Fabrication And Evaluation Of A Novel Kind Of Decellularized Nerve Allograft With Axial Multiple Channels

Objective:Peripheral nerve injury that can lead to disability affects millions of people worldwide annually.As the gold standard treatment of peripheral nerve injury,autologous nerve grafts are the most widely used,but the clinical application of the treatment is greatly limited by many disadvantages such as limited availability and morbidity at donor site.Due to low immunogenicity and preservation of inherent extracellular matrix,decellularized nerve allografts emerged as a promising alternative to autologous nerve grafts with special attention.However,the internal microstructure of decellularized nerves prepared by conventional decellularization method is very dense,with small pore size and low porosity,which is difficult to meet the needs of cell migration and nutrient exchange during nerve regeneration.Thus,curative effects of autologous nerve grafts are variable and still have a gap with autologous nerve grafts.In order to deal with these limitations of autologous nerve grafts,novel multichannel decellularized nerve allografts were prepared by the modified decellularization method which combined osmotic effect,unidirectional freeze-drying,axial puncture and mild detergent treatment.1.Evaluate the decellularization effectiveness,histomorphological characteristics,preservation of extracellular matrix components,bioactive molecules and factors and mechanical properties of novel decellularized nerve allografts.2.Analyze the effect on biological behaviors of Schwann cells and biocompatibility of the novel decellularized nerve allografts through the in vitro experiments.3.Assess the ability of the novel decellularized nerve allografts to promote nerve regeneration in vivo by repairing sciatic nerve defect of rats.Methods:1.The novel multichannel decellularized nerve allografts were prepared by the modified decellularization method and compared with the conventional decellularized nerve allografts prepared by Sondell decellularization method?SDNA?and native nerves?Native?.HE,DAPI and modified PAGE staining of transverse sections were performed to evaluate the residual degree of cellular components and myelin sheaths.The content and fragment size of residual DNA were determined by DNA quantitative detection and agarose gel electrophoresis,respectively.He and Masson staining of longitudinal sections were used to analyze histomorphological characteristics.The internal microstructure and pore characteristics were further analyzed by scanning electron microscopy.The porosity was measured by mercury intrusion porosimeter.The content of total collagen and GAGs were assessed by biochemical quantitative detection.The preservation of bioactive molecules?COI-I,COL-IV,LN and FN?and regenerative factors?NGF,BDNF and VEGF?were analyzed by Western Blot and ELISA,respectively.Swelling behavior was tested to assess water absorption ability.Mechanical property test was utilized to evaluate mechanical performance.2.The primary Schwann cells of rats were seeded and cultured at the ends of MDNA and SDNA for 3 and 7 days.The proliferation and infiltration of Schwann cells into allografts were analyzed by DAPI staining and the biocompatibility of MDNA and SDNA was evaluated by measuring the amount of DNA released The quantification of DNA released from damaged or dead cells into culture medium.RSC96cells were cultured in extract medium of MDNA and SDNA,and CCK-8 test was performed to assess their cytotoxicity.3.A rat model of sciatic nerve defect was established,and the rats were randomly divided into three groups according to nerve grafts:autologous nerve grafts?ANG?,conventional decellularized nerve allografts?SDNA?,and modified decellularized nerve allografts?MDNA?.At 6 and 12 weeks after surgery,the circumferences measurement of hind limbs and footprint analysis were conducted to assess recovery of muscle and motor function.The electrophysiological test was performed to evaluate the conduction function of regenerative nerve.The density of myelinated axons and ratio of NF200 positive area were analyzed by toluidine blue staining and immunofluorescence detection to evaluate the quality of regenerative nerves.Results:The HE,DAPI and modified PAGE staining of transverse sections indicated that no visible cell nuclei and myelin sheaths in MDNA can be detected as well as SDNA.The DNA quantitative assay suggested that both MDNA and SDNA showed a significant reduction of DNA content compared with Native?p<0.05?.The residual DNA contents of MDNA and SDNA had no significant difference?p>0.05?and were considered to meet the internationally recognized criterion of 50ng/mg.The agarose gel electrophoresis revealed that there was no visible DNA band in MDNA and SDNA.The HE and Masson staining of longitudinal sections showed that unlike the dense internal structure of SDNA,the structure of MDNA was relatively loose.There were several microchannels and macrochannels aligned axially between collagen fibers.The further analysis of scanning electron microscopy revealed that axons and myelin sheaths of MDNA and SDNA were efficiently removed.In SDNA,basal lamina tubes were preserved.However,in MDNA,it can be observed the presence of the optimized microstructure with microchannels and macrochannels.The results of pore analysis indicated that the average pore size of microchannels formed by unidirectional freeze-drying was significantly larger than that of basement lamina tubes?p<0.05?.The quantitative assay of total collagen suggested that MDNA and SDNA had no significant