医用NiTi合金表面TiO_2膜层的制备及生物活性研究
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摘要
本论文采用微弧氧化法对NiTi合金进行表面改性，提高其耐腐蚀性，降低Ni离子的析出量，改善NiTi合金的生物活性。利用X射线衍射仪(XRD)、扫描电子显微镜(SEM)、能谱(EDS)和X射线光电子谱(XPS)研究膜层的相结构、形貌和膜层组成成分。利用电化学方法和Hank’s溶液浸泡实验测试和评价了微弧氧化膜层的耐腐蚀性和对镍离子析出的抑制作用。通过SBF浸泡和成骨细胞培养研究了微弧氧化膜层诱导磷灰石的能力和膜层表面的细胞生长行为，评价微弧氧化膜层的生物活性。在SEM和荧光显微镜下观察了细胞在膜层表面的生长状态和繁殖行为，评价膜层的生物相容性。另外还通过对碱性磷酸的监测，评价细胞在膜层表面的活性。
以浓硫酸为电解液对NiTi合金进行微弧氧化处理，在NiTi合金表面制备了一层厚度大约在0.4-0.5μm之间、与基体结合良好、能有效抑制镍离子析出的多孔TiO_2膜层。以浓磷酸为电解液对NiTi合金进行微弧氧化处理时，在合金表面形成了含P元素的TiO_2膜层。膜层内的TiO_2成分主要是以非晶态形式存在，水热处理之后，非晶态的TiO_2可以晶化形成锐钛矿型TiO_2。膜层厚度随着微弧氧化时间的增长先增大后减小，粗糙度随着处理时间的增长而增大。电流密度的增大使膜层厚度增厚。在浓磷酸中形成的微弧氧化膜层其耐腐蚀性能和抑制镍离子析出的能力都随着微弧氧化时间的增长或电流密度的增大先增大后减小。
在含氧化钛溶胶的乙二醇电解液(Ti-Sol)中对NiTi合金进行微弧氧化，表面形成含有P元素的TiO_2膜层，膜层厚度相对于浓酸体系明显增厚。膜层内各物质是以非晶态形式存在的，水热处理之后，膜层内部非晶态形式的TiO_2结晶形成锐钛矿型TiO_2。随着微弧氧化时间的增长或者电流密度的增大，膜层的厚度和粗糙度显著增加。膜层的耐腐蚀性、抑制Ni离子析出能力以及膜层与基体间的结合强度随着微弧氧化时间的增长或电流密度的增大先提高后降低。在进一步添加甘油磷酸钙(Ca-Gp)的电解液(TiO_2-Sol-Ca)中对NiTi合金进行微弧氧化，制备的TiO_2膜层中含有Ca、P元素。XRD结果显示膜层中存在Ca_2P_4O_(12)4H_2O。电解液中Ca元素的添加使制备的微弧氧化膜层厚度和粗糙度增大，耐腐蚀性和抑制Ni离子析出能力降低，膜层与基体间的结合强度降低。
NiTi合金在不同电解液中制备的微弧氧化膜层均能在SBF溶液中诱导形成羟基磷灰石，说明微弧氧化处理能够提高NiTi合金表面的生物活性。XRD、EDS、XPS和FT-IR结果表明诱导生成的磷灰石是一种含有碳酸根的类骨磷灰石。膜层中Ca、P元素可以提高膜层的诱导磷灰石沉积的能力。不同电解液中制备的膜层诱导磷灰石沉积能力由高到低依次为：TiO_2-Sol-Ca>TiO_2-Sol>浓磷酸>浓硫酸。
NiTi合金表面生成的微弧氧化膜层有利于成骨细胞繁殖。成骨细胞培养结果表明，随着培养时间增长，成骨细胞在微弧氧化膜层表面增殖良好，细胞数量增多。和未处理的NiTi合金基体相比，成骨细胞在微弧氧化膜层表面的增殖速度上升，细胞生长更为旺盛。SEM观察发现细胞很好地铺展于涂层表面，紧密贴壁，形态完整，呈现多边形形态。细胞染色后的荧光显微镜图片显示成骨细胞在膜层表面存活完好且成长旺盛，说明微弧氧化层具有良好的细胞相容性。成骨细胞繁殖行为和碱性磷酸酶检测结果显示，在浓硫酸、浓磷酸和TiO_2-Sol体系中制备的陶瓷膜表面成骨细胞活性较高，繁殖能力较强，说明在这三种电解液体系中制备的膜层可以提高NiTi合金表面的细胞活性和生物相容性。但含钙TiO_2-Sol体系制备的微弧氧化膜层由于较高的Ni离子析出量抑制了成骨细胞在其表面的吸附和繁殖，和其他三种膜层相比生物相容性有所下降。
The micro-arc oxidation (MAO) method has been employed for the surfacemodification of NiTi alloy to improve its corrosion resistance, reduce the releasingamount of the Ni ions, and improve the biological activity of the NiTi alloy. Thesurface morphology, phase composition, element component and valence of thecoatings have been characterized by SEM, XRD, EDS, and XPS. The corrosionresistance and the Ni ion releasing rate of the samples have been evaluated byelectrochemical method and Hank's solution immersion test. SBF soakingexperiment has been employed to study the apatite-induce ability, and evaluate thebioactivity of the micro-arc oxidation coating. The osteoblast cell culture studyhas been employed to study the biocompatibility of the coating. The SEM andfluorescence microscopy have been employed to observe the growth andreproductive behavior of the cells on the surface of the MAO coating. The cellbioactivity on coating surface has been also evaluated by monitoring alkalinephosphatase activity.
