载EPO腺病毒的PLGA纳米纤维支架在体内促进骨缺损修复作用

目的: 将促红细胞生成素(EPO)腺病毒局限在应用部位,观察其在体内的促进骨形成作用。方法: 采用冷冻干燥法,将增强型绿色荧光蛋白(EGFP)腺病毒与聚乳酸羟基乙酸共聚物(PLGA)纳米纤维支架复合作为实验组(PLGA/Ad-EGFP组),以等剂量的EGFP腺病毒为对照组,分别感染骨髓基质细胞(BMSCs),以BMSCs感染EGFP的荧光细胞面积比例检测病毒活力。采用Picogreen dsDNA定量检测试剂盒检测病毒释放动力学。将EPO腺病毒与PLGA纳米纤维支架冻干复合作为实验组(PLGA/Ad-EPO组),以单纯骨缺损为对照组,植入大鼠颅骨缺损处,在第4和8周采用Micro CT及组织学HE染色检测EPO腺病毒的新生骨面积百分比。结果: PLGA/Ad-EGFP组荧光细胞面积比例明显高于对照组(P<0.05);腺病毒在PLGA上呈突释曲线,第1小时存留53.0%±5.6%,第16小时存留20.0%±3.3%。与对照组比较,PLGA/Ad-EPO组在第4周促进红细胞生成,并在第4及8周大鼠颅骨缺损修复,新生骨面积百分比达到25.0%±5.7%及35.0%±6.3%(P<0.05)。结论: 应用冻干法将腺病毒与PLGA复合能够有效保存病毒活性, PLGA /Ad-EPO纳米纤维支架能够在一定程度上促进骨缺损修复。

Promotive effect of PLGA nanofiber scaffold adsorped Epo gene mediated by adenovirus on bone defect repair

Objective To investigate the influence of erythropoietin(EPO) in the bone defect after localized on polylactic-co-glycolic acid(PLGA) surface. Methods The bone marrow stromal cells (BMSCs) were isolated and cultured,and the adenovirus vector AdCMV-EGFP was used for gene transfection. The lyophilization method was used to compound AdCMV-EGFP and PLGA nanofiber scaffold as experiment group(PLGA/Ad-EGFP group),and the equivalent Ad-EGFP as control group;the adenovirus viability was detected using the percentage of fluorescence cell area of BMSCs infected with EGFP.Quantitative Picogreen dsDNA kit was used to detect the release kinetics of adenovirus.The Ad-EPO and PLGA nanofiber scaffold was compounded as experiment group(PLGA/Ad-EPO group),and single bone defect as control group;the PLGA/Ad-EPO were implanted into the critical skull bone defect of the rats,and the percentages of new bone areas at the 4th week and 8th week were detected by muro-CT and HE staining. Results Compared with control group,the percentage of fluorescentce cell area of the rats in PLGA/Ad-EGFP group was increased(P<0.05).The release kinetics of adenovirus on PLGA was a burst release curve,the retention on PLGA of adenovirus in the first hour was 53.0%±5.6%,and in the 16th hour was 20.0%±3.3%. Compared with control group, the erythrocytopoiesis was promoted at the 4th week, and the percentages of new bone areas at the 4th and 8th week were increased to 25.0%±5.7% and 35.0%±6.3% in PLGA/Ad-EPO group(P<0.05). Conclusion The lyophilizaion method could reserve the adenovirus activity,and PLGA/Ad-EPO nanofiber scaffold could promote the bone defect repair in a certain extent.