增强型生物活性玻璃/胶原复合材料的促血管化作用

PROMOTED VASCULARIZATION OF ENHANCED BIOACTIVE GLASS/COLLAGEN COMPOSITE SCAFFOLD

目的骨组织工程所使用的支架材料能否快速、有效地血管化是骨修复的关键。研究增强型生物活性玻璃/胶原复合材料对血管化的协同和促进作用,为构建血管化组织工程骨修复骨缺损寻找适宜的支架材料。方法体外分离获取人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs),并通过血管性血友病因子(von Willebrand factor,vWF)与CD34抗原鉴定,取第1代细胞用于实验。将细胞接种于增强型生物活性玻璃/胶原复合材料上,扫描电镜观察细胞黏附情况。取细胞接种于增强型生物活性玻璃/胶原复合材料作为实验组,等量细胞直接接种于培养板常规培养作为对照组,采用alarmarBlue法动态检测细胞增殖率,实时荧光定量PCR检测内皮细胞相关基因VEGF、血管内皮生长因子受体1(fms-related tyrosine kinase1,Flt-1)、血管内皮生长因子受体2(kinase insert domain receptor,Kdr)的mRNA表达。取SD大鼠6只,将支架材料包埋于大鼠股内侧皮下,实验组采用增强型生物活性玻璃/胶原复合材料、中央轴向植入血管束,对照组采用非增强型生物活性玻璃/胶原复合材料,分别于植入5、10d取出,行冰冻切片并HE染色动态观察支架材料内部的血管化状态。结果分离的细胞通过形态学及vWF、CD34免疫荧光鉴定为HU VECs。扫描电镜示HUVECs在支架材料上黏附较好。HUVECs在实验组支架材料上增殖明显,在第3天后细胞增殖率开始高于对照组,11d达到增殖平台期时细胞数仍多于对照组(P<0.05)。实时荧光定量PCR检测示,培养3d实验组VEGF、Flt-1、Kdr的mRNA表达均显著高于对照组(P<0.05)。包埋大鼠皮下实验可见,实验组5、10d时植入的血管仍通畅,其周围新生血管随时间延长而增多,材料周缘可见宿主血管浸润生长,但新生血管稀疏;对照组仅有纤维组织生长,10d时材料已大部分降解,组织难以长入。结论增强型生物活性玻璃/胶原复合材料在大鼠体内外可促进血管化,可能适于作为构建血管化组织工程骨的支架材料。

Objective Rapid and effective vascularization of scaffolds used for bone tissue engineering is critical to bony repair.To study the cooperative and promotion effects of enhanced bioactive glass/collagen composite scaffold on vascularization for searching for a kind of eligible vascularized scaffold to repair bone defect.Methods The human umbilical vein endothelial cells（HUVECs） were collected from human umbilical core,and identified through von Willebrand factor（vWF） and CD34 immunofluorescence.The 1st passage of HUVECs were suspensed and seeded into the scaffold.The attachment and proliferation of HUVECs on the scaffold were observed through scanning electron microscope（SEM）.HUVECs were seeded on the scaffold as the experimental group,and on 96-well plate as the control group.The growth rate of HUVECs was detected through alarmarBlue at 1,3,5,7,9,and 11 days.Meanwhile,the mRNA expression levels of VEGF,fms-related tyrosine kinase 1（Flt-1）,and kinase insert domain receptor（Kdr） were detected through real-time fluorescence quantitative PCR.Twelve scaffolds were embedded subcutaneouly into 6 Sprague-Dawley rats.The enhanced scaffolds were used and the arteria and vein saphena bundle were embedded straightly through the central slot of scaffold in experimental group,and the common scaffolds were used in control group.Frozen section and HE staining of scaffolds were performed at 5 days and 10 days to observe the vascularization of embedded scaffold.Results HUVECs were identified through morphology,vWF and CD34 immunofluorescence.SEM results showed HUVECs could attach to the scaffold tightly and viably.HUVECs proliferated actively on the scaffold in experimental group;the growth rate in experimental group was higher than that in control group at 3-11 days,showing significant differences within 5-11 days（P 0.05）.The real-time fluorescence quantitative PCR results showed that the mRNA expression levels of VEGF,Flt-1,and Kdr in experimental group were higher than those in control group at 3 days,showing significant differences（P 0.05）.Frozen section and HE staining of the scaffolds in experimental group showed that the embedded vessel bundle were still patency at 5 days and 10 days,that many new vessels were observed around the embedded vessel bundle and increased with time,host vessels infiltrated in the surrounding area of scaffold and fewer neo-vessels at the distant area.But there was only some fibrous tissue appeared in control group,and at 10 days,the common scaffold degradated,so few normal tissue appeared at the embedded area.Conclusion Enhanced bioactive glass/collagen composite scaffold can promote vascularization in vitro and in vivo,and may be used in bone tissue engineering.