压力刺激对组织工程骨替代物构建的促进作用研究  

The effects of pressure stimulation on enhancing the construction of tissue engineering bone substitute

作　　者：吕兰欣 颜晓庆 杨红宁 韩东 胡书群 LV Lan-Xin;YAN Xiao-Qing;YANG Hong-Ning;HAN Dong;HU Shun-Qun(Institute of Health Emergency,Xuzhou Medical University,Emergency Center,Affiliate Hospital of Xuzhou Medical University Xuzhou,Xuzhou 221002,China)

机构地区：[1]徐州医科大学卫生应急研究所,徐州医科大学附属医院急救中心,江苏徐州221002

出　　处：《转化医学电子杂志》2018年第11期1-5,共5页E-Journal of Translational Medicine

基　　金：国家自然科学基金项目(31800810);江苏省自然科学基金(BK20180998);江苏省博士后基金(2018K055B);江苏省高校自然科学研究面上项目(16KJB310018);徐州市科技计划项目(KC16SY156)

摘　　要：目的:构建仿生三维多孔支架-细胞复合物,探讨压力刺激对其成骨作用的影响。方法:用颗粒沥滤法制备聚乳酸-聚乙醇酸共聚物(PLGA)三维多孔支架,与人骨髓间充质干细胞(hMSCs)和人脐静脉内皮细胞(HUVECs)共培养,形成支架-细胞复合物,用PKH26染料对hMSCs进行细胞膜染色示踪,在第1、3、7天用激光共聚焦显微镜观察,在第3、5、7、9天用CCK-8试剂盒分别检测细胞增殖情况;培养10 d后将此复合物置于压力型生物反应器中进行压力刺激,检测其成骨及血管化情况;然后将压力刺激后的复合物植入鸡尿囊胚模型(CAM)中,7 d后取出,用micro-CT检测其成骨情况。结果:细胞示踪及CCK-8检测显示细胞增殖良好; micro-CT结果显示压力刺激后矿化显著增多,但总量较少; CD31免疫荧光染色结果显示压力刺激对血管生成作用不明显; CAM实验显示,压力刺激后的支架-细胞复合物对于体内骨生成有显著促进作用。结论:构建的支架-细胞复合物在压力刺激下可以快速骨化,是一种有应用潜力的组织工程骨替代物。Objective: To construct biomimetic 3D porous scaffold-cell composite and study the effect of pressure stimulation on its bone formation. Methods: Poly lactic-co-glycolic acid( PLGA) porous scaffolds were prepared by particles leaching method and co-cultured with human mesenchymal stem cells( hMSCs) and human umbilical vein endothelial cells( HUVECs) to form scaffold-cell composite,on which h MSCs were stained by PKH26,one type of cell membrane staining dye with red fluorescence. Confocal microscopy were used to observe cells after 1,3 and 7 days of culture. CCK-8 experiments were done to detect the proliferation of cells after culturing for 3,5,7 and 9 days. After 10 days culture in normal condition( static culture),scaffold-cell composite were transferred into pressure bioreactor for one hour( loading culture) and assessed the bone and vascular formation after one day static culture. In order to mimic in vivo condition,loading cultured composites were transplanted into chicken allantoic model( CAM) for 7 days,and then detected the bone formation by micro-CT. Results: Cell tracking and CCK-8 results showed that cells on scaffolds had good viability; Micro-CT showed that loading culture had more calcium deposit than static culture,while the total amount of calcium deposit in both groups was low. CD31 immunofluorescence staining showed that loading culture had no effect on vascularization. CAM results indicated that loading culture had significant effects on bone formation of scaffold-cell composite in vivo. Conclusion: The scaffold-cell composites we fabricated can ossify quickly under loading culture condition and is one kind of potential tissue engineered bone substitute.

关 键 词：多孔支架 压力刺激 共培养 

分 类 号：R318[医药卫生—生物医学工程]