Studies On Angiogenesis And Neural Recovery In A Spinal Cord Hemisection Injury Model Based On Co-delivery Of VEGF And NT-3 In Combination With Tissue Engineered Constructs

Objective:To evaluate the role of co-release of VEGF and NT-3 in vascular repair and nerve regeneration in rat spinal cord hemisection.Methods:1.Blank-PLGA nanospheres(B-NPs)?VEGF-PLGA nanospheres(V-NPs)?NT3-PLGA nanospheres(N-NPs)were prepared by the emulsion-solvent evaporation method,Calculated the Characterization?entrapment efficiency?Release kinetics of V-NPs and N-NPs.2.Isolation,culture and identification of rat BMSCs.3.Preparation of ASCS and co-culture with BMSCs.4.Preparation of hemisection model of rat.5.Identify the experimental group.6.The rats in each group were sacrificed at the 1st and 8th weeks post-injury,the spinal cords were harvested,Immunofluorescence?H&E and Nissl/myelin staining were used to assess the blood vessel changes?expressions of axonal?neurogenesis and astrogliosis?Cavity formation and Remyelination within the spinal cords in different groups.The Basso,Beattie,and Bresnahan(BBB)locomotor rating scale was used to evaluate functional recovery of the hindlimbs at 3,7,14,28,42,56 day.Result:1.V-NPS,N-NPS and B-NPS were successfully prepared,which the drug encapsulation efficiency is high and the release rate is good.2.BMSCs were successfully cultured and confirmed to have multiple differentiation potentials.3.Morphological observation of ASCS and co-culture of ASCS and BMSCs.After decellularization and lyophilization,the cellular components within the spinal cord were completely wiped off and a regular reticular structure was formed and observed by H&E staining.Under SEM observation,ASCS exhibited an interconnected porous structure,After co-culture of ASCS and BMSCs for 2 weeks in vitro,the constructs were examined by histology and SEM,verified the adhesion and proliferation of BMSCs in the 3D porous scaffolds.4.Experimental groups.4 experimental groups were established:(1)Control group (n=8)in which the rats underwent laminectomy and hemisection SCI;(2)Sham roup(n=8)in which the rats underwent laminectomy only;(3)ABC group(n=12) n which the rats underwent hemisection SCI followed by injection of -NPs-suspension and implantation of ASCS/BMSCs-constructs;(4)ABVN roup(n=12)in which the rats underwent hemisection SCI followed by injection f V-NPs&N-NPs-suspensions and implantation of ASCS/BMSCs-constructs.5.Immunofluorescence?H&E and Nissl/myelin staining.At 1 W post-injury,relative o Control and Sham groups,the densities of RECA1-positive blood vessels in BC and ABVN groups were obviously higher;NF-H was used to stain the euron-specific cytoskeletal components at 8 W post-injury.Immunolabeling for F-H revealed that NF-H positive axons within the lesion site increased mazingly due to the treatment of ABC or ABVN.Relative to ABC-treated rats, he reduction in size of cavities was observed in ABVN group;double mmunofluorescent staining of NeuN and GFAP centered at the lesion site was pplied at 8 weeks,In ABVN group,the size and density of neurons were much etter than that of ABC group,GFAP-positive astrocytes in ABC group were bviously denser than in the ABVN group.according to the H&E stained images.The rats treated with a hemisection SCI without implants formed the largest avities among 4 groups,the smaller cavities were observed in ABVN group,.And rats in ABVN group exhibited a better integration between the implant and ost spinal cord.by using the double-compound staining dyes,Cavity sizes were he largest in Control group[(0.61±0.05)mm~2 per unit area],while the data in ham,ABC and ABVN groups was(0.13±0.03),(0.42±0.06)and(0.32±0.05) espectively.Quantification of yellow-stained myelin revealed the most prominent emyelination in ABVN group[(0.25±0.05)mm~2 per unit area],while the data in BC,Control and Sham groups was(0.19±0.05),(0.09±0.03)and(0.40±0.06) espectively.6.Locomotor recovery determined by BBB scores.rats in ABVN group reached ignificantly higher locomotor scores than Control group at all time points ost-operatively,comparisons between the ABVN and ABC groups revealed that o-delivery of VEGF and NT-3 promoted significantly better locomotor recovery n rats at 28,42 and 56 days post-operatively.Conclusion:The tissue-engineered co-release of VEGF and NT3 significantly promoted the repair of nerves and the regeneration of blood vessels in rats with spinal cord emisection injury.It can also be seen from the BBB score that the motor function of rats with spinal cord hemisection injury is significantly improved and restored.