After NiTi alloy had been MAO treated in the concentrated sulfuric acidelectrolyte, a porous TiO_2ceramic coating with the thickness of0.4-0.5μm couldbe prepared on its surface. The coating was tightly adherented to the NiTi alloy,and could effectively inhibit the Ni releasing. When the concentrated phosphoricacid was employed as the MAO electrolyte to treat the NiTi alloy, a P-containingTiO_2coating could be formed on the surface of NiTi alloy. TiO_2in the film wasmainly in the amorphous state, and the amorphous TiO_2could be transformed toanatase after the hydrothermal treatment. The thickness of the MAO coating firstincreased and then decreased with the increase of the MAO processing time, andthe roughness increased with the growth of the processing time. The increase ofcurrent density could make the coating thickness thicker. Both the corrosionresistance and Ni ions inhibition ability of the MAO coating formed inconcentrated phosphoric acid electrolyte first increased and then decreased withthe increase of the MAO processing time or the increase of current density.
When the NiTi alloy was MAO treated in the ethylene glycol electrolytewhich contained TiO_2Sol (TiO_2-Sol), a P-containing TiO_2coating could form onthe surface of the alloy, and the thickness of the coating was significantly thickerthan the coating formed in the concentrated acid system. The substance in thecoating was mainly in the amorphous state, and the amorphous TiO_2in the coatingcould be transformed to anatase after the hydrothermal treatment. The thicknessand roughness of the MAO coating increased with the increase of MAO processing time or the increase of current density. The corrosion resistance and Ni ionsinhibition ability of the MAO coating first increased and then decreased with theincrease of the MAO processing time or the increase of current density. When thecalcium glycerophosphate was added to the Ti-Sol electrolyte, Ca and P elementscould be found in the prepared TiO_2coating. The XRD results showed thatCa_2P_4O_(12)4H_2O had been formed in the coating. The addition of the Ca elementsin the MAO electrolyte increased the thickness and roughness of the MAO coating,reduced the corrosion resistance and the Ni ions inhibition ability of the MAOcoating, and also reduced the binding-strength between the coating and substrate.
All the MAO coatings prepared in different electrolytes could induce theformation of hydroxyapatite when soaked in SBF solution, which indicated theMAO treatment could improve the bioactivity of the NiTi alloy. XRD, EDS, XPS,and FT-IR results showed that the induced apatite was a kind of carbonatecontaining bone-like apatite. Ca and P elements combination could improve theapatite-induced ability of the MAO coating. The order of the apatite-inducedability of the coatings form in different electrolytes was: TiO_2-Sol-Ca> TiO_2-Sol>concentrated phosphoric acid> concentrated sulfuric acid.
The MAO coatings had the appropriate condition for the cell proliferation.The results of in-vitro cell culture showed that the proliferation of osteoblast cellhas rosen with the increase of the incubation time, and the proliferation ofosteoblasts was significantly higher than the untreated NiTi alloy substrate. SEMshowed that the cells spread well on the surface of the coating and tightlyadherented to the surface and it could also be found that the cells were intact andfull. Fluorescence microscopy revealed that the cells lived well in the surface ofthe coating and growed strongly. That indicated the micro-arc oxidation layer hasgood cell compatibility. Osteoblasts multiplication behavior and alkalinephosphatase results showed that osteoblasts had good bioactivity and proliferationablity on the MAO coatings prepared in concentrated sulfuric acid, concentratedphosphoric acid, and the TiO_2-Sol electrolyte. It could be included that NiTi alloysafter MAO treatment in these three electrolyte can improve its activity andbiocompatibility. But due to the high amount of Ni ions released from the MAOcoating prepared in TiO_2-Sol-Ca electrolyte, the osteoblast adsorption andreproduction was inhibited, and the biocompatibility of the coating declined.
