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目的:观察电化学沉积的纳米结构掺锶羟基磷灰石涂层表面对大鼠骨髓间充质干细胞早期粘附行为的影响。创新点:首次观察电化学沉积的掺锶羟基磷灰石涂层表面骨髓间充质干细胞的早期粘附行为,并对相关基因进行了检测。方法:纯钛表面经过喷砂和双重酸处理,形成多孔粗糙结构。用电化学方法在其粗糙表面沉积羟基磷灰石涂层(HA)和掺锶羟基磷灰石涂层(Sr-HA)。用贴壁法将4周大鼠股骨骨髓间充质干细胞分离进行原代培养后将细胞接种到多孔纯钛,HA和Sr-HA表面培养1、6和24小时。用场发射扫描电镜(SEM)观察细胞的形貌特点。用异硫氰酸荧光素(FITC)标记的鬼笔环肽进行免疫荧光染色标记细胞骨架,Hoechst 33258进行细胞核染色,激光共聚焦荧光显微镜进行拍照后使用Image J进行细胞的计数和图形分析。用实时荧光定量逆转录聚合酶链式反应(RT-q PCR)测定24、48和72小时不同实验组中骨髓间充质干细胞中FAK、vinculin、integrin β1和integrin β3的基因表达,并进行统计学分析。结论:SEM观察结果显示,骨髓间充质干细胞在三种表面都能正常黏附、生长和增殖。在Sr-HA组表面,细胞粘附、细胞活力和细胞骨架的铺展都较粗糙表面组和HA组有较显著的提高。RT-q PCR结果显示,在各个时间点Sr-HA组表面骨髓间充质干细胞的FAK、vinculin、integrin β1和integrin β3的基因表达与粗糙组有显著性差异,且在24小时后与HA组亦有显著性差异。综上所述,掺锶羟基磷灰石纳米涂层具有较好的生物相容性,可以促进大鼠骨髓间充质干细胞在纯钛表面的早期粘附。
OBJECTIVE: To observe the effect of electrochemically deposited nano-structured strontium-doped hydroxyapatite coating on the early adhesion of rat bone marrow mesenchymal stem cells. Innovative point: the first observation electrochemical deposition of strontium hydroxyapatite coating surface adhesion of early bone marrow mesenchymal stem cells, and related genes were detected. Methods: Pure titanium surface after sandblasting and double acid treatment, the formation of porous rough structure. Hydroxyapatite (HA) and strontium-doped hydroxyapatite (Sr-HA) coatings were electrochemically deposited on their rough surfaces. After 4 weeks of primary rat femur bone marrow mesenchymal stem cells were isolated and primary cultured with adherent method, the cells were inoculated into porous titanium, HA and Sr-HA were cultured on the surface for 1, 6 and 24 hours. Field emission scanning electron microscopy (SEM) observed cell morphology. Cytoskeleton was labeled by immunofluorescence staining with fluorescein isothiocyanate (FITC) labeled phalloidin. Hoechst 33258 cells were stained with nuclei. Laser scanning confocal microscopy was used for image counting and image analysis with Image J. The gene expression of FAK, vinculin, integrin β1 and integrin β3 in bone marrow mesenchymal stem cells in different experimental groups at 24, 48 and 72 hours were determined by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-q PCR) Analysis. Conclusion: The results of SEM showed that BMSCs could adhere, grow and proliferate normally on all three surfaces. On the surface of Sr-HA group, cell adhesion, cell viability and cytoskeletal spreading were more significant than those of rough surface group and HA group. RT-qPCR results showed that the gene expression of FAK, vinculin, integrin β1 and integrin β3 on the surface of BMSCs in Sr-HA group were significantly different from that in rough group at each time point, and after 24 hours, There are also significant differences. In summary, the strontium-doped hydroxyapatite nano-coating has good biocompatibility and can promote the early adhesion of rat bone marrow mesenchymal stem cells on the surface of pure titanium.