Preparation,Characterization And Biocompatibility Studies On Acellular Spinal Cord Scaffolds With Sustained Delivery Of Vascular Endothelial Growth Factor

Objectives:The objectives of the present study was first,to construct a novel sustained release system of VEGF-PLGA Crosslinked acellular spinal cord scaffold;and analysis the performance of this sustained release system,including preliminary study on the biocompatibility of nanospheres(B-NPs),acellular spinal cord scaffold(ASCS),PLGA nanosphere Acellular scaffold(B-ASCS)and bone marrow mesenchymal stem cell(BMSCs);secongd,to provide new materials,new methods and theoretical basis for the combined transplantation in the treatment of spinal cord injury(SCI).Methods:1.B-NPs ? VEGF-PLGA nanospheres(V-NPs)were prepared by the emulsion-solvent evaporation method,ASCS was prepared by the chemical extraction.And then,the performance of B-NPs,VEGF-PLGA,ASCS was evaluated.2.At the same time of crosslinking modification of ASCS,the B-NPs and V-NPs were cross-linked to ASCS through crosslinking modification of the genipin,which construct a novel sustained release system of VEGF-PLGA Crosslinked acellular spinal cord scaffold.The morphology,crosslinking rate and drug release curve of this system were used to evaluate thier performance.3.The B-NPs ? ASCS ? B-ASCS was co-cultured respectively with P3 generation BMSCs.The proliferation of BMSCs cells was tested by the CCK-8 assay on day 1,day 3,day 5 and day 7.Results:1.Under the Scanning electron microscopy,the B-NPs and VEGF-PLGA nanospheres(V-NPs)prepared by the emulsion solvent evaporation method showed that the microspheres had good morphology and smooth surface,but the average particle size of V-NPs(203.2 + 4.7nm)was slightly larger than B-NPs(180.3 + 3.9nm),the particle size distribution of V-NPs(150-250nm)was more uniform than that of B-NPs(100-280nm);The encapsulation efficiency of V-NPs was 90.8±3.1 %,which detected by the UV Spectrophotometry and in vitro,the sustained release time of the VEGF was up to 30 days.The HE staining,of the ASCs prepared by chemical extraction revealed that the scaffolds showed a pore like structure with different sizes and cellular components removed.Under the SEM scan,the scaffold showed irregular three-dimensional network structure;the surface of the extracellular matrix was rough with lamellar arrangement,and formed irregular cavity;its pore size was about 45.6±10.9 ?m,and its porosity was 82.2±2.9%.2.At the same time of crosslinking modification of ASCS,the B-NPs and V-NPs were cross-linked to ASCS through crosslinking modification of the genipin,which formed the scaffold of B-ASCS?V-ASCS.Under the SEM scan,the B-ASCS?V-ASCS maintain a good three-dimensional network pore structure;its porosity was 75.0±1.9%,73.9±2.1%,respectively;Crosslinking rate reached 71.9±3.9%,71.4±4.7%,respectively.UV spectrophotometry was used to measure the release property of V-NPs in V-ASCS bracket,and the sustained release time of VEGF165 was 30 days.3.The B-NPs?ASCS?B-ASCS were co-cultured with P3 generation BMSCs,respectively.The proliferation of BMSCs cells was tested by the CCK-8 assay on day 1,day 3,day 5 and day 7.The results showed that the BMSCs in the three groups had obvious proliferation and growth in good condition,and no significant statistically difference between groups was observed in BMSCs proliferation(p>0.05).Conlusions:1.The ideal lV-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;what's more,it can maintain the integrity of the extracellular matrix,which is consistent with the requirements of the present study,that is,maximum maintain its natural three-dimensional space structure.2.At the same time of crosslinking modification of ASCS,the B-NPs and V-NPs were cross-linked to ASCS through crosslinking modification of the genipin,which formed a novel sustained release system system of VEGF-PLGA crosslinked acellular spinal cord scaffold.The sustained release system not only keeps the original three-dimensional structure of ASCS,but also retains the slow release performance of V-NPs.3.B-NPs,ASCS,BMSCs and B-ASCS formed by the genipin have good biocompatibility without significant cytotoxicity,which can provide new materials,new methods and theoretical basis for the combined transplantation in the treatment of spinal cord injury(SCI).