【作者】 黄丹；

【导师】 王贻宁；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2018， 博士

【摘要】 [研究背景和目的]随着钛材料骨整合现象的发现,钛及钛合金作为植入材料在生物医学相关领域的应用越来越为普遍。然而,钛本身属于惰性金属,在一些条件欠佳的情况下(患者抵抗力低下、损伤为开放性、机体患有系统性疾病等),其应用仍然面临着两个不可忽视的挑战:细菌感染和骨结合的不足。当材料植入组织内时,周围的细胞会粘附在材料表面并发生增殖和成骨分化,但如果此时周围微环境内有细菌存在,细菌就会和细胞竞争粘附有限的材料表面,从而影响细胞的成骨活动,最终影响骨生成及骨结合的形成,所以严格的控制细菌感染对形成骨结合至关重要。此外,植体周围同时需要充足的新生血管网络给细胞提供氧气和营养物质,让细胞生长、增殖从而促进骨再生并形成骨结合。为了得到理想的钛生物材料,大量研究期望通过表面的结构和化学改性来赋予其抗菌性能、促血管化性能以及促成骨性能。在本课题组前期的研究中,通过简便的一步法电沉积技术成功地制备了壳聚糖/明胶(chitosan/gelatin,CSG)涂层,并证实了这种三维多孔的涂层具有优良的机械力学特性、可降解性、生物相容性和促进骨结合性能。此外这种有机涂层还能作为载体,载入其他具有生物学作用的活性分子或者金属离子。在众多的功能性物质中,我们发现金属铜(Cu)离子在一定条件下可能会同时具有抗菌性、促血管化性能、促成骨性能。综合以上考虑,第一部分实验通过电沉积(Electrophoreticdeposition,EPD)技术将铜离子载入到CSG涂层中,制备出铜/壳聚糖/明胶(Cu/CSG)复合涂层,并对其表面形貌、理化性质、铜离子的载入与缓释、溶胀、降解性能、机械力学性能进行了系统表征,同时利用革兰氏阴性大肠杆菌和革兰氏阳性金黄色葡萄球菌定性及定量地检测涂层的抗菌性能,并以骨髓基质细胞(BMSCs)为种子细胞研究涂层的生物相容性、促血管化性能及促成骨性能。本研究将会为今后载铜多功能涂层在临床医学领域的相关应用奠定一定的理论及实验基础。此外,本课题组前期的实验已证实锶(Sr)离子及银(Ag)离子可以分别载入CSG涂层中,并分别发挥促成骨作用及抗菌效应。第二部分实验中,我们在此基础上,将锶离子及银离子共同载入到了 CSG涂层中,制备了锶/银/壳聚糖/明胶涂层(Sr/Ag/CSG),表征其各种材料学性能,并利用细菌及细胞对其生物学性能进行了评价,以期为该多功能涂层将来在临床上的应用提供一定的基础。第一部分铜/壳聚糖/明胶复合涂层的制备及性能研究实验一铜/壳聚糖/明胶复合涂层的制备[材料与方法]配制壳聚糖/明胶混合溶液,向此溶液中加入不同浓度的氯化铜,得到含不同浓度铜离子(0、0.01mM、0.1mM、1mM、10mM)的电沉积液。采用电沉积技术在SLA钛片表面制备铜/壳聚糖/明胶涂层,分别记为CSG、CuⅠ、CuⅡ、CuⅢ、CuⅣ组。[结果]随着电沉积液中铜离子浓度的升高,溶液颜色由无色透明逐渐加深至浅蓝色。电沉积得到的新制涂层为含气泡的凝胶状物质,干燥后,涂层呈均匀的薄膜样结构。随着实验组中铜离子浓度的升高,所制备的涂层由无色透明逐渐变紫。实验二铜/壳聚糖/明胶复合涂层的材料学性能表征[材料与方法]透射电子显微镜(Transmission Electron Microscope,TEM)及荧光显微镜观察新制涂层的微观结构,场发射扫描电子显微镜(Field emission scanning electron microscopy,FESEM)观察干燥涂层的微观结构。能谱分析仪(Energy-dispersive X-ray spectrometry,EDS)分析涂层的元素组成,衰减全反射傅里叶变换红外光谱仪(Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy,ATR-FTIR)分析涂层的化学组成和化学键的变化,X射线光电子能谱(X-ray photoelectron spectroscopy,XPS)检测涂层的元素组成及其中铜的含量和化学价态。原子吸收光谱仪(Atomic absorption spectrophotometer,AAS)分析涂层中铜离子的载入情况和在溶液中的缓释性能,并用TEM观察缓释出来的结构。分析涂层在溶液中的溶胀及质量损失情况。在力学测试平台上检测涂层的机械力学性能(拉伸粘接强度和剪切粘接强度)。[结果]TEM显示在涂层制备过程中,有许多纳米级颗粒样结构沉积在钛表面,荧光显微镜下新制涂层呈三维多孔结构。干燥后的涂层在FESEM上观察可见许多微米级颗粒,而且这些颗粒是由更多的纳米结构组成。EDS检测证实了涂层中铜离子的存在。ATR-FTIR结果显示所有涂层都含有壳聚糖和明胶的特征峰。XPS检测说明涂层中的铜元素是以单质铜的形成存在。AAS结果说明涂层中铜的载入量与电沉积液中铜的浓度成正比,所有涂层中铜的缓释情况表现为前期的突释和后期的缓释,TEM观察到缓释出来的结构为纳米级的颗粒。溶胀实验证实所有组别涂层在溶液中很快便可发生吸水膨胀从而恢复其三维多孔结构。涂层的降解实验显示,所有涂层均表现为先快后慢的降解模式。机械力学检测说明铜的载入提高了涂层的拉伸粘接强度,而涂层的剪切粘接强度未受显著影响。实验三铜/壳聚糖/明胶复合涂层的体外抗菌性能研究[材料与方法]用FESEM观察细菌在涂层上的粘附情况,再用活死菌染色液对共培养后的菌液进行染色观察,并对其中的活菌和死菌分别进行定量分析。利用平板计数法对粘附于涂层表面和悬浮于涂层周围菌液内的活菌量进行统计,以此来定量分析涂层的抗菌性。此外,用BCA试剂盒测定共培养后菌液里的蛋白含量。[结果]FESEM结果显示随着涂层中铜浓度的升高,涂层上粘附的细菌量逐渐减少,而且高浓度载铜涂层上的细菌发生了皱缩变形。菌液的活死菌染色结果也说明随着实验组中所载入铜离子浓度的升高,绿色荧光(活菌)的密度逐渐降低,而红色荧光(死菌)的密度逐渐增强。平板计数法的定量结果更一步说明X涂层对粘附于其表面和悬浮于周围菌液内的细菌均有抑制作用,且其抗菌性大小与涂层中铜元素的载入量成正相关关系。此外,BCA蛋白含量的测定结果说明载铜涂层能使细菌细胞膜的渗透性增强,从而导致细菌体内的蛋白外渗。实验四铜/壳聚糖/明胶复合涂层的体外细胞生物学性能研究[材料与方法]FESEM及荧光显微镜观察骨髓基质细胞在涂层上的粘附和伸展情况,利用CCK-8试剂盒检测细胞在各组涂层上的增殖情况,并比较涂层缓释出来的铜有机复合物与单纯无机铜离子的细胞毒性。利用实时荧光定量PCR检测细胞成血管相关基因(包括HIF-1a、VEGF)的表达。此外,用pNPP法检测细胞的碱性磷酸酶(alkaline phosphatase,ALP)活性,并用实时荧光定量PCR检测细胞成骨相关基因(包括ALP、OCN、Runx2、Col-1)的表达。