改性3D打印钛支架对脂肪间充质干细胞成骨分化的影响  

Effect of modified three-dimensional-printed titanium scaffold on osteogenic differentiation of adipose-derived mesenchymal stem cells

作　　者：王静[1] 杨九菊[1] 王宁宁 刘超[2] Wang Jing;Yang Jiuju;Wang Ningning;Liu Chao(Department of Stomatology,Cangzhou Medical College,Cangzhou 061001,Hebei Province,China;Department of Oral and Maxillofacial Surgery,Qilu Hospital of Shandong University,Jinan 250012,Shandong Province,China)

机构地区：[1]沧州医学高等专科学校口腔系,河北省沧州市061001 [2]山东大学齐鲁医院口腔颌面外科,山东省济南市250012

出　　处：《中国组织工程研究》2022年第27期4265-4271,共7页Chinese Journal of Tissue Engineering Research

基　　金：沧州医专自然科学课题(19Z015),项目负责人:王静。

摘　　要：背景:3D打印钛支架克服了传统钛支架内部结构可控性差及外形不匹配等缺陷,但由于钛的生物惰性较大,使其在植入体内后难以与周围组织快速稳定地结合。目的:采用喷砂酸蚀和阳极氧化方法改性3D打印钛支架表面,观察其对脂肪间充质干细胞黏附、增殖和成骨分化能力的影响。方法:采用喷砂酸蚀联合阳极氧化方法在3D打印钛支架表面构建微纳米多孔结构,扫描电镜观察其表面特征。将第3代大鼠脂肪间充质干细胞分别接种于3D打印钛支架组(A组)、3D打印钛支架+成骨诱导液(B组)、改性3D打印钛支架(C组)上,通过细胞骨架与CCK-8实验检测细胞的黏附和增殖能力,碱性磷酸酶与茜素红染色观察细胞的成骨分化能力,实时定量PCR分析细胞成骨相关基因的表达,免疫荧光染色观察细胞骨钙素与骨桥蛋白的表达。结果与结论:①扫描电镜下可见,改性后的3D打印钛支架表面出现微米及亚微米级凹坑和沟槽,内部有直径为70-100 nm的纳米级孔隙,孔隙之间相互连通;②共聚焦显微镜下可见,A、B组细胞伪足及触角较少,尚未铺展;C组细胞完全铺展在材料表面,可见大量明显的细胞伪足和触角,紧密附着于材料表面;CCK-8实验显示,改性3D打印钛支架可促进脂肪间充质干细胞的增殖;③碱性磷酸酶与茜素红染色显示,B、C组碱细胞性磷酸酶活性与矿化水平高于A组(P<0.05);④B、C组细胞骨钙素、RUNX2、碱性磷酸酶及Ⅰ型胶原mRNA表达均高于A组(P<0.05),骨桥蛋白与骨钙素蛋白表达均高于A组(P<0.05);⑤结果表明,喷砂酸蚀联合阳极氧化改性3D打印钛支架具有良好的生物相容性,可促进脂肪间充质干细胞的黏附、增殖和成骨分化。BACKGROUND:The three-dimensional(3D)-printed titanium scaffold overcomes the disadvantages of traditional titanium scaffolds,such as poor controllability of the internal structure and mismatch in shape.However,due to the relatively large biological inertia of titanium,it is difficult to combine with surrounding tissues quickly and stably after implantation in the body.OBJECTIVE:To modify the surface of 3D-printed titanium by sandblasting acid etching and anodic oxidation and observe its effect on the adhesion,proliferation,and osteogenic differentiation of adipose-derived mesenchymal stem cells.METHODS:Micro-nano porous structure was constructed on the surface of 3D-printed titanium by sandblasting acid etching and anodic oxidation.The surface was characterized by scanning electron microscopy.Adipose-derived mesenchymal stem cells at passage 3 were inoculated in the Ti group(group A),Ti+osteogenic induction group(group B),and modified Ti group(group C).Cytoskeleton and CCK-8 assay were used to detect cell adhision and proliferation.Alkaline phosphatase staining and Alizarin red staining were used to observe osteogenesis.Real-time PCR was applied to analyze the expression of osteogenic genes.Immunofluorescence staining was employed to observe the expression of osteocalcin and osteopontin in cells.RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that micro and submicron scale pits and grooves could be seen on the surface of the modified 3D-printed titanium scaffold.There were nanoscale pores with a diameter of 70-100 nm inside the scaffold and the pores were connected with each other.(2)Under a confocal microscope,there were few pseudopods and antennae in groups A and B,and they had not been spread;the cells in group C were completely spread on the surface of the material,and a large number of obvious pseudopods and antennae were visible,which were closely attached to the surface of the material.CCK-8 assay showed that modified 3D-printed titanium scaffolds could promote the proliferation of adipose-de

关 键 词：3D打印钛 脂肪间充质干细胞 黏附 增殖 生物相容性 成骨蛋白 成骨基因 成骨分化 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]