difference?p>0.05?.The quantitative assay of GAGs indicated that MDNA and SDNA exhibited a significant reduction compared with Native?p<0.05?.MDNA had a significantly higher GAGs content than SDNA?p<0.05?.The results of Western Blot analysis revealed that for LN and FN,the intensity of MDNA was higher compared with that of SDNA?p<0.05?.COL-I and COL-IV in two decellularization groups were retained at about the same intensity?p>0.05?.The results of ELISA showed that the contents of VEGF and NGF were significantly higher in MDNA than those in SDNA?p<0.05?,but there was no significant difference in the content of BDNF between them?p>0.05?.The detection of porosity indicated that the porosity of MDNA and SDNA increased significantly compared with that of Native?p<0.05?,and MDNA achieved significant higher porosity than SDNA?p<0.05?.The assessment of water absorption showed that the water absorption value of MDNA and SDNA increased significantly compared with that of Native?p<0.05?,and MDNA was significantly higher than SDNA?p<0.05?.The results of mechanical property test indicated that MDNA was significantly higher in Young's modulus than Native?p<0.05?,but there was no significant difference in stress at fracture,strain at fracture and suture retention strength between them?p>0.05?.The Young's modulus and stress at fracture of MDNA were significantly lower than those of SDNA?p<0.05?,and they had no significant difference in other terms?p>0.05?.2.In the in vitro experiments,it was observed that after 3d of incubation,there was no obvious cell migration into samples of SDNA,with most cells remaining at the initial seeding site.However,in MDNA,numerous cells penetrated into samples through macrochannels.After 7d of incubation,the cells in all groups appeared to have proliferated.In SDNA,a majority of cells were still distributed in the superficial area of samples.However,in MDNA,it was observed that lots of cells proliferated and migrated further along macrochannels.Meanwhile,several cells were found to migrate into samples through microchannels.In addition,the DNA content of medium in MDNA and SDNA exhibited no significant difference to each other?p>0.05?and did not differ significantly from that in the negative control group?p>0.05?,but were significantly lower than that in the positive control group?p<0.05?.The results of CCK-8 revealed that at three time points,the OD₄₅₀50 value of MDNA and SDNA were comparable?p>0.05?and exhibited no significant difference compared with that of the negative control group?p>0.05?.Nevertheless,they were significantly higher than that of the positive control group?p<0.05?.3.In the in vivo experiments,for the recovery of muscle and motor function,it can be found that the circumferences of hind limbs and sciatic functional index?SFI?of all groups exhibited tendencies to increase over time.At 6 weeks,there were no significant difference among MDNA,SDNA and ANG?p>0.05?.At 12 weeks,the circumferences and SFI of MDNA were significantly higher in than those of SDNA?p<0.05?,but significantly lower than those of ANG?p<0.05?.The results of electrophysiological test showed that at 6 weeks,there was no significant difference in the nerve conduction velocity?NCV?and amplitude between MDNA and SDNA?p>0.05?.At 12 weeks,MDNA had significantly higher NCV and amplitude compared with SDNA?p<0.05?.ANG exhibited the highest NCV and amplitude at two time points?p<0.05?.The results of toluidine blue staining showed that at 6 weeks,comparing with SDNA,the myelinated axons density of MDNA was significantly higher at the central grafts?p<0.05?,but had no significant difference at the distal stumps?p>0.05?.The densities at the central grafts and distal stumps were significantly lower in MDNA than those in ANG?p>0.05?.At 12 weeks,the densities at the central grafts and the distal stumps in MDNA were significantly higher than those of SDNA?p<0.05?.Comparing with ANG,the density of MDNA had no significant difference at the central grafts?p>0.05?,but was significantly lower at the distal stumps?p<0.05?.The results of immunofluorescence analysis indicated that at 6weeks,comparing with SDNA,the ratio of NF200-positive area in MDNA was significantly higher at the central grafts?p<0.05?,but had no significant difference at the distal stumps?p>0.05?.The ratios at the central grafts and distal stumps were significantly lower in MDNA than those in ANG?p<0.05?.At 12 weeks,the ratios at the central grafts and the distal stumps in MDNA were significantly higher than those in SDNA?p<0.05?,and significantly lower compared with those in ANG?p<0.05?.Conclusions:1.The modified decellularization method can not only remove cellular components efficiently,but also play a better effect on preservation of extracellular matrix components,bioactive ECM molecules and regenerative factors.Meanwhile,the method can increase porosity and water absorption by introducing macrochannels and microchannels,which optimizes the internal microstructure of allografts,but the mechanical properties were not significantly influenced.2.The in vitro experiments demonstrated that the novel multichannel decellularized nerve allografts prepared by the modified method showed excellent biocompatibility and possessed a better ability of facilitating proliferation and penetration of Schwann cells.3.In the in vivo experiments,the novel decellularized nerve allografts exhibited more capable to guide regenerative nerve fibers through defective segment and restore innervation of target organs,thus achieving better recovery of muscle and motor function,compared with conventional decellularized nerve allografts.