以浓硫酸为电解液对NiTi合金进行微弧氧化处理，在NiTi合金表面制备了一层厚度大约在0.4-0.5μm之间、与基体结合良好、能有效抑制镍离子析出的多孔TiO_2膜层。以浓磷酸为电解液对NiTi合金进行微弧氧化处理时，在合金表面形成了含P元素的TiO_2膜层。膜层内的TiO_2成分主要是以非晶态形式存在，水热处理之后，非晶态的TiO_2可以晶化形成锐钛矿型TiO_2。膜层厚度随着微弧氧化时间的增长先增大后减小，粗糙度随着处理时间的增长而增大。电流密度的增大使膜层厚度增厚。在浓磷酸中形成的微弧氧化膜层其耐腐蚀性能和抑制镍离子析出的能力都随着微弧氧化时间的增长或电流密度的增大先增大后减小。
在含氧化钛溶胶的乙二醇电解液(Ti-Sol)中对NiTi合金进行微弧氧化，表面形成含有P元素的TiO_2膜层，膜层厚度相对于浓酸体系明显增厚。膜层内各物质是以非晶态形式存在的，水热处理之后，膜层内部非晶态形式的TiO_2结晶形成锐钛矿型TiO_2。随着微弧氧化时间的增长或者电流密度的增大，膜层的厚度和粗糙度显著增加。膜层的耐腐蚀性、抑制Ni离子析出能力以及膜层与基体间的结合强度随着微弧氧化时间的增长或电流密度的增大先提高后降低。在进一步添加甘油磷酸钙(Ca-Gp)的电解液(TiO_2-Sol-Ca)中对NiTi合金进行微弧氧化，制备的TiO_2膜层中含有Ca、P元素。XRD结果显示膜层中存在Ca_2P_4O_(12)4H_2O。电解液中Ca元素的添加使制备的微弧氧化膜层厚度和粗糙度增大，耐腐蚀性和抑制Ni离子析出能力降低，膜层与基体间的结合强度降低。
NiTi合金在不同电解液中制备的微弧氧化膜层均能在SBF溶液中诱导形成羟基磷灰石，说明微弧氧化处理能够提高NiTi合金表面的生物活性。XRD、EDS、XPS和FT-IR结果表明诱导生成的磷灰石是一种含有碳酸根的类骨磷灰石。膜层中Ca、P元素可以提高膜层的诱导磷灰石沉积的能力。不同电解液中制备的膜层诱导磷灰石沉积能力由高到低依次为：TiO_2-Sol-Ca>TiO_2-Sol>浓磷酸>浓硫酸。
NiTi合金表面生成的微弧氧化膜层有利于成骨细胞繁殖。成骨细胞培养结果表明，随着培养时间增长，成骨细胞在微弧氧化膜层表面增殖良好，细胞数量增多。和未处理的NiTi合金基体相比，成骨细胞在微弧氧化膜层表面的增殖速度上升，细胞生长更为旺盛。SEM观察发现细胞很好地铺展于涂层表面，紧密贴壁，形态完整，呈现多边形形态。细胞染色后的荧光显微镜图片显示成骨细胞在膜层表面存活完好且成长旺盛，说明微弧氧化层具有良好的细胞相容性。成骨细胞繁殖行为和碱性磷酸酶检测结果显示，在浓硫酸、浓磷酸和TiO_2-Sol体系中制备的陶瓷膜表面成骨细胞活性较高，繁殖能力较强，说明在这三种电解液体系中制备的膜层可以提高NiTi合金表面的细胞活性和生物相容性。但含钙TiO_2-Sol体系制备的微弧氧化膜层由于较高的Ni离子析出量抑制了成骨细胞在其表面的吸附和繁殖，和其他三种膜层相比生物相容性有所下降。
The micro-arc oxidation (MAO) method has been employed for the surfacemodification of NiTi alloy to improve its corrosion resistance, reduce the releasingamount of the Ni ions, and improve the biological activity of the NiTi alloy. Thesurface morphology, phase composition, element component and valence of thecoatings have been characterized by SEM, XRD, EDS, and XPS. The corrosionresistance and the Ni ion releasing rate of the samples have been evaluated byelectrochemical method and Hank's solution immersion test. SBF soakingexperiment has been employed to study the apatite-induce ability, and evaluate thebioactivity of the micro-arc oxidation coating. The osteoblast cell culture studyhas been employed to study the biocompatibility of the coating. The SEM andfluorescence microscopy have been employed to observe the growth andreproductive behavior of the cells on the surface of the MAO coating. The cellbioactivity on coating surface has been also evaluated by monitoring alkalinephosphatase activity.
