【作者】 吴志强；

【导师】 许卫红；

【作者基本信息】 福建医科大学 ， 外科学（骨外）， 2017， 硕士

【摘要】 目的构建一种新型VEGF-PLGA交联脱细胞脊髓支架的缓释系统,分析该缓释系统性能;初步研究纳米微球(B-NPs)、脊髓脱细胞支架(ASCS)、PLGA纳米微球脱细胞支架(B-ASCS)与骨髓间充质干细胞(BMSCs)的生物相容性,为后续联合移植治疗脊髓损伤(SCI)提供新材料、新方法和理论依据。方法1.分别采用乳化-溶剂挥发法制备B-NPs、VEGF-PLGA纳米微球(V-NPs)、化学萃取法制备ASCS,并评价其性能;2.通过京尼平的交联改性作用,在对ASCS交联改性的同时,分别将B-NPs、V-NPs结合到ASCS上,制备纳米微球交联脱细胞脊髓支架的缓释系统,并用形态学、交联率、药物释放曲线等评价其相关性能;3.将B-NPs、ASCS、B-ASCS分别与P3代BMSCs共培养,于1天、3天、5天、7天用CCK-8检测BMSCs细胞增殖情况。结果1.通过乳化-溶剂挥发法制备的B-NPs、V-NPs,扫描电镜检测示微球形态均良好、表面光滑,但V-NPs的平均粒径(203.2±4.7nm)稍大于B-NPs(180.3±3.9nm),V-NPs的粒径分布(150-250nm)比B-NPs(100-280nm)更为均匀;用紫外分光光度法检测V-NPs包封率为90.8±3.1%,体外VEGF165持续缓释时间可达30天。采用化学萃取法制备的ASCS,HE染色见支架呈现大小不等的网孔状结构,细胞成分基本去除;SEM扫描见支架呈不规则网孔状三维结构,细胞外基质表面粗糙状,并呈层状排列,形成不规则空腔,孔径约45.6±10.9μm,孔隙率达82.2±2.9%。2.通过京尼平的交联改性作用,在对ASCS交联改性的同时,分别将B-NPs、V-NPs结合到ASCS上,形成B-ASCS、V-ASCS支架。SEM扫描见B-ASCS、V-ASCS仍保持良好的三维网孔状结构,孔隙率分别为75.0±1.9%、73.9±2.1%,交联率分别达71.9±3.9%、71.4±4.7%;紫外分光光度法检测V-ASCS支架中的V-NPs缓释性能良好,VEGF165缓释持续时间亦可达30天。3.将B-NPs、ASCS及用京尼平交联形成的B-ASCS分别与P3代BMSCs共培养后,并于1天、3天、5天、7天用CCK-8检测,结果显示三组材料中BMSCs均增殖明显、生长状态良好,且组间BMSCs增殖性均无明显差异,无统计学意义(P>0.05)。结论1.通过乳化-溶剂挥发法可制备理想的V-NPs,其药物包封率高、缓释性良好;采用化学萃取法制备的ASCS程序简便,去除细胞较彻底,并能尽量保持细胞外基质的完整性,最大限度的保持其天然三维空间立体结构,符合本课题前期要求。2.通过京尼平的交联改性作用,在对脱细胞支架改性的同时,成功地将缓释纳米微球结合到脱细胞支架上,形成新型缓释纳米微球交联脱细胞脊髓支架的缓释系统;该缓释系统不仅保留了ASCS原有的天然三维空间立体结构,而且保存了纳米微球良好的缓释性能。3.B-NPs、ASCS及用京尼平交联形成的B-ASCS均与BMSCs具有良好的生物相容性,无明显细胞毒性,可为后续联合移植治疗SCI提供新材料、新方法和理论依据。更多还原

【Abstract】 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 μm,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).更多还原