摘要
目的探讨纳米钛酸钡@金肖特基结涂层(nBTO@Au)的制备方法并进行系统表征,通过体外实验评估其生物安全性及对大鼠骨髓间充质干细胞(BMSCs)成骨性能的影响,以开发具有优异促成骨活性的种植体涂层。方法以纯钛钛片(纯钛组)为对照组,采用阳极氧化在纯钛钛片表面制备二氧化钛涂层,进一步通过水热法制备纳米钛酸钡涂层(nBTO)钛片(nBTO组),并利用柠檬酸钠还原四氯金酸,使金在高温下原位生长于nBTO上,制备nBTO@Au钛片(nBTO@Au组)。通过扫描电镜观察各组涂层表面形貌,能量色散X射线谱(EDS)和X射线光电子能谱(XPS)分析各组钛片涂层的元素组成,X射线衍射(XRD)及拉曼光谱分析各组钛片涂层的晶体结构,通过水接触角分析各组钛片涂层的亲水性。将各组钛片分别与大鼠BMSCs共培养,通过细胞计数法检测培养1、3、5、7 d的细胞增殖情况,活死细胞染色实验评估各组钛片对鼠BMSCs的细胞毒性,细胞骨架染色法观察BMSCs形态和黏附情况,碱性磷酸酶(ALP)染色及定量试剂盒检测成骨诱导7 d后BMSCs的ALP表达情况,茜素红染色及定量实验检测成骨诱导14 d后BMSCs细胞外基质矿化情况,每项实验每组各3个钛片。结果nBTO@Au组扫描电镜显示,直径为(14.838±0.718)nm、尺寸均一且分布均匀的金纳米颗粒成功原位生长于nBTO表面。EDS和XPS显示,nBTO@Au中含Ba、Ti、O和Au元素。XRD和拉曼光谱分析显示,水热法可合成四方相钛酸钡。水接触角结果显示,纯钛组水接触角为66.8°±0.45°,nBTO组为22.55°±0.42°,nBTO@Au组为26.78°±1.15°,即nBTO和nBTO@Au组涂层亲水性良好。培养1、3 d,nBTO组细胞增殖A值显著小于纯钛组(P<0.05);nBTO@Au组细胞增殖A值与纯钛组、nBTO组差异均无统计学意义(均P>0.05)。培养5 d,nBTO@Au组细胞增殖A值显著小于纯钛组(P<0.05),nBTO组细胞增殖A值显著小于nBTO@Au组和纯钛组(P<0.05)。培养7 d,3组细胞增殖A值差异无统计学意义(F=1.62,P>0.05)。活死细胞染色显示各组细胞存活率均超过90%,形态正常,死细胞少,说明nBTO@Au有良好的生物相容性。与纯钛组相比,nBTO和nBTO@Au组均可促进细胞贴壁和铺展,但两组细胞形态无明显差别。ALP染色显示,nBTO@Au组染色面积更大、颜色更深;定量结果显示,nBTO@Au组ALP活性显著大于nBTO组和纯钛组(P<0.05),nBTO组也显著大于纯钛组(P<0.05)。茜素红染色显示,nBTO@Au组染色最深,nBTO组次之,纯钛组最浅;定量分析显示,nBTO@Au组钙结节沉积量显著大于其余两组(P<0.05),nBTO组也显著大于纯钛组(P<0.05)。结论本项研究成功制备的nBTO@Au涂层具备良好的生物相容性和促成骨活性。
ObjectiveTo prepare a nano-barium titanate@gold Schottky junction(nano-BaTiO_(3)@Au)coating and investigate its effects on the adhesion,proliferation,and osteogenic differentiation of bone marrow stem cells(BMSCs),aiming to explore a titanium surface modification strategy with superior osteogenic activity.MethodsPure titanium specimens served as the control group(Ti group).Titanium dioxide coatings were prepared on their surfaces via anodic oxidation.Nano-barium titanate(nBTO group)was further synthesized using the hydrothermal method.Gold nanoparticles were grown in situ on the nano-BaTiO_(3) via high-temperature reduction of chloroauric acid using sodium citrate,yielding the nano-barium titanate@gold Schottky junction coating(nBTO@Au group).Surface morphology was observed by scanning electron microscopy(SEM).Elemental composition was analyzed using X-ray energy dispersive spectrum(EDS)and X-ray photoelectron spectroscopy(XPS).Crystal structure was analyzed using X-ray diffraction(XRD)and Raman spectroscopy.Hydrophilicity was assessed via water contact angle measurement.Specimens were co-cultured with BMSCs to evaluate biocompatibility and osteogenic properties.Cell proliferation on days 1,3,5,and 7 was assessed using the cell counting kit-8(CCK-8)assay.Cytotoxicity towards BMSCs was assessed using live/dead cell staining.Cell morphology and adhesion were observed using cytoskeleton staining.Alkaline phosphatase(ALP)expression in BMSCs after 7 days was quantified using an ALP activity assay and ALP staining.Extracellular matrix mineralization after 7 days was evaluated using alizarin red staining and quantification assay.Each experiment was performed using three specimens per group.ResultsScanning electron microscopy revealed that gold nanoparticles with the diameter of(14.838±0.718)nm,uniform in size and homogeneously distributed,were successfully grown in situ on the surface