[结果]FESEM结果显示细胞能在涂层表面正常粘附与伸展。荧光显微镜下可见,细胞充分铺展呈多角形,并通过边缘发出的许多丝状伪足互相关联,其中CuⅣ组涂层上的细胞密度较其他组明显偏小。CCK-8检测结果说明,CuⅠ、CuⅡ和CuⅢ组均能支持细胞的正常增殖,而Cu Ⅳ组表现出了一定的细胞毒性。铜有机复合物与单纯铜离子的细胞毒性比较的结果显示,所有铜有机复合物组的细胞数目都显著性地高于相应的单纯铜离子组。同时,随着铜离子浓度的增高,细胞数目呈现出下降的趋势。荧光定量PCR结果说明,Cu Ⅱ和Cu Ⅲ组细胞成血管相关基因的表达水平显著性地高于对照CSG组,而Cu Ⅳ组细胞相关基因的表达显著性地低于其他组。ALP活性检测的结果说明,CuⅠ、CuⅡ和CuⅢ组细胞ALP活性随着铜浓度的升高而逐渐增加,其中,Cu Ⅲ组与CSG组的差异具有统计学意义。同时PCR结果显示,Cu Ⅲ组成骨相关基因的表达均显著性的高于其他组(14天时差异明显)。另外Col-1基因在Cu Ⅳ组的表达显著低于CSG组。[结论]1.本研究首次通过电沉积技术在钛表面成功地制备出了载铜的无机-有机(铜/壳聚糖/明胶)复合涂层,此涂层呈三维多孔结构;2.涂层的材料学性能分析表明,二价铜离子在电沉积过程中被还原为了铜单质,并与壳聚糖/明胶有机复合物形成了纳米结构后被沉积在钛表面,最后铜复合物又以纳米颗粒的形式释放出来发挥作用,铜的载入未明显改变涂层的溶胀性能,但减慢了涂层的降解速率,并提高了涂层的机械力学性能;3.体外抗菌实验说明,铜的抗菌性能在涂层中得以保存,涂层对粘附于其表面和悬浮于其周围的细菌都有一定的抗菌性,且其抗菌性与铜载入量成正相关关系;4.体外细胞实验说明,Cu Ⅱ和Cu Ⅲ组涂层有一定的促血管化性能及促成骨性能,而CuⅣ组涂层表现出了一定的细胞毒性;5.综合考虑涂层的材料学性能、细菌及细胞生物学性能,ChⅢ组涂层具有多功能性,它可以同时具有一定的抗菌性、促血管化性能、及促成骨性能,本研究将会为今后钛表面载铜功能性涂层的临床应用提供一定的理论及实验基础。第二部分锶/银/壳聚糖/明胶复合涂层的制备及性能研究实验五锶/银/壳聚糖/明胶复合涂层的制备及材料学性能表征[材料与方法]向CSG溶液中加入不同浓度的锶/银混合溶液,得到含不同浓度锶/银离子(0、10/1mM、50/1mM、50/5mM)的电沉积液。分别记为 CSG、Sr10/Ag1、Sr50/Ag1、及Sr50/Ag5组。按前述方法电沉积制备涂层。利用荧光显微镜、FESEM、EDS、XRD、AAS、TEM等方法表征涂层的材料学性能。[结果]电沉积过程中可见凝胶状涂层逐渐均匀沉积在阴极钛片表面。荧光显微镜下,实验组涂层组在CSG涂层的基础上出现了许多粒径极小的颗粒样物质。扫描电镜下,实验组涂层可见散在分布的微米球,这些微米球是由更小的纳米颗粒所组成,且这些微球上分布了 一一些亮度比周围结构更高的纳米颗粒。EDS证实了实验组涂层中锶、银的存在,而XRD证明这两种元素主要以SrC03及AgCl的形式存在。AAS结果说明涂层中锻/银的载入量与电沉积液中离子的浓度成正比,所有涂层中银/银的缓释情况表现为前期的突释和后期的缓释,TEM观察到这些缓释出来的结构为纳米级的颗粒。所有组别的涂层均表现为先快后慢的降解模式。实验六锶/银/壳聚糖/明胶复合涂层的体外抗菌性能研究[材料与方法]用活死菌染色液对共培养后的菌液进行染色观察;用平板计数法对粘附于涂层表面和悬浮于涂层周围的活菌量进行统计,以此来定量分析涂层的抗菌性。[结果]菌液的活死菌染色结果说明随着实验组中银离子载入量的升高,绿色荧光(活菌)的密度逐渐降低。平板计数法的定量结果更一步说明了涂层对粘附于其表面和悬浮于其周围菌液内的细菌均有抑制作用,且其抗菌性大小是与涂层中载入银离子的量成正相关关系的。实验七锶/银/壳聚糖/明胶复合涂层的体外细胞生物学性能研究[材料与方法]FESEM及荧光显微镜观察细胞在涂层上的粘附和伸展情况。利用CCK-8试剂盒检测细胞在各组涂层上的增殖情况,此外检测细胞的ALP活性,并用RT-qPCR检测细胞成骨相关基因的表达。[结果]FESEM结果显示除Sr50/Ag5组外细胞都能在涂层表面正常粘附与伸展。荧光显微镜下可见,细胞充分铺展呈多角形,并通过边缘发出的许多丝状伪足互相关联,而Sr50/Ag5组仅见少许细胞碎片。CCK-8结果说明,Sr10/Ag1及Sr50/Agl组涂层能促进细胞增殖,而Sr50/Ag5组表现出明显的细胞毒性。ALP活性分析及成骨相关基因的PCR结果说明Sr10/Ag1以及Sr50/Ag1组涂层均能促进细胞的成骨向分化,且其中Sr50/Ag1组的效果更为显著。[结论]1.通过电沉积技术在钛表面成功地制备了载锶/银的复合涂层(锶/银/壳聚糖/明胶);2.该涂层中两种离子分别主要以SrC03及AgCl的形式存在,并以纳米复合物的形式释放出来发挥作用,离子的载入减慢了涂层的降解速率;3.锶/银/壳聚糖/明胶涂层对粘附于其表面和悬浮于其周围的细菌都有一定的抗菌性,且其抗菌性与银载入量成正相关关系;4.体外细胞实验说明,Sr10/Ag1以及Sr50/Ag1组涂层均能促进细胞的增殖和成骨向分化,且其中Sr50/Ag1组的效果更为显著,而Sr50/Ag5组有明显的细胞毒性;5.综合考虑涂层的各项性能,Sr50/Ag1组涂层具有多功能性,它能在保证一定抗菌性的同时对细胞的增殖及成骨分化起明显促进作用。本研究将会为今后锶/银/壳聚糖/明胶涂层的临床应用提供了一定的实验基础。更多还原

【Abstract】 [Background and objective]Since the discovery of osseointegration of titanium,titanium and its alloys have been increasing used in clinic for biomedical application.However,their bio-inert surface challenges application in patients with compromised surgical conditions(weakened immunity,open injury,systemic diseases),and their use still faces two risk factors:bacterial infections and lack of local tissue integration.If bacteria existed in the microenvironment,bacteria will compete with cell to occupy the surface once the material is