Implant Of The VEGF Sustained Delivery Acellular Spinal Cord Scaffold Promotes Vascular Remodeling In A Rat Spinal Cord Hemisection Model

Objective:To investigate the effect ofimplant of the VEGF sustained delivery acellular spinal cord scaffold promotes vascular remodeling in a rat spinal cord hemisection model,and preliminary evalute the spinal cord function recovery after spinal cord injury.Methods:1.Blank-PLGA nanospheres(B-NPs)?VEGF-PLGA nanospheres(V-NPs)were prepared by the emulsion-solvent evaporation method,ASCS was prepared by the chemical extraction.And B-NPs and V-NPswere cross-linked with ASCS by genipin.The performance of B-NPs,VEGF-PLGA,ASCS was observed by scanning electron microscopy.2.Preparation of hemisection model of SD rat.Forty-eight male SD rats were randomly divided into four groups:(1)Control group,where rats received laminectomy and hemisection of the spinal cord;(2)Sham group,where rats received laminectomy only;(3)B-ASCS group and(4)V-ASCS group,where rats received abovementioned standard surgery and transplantation of B-ASCS and V-ASCS,respectively.3.At 1,4 and 8 weeks post-injury,rats in each group were transcardially perfused withMicrofil contrast agentprior to MicroCT examination.And using ahigh-resolution Micro CT to study the 3D morphology of the spinal cord microvasculature,and quantitative analysis of 3D vascular morphometric parameters.Histopathology was observed by HE staining after 8 weeks.Nissl bodies and myelin was observed by Nissl bodies and myelin staining after 8weeks.The Basso,Beattie,and Bresnahan(BBB)locomotor rating scale was used to evaluate functional recovery of the hindlimbs at 3,7,14,21,28,35,42,49,56 day.Result:1.B-NPs and V-NPs can be prepared by the emulsion solvent evaporation method,which the drug encapsulation efficiency is high and the release rate is good.ASCS prepared by chemical extraction is simple,removing cells more thoroughly,and can maintain the integrity of the extracellular matrix.The method that B-NPs and V-NPs were cross-linked with ASCS by genipin,which note only formed a novel sustained release system of VEGF-PLGA cross-linked acellular spinal cord scaffold,but also kept the original three-dimensional structure of ASCS.2.Three-dimedsional visualization of spinal cord microvasculature: The capillary network was destroyed in left-side of the cord and remained in a delayed reconstruction in Control group,whereas it was persistently intact in Sham group during the post-operative stage.In B-ASCS and V-ASCS groups,the capillaries developed obviously from vascular sprouts,especially in the latter.3.Quantitative analysis of 3D vascular morphometric parameters :Relative to Control group,Percent vessel volume(vessel volume/total tissue volume,VV/TV)in B-ASCS and V-ASCS groups were significantly increased at 1 W post-injury(P<0.01).Especially in V-ASCS group,VV/TV was significantly higher than in other groups(P<0.01).At 4 W,VV/TV was sharply declined in V-ASCS group,whereas it was slightly changed in B-ASCS group.No statistically significant differences were seen between B-ASCS and V-ASCS groups at 8W post-injury.Moreover,the data in V-ASCS group was significantly lower than in Sham group(P<0.05).Results of Vessel density(VDn)were similar to VV/TV in comparison among the 4 groups at 1 W post-injury.Briefly,VDn was the highest in V-ASCS group,higher in B-ASCS group,and the lowest in Control group(P<0.01).At 4 W post-injury,VDn was decreased in B-ASCS and V-ASCS groups.There was no statistical difference among the 4 groups except between Control and Sham groups(P<0.05).At 8 W,VDn in V-ASCS group was getting close to that in Sham group(P>0.05),and was higher than in B-ASCS group(P<0.05).After 1 W of injury,rats received hemisection in Control,B-ASCS and V-ASCS groups were all significantly increased in average vessel diameter(VD)(P<0.01).However,there was no significant difference within each of the two groups.Then,VD in the groups was decrease with time.There was no significant difference in comparison between B-ASCS or V-ASCS group and Sham group(P>0.05)at 4 W and 8 W post-injury.Vessel-branches density(VBDn)was the highest in V-ASCS group,higher in B-ASCS group,and the lowest in Sham group(P<0.01)at 1 W post-injury.Then,it was sharply declined in V-ASCS group at 4 W and 8 W post-injury,while declined slower in B-ASCS group.VBDn in V-ASCS group was getting close to Sham group after 8 W of hemisection and transplantation(P>0.05).4.The BBB scores: There were significant differences between Sham group and all the others at all time points(P<0.01).The BBB scores of Sham group were stable throughout the experiment.Also,there were significant differences between Control group and V-ASCS group at all time points(P<0.01).Between Control group and B-ASCS group,statistical differences were observed at all time points(P <0.01,except Day 28 of P<0.05)except Day 35(P>0.05).Relative to B-ASCS group,the BBB scores in rats received V-ASCS grafts were significantly higher at Day 28,35 and 42(P<0.05).5.HE staining:At 8 weeks post-injury,the scaffolds implant in both B-ASCS and V-ASCS groups could integrate with the host tissue by bridging the rostral and caudal spinal cord gaps.However,V-ASCS was better integrated with the host tissue with comparatively smaller cavities.Relative to Sham group,cavities in Control group were the biggest with no tissue to bridge the gap.6.Nissl bodies and myelin staining:At 8 weeks post-injury,Sham group had normal Nissl bodies and myelin.Relative to Sham group,there were no Nissl bodies and myelinin Control group,while the scaffolds implant in both B-ASCS and V-ASCS groups could find both smaller Nissl bodies anddisorganized myelin.However,V-ASCS had more Nissl bodies and myelin.Conclusion:1.Implantion of the V-ASCS or B-ASCS can accelerate the vascular remodeling in a rat spinal cord hemisection model,but the V-ASCS is better.2.VEGF can promote the recovery of axons and myelin in the injured segmentin rats in histology,and can promote the limited recovery of the lower limb nerve function in rats.