second set was longitudinally sectioned for neuromuscular junction staining with a-Bungarotoxin (a-BTX) as previously described The area of the junction covered field was calculated. All quantifications were done in six random non-overlapping fields of every eighth section of each subject using Image-Pro Plus software.ResultsFunctional recovery of the injured hindlimb.When the locomotor function of the injured hindlimb was assessed by foot print testing (SFI), we found almost 90% of steps of injured hindlimbs in the NG group could not leave an adequate foot print on the paper covered runway. Quantification data from the subjects with implants showed the percentage of palm supported was increasing in three kinds of artificial nerve groups and PN group. Next, six palms supported foot prints of each subject were selected to measure the Rotation Angle of Foot injured (RAFi) and the Injured Hindlimb Synergia (IHS). Due to the motor function impairment, both degree of RAFi and IHS were increased after injury. If either value of RAFi and IHS reverts toward the normal value (prior to the injury), it means locomotor function is improved after treatments. Statistical analysis revealed both of RAFi and IHS slightly increased in three kinds of artificial nerve groups and PN group compared to sham group.Electrophysiology assessment.The effect of latency and amplitude in PLGA+collagen group and PLGA+SAP+SCs group is better than PLGA group, Proof that these two kinds of artificial nerve have shown the obvious effect in the functional recovery after peripheral nerve injury.Axonal regeneration in the conduit.Three kinds of artificial nerve groups and PN group integrated properly with the host nerve which was absent of any significant scarring occurrence. On the longitudinal sections stained by fluorescent immunohistochemistry, robust NF-200 positive axons were demonstrated in the two groups. Of note, axons grew with a linear profile. On the transversal sections, the axons almost fully filled in the entire conduit. In contrast, only the proximal part of the PLGA group implant was mode rately filled with axons.The remyelination of regenerated axons. After double staining with NF-200 and MBP antibodies, it was shown that a lot of NF-200 positive axons in the implants were wrapped with MBP positive myelin. The size of the regenerated axons and myelin in three kinds of artificial nerve groups and PN group were smaller than that of the sham sciatic nerve, but the regenerated axons almost filled up the implants with a lot of them being myelinated. In the PLGA group, the regenerated axons were scattered mainly around the edges of the conduit. In the first part, we assessed the ultrathin transverse sections of the middle segment of the implants with TEM. Quantification demonstrated that the remyelination rate of axons was significantly higher in the PLGA+SAP group and than that in the PLGA group, but lower than the sham and PN groups. Tthe axon diameter and myelin thickness in the PLGA+SAP and PLGA groups were significantly lower than those in sham and PN groups. Additionally, the G-ratio of myelinated fibers was lowest in the sham group, and highest in the PLGA group.Survival and regeneration of injured neurons.As the motoneurons in the lumbar spinal cord extended their axons into the sciatic nerve, these motoneurons were injured during the sciatic nerve transection which could result in death. Unexpectedly, there were no significant differences among three kinds of artificial nerve groups and PN group. However, the FG retrograde-labeling demonstrated motoneurons have regenerated axons in the ipsilateral ventral have statistical differences among groups.Gastrocnemius muscle wet weight and muscular morphology.After sciatic nerve transection, the gastrocnemius muscle lost weight dramatically and showed serious steatosis and myoatrophy in the NG group, which has not any treatment for the nerve defect and the targetedgastrocnemius muscle suffered from chronic denervation. In contrast, steatosis and myoatrophy were ameliorated with the implants. Quantification showed that the myofiber’s transverse area ratio and the whole gastrocnemius muscle’s wet weight ratio in other groups have statistically significant differences.Quantification of neuromuscular junction.Using a-BTX stained longitudinal gastrocnemius muscle sections, neuromuscular junctions showed attachment onto the myo fibers. In nerve injury involved groups, the sizes of junctions were decreased in various degrees, all of which were significantly lower than that of sham group. Among those, the lowest was in NG group and increasing from PLGA group, three kinds of artificial nerve groups to PN group.ConclusionIn the present study we developed a novel artificial nerve graft named SAPNS containing PLGA conduit (PLGA+SAP), which was made of an outer porous Poly (lactic-co-glycolic acid) (PLGA) conduit and internal amphiphilic hydrogel of self-assembling peptide nano fiber scaffold (SAPNS). Once a transection surgery was performed to prepare a sciatic nerve defect model using rats, the PLGA+SAP group was transplanted to bridge the 10-mm gap of defected nerve. Allogeneic peripheral nerves (PN) or empty PLGA conduits (PLGA) were grafted as positive or negative controls. By the assessments of retragrade tracing, behavioural testing, and a series of histomorphometries, our results indicated that PLGA+SAP group has good capability to promote the axonal regeneration and remyelination in the defected nerve. Compared with the PLGA group, there were much more axons regenerated into and across the PLGA+SAP group. Moreover, the diameter of regenerated axons, thickness of the myelin in the conduit, size of neuromuscular junctions, and diameter of myofibers in the target muscle were all significantly increased when bridged with PLGA+SAP group. Based on the above data the present SAPNS containing PLGA conduit demonstrated high potential to work as an artificial nerve graft for the repairing of peripheral nerve injury.