implanted,thus function of osteoblast will be influenced and finally disturb osseointegration.So,strict control of bacterial infection is vital for a successful implant.In addition,sufficient vascular network is also prerequisite for osseointegration,which can provide oxygen and nutrient to guarantee the proliferation,differentation of osteoblast.In order to get the ideal titanium biomedical material,numerous studies have been attempted to modify its surface topography and chemical composition for the purpose of obtaining multifunctional properties of antibacterial ability,angiogenic and osteogenic activity.In our previous work,we have successfully prepared the chitosan/gelatin(CSG)composite coatings on titanium via single-step electrophoretic deposition(EPD),which has three-dimensional porous scaffold like structure,and showed favorable mechanical properties,biodegradability,biocompatibility and osteogenic properties.What’s more,this CSG coating was a promising candidate for further loading of functional agents like bioactive molecules and metal ions.Among the variety of functional agents,we have found that metal copper(Cu)ion may have antibacterial,angiogenic,and osteogenic activities simultaneously at certain condition.Taking the above background into consideration,the present study aim to introduce copper element into the CSG coating via EPD,thus fabricating the Cu/CSG composite coatings.The surface morphology,physicochemical properties,copper entrapment and release profile,swelling and degradation property,and mechanical property were investigated.The antibacterial ability of the coatings against E.coli and S.aureus was examined.Furthermore,rat bone marrow stromal cells were used to investigate the cytocompatibility,angiogenic,and osteogenic potential of the coatings.This study may provide theoretical and experimental basis for the potential use of the Cu-doped CSG coatings in clinical application.In addition,in our previous study,we have already found that strontium and silver can be loaded into the CSG coatings,respectively,and the Sr-containing coatings have osteogenic property,while the Ag-doped coatings have antibacterial activity.Under this background,in the second part,strontium and silver had been co-deposited into the CSG coatings,thus preparing Sr/Ag/CSG coatings.The physicochemical properties,ion entrapment and release profile,degradation ratio were analyzed.In addition,bacteria and BMSCs were used to evaluate its biological activity.This investigation may provide certain basis for its future use.Part I Fabrication and characterization of Cu-doped CSG coatings Exp.l Fabrication of Cu-doped CSG coatings[Materials and Methods]The chitosan/gelatin solution was prepared as our previous study.Copper chloride was added into the CSG solution to obtain the final electrophoretic solution with Cu concentrations of 0,0.01,0.1,1,and 10 mM.The obtained Cu/CSG coatings were labeled as CSG,Cu Ⅰ,Cu Ⅱ,Cu Ⅲ,and Cu Ⅳ.