Repair Of Long Segmental Defects Of Sciatic Nerve By Hyaluronic Acid Hydrogel Sustained-Release Schwann Cell Exosome

Objective:(1)Isolation and culture of Schwann cells from newborn SD rats in vitro,and extracting exosomes secreted by Schwann cells by ultracentrifugation and identifying specific proteins.Transmission electron microscopy was used to observe the particle size and distribution of Schwann cell exosomes.(2)To study the biological effects of Schwann cell exosomes on neurons and Schwann cells.Observe and study the biological characteristics of hyaluronic acid gel sustained-release exosomes.(3)To investigate the morphological and functional effects of hyaluronic acid hydrogel-coated slow-release Schwann cell exosomes filled with PCL nerve conduit for repairing long segmental defects of rat sciatic nerve.Methods:(1)Primary rat Schwann cells were extracted by 0.1%type IV collagenase digestion and cultured and purified by DMEM/F12 complete medium.Purity identification was performed using S100 and Sox10.The proliferation status of Schwann cells was detected using a CCK-8 kit.The exosomes secreted by Schwann cells were extracted by ultra-high speed centrifugation,and the content of protein in exosomes was detected by BCA kit.The exosome surface markers CD63,TSG101,CD9,Rab5 and Na~+/K~+ATPase secreted by Schwann cells were identified by Western blot.(2)Construct a 1%strength hyaluronic acid hydrogel and wrap the Schwann cell exosomes.The sustained release profile of the exosomes was detected by placing the BCA kit in vitro in PBS.The Schwann cell exosomes were used to intervene in Schwann cells and neurons,and the CCK-8 kit was used to detect the proliferation of Schwann cells by exosomes and the secretion of bioactive proteins of the two cells.After 1,3,and 5 days of culture,the exosome-intervened Schwann cells expressed levels of NGF,BDNF,myelin zero protein(P0),and cell polar protein Par-3.At the same time,ELISA was used to detect growth-associated protein-43(GAP-43)and neurofilament-200(NF-200)in neurons on day 1,3 and 5 after exosome intervention.The expression level of?III-tubulin.(3)PCL nerve catheter was prepared by electrospinning,and the general morphology and microstructure of the catheter were observed by scanning electron microscopy.The diameter of the fiber filament was measured.The mechanical parameters such as Young's modulus,maximum stress,maximum load and maximum breaking tension of PCL nerve conduit were detected by a small-scale mechanical instrument.A 15 mm long segmental rat sciatic nerve defect was prepared by hyaluronic acid hydrogel-encapsulated Schwann cell exosomes filled with PCL nerve conduit,and end-to-end anastomosis was performed.A PCL catheter filled with hyaluronic acid alone was used as a control group,and a control was also performed in the autologous nerve transplantation group.Rat sciatic nerve index and regenerative nerve electrophysiological function were measured at 4,8,and 12 weeks after surgery.Fluorescent gold retrograde tracing was used to detect the survival of neurons and the regeneration of nerve cells in rats.The diameter of myelinated axons,the number of myelinated axons,and the area of regenerated axons were observed by transmission electron microscopy.NF-200 and S100 immunofluorescence staining of each group of regenerative nerves,and the regeneration of nerve structure during observation period.HE staining was used to observe the difference of muscle fiber diameter of gastrocnemius in three groups of rats.Results:(1)Schwann cells can be seen as a shuttle type under light microscope,the volume is not large,there are two poles,the nucleus is in the middle,relatively obvious,and has refractive index.The cells are mostly in a longitudinal arrangement.After 2 hours of inoculation,the rate of adherence of Schwann cells was about 23.5%,the adherence ratio increased to 47.9%at 6 hours,and 88.9%of cells adhered at 12hours,and the cells were attached after 16 hours.The ratio of the walls is higher than99%.At 3 days of culture,the number of Schwann cells increased by 3.9 times compared with the original,and increased by 11.4 times when cultured for 7 days.The purity of S-100 and Sox-10 double-labeled fluorescent staining for Schwann cells was 97%.The exosome particle size is mainly distributed between 100-160 nm,and the average diameter of the exosomes measured in this study is 125 nm.Western blot analysis showed that exosomal specific proteins CD63,TSG101 and CD9 were expressed in the Schwann cell exosomes extracted from this study,but not in the exosome expressing proteins Rab5 and Na~+/K~+ATPase.