Acellular Nerve Scaffolds Improve The Functional Recovery Stimulated By Cyclic Tensile Strain(CTS)

Objective: 1. To improve the acellular nerve scaffolds by the technique of freeze-drying and hypotonic buffer. To advance the method of rabbit SCs isolated and purified with the existing methods. And to build a new type peripheral nerve graft with tissue engineering technique by seeding the SCs into the improved acellular nerve scaffold in vitro. 2. After the cell-scaffolds composites structured in vitro were stimulated by the different deformations(0%, 5%, 10% and 15%), those composites were implanted into the model of rabbit sciatic nerve defect to repair the nerve defect. To explore the degree of nerve functional recovery and the molecular biology change of SCs during in vivo.Methods: 1 The traditional porosity of acellular nerve scaffolds was advanced by the technique of freeze-drying and hypotonic buffer. And then The histological characterizations of scaffolds were showed by the HE staining and scanning electron microscope(SEM) to make sure the immunogenic cells removed and the ECM components reserved. Based on variety methods of SCs isolated and purified, we explore the method which acquire the primary SCs mixed with fibroblasts by the enzyme digestion, and antimetabolites(Ara-C) were used to inhibit the proliferation of fibroblast, the proliferation of first purified SCs were stimulated by HRG1-β1, and then SCs were purified again combined with the differential reactions of enzyme and differential time of sticking wall between SCs and fibroblast. The SCs marked by the PKH26 were seeded into the scaffolds under the stereomicroscope by the method of microinjection, and the condition of internal SCs were observed by HE staining. 2. The cell-scaffolds composites were stimulated by the continued for 3h using the Electro Force 3200 test instrument and the Bio Dynamic chamber(BO SE) by the cyclic tensile strain at the frequency of 0.25 Hz and different deformations(0%, 5%, 10% and 15%). And then the cell-scaffolds composites were used to implant into the model of rabbit sciatic nerve defect(1cm-long). The continuity and morphological changes were assessed by ultrasound examination at 4 and 8 weeks postoperatively. Sciatic nerve function was evaluated by neurophysiological examination and calculated the nerve conduction velocity(NCV) at 8 weeks postoperatively. Recovery of gastrocnemius musculus were assessed by the ratio of wet weight at 8 weeks postoperatively. After that the scaffolds were took out and conducted the HE staining, toluidine blue staining to show the cellular distribution and count the myelin sheath ring. Immunofluorescence staining of-100, GFAP and NF-200 were observed and the integrated optical density(IOD) of each groups were measured by IPP. The fluorescent tracer(PKH26) was observed at the distal anastomosis under the fluorescence microscope.Results: 1.The results of HE staining and SEM showed that the myelin sheath and SCs were removed, and abundant pore structure exist in the interior of advanced acellular nerve scaffolds. The new method of SCs purified could acquire abundance SCs with high proliferative activity and purity rapidly. The result of HE staining showed that the SCs survived well in the interior of advanced acellular nerve scaffolds. 2. The results showed that the continuity of epineurium was integral at 8 weeks postoperatively from the long-axis of the ultrasound images. From the short-axis images, the nerve fiber bundle were separated by epineurium, and the contours of nerve fiber bundles were clear and integral. At 8 weeks postoperatively, the group of 10% deformations demonstrated the higher value in all scaffolds groups(0%, 5%, 10% and 15% deformations), and lower than the autologous nerve group or control group. And nascent potential was seen in the spontaneous potential of 0%, 5% and 15% deformations group, the regenerative potential and positive sharp waves were seen in the 10% deformations group. The ratio of wet weight showed that the 10% deformations group was higher than other three deformations groups. After 8 weeks postoperatively, the results of HE staining showed that the basal lamina and fibers of different deformations groups(0%, 5% and 10%) and autologous nerve group was arranged in a wave-like structure, but the internal structure of 15% deformations group was partly disintegrated. The numbers of myelinated nerve fibers in 10% deformations group was higher than 0%, 5% deformations group, but less than autologous nerve group. The IOD of S-100 and GFAP in 10% deformations group were higher than 0%, 5% deformations group and autologous nerve group, but less than control group. The IOD of NF-200 in 10% deformations group was higher than 0%, 5% deformations group, but less than autologous nerve group and control group. Red fluorescent cells labeled with PKH26 were visible in the distal anastomosis of the scaffolds except the 15% deformations group.Conclusion: The histological analyses have confirmed the appropriate CTS could stimulate the migration of Schwann cells. The electrophysiological analyses confirmed that the appropriate CTS could promote the functional recovery of peripheral nerve defect repaired with the advanced acellular nerve scaffolds.