[Results]With increasing amount of Cu in the EPD suspension,the color of the solution changed gradually from transparent to light blue,while color of the coating changed from transparent to purple.Porous hydrogel coatings were uniformly deposited on the substrates after EPD.After being air-dried,the coatings showed a thin film-like structure on the Ti disks.Exp.2 The physicochemical properties of Cu-doped CSG coatings[Materials and Methods]Microstructure of the freshly deposited coatings was observed by TEM and fluorescence microscopy.After being air-dried,the coatings were examined by FESEM.For qualitative elemental analysis of the coatings,EDS was used.Surface chemistry was investigated with ATR-FTIR;In addition,the chemical state of Cu in the coatings was studied by XPS.The copper entrapment and release profile were analyzed by AAS,and the morphology of the release compounds was observed by TEM.The swelling ratio and weight loss of the coating in the PBS solution were measured.Mechanical tests were conducted to evaluate the mechanical property of the coatings(tensile bond strength and shear bond strength).[Results]TEM results showed that nanoscale particles were deposited onto the titanium surface during EPD.Fluorescence images of the wet coatings displayed macroporous morphology.Under FESEM,microscale particles were distributed in the Cu-doped coatings,which were composed of smaller nanoparticles.EDS analysis confirmed the presence of Cu in the coatings.The ATR-FT1R spectra of all coatings exhibited characteristic peaks of chitosan and gelatin.XPS result showed bivalent copper ions were reduced to metallic Cu in the coating.AAS results showed that Cu entrapment was positively correlated to the Cu concentration in the EPD solutions.All Cu-containing nanocomposite coatings presented a biphasic release of Cu with an initial burst release followed by sustained slow release.TEM images display the morphology of the nanocomposites released from the coatings.The study on the swelling ratio of the coatings indicated that all the coatings could recover to three-dimensional macroporous structure.Degradation results showed that the weight of all the samples decreased sharply in the first few days,followed by relatively little change in the following days.Mechanical tests showed that the tensile bond strength of the coatings increased when Cu was introduced,while there was no significant difference for the shear bond strength.Exp.3 In vitro antibacterial study of the Cu-doped CSG coatings[Materials and Methods]FESEM was used to observe the bacteria adhered on the specimens,in addition,Live/Dead staining assay was applied to analyze the bacterial solution,and the number of live and dead bacterial cells was quantitatively measured by software.The bacteria adhered on the sample surface and suspended in the surrounding medium were counted by spread plate method,thus quantitatively calculated the antibacterial rate.The protein content in the medium after co-culture was measured by bicinchoninic acid(BCA)protein assay kit.