After NiTi alloy had been MAO treated in the concentrated sulfuric acidelectrolyte, a porous TiO_2ceramic coating with the thickness of0.4-0.5μm couldbe prepared on its surface. The coating was tightly adherented to the NiTi alloy,and could effectively inhibit the Ni releasing. When the concentrated phosphoricacid was employed as the MAO electrolyte to treat the NiTi alloy, a P-containingTiO_2coating could be formed on the surface of NiTi alloy. TiO_2in the film wasmainly in the amorphous state, and the amorphous TiO_2could be transformed toanatase after the hydrothermal treatment. The thickness of the MAO coating firstincreased and then decreased with the increase of the MAO processing time, andthe roughness increased with the growth of the processing time. The increase ofcurrent density could make the coating thickness thicker. Both the corrosionresistance and Ni ions inhibition ability of the MAO coating formed inconcentrated phosphoric acid electrolyte first increased and then decreased withthe increase of the MAO processing time or the increase of current density.
When the NiTi alloy was MAO treated in the ethylene glycol electrolytewhich contained TiO_2Sol (TiO_2-Sol), a P-containing TiO_2coating could form onthe surface of the alloy, and the thickness of the coating was significantly thickerthan the coating formed in the concentrated acid system. The substance in thecoating was mainly in the amorphous state, and the amorphous TiO_2in the coatingcould be transformed to anatase after the hydrothermal treatment. The thicknessand roughness of the MAO coating increased with the increase of MAO processing time or the increase of current density. The corrosion resistance and Ni ionsinhibition ability of the MAO coating first increased and then decreased with theincrease of the MAO processing time or the increase of current density. When thecalcium glycerophosphate was added to the Ti-Sol electrolyte, Ca and P elementscould be found in the prepared TiO_2coating. The XRD results showed thatCa_2P_4O_(12)4H_2O had been formed in the coating. The addition of the Ca elementsin the MAO electrolyte increased the thickness and roughness of the MAO coating,reduced the corrosion resistance and the Ni ions inhibition ability of the MAOcoating, and also reduced the binding-strength between the coating and substrate.
All the MAO coatings prepared in different electrolytes could induce theformation of hydroxyapatite when soaked in SBF solution, which indicated theMAO treatment could improve the bioactivity of the NiTi alloy. XRD, EDS, XPS,and FT-IR results showed that the induced apatite was a kind of carbonatecontaining bone-like apatite. Ca and P elements combination could improve theapatite-induced ability of the MAO coating. The order of the apatite-inducedability of the coatings form in different electrolytes was: TiO_2-Sol-Ca> TiO_2-Sol>concentrated phosphoric acid> concentrated sulfuric acid.
The MAO coatings had the appropriate condition for the cell proliferation.The results of in-vitro cell culture showed that the proliferation of osteoblast cellhas rosen with the increase of the incubation time, and the proliferation ofosteoblasts was significantly higher than the untreated NiTi alloy substrate. SEMshowed that the cells spread well on the surface of the coating and tightlyadherented to the surface and it could also be found that the cells were intact andfull. Fluorescence microscopy revealed that the cells lived well in the surface ofthe coating and growed strongly. That indicated the micro-arc oxidation layer hasgood cell compatibility. Osteoblasts multiplication behavior and alkalinephosphatase results showed that osteoblasts had good bioactivity and proliferationablity on the MAO coatings prepared in concentrated sulfuric acid, concentratedphosphoric acid, and the TiO_2-Sol electrolyte. It could be included that NiTi alloysafter MAO treatment in these three electrolyte can improve its activity andbiocompatibility. But due to the high amount of Ni ions released from the MAOcoating prepared in TiO_2-Sol-Ca electrolyte, the osteoblast adsorption andreproduction was inhibited, and the biocompatibility of the coating declined.
引文
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