载锶纳米钛表面促进骨质疏松大鼠骨髓间充质干细胞成骨分化的体外研究

An in vitro study on strontium-loaded nano-titanium surface promoting osteogenic differentiation of bone marrow mesenchymal stem cells of osteoporotic rats

目的:研究钛种植体载锶纳米表面的生物学效应,及其对骨质疏松大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用。方法:以纯钛(titanium,Ti)片为对照组(control-Ti),用氢氧化钠(NaOH)水热处理纯钛片后获得纳米组(nanometric-Ti, nm-Ti),用Sr(OH)2水热处理纯钛片获得载锶(strontium,Sr)纯钛组(Sr-Ti),将纯钛片先后以NaOH、Sr (OH)_(2)两步法水热处理获得载锶纳米组(nm/Sr-Ti)。通过扫描电镜(scanning electron microscope,SEM)、X射线光电子能谱(X-ray photoelectron spectroscopy,XPS)和X射线衍射(X-ray diffraction,XRD)技术等检测各组表面性能。采用双侧卵巢切除术(ovariectomy,OVX)建立骨质疏松大鼠模型,10周后提取BMSCs,通过CCK-8法和实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)评价各组钛表面对正常及骨质疏松大鼠BMSCs成骨能力的影响。结果:与control-Ti组相比,nm-Ti组和nm/Sr-Ti组表面均呈现成簇片状纳米结构,Sr-Ti组和nm/Sr-Ti组表面负载Sr离子。CCK-8结果显示,正常及骨质疏松大鼠BMSCs在各组钛片表面均呈现良好的黏附和增殖能力,其中nm/Sr-Ti组细胞增殖最活跃。RT-qPCR结果显示,nm-Ti组和nm/Sr-Ti组成骨基因OCN、OPN及BMP-2表达随时间推移升高,且与对照组相比,差异显著。在骨质疏松大鼠模型中,nm/SrTi组显著促进了BMSCs早期BMP-2的表达。结论:载锶纳米钛表面有利于BMSCs的黏附、增殖和分化,并可能对骨质疏松大鼠的种植体周围骨整合有促进作用。

Objective:To study the biological effect of strontium-loaded nano-titanium surface and its effects on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)of osteoporotic rats in vitro.Methods:The cleaned and dried titanium sheets were used as the control group(control-Ti).Using a hydrothermal method,the cleaned titanium sheets were treated with NaOH solution to achieve nanometric-Ti group(nm-Ti).And by hydrothermally treating titanium sheets using Sr(OH)_(2) solution,we achieved strontium-loaded titanium group(Sr-Ti).At last,we achieved strontium-loaded nano-titanium group(nm/Sr-Ti)by a two-step hydrothermal treatment of NaOH and Sr(OH)_(2) solution.Then,surface charac teristics of these groups were analyzed by scanning electron microscope(SEM),X-ray photoelectron spectroscopy(XPS)and X-ray diffraction(XRD)measurement.A rat model of osteoporosis was also established by bilateral ovariectomy(OVX)in female rats.After 10 weeks,the BMSCs were extracted and seeded on the surfaces of different titanium sheets.CCK-8 and real-time quantitative polymerase chain reaction(RT-qPCR)were used to evaluate the effects of different titanium sheet surfaces on the osteogenesis of BMSCs of osteoporotic rats.Results:According to the SEM results,the surfaces of nm-Ti and nm/Sr-Ti group compared with that of control-Ti group showed nano-sheet structures,and Sr element was detected on the surfaces of the Sr-Ti group and the nm/Sr-Ti group.The result of CCK-8 showed that the number of BMSCs from normal and osteoporotic rats increased with time on the surface of each titanium sheets,and nm/Sr-Ti group proliferated the most.The result of RT-qPCR showed that the expression levels of OCN,OPN and BMP-2 genes in nm-Ti group and nm/Sr-Ti group significantly increased with time compared to control-Ti group,and the changes in nm/Sr-Ti group were the most significant.Conclusion:Strontium-loaded nano-titanium surface is beneficial to the adhesion,proliferation and differentiation of BMSCs,and has the potential to promote osseointegration around implants in osteoporotic rats.