[Results]FESEM showed that the amounts of bacteria on the surfaces decreased with increasing Cu concentration in the coatings,in addition,the bacteria were shriveled and deformed at high Cu concentration groups.Live/Dead staining assay displayed that as Cu concentration increased,the green fluorescent intensity(live bacterial colonies)decreased,while the red fluorescent intensity(dead bacterial colonies)increased.The bacteria spread counting method also verified the antibacterial activity of the coatings against the bacteria adhered on the sample surface and suspended in the surrounding medium,and the antibacterial rate was positively correlated with the copper concentration.BCA kit assay showed that Cu-doped coatings undermined the permeability of bacterial membranes,and led to the leakage of bacterial proteins.Exp.4 In vitro cellular study of the Cu-doped CSG coatings[Materials and Methods]FESEM and fluorescence microscopy were used to observe the cell morphology on the coated Ti samples.Cell counting kit-8(CCK-8)was used to analyze the cell proliferation.In addition,cytotoxicity of copper-organic compounds and pure copper ion were compared by the CCK-8 kit.Real-time quantitative polymerase chain reaction(RT-qPCR)was executed to assess angiogenesis-related gene(including HIF-la and VEGF)expressions of various samples in BMSCs.Moreover,ALP(alkaline phosphatase)activity of the cells were measured by pNPP method,and the osteogenesis-related gene(including ALP,OCN,Runx2,and Col-1)expressions were examined by RT-qPCR.[Results]FESEM results indicated that Cu-incorporated coatings could support BMSCs attachment and spreading.In addition,immunofluorescence staining of all groups exhibited a polygonal shape with extended filopodia,through which the cells were connected.Cell density of the Cu Ⅳ group was less than all the other groups.CCK-8 assay showed that Cu Ⅰ,Ⅱ,and Ⅲ groups could support cell proliferation,while Cu IV group displayed certain cytotoxicity.CCK-8 tests showed that cell activity of the Cu organic compounds group was significantly higher than the corresponding Cu ion group.In addition,the cell activity decreased as the concentration of copper increased.In the RT-qPCR assay,Cu Ⅱ and Cu Ⅲ groups demonstrated significantly up-regulated gene expressions of angiogenic factors compared with the CSG group,while the Cu IV group showed remarkably lower expressions of these genes.ALP activity of BMSCs in Cu Ⅰ,Cu Ⅱ,and Cu Ⅲ groups increased as Cu loading ascended,and it was significantly higher in the Cu Ⅲ group than CSG group.Meanwhile,RT-qPCR analysis showed that BMSCs expressions of osteogenesis-related genes were dramatically up regulated in Cu Ⅲ samples compared with other groups,especially on day 14.In addition,Col-1 expression was apparently lower in the Cu Ⅳgroup than in the CSG group.