(2)The release rate of exosomes in the hyaluronic acid gel was higher in the first 3 days,the release rate was34%on the first day and 41%on the third day,and the release gradually became slower with the passage of time.The cumulative release ratio at days 4,5,6,8,16,and 32 was 43%,47%,51%,53%,75%,90%,and the cumulative release rate was measured at day 64.99%,almost completely released.The number of axons in the control group was 35.4±4.3,and that in the exosomal stimulation group was 126.3±14.4(P<0.05).In terms of axon growth length,the average length of the exosomes group was as long as(1563.0±132.5)?m,which was significantly higher than that of the control group(864.3±77.4)?m(P<0.05).The cell proliferation level of Schwann cells in the exosomes intervention group was significantly higher than that in the control group on the 3rd,5th and 7th day after culture(P<0.05).The levels of NGF,BDNF,myelin zero protein(P0)and cell polar protein Par-3 secreted by Schwann cells after exosome stimulation were significantly increased on the 3rd and 5th day after culture compared with the first day.The expression level on day 5 was also significantly higher than that on day 3(P<0.05).The marker proteins secreted by exosome-stimulated DRG neurons were detected,and the levels of GAP-43,NF-200,and?III-tubulin were significantly increased on the 3rd and 5th day after culture compared with the first day.High,and the expression level on day 5 was also significantly higher than that on day 3(P<0.05).(3)The diameter of the inner hole of the PCL nerve conduit is about 1500?m,and the thickness of the tube wall is about500?m,the surface fiber filaments are evenly distributed,and the average diameter of the fiber filament is 4.9?m.The porosity of PCL nerve conduit is(89.7±2.6)%,the maximum stress is(7.5±0.6)MPa,the Young's modulus is(22.4±2.1)MPa,the maximum breaking tension is(596.4±33.8)%,and the maximum load is(5.6±0.8)N,in line with nerve regeneration requirements.At 4,8,and 12 weeks after surgery,the diameter of regenerated nerve myelinated axons,the number of myeloid axons,and the area of regenerative axons in the PCL/HA-EXO group were significantly higher than those in the PCL/HA group.The-ratio value was significantly lower than the PCL/HA group(P<0.05).At three time points,the number of myelinated axons,the area of regenerated axons and G-ratio values of PCL/HA-EXO were not significantly different from those of autologous nerve transplantation group(P>0.05).At 12 weeks after surgery,the axons of the PCL/HA-EXO group grew antegrade along the catheter with obvious directionality.The expression of axons and Schwann cells was significantly higher than that of the PCL/HA group,which was not significantly different from the autologous nerve transplantation group.The postoperative action potential amplitude,nerve conduction velocity and latency of the rats in each group improved with time,but the PCL/HA-EXO group and the autologous nerve group were more effective than the PCL/HA group.The three time points after 4,8,and 12weeks were significantly higher than those in the PCL/HA group(P<0.05).At the 8th week after surgery,there was no significant difference in nerve conduction velocity and latency between PCL/HA-EXO group and autologous nerve transplantation group,but there were still some differences in compound muscle action potential(P<0.05).At 12 weeks postoperatively,there were no significant differences between the three indicators in the PCL/HA-EXO group and the autologous nerve transplantation group(P>0.05).The SFI index of PCL/HA-EXO group and autologous nerve transplantation group was significantly higher than that of PCL/HA group at three time points after operation(P<0.05),and PCL/HA-EXO at the 8th and12th week after operation.The SFI index of the group and the autologous nerve transplantation group were similar,and there was no statistical difference(P>0.05).The diameters of the gastrocnemius muscles in the PCL/HA-EXO group and the autologous nerve graft group were(68.4±6.2)?m and(72.3±7.6)?m,which were significantly higher than those in the PCL/HA group(P<0.05),and PCL.There was no significant difference between the/HA-EXO group and the autologous nerve transplantation group(P>0.05).Conclusion:(1)This study successfully isolated and extracted the neonatal SD rat Schwann cells with high purity,which meets the requirements of subsequent experiments.Ultracentrifugation can successfully extract exosomes secreted by Schwann cells,which are identified as exosomes secreted by Schwann cells with high purity.(2)Hyaluronic acid hydrogel can effectively encapsulate and release exocrine secreted by Schwann cells,and its sustained-release curve is similar to the time of Schwann cell proliferation and migration and axon regeneration in vivo,which can be better in In vivo application.Exosomes secreted by Schwann cells can effectively promote the proliferation of Schwann cells,promote the secretion of Schwann cells and neuron-specific proteins,and promote nerve regeneration.(3)PCL/HA-EXO nerve conduit can effectively promote the recovery of motor function and histological and electrophysiological improvement of long segmental nerve defects in rats.