Sustained Release Of Collagen ? Potentiates Sciatic Nerve Regeneration By Polarizing Macrophage Phenotype

Objective:(1)Synthesis of RADA16-I self-assembled polypeptides by Fmoc solid phase method,identification and characterization of their physicochemical properties.Preparation of PCL nerve conduits by electrospinning and determination of mechanical properties.(2)RADA16-I self-assembled hydrogel was used to encapsulate sustained-release type ? collagen,and its release curve was observed in vitro.The macrophages were induced by collagen type VI and the effect of in vitro polarized macrophages was observed.The effects of polarized macrophages on the proliferation and function of Schwann cells were studied by co-culture model.(3)To investigate the effect of RADA16-I self-assembled hydrogel encapsulated sustained-release type ? collagen filled into PCL nerve tube on repairing long sciatic nerve defect in rats.Methods:(1)Synthesis of RADA16-I self-assembled polypeptides by Fmoc solid-phase synthesis.The synthetic polypeptides were purified by RP-HPLC.The synthesis and purification of the small molecule compound RADA16-I were characterized by 1HNMR nuclear magnetic resonance(NMR).Infrared Absorption was detected by Infrared Spectroscopy.Observation of Hydrogel Fiber Morphology by Transmission Electron Microscopy.The rheological properties of self-assembled hydrogels were measured using a rheometer.The PCL conduit was prepared by electrospinning.The average diameter of the fiber was observed by scanning electron microscopy.The porosity of the fiber was measured by the modified specific gravity method.The stress-strain curve was measured by a small scale mechanical extensometer.The Young's modulus,Maximum stress,maximum load and maximum breaking tension were calculated.(2)A mixed hydrogel containing 0.1% ? collagen and 1% RADA16-I mixture was prepared and allowed to polymerize at room temperature.25 ?l of hydrogel was filled in PCL conduit and placed in PBS.The supernatant of culture was collected at 0.5,1,2,3,4,5,6,7,14,28 th day,The cumulative release rate of type ? collagen was detected by ELISA.Separation,purification,identification of Schwann cells.The rat RAW264.7 macrophages were inoculated into 24-well plates with RPMI1640 medium containing 0.1% ? collagen.After 72 hours of incubation and intervention,CD68,CCR7,iNOS,CD206 and CD163 were used to identify macrophage phenotype.The culture medium of macrophages induced by ? collagen was mixed with fresh DMEM / F12 medium in a ratio of 1:1,and the mixed medium was used to culture Schwann cells.The model was cultured and the proliferation of Schwann cells was detected by CCK-8 after 5 days of intervention.RT-PCR was used to detect the expression of GDNF,NGF,NT3 and MBP mRNA in Schwann cells under co-culture conditions.(3)A mixed hydrogel containing 0.1% ? collagen and 1% RADA16-I was prepared,filled with 17 mm PCL nerve conduit and bridged the sciatic nerve 15 mm long segment defect.Using a simple RADA16-I hydrogel filled with PCL conduit and autologous nerve grafts as a control.At 4,8 and 12 weeks postoperatively,the nerve regeneration effect was evaluated by retrograde tracing,sciatic nerve index,electrophysiological examination,immunofluorescence,regenerative nerve and muscle morphological analysis.Results:(1)The yield of RADA16-I was 85.6%.After the formation of hydrogel by RADA16-I,the peak of infrared spectrum was displaced and the hydrogel was formed in the form of ionic bond.The concentration of RADA16-I increases,and the temperature of the gel is increased.RADA16-I concentration increased,the pH value of gelation increased.(2)? collagen release cycle is very long,up to 56 days.3 days: 47.8 � 5.7%;4 days: 52.5 � 6.1%;2 days: 43.1 � 3.8%;3 days: 47.8 � 5.7%;4 days: 52.5 � 6.1% 5 days: 56.0 � 5.2%;6 days: 62.2 � 4.5%.In the 6th day to the 56 th day,the release gradually slowed down,7 days: 65.1 � 5.3%;14 days: 71.6 � 5.0%;28 days: 78.8 � 4.1%;56 days: 98.7 � 0.8%.S100 and Sox10 fluorescent double staining showed that the purity of Schwann cells was as high as 96.3%.After 72 h of collagen type VI induction,M2 macrophages accounted for 55.2% of CD68 cells.The proliferation of Schwann cells was significantly higher than that of the control group at the 3rd and 5th day(P<0.05).The expression of GDNF,NGF,NT3 and MBP in Schwann cells was significantly higher than that in the control group(P<0.05).(3)The proportion of M1 macrophages in PCL group(CCR7 + / CD68 + or iNOS + / CD68 +)was significantly higher than that in PCL / Collagen ? group at 3 days and 14 days after stent implantation(P <0.05).In the PCL / Collagen ? group,the percentage of M2 macrophages(CD206 + / CD68 + or CD163 + / CD68 +)was significantly higher than that in the PCL group(P <0.05)at 3 days and 14 days after stent implantation.The regenerated axonal area,the number of myelinated axons,myelin diameter,G-ratio and myelin thickness in PCL/Collagen VI group were significantly better than those in PCL group(P<0.05)at 3 time points.At 12 weeks,there was no significant difference between the number of medullary axons and the G-ratio between the autologous nerve group and the PCL/Collagen VI group(P> 0.05).Immunofluorescence showed that the nerve fibers and Schwann cells in the PCL/Collagen VI group were more ordered to grow,showing that the Schwann cells were wrapped along the growth direction of the axons,and also showed good regeneration effect.The number of sensory and motor neurons,sciatic nerve index,CMAP,nerve conduction velocity and CMAP latency of PCL/Collagen VI group and autologous nerve group were significantly better than those of PCL group(P<0.05).There was no significant difference in the above indexes between PCL/Collagen VI group and autologous nerve group(P> 0.05).Muscle and vascular staining analysis also showed that PCL/Collagen VI had higher muscle fiber area ratio and microvessel number.Conclusion:(1)We successfully prepared RADA16-I polypeptide which could self-assembled in to hydrogel in physiological environment.This property can use for protein encapsulation.(2)RADA16-I hydrogel can effectively encapsulate and slow release type VI collagen,and its sustained release curve is similar to that of macrophage recruitment and migration in vivo,and can be used in vivo.Type VI collagen can effectively induce the transformation of macrophages to M2 in vitro,and it can promote the biological function of Schwann cells.(3)RADA16-I hydrogel-encapsulated sustained-release type VI collagen can effectively transform macrophages into M2 in vivo,and can effectively improve the quality of nerve regeneration and recovery of motor function.