RGD多肽修饰多孔钽对MG63成骨样细胞-钽界面形态及成骨因子表达的影响  

Effects of RGD peptides-grafted porous tantalum on morphological change of MG63 osteoblasts-tantalum conjunctive interface and expression of osteogenesis factors

作　　者：甘洪全 王茜 张辉[3] 刘鑫[4] 邓华民 宋会平 王志强 李琪佳 GAN Hong-quan;WANG Qian;ZHANG Hui;LIU Xin;DENG Hua-min;SONG Hui-ping;WANG ZHi-qiang;LI Qi-jia(Department of Orthopaedics, Affiliated Hospital, North China University of Science and Technology;Department of Anatomy, Basic Medical College, North China University of Science and Technology;Department of Joint Surgery, the Second Hospital of Tangshan;Department of Clinical Laboratory, Kailuan General Hospital;Experimental Center,North China University of Science and Technology, Tangshan 063000, Hebei, China)

机构地区：[1]华北理工大学附属医院骨科 [2]华北理工大学基础医学院人体解剖系 [3]唐山市第二医院关节科 [4]开滦总医院检验科 [5]华北理工大学医学实验中心,河北唐山063000

出　　处：《北京大学学报（医学版）》2018年第1期176-182,共7页Journal of Peking University:Health Sciences

基　　金："十二五"国家科技支撑计划项目(2012BAE06B03);河北省科技支撑项目(16277776D);河北省卫生和计划生育委员会2015年临床医学优秀人才培养项目(361036);河北省医学科学研究重点课题计划(20160225)资助~~

摘　　要：目的:通过观察精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp polypeptedes,RGD)多肽表面修饰多孔钽对MG63成骨样细胞-多孔钽结合界面形态变化以及相关成骨因子表达的影响,评价RGD修饰多孔钽对钽-骨界面骨整合所起的重要作用。方法:直径10 mm、厚3 mm的多孔钽片通过表面吸附法分别将3种质量浓度的RGD溶液(1 g/L、5g/L、10 g/L)吸附于多孔钽表面。取第2代MG63成骨样细胞与钽-RGD复合培养成为成骨细胞/钽/RGD复合物,分为4组:未修饰Ta组(对照组),1 g/L成骨细胞/Ta/RGD组,5 g/L成骨细胞/Ta/RGD组及10 g/L成骨细胞/Ta/RGD组。扫描电子显微镜观察多孔钽及复合物外观特征、成骨细胞与Ta/RGD界面的相互作用,测定成骨细胞在Ta/RGD材料上的黏附率,免疫细胞化学染色检测各组成骨蛋白骨钙蛋白(osteocalcin,OC)、纤维连接蛋白(fibronectin,FN)及丝状肌动蛋白(filamentous actin,F-actin)的表达。结果:扫描电子显微镜观察,成骨细胞在RGD修饰后多孔钽界面铺展并向孔隙内伸延,分泌细胞外基质覆盖于多孔钽微孔内及界面。24 h、48 h及72 h各组成骨细胞在Ta/RGD材料界面的黏附率均高于未修饰组(对照组),其中5 g/L成骨细胞/Ta/RGD组在48 h的黏附力最强[(68.07±3.80)vs.(23.40±4.39),P<0.05]。免疫细胞化学染色发现,成骨细胞与Ta/RGD复合培养48 h成骨细胞OC、FN及F-actin的阳性表达均高于未修饰组,其中5 g/L成骨细胞/Ta/RGD组高于10 g/L组及1 g/L组[OC:(18.08±0.08)vs.(15.14±0.19),P<0.05;(18.08±0.08)vs.(14.04±0.61),P<0.05。FN:(24.60±0.98)vs.(15.90±0.53),P<0.05;(24.60±0.98)vs.(15.30±0.42),P<0.05。F-actin:(29.20±1.31)vs.(24.50±1.51),P<0.05;(29.20±1.31)vs.(16.92±0.40),P<0.05]。细胞骨架蛋白F-actin呈丝状分布于胞质,沿细胞胞突纵向排列,其蛋白表达高于OC及FN。结论:RGD多肽有利于成骨细胞在多孔钽界面黏附、铺展及细胞骨架蛋白的重组,从而促进成骨细胞-多孔钽界面改建及骨整合。Objective： To investigate the effects o f the A rg -G ly-Asp polypeptedes （R G D ） peptides- modified porous tantalum surface on osteoblasts morphology and expressions o f osteogenesis fa c to rs, and to evaluate RGD peptides promotes ju n c tu ra ossium o f tantalum-bone interface in vivo. M e th o d s ： RGD peptides of different concentrations （1 g/L , 5 g/L , and 10 g/L ） were loaded to porous tantalum slices with a diameter of 10 mm and a thickness o f 3 mm by physical absorption. The 3rd generation o f MG63 cells were co-cultured wi th tantalum and d iv ide d in to 4 groups： T a-cel ls （c o n t ro l） g ro u p, 1 g / L cells/ Ta/RGD group, 5 g/L cells/Ta/RGD g ro u p, and 10 g/ L cells/T a /R G D group. Porous tantalum compo-sites and osteoblasts-tantalum interface were observed by scanning electron microscopy. The adhesion rate of osteoblasts was detected and immunocytochemistry was used to detect the expressions of filamentous ac- tin （ F-actin） , osteocalcin （ OC ） and fibronectin （ FN） . R e sults ： The scanning electron microscope （SEM） revealed that osteoblasts distributed on the surface of porous tantalum and secreted extracellular matrix on outside and inner of micro-pores. The osteoblasts adhesion rate on porous tantalum modified with RGD was higher than that in the unmodified porous tantalum at the end of 24, 48,and 72 hours. The best adhesion effect was got in 5 g/L cells/Ta/RGD group at hour 48 [（68. 07 ± 3. 80） 仍. （23？ 40 ±4. 39）,P 〈 0. 05 ] ？ The results of immunocytochemistry showed that the expressions intensity of F-actin, OC and FN in osteoblasts on porous tantalum modified groups with RGD were stronger than that in the unmodified groups, and the expressions of 5 g/L cells/Ta/RGD group were significantly higher than those in the 10 g/L group and 1 g/L group [ OC ： （1 8 .0 8 ± 0 . 08） （15. 14 ± 0 . 19） , P 〈 0 . 05 ;（18.08 ±0.08） i （14.04 ±0. 61）, P 〈0.05. FN： （24.60 ±0. 98）

关 键 词：RGD多肽 钽 细胞黏附 成骨细胞 

分 类 号：R318.08[医药卫生—生物医学工程]