of the nBTO coating.EDS and XPS confirmed the presence of Ba,Ti,O,and Au elements in the nBTO@Au composite coating.XRD and Raman spectroscopy analysis indicated that the nanostructured barium titanate(nBTO)coating was synthesized via a hydrothermal method.Water contact angle measurements showed that the contact angle was 66.8°±0.45°for the control group,22.55°±0.42°for the nBTO group,and 26.78°±1.15°for the nBTO@Au group,indicating good hydrophilicity of both nBTO and nBTO@Au coatings.On day 1 and day 3 of culture,the cell proliferation in the nBTO group was significantly lower than that in the control group(P<0.05).In contrast,no significant differences were observed between the nBTO@Au group and either the control group or the nBTO group(all P>0.05).By day 5,the cell proliferation of nBTO@Au groups was significantly lower than that of the control group(P<0.05),and the cell proliferation of nBTO group was significantly lower than that of the control group and that of the nBTO@Au group(P<0.05).By day 7,there were no statistically significant differences in cell proliferation among all experimental groups(F=1.62,P>0.05).Live/dead cell staining demonstrated that the cell survival rate exceeded 90%in all groups,with normal morphology and few dead cells,indicating good biocompatibility of the nBTO@Au coating.Compared to the control group,both nBTO and nBTO@Au groups promoted cell adhesion and spreading,although no significant difference in cell morphology was noted between the two modified groups.ALP staining revealed a larger stained area and deeper coloration in the nBTO@Au group.Quantitative results showed that ALP activity in the nBTO@Au group was significantly higher than that in both the nBTO and control groups(P<0.05),and the nBTO group also exhibited significantly higher activity than the control group(P<0.05).Alizarin red staining indicated the deepest coloration in the nBTO@Au group,followed by the nBTO group,and the lightest in the control group.Quantitative analysis further confirmed that the amount of calcium nodule deposition in the nBTO@Au group was significantly greater than that in the other two groups(P<0.05),and the nBTO group also showed significantly more deposition than the control group(P<0.05).ConclusionsThis study successfully prepared an nBTO@Au coating possessing good biocompatibility and enhanced osteogenic properties.
作者
郭小灵
孙丹鹤
李露颖
钟林峰
汪晓艳
周荃
赵领洲
Guo Xiaoling;Sun Danhe;Li Luying;Zhong Linfeng;Wang Xiaoyan;Zhou Quan;Zhao Lingzhou(Air Force Clinical College,The Fifth School of Clinical Medicine,Anhui Medical University,Hefei,230032,China;School of Biological Science and Medical Engineering,Beihang University,Beijing 100191,China;School of Materials Science and Engineering,University of Science and Technology Beijing,Beijing 100083,China;Department of Stomatology,Air Force Medical Center,People′s Liberation Army,Beijing 100142,China)
出处
《中华口腔医学杂志》
北大核心
2025年第11期1264-1273,共10页
Chinese Journal of Stomatology
基金
空军特色医学中心快响课题(2024ZXKXKT012)。
关键词
钛
牙种植体
骨生成
骨髓间充质干细胞
钛酸钡
肖特基结
Titanium
Dental implants
Osteogenesis
Bone marrow mesenchymal stem cells
Barium titanate
Schottky junction
