| Peripheral nerve injury and pathological changes caused by traffic accidents,natural disasters,wars and clinical operation accidents have resulted in the decline of patients’ quality of life,the increase of family burden,and also caused huge losses to society.According to statistics,there are about 1 million new cases of peripheral nerve injury in our country every year,including about 300,000 cases of peripheral nerve defects.The repair of nerve defect and its functional reconstruction is a clinical problem,especially the repair of long-distance peripheral nerve defect(the length of the nerve defect is greater than or equal to 4 times the diameter).At present,the gold standard for the clinical treatment of nerve defects is still autologous nerve transplantation.However,due to the size mismatch of autologous nerve transplantation and new trauma,its clinical application is limited.Studies have shown that the biomimetic properties of biomaterials play a crucial role in promoting nerve tissue repair.After implantation,biomimetic materials can effectively regulate the cell behavior of the microenvironment of the trauma site and surrounding tissues,and are expected to achieve nerve tissue regeneration and functional recovery.Therefore,how to find a repair method that can replace autologous nerve transplantation has become a worldwide problem in the field of clinical treatment of peripheral nerve defects.The emergence of tissue-engineered nerve grafts has provided new ideas for the treatment of peripheral nerve defects.So far,its ability to repair nerve defects is still limited,and there is still a large gap between it and autologous nerve transplantation as the gold standard.Therefore,it is urgent to design a tissue-engineered nerve graft with excellent function of repairing peripheral nerve defects.With the advancement of tissue decellularization technology,xenogeneic decellularized nerve matrix(x DNM)has become a promising biomaterial for the treatment of peripheral nerve defects.Decellularization technology retains the extracellular matrix components of natural tissues,and effectively removes antigenic substances to avoid immune rejection between individuals simultaneously.Electrospinning technology builds a three-dimensional fiber network structure that simulates the extracellular nerve matrix,thereby providing a natural microenvironment closer to the growth of nerve cells.Therefore,the decellularized nerve grafts prepared by electrospinning technology have received extensive attention from researchers.At present,there are many studies on the preparation of decellularized nerve matrix by electrospinning technology:(1)Firstly,the decellularized nerve matrix is digested into slightly smaller molecular proteins by biological enzymes,and then the enzymatically hydrolyzed proteins are prepared into nerve grafts by electrospinning technology.(2)In the process of electrospinning of decellularized nerve matrix,high molecular polymers are mixed to different degrees to process and prepare nerve grafts.Currently,there is no report on the preparation of neural grafts by electrospinning with pure decellularized nerve matrix protein.Theoretically,the electrospinning decellularized nerve matrix graft is closest to the autologous nerve in composition,and also mimics the extracellular matrix structure in terms of spatial structure.Based on the above background,in order to better use xenogeneic decellularized nerve matrix for the treatment of peripheral nerve defects,this project proposes:(1)The unique mild decellularization technique was adopted to avoid excessive elution of chemical detergent and remove the antigenic substances as much as possible;(2)The problem of independent electrospinning of decellularized nerve matrix was solved without enzymatic digestion,and the pure decellularized nerve matrix grafts were prepared based on electrospinning technology;(3)The repair of peripheral nerve defects by electrospinning decellularized nerve matrix grafts was verified by in vitro and in vivo experiments.This topic mainly includes the foll owing research contents and conclusions:(1)Preparation and characterization of neural acellular matrix based on “physical freeze-thaw + chemical washing” technology.In order to reduce the damage of chemical detergents to x DNM,a combination of gentler methods for decellularization is required.Our study showed that after freezing and thawing at-20℃,-80℃ and liquid nitrogen(-196℃),there was a certain reduction of cells in neural tissue.Especially after liquid nitrogen treatment,the number of cells was significantly reduced,and the removal