[负载血管内皮生长因子165的多孔聚己内酯支架对脂肪干细胞成骨分化的影响]
[Effect of vascular endothelial growth factor 165-loaded porous poly (ε-caprolactone) scaffolds on the osteogenic differentiation of adipose-derived stem cells].
作者信息
Xu Wanlin, Lu Hao, Ye Jinhai, Yang Wenjun
机构信息
Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200011, P.R.China;Shanghai Key Laboratory of Stomatology/Shanghai Research Institute of Stomatology, National Clinical Research Center of Stomatology, Shanghai, 200011, P.R.China.
Jiangsu Provincial Key Laboratory of Oral Diseases, Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Stomatology, Nanjing Medical University, Nanjing Jiangsu, 210029, P.R.China.
出版信息
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018 Mar 15;32(3):270-275. doi: 10.7507/1002-1892.201710064.
OBJECTIVE
To explore the effect of vascular endothelial growth factor 165 (VEGF )-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).
METHODS
The VEGF -loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs . Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models ( =12). The 12 calvarial bone defects were randomly divided into 3 group ( =4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects .
RESULTS
The morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group ( =10.761, =0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group ( <0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group ( <0.05).
CONCLUSION
The VEGF -loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both and .
目的
探讨负载血管内皮生长因子165（VEGF）的多孔聚己内酯（PCL）支架对脂肪来源干细胞（ADSCs）成骨分化的影响。
方法
通过溶剂浇铸/盐析和热致相分离技术相结合制备负载VEGF的多孔PCL支架（记作Sf-g/VEGF），然后在扫描电子显微镜（SEM）下观察。用ELISA试剂盒测定释放动力学。从15只Sprague Dawley大鼠的腹股沟脂肪垫中分离并培养ADSCs。将第3-4代ADSCs接种到支架上，然后培养7天。将第3-4代ADSCs接种到多孔PCL支架（记作Sf-g）上作为对照。进行茜素红S（ARS）染色、ARS活性测定和实时定量PCR（RT-PCR）以检测ADSCs的成骨分化。招募6只Sprague Dawley大鼠制备双侧颅骨缺损模型（n = 12）。将12处颅骨缺损随机分为3组（n = 4）。阴性对照组缺损不做处理；Sf-g组和Sf-g/VEGF组的缺损分别用ADSCs-Sf-g支架复合物和ADSCs-Sf-g/VEGF支架复合物修复。移植后8周，进行Micro-CT和HE染色以评估成骨效果。
结果
Sf-g/VEGF支架形态多孔且连接良好，120小时内累积释放率约为80%。ARS染色显示Sf-g/VEGF组的ARS活性强于Sf-g组（F = 10.761，P = 0.000）。Sf-g/VEGF组中，成骨特异性标志物[富含AT序列的特殊蛋白2（Satb2）、碱性磷酸酶（ALP）、骨钙素（OCN）和骨桥蛋白（OPN）]的mRNA表达显著高于Sf-g组（P < 0.05）。Micro-CT和HE染色结果也证实了Sf-g/VEGF支架的促进作用。两组的所有缺损均有新骨组织部分修复，尤其是在Sf-g/VEGF组。Sf-g/VEGF组新骨组织的体积和面积显著高于Sf-g组（P < 0.05）。
结论
负载VEGF的支架在体内和体外均可显著促进ADSCs的成骨分化。
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