[Conclusions]1.For the first time,copper-doped inorganic-organic(Cu/CSG)composite coatings were successfully deposited on titanium substrates via EPD,and the coatings had a three-dimensional porous structure;2.Through physical-chemical analysis of the coatings,it was verified that the bivalent copper ions were reduced to metallic Cu,the combined Cu-CSG nanocomposites were deposited on the Ti substrate,and Cu compounds released from the coatings displayed nano-scale structure.Incorporation of Cu into CSGcoatings did not influence the swelling property significantly,but decrease the degradation ratio,and improve the mechanical property;3.In vitro antibacterial study showed that the antibacterial property of copper was preserved.The coatings have antibacterial ability against the bacteria adhered on the sample surface and suspended in the surrounding medium.In addition,the antibacterial rate was positively correlated with the copper concentration;4.As for the in vitro cellular study,Cu Ⅲ group displayed improved angiogenic and osteogenic activities,while the Cu IV group showed certain cytotoxicity;5.Considering the performance of coatings systemically,multifunctional Cu Ⅲgroup coatings could guarantee considerable antibacterial activity and benefit angiogenic and osteogenic differentiation,this study may provide theoretical and experimental basis for the potential use of the Cu-doped CSG coatings in clinical application.Part Ⅱ Fabrication and characterization of Sr/Ag-doped CSG coatings Exp.5 Fabrication and material property of Sr/Ag-doped CSG coatings[Materials and Methods]Sr/Ag solution was added into the CSG solution to get the final electrophoretic solution with Sr/Ag concentrations of 0,10/1 mM,50/1 mM,and 50/5 mM,the obtained coatings were labeled as CSG,Sr10/Agl,Sr50/Agl,and Sr50/Ag5.Fluorescence microscopy FESEM,EDS,XRD,AAS,and TEM were used to evaluate their property.[Results]After EPD,hydrogel coatings were uniformly deposited on the Ti surface.Homogeneously distributed white particles appeared in the Sr/Ag/CSG coatings under fluorescence microscopy.Under FESEM,microscale particles were distributed in the Sr/Ag/CSG coatings,and some brighter nanoparticles appeared in the microparticles.EDS verified the presence of Sr/Ag in the coatings,while the XRD showed that these two elements mainly existed in the form of SrCO3 and AgC1.AAS results showed that Sr/Ag entrapment was positively correlated to the Sr/Ag concentration in the EPD solutions.All coatings displayed.a biphasic release of Sr/Ag with an initial burst release followed by sustained slow release.TEM images presented that nanocomposites released from the coatings.Degradation results showed that the weight of all