rate could reach 58.8%.Simultaneously our study attempted 20-40 k Hz ultrasonic concussion to treat nerves,but it did not show significant decellularization effects.The study also compared different chemical elution methods,and the results showed that the Sondell method using octylphenoxypolyethoxyethanol(Triton X-100)and sodium deoxycholate had better decellularization effect,and the elution rate of genetic material was up to 97%.We combined “physical freezing-thaw + chemical washing” to decellularize nerves.The specific methods are as follows:(1)Firstly,porcine nerve were completely frozen in liquid nitrogen at-196℃ by “ultra-low temperature freezing-rapid melting”,and then rapidly melted a nd shaken in pure water at 37 ℃.The cycle was repeated for 6 times.The ice crystals formed in the cells and the increase of cell fluid concentration during freezing and thawing will lead to cell rupture.(2)The concentrations of Triton X-100 and sodium deoxycholate solution in Sondell method were both adopted the lowest concentration of 4% in literature,and the washing time of the two was shortened to 6 and 12 hours respectively for chemical elution.(3)The results of “physical freeze-thaw + chemical washing”showed that the DNA residue in x DNM was only 21.33 ng/mg,the pig-specific antigen α-Gal was significantly reduced to 23.4 ng/mg,and the main protein components were also well preserved.Studies have shown that the combined“physical freeze-thaw + chemical washing” method can successfully prepare decellularized nerve matrix that meet the requirements of national biosafety,while its conditions are mild,the destruction of the decellularized nerve matrix is reduced as much as possible,and the composition of the decellularized nerve matrix is maintained.(2)Exploration and characterization of conditions for electrospinning pure decellularized nerve matrix grafts.Preparation of pure decellularized nerve matrix electrospinning solution: The study attempted the direct dissolution of x DNM by various solvents such as dimethyl sulfoxide,dimethylformamide,dichloromethane,chloroform,formic acid,trifluoroacetic acid,and hexafluoroisopropanol.The study found that x DNM took 2-5days to dissolve in formic acid and trifluoroacetic acid,and the obtained x DNM concentration was 5-20%;it took 5-20 days to dissolve in hexafluoroisopropanol,and the concentration of x DNM was 5-15%,the higher the concentration,the longer the dissolution time.The dissolution time for a concentration of 20% was 30 days.Study on electrospinning conditions of pure decellularized nerve matrix grafts:We successively conducted a large number of electrospinning experiments with x DNM spinning solutions with different concentrations of formic acid,trifluoroacetic acid and hexafluoroisopropanol under different conditions.It was found that only when x DNM was dissolved in hexafluoroisopropanol and the concentration reached10-20 %,the fibrous structure could be successfully formed.Afterwards,we studied three x DNM concentrations of 10%,15%,and 20%,four spinning voltages of 11,14,17,and 20 k V,four spinning receiving distances of 7,10,13,and 16 cm,and three spinning solution push rates of 0.3,0.6,and 0.9 m L/h to explore the optimal conditions for x DNM electrospinning.The results showed that when the x DNM concentration was 15% w/v,the electrostatic field voltage was 14 k V,the solution pushing speed was 0.6 m L/h,and the receiving distance was 10 cm,the distribution of x DNM electrospinning fibers was the most uniform and stable.The pure x DNM electrospinning nerve graft(x DNME)was prepared under the same conditions.x DNME was rinsed 3 times with 95% ethanol for 1 h each time.Then x DNME was rinsed with pure water 3 times,1 h each time.Finally,it was dried in a 37°C oven to completely remove the residual solvent.x DNME has certain mechanical properties,but its mechanical properties need to be improved in order to full y meet the clinical requirements for nerve defect repair with higher mechanical strength.At the same time,the degradation rate of x DNME is too fast to be controlled,so it needs to be further optimized.