掺锶聚磷酸钙对成骨细胞分泌血管内皮生长因子的影响

Effect of strontium-doped calcium polyphosphate on the secretion of vascular endothelial growth factor from osteoblastic cells

背景:掺锶聚磷酸钙作为一种新型骨修复材料,具有良好的生物相容性和可控降解性。课题组前期研究表明其具有促进血管生成的作用,但其机制尚不清楚。目的:将ROS17/2.8成骨细胞与掺锶聚磷酸钙支架在体外共培养,观察细胞增殖情况和促血管生成因子血管内皮生长因子的分泌情况。设计、时间及地点:对比观察实验,于2008-10/2009-06在四川大学组织工程研究室完成。材料:制备掺锶量为1%,2%,5%,8%,10%的掺锶聚磷酸钙和未掺锶聚磷酸钙骨组织工程支架材料。ROS17/2.8成骨细胞株购自于四川大学华西医院移植免疫与移植工程实验室。方法:①制备细胞支架复合物:将材料置于24孔培养板中,每孔接种300μL细胞悬液(细胞浓度为2×107L-1),培养14d,隔天换液。②分别于1,3,5,7,10,14d行MTT法观察成骨细胞的增殖情况。③将培养7d的细胞支架复合物,离心取上清液,用夹心ELISA法检测血管内皮生长因子的分泌情况。主要观察指标:观察掺锶聚磷酸钙和未掺锶聚磷酸钙支架上成骨细胞的增殖情况及血管内皮生长因子的分泌情况。结果:MTT法实验结果表明,与未掺锶聚磷酸钙组相比,掺锶聚磷酸钙组能促进成骨细胞的增殖,且8%掺锶聚磷酸钙效果最好;ELISA结果表明,与未掺锶聚磷酸钙组相比,掺锶聚磷酸钙组能上调血管内皮生长因子的蛋白分泌量,其中以8%掺锶聚磷酸钙促进作用最为明显(P<0.05)。结论:锶能有效促进成骨细胞增殖,并能明显上调血管内皮生长因子的分泌,且以8%掺锶聚磷酸钙效果最好,在一定程度上揭示了其具有促血管化作用的机制。

BACKGROUND： Strontium-doped calcium polyphosphate （SCPP） is a new type of bone repair materials with good biocompatibility and controlled degradation. The preliminary studies of our group indicate their role in promoting angiogenesis, but its mechanism is unclear. OBJECTIVE： By co-culturing osteoblasts ROS17/2.8 with SCPP in vitro to observe cell proliferation and the secretion of vascular endothelial growth factor （VEGF）. DESIGN, TIME AND SETTING： A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2008 to June 2009. MATERIALS： A series of calcium polyphosphate （CPP） respectively containing 0%, 1%, 2%, 5%, 8%, and 10% Sr2＋ were prepared. ROS17/2.8 osteoblastic cell strain was provided by Laboratory of Transplantation Immunity and Transplantation Engineering, West China Hospital, Sichuan University. METHODS： ① Preparation of cell scaffold complexes： The materials were placed in 24-well plates, then 300 μL cell suspension with a concentration of 2×107 cells/L was inoculated into each hole. These complexes were cultured for 14 days and the liquid was changed every two days. ② These complexes were measured by MTT assay to observe the proliferation of osteoblasts on the 1st, 3rd, 5th, 7th, 10th and 14th days, respectively. ③ The centrifugal supernatant of the complex cultured for seven days was measured by ELISA assay to check the secretion of VEGF. MAIN OUTCOME MEASURES： The proliferation of osteoblastic cells on SCPP and CPP was observed. The amount of VEGF protein secreting from osteoblastic cells was detected. RESULTS： The results of MTT showed that, compared with the CPP group, SCPP groups could promote the proliferation of osteoblasts, and 8% SCPP group was the best; ELISA results showed that, compared with the CPP group, SCPP groups could increase the amount of VEGF protein secretion, of which the promoting role of 8% SCPP was the most obvious （P 〈 0.05）. CONCLUSION： When cultured with osteoblasts, SCPP can promote cell proliferation, and can significantly increase the secretion of VEGF; moreover, 8% SCPP is the best, which reveals a certain mechanism of its promoting angiogenesis.