the samples decreased greatly in the first few days,followed by relatively little change in the following days.Exp.6 In vitro antibacterial study of the Sr/Ag-doped CSG coatings[Materials and Methods]Live/Dead staining assay was used to obeserve the bacterial solution.Spread plate method was applied to count the bacteria on the sample surface and suspended in the surrounding medium,thus quantitatively calculated the antibacterial rate.[Results]Live/Dead staining assay showed that as Ag concentration increased,the green fluorescent intensity(live bacterial colonies)decreased.The bacteria spread counting method also verified the antibacterial activity of the coatings against the bacteria adhered on the sample surface and suspended in the surrounding medium.In addition,the antibacterial rate was positively correlated with the silver concentration.Exp.7 In vitro cellular study of the Sr/Ag-doped CSG coatings[Materials and Methods]FESEM and fluorescence microscopy were used to observe the cell morphology.Cell counting kit-8(CCK-8)was used to analyze the cell proliferation after seeding on the coatings.Moreover,ALP activity of the cells was measured,and the osteogenesis-related gene expressions were examained by RT-qPCR.[Results]FESEM results indicated that Sr/Ag-incorporated coatings could support BMSCs attachment and spreading except for the Sr50/Ag5 group.Under fluorescence microscopy,cells exhibited a polygonal shape with extended filopodia,while only some debris could be observed in the Sr50/Ag5 group.CCK-8 assay showed that Sr10/Agl and Sr50/Agl groups could promote proliferation of the cells,while the Sr50/Ag5 group displayed significantly cytotoxicity.ALP activity assay and RT-qPCR analysis showed that expressions of osteogenesis-related genes in BMSCs were enhanced in Sr10/Agl and Sr50/Agl groups,and the effect of Sr50/Agl group is greater than Sr10/Agl group.[Conclusions]1.Sr/Ag-doped inorganic-organic(Sr/Ag/CSG)composite coatings were successfully fabricated on titanium substrates via EPD;2.Sr/Ag mainly existed in the coatings as the form of SrCO3 and AgCl,and the compounds released from the coatings displayed nano-scale structure.Incorporation of Sr/Ag into CSG coatings decrease the degradation ratio;3.Sr/Ag/CSG coatings could inhibite the bacteria adhered on the sample surface and suspended in the surrounding medium,and the antibacterial rate was positively correlated with the silver concentration;4.Sr10/Agl and Sr50/Agl group could promote proliferation and osteogenic differentiation of the cells,and the effect of Sr50/Agl group is greater than Sr10/Agl group.In addition,Sr50/Ag5 group displayed significantly cytotoxicity;5.In total,Sr50/Agl group coating is multifunctional,which could have antibacterial activity and osteogenic property simultaneously,this investigation may provide certain basis for the future use of Sr/Ag/CSG coatings.更多还原