(3)Effect of proanthocyanidin cross-linking on x DNME nerve grafts.Proanthocyanidin(OPC)is a natural plant-derived antioxidant,with good biological safety,strong antioxidant and free radical elimination effects,so it has a good protective effect on tissues and is an excellent biological cross-linking agent.We used OPC to cross-link the prepared x DNME nerve grafts,and studied the conditions of cross-linking: we systematically investigated different concentrations of 5,10,15,20,30 mg/m L to cross-link x DNME.The results of Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD)showed that OPC could cross-link with x DNM protein.It was found that OPC cross-linking treatment could significantly improve the mechanical properties of x DNME,and the 15 mg/m L cross-linking group had a maximum tensile strength of 1.492 N.The final study showed that 15 mg/m L OPC solution prepared x DNME-OPC nerve graft has the best mechanical properties.OPC cross-linking could also significantly prolong the degradation time of x DNME,and its degradation rate could be regulated by the concentration of the cross-linking agent.(4)In vitro cell evaluation of x DNME and x DNME-OPC neural grafts(1)Cytotoxicity analysis: According to the method in Chinese medical device evaluation standard GB/T 16886,L929 fibroblasts were cultured with graft extract.The MTT results showed that the cell viability of L929 in the x DNME and x DNME-OPC groups could reach 97.7% and 95.3%,respectively,proving that the neural grafts prepared in this study had no cytotoxicity.(2)In vitro cell co-culture: Primary Schwann cells were cultured on nerve grafts.After 2 days,CCK-8 results showed that the viability of Schwann cells on x DNME and x DNME-OPC was 88% and 86%,respectively.In addition,after DRG was cultured on x DNME and x DNME-OPC for 3 days,the growth length of nerve fibers could reach 764.9 μm and 643.5 μm,respectively,showing a good growth state.(3)Whole transcriptome sequencing: Whole transcriptome sequencing analysis was performed on DRG cultured for 3 days,and the results showed that the gene expression of DRG in each group was significantly different.x DNME enhanced the expression of genes in cell migration,adhesion,cytoskeleton,and cell surface receptor signaling pathways.x DNME-OPC significantly enhanced cellular nucleic acid metabolism,ribonucleic acid biosynthesis and other biological processes,and regulated molecular pathways such as immune response and anti-infection.(5)Research on repairing peripheral nerve defects with x DNME and x DNME-OPC nerve grafts.The x DNME and x DNME-OPC nerve grafts were applied to repair 8 mm sciatic nerve defects in rats.12 weeks after surgery:(1)The morphological observation results showed that the regenerated nerve fibers in x DNME and x DNME-OPC grafts completely connected both ends of the defect,and the average thickness of the newly regenerated myelin sheath was 0.64 μm and 0.54 μm,respectively.The two nerve grafts had good histocompatibility,and neither caused obvious immune rejection.(2)The evaluation of regenerative nerve function recovery proved that the compound muscle action potential(CMAP)of the x DNME and x DNME-OPC groups were 9.2m V and 8.1 m V,respectively,and the electrical signal conduction velocity reached8.86 m/s and 7 m/s,respectively.The response times to thermal pain stimuli were 15.5s and 17.2 s,respectively.All these results proved that the regenerated nerves had the functions of sensing stimulation,conducting signals and controlling movement.To sum up,we draw the following conclusions:(1)We prepared porcine decellularized nerve matrix by a mild decellularization technique of “physical freeze-thaw + chemical washing”,in which the elution rate of genetic material was as high as 95.6%,which met the biosafety requirements;(2)We screened out hexafluoroisopropanol as a solvent for dissolving decellularized nerve matrix.When the decellularized nerve matrix concentration was 15% w/v,the electrostatic field voltage was 14 k V,and the solution pushing rate was 0.6 m L/h,the receiving distance was 10 cm,the distribution of x DNM electrospinning fibers was the most uniform and stable.Hence,we prepared pure decellularized nerve matrix electrospinning nerve graft(x DNME)under the same condition;(3)We used natural biological cross-linking agent proanthocyanidin to cross-link x DNME,which could significantly improve its mechanical properties,and the degradation rate of the graft could be regulated by the cross-linking concentration at the same time;(4)In vitro cell culture experiments showed that both x DNME and x DNME-OPC had excellent cell safety,and had good biocompatibility with nerve cells;(5)Animal experiments showed that both x DNME and x DNME-OPC had good histocompatibility when implanted in vivo.Both grafts were preferable repairing 8 mm sciatic nerve defect in rats.The above results reveal that both x DNME and x DNME-OPC have good performance in repairing peripheral nerve defects,which provides a new idea and a solid foundation for clinical repair of peripheral nerve defects. |
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