四级钛表面纳米锌离子注入沉积改性的基础研究
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摘要
研究背景和立题依据
种植体的表面微形貌是影响种植体及种植体长期存活的重要因素。由于钛自身不具备抗菌性能,而种植体周围炎是导致种植义齿失败的主要原因之一。
几乎所有常用的牙科种植材料都没有诱导成骨的效用,或者诱导成骨的能力很弱,不能达到理想的骨结合效果。
现有的种植体表面处理方法存在着各自的弱点与不足,同时具有良好的生物相容性和抗菌性能的种植材料国内外尚未见报道。
理想的种植体表面形貌尚未明确且表面处理技术均来自于国外。
意义:扩大种植义齿适应症、提高种植义齿的长期成功率、减轻患者的经济负担、提高病人的生存质量。
材料和方法
1.全方位离子注入与沉积装置是哈尔滨工业大学现代焊接生产技术国家重点实验室自主研发的第四代等离子体浸没离子注入沉积装置;
2.首都医科大学北京口腔医学院研究所进行预实验且已完成部分实验;
3.首都医科大学和中科院化学研究所具备先进的仪器设备,可进行改性后表面形貌的检测,并对此项研究提供相关的技术指导;
4.首都医科大学附属北京口腔医院研究所设备先进,是全国最具权威的菌库基地之一,技术成熟,能支持并完成本实验的微生物部分及细胞分子水平的实验;
5.动物模型选用小型猪,这也是首医王松灵教授领导的课题小组的一大特色,掌握一定的技术和方法;
纯钛表面处理方法及其特性的研究:
应用全方位离子注入与沉积技术,通过扫描电镜(SEM)、激光共聚焦显微镜(CO-FOCAL)、x线衍射仪(XRD)、X线光电子能谱分析(XPS)、透射电镜(TEM)等分析表面特性及成分。
生物安全性研究:
成骨细胞的复苏和培养、细胞毒性试验(MTT)、细胞粘附和增殖实验、扫描电镜观察(SEM)、丫啶橙染色法、PI流式细胞仪分析、成骨细胞粘着斑的形成、成骨细胞细胞外基质的形成和表达、免疫荧光法检测I型胶原的形成、Realtime-PCR检测I型胶原mRNA的表达、蛋白印迹法(Western Blotting)检测I型胶原蛋白的表达。动物体内对比研究
建立动物模型,将实验组鈦片(Zn-Ti)与对照组(cp-Ti)分别植入小型猪颌骨内,不同时间(30d;60d;90d)采集标本,进行组织形态学及扫描电镜观察。
结果:
1.纯钛表面处理方法及其特性的评价
(1) X线光电子能谱分析
X线光电子能谱分析表明,经过PIIID(全方位等离子体浸没离子注入与沉积)改性后的钛片表面新出现了锌和氧元素的信号。同时,随着锌离子注入沉积时间的延长,锌含量逐渐增加,在Zn-Ti-80 min组时,锌含量最高。
(2)扫描电镜(SEM)
通过扫描电镜(SEM)可见在纯钛组(cp-Ti),材料表面有少量的微小裂隙,锌离子注入沉积组,Zn-Ti-20 min和Zn-Ti-40 min组,改性后材料表面整体呈现云雾状的光滑表面。在锌离子注入沉积后的Zn-Ti-60 min and Zn-Ti-80 min组的钛片上表面呈现出均匀的“蜂窝状”纳米级样结构,直径约60-100nm,深度随着注入沉积时间的延长而增加,到80min组时深度约为200nm;该表面光滑9级,粗糙度0.4um。
2.抑菌实验
锌离子注入沉积钛表面可以减少变形链球菌的粘附,间接的提升其有抑菌功能。变形链球菌定植在钛表面的数量及菌体形态显示;在Zn-Ti-60 min和Zn-Ti-80 min组时变形链球菌的数量远远少于Zn-Ti-20 min和Zn-Ti-40 min组。Zn-Ti-80 min组的钛片表面几乎没有菌体附着,只能看见少数残存的菌落碎片。
3.生物安全评价
(1) MG-63细胞形态变化
在体外实验中,经过全方位离子注入沉积改性后的锌、钛离子表面对MG - 63细胞的形貌的影响,通过扫描电镜分析表明;在纯钛组表面细胞数量及伸展均不足,改性后的钛片组表面细胞数量远远比纯钛组密度高,且伸展充分,Zn-Ti-80 min组最明显。
(2)在6小时时间点上,各组间(纯钛组与改性组)比较分析,细胞增殖数量经统计学分析p>0.05,无统计学意义;在24小时时间点上,改性后的各组与纯钛组经统计学分析p<0.05,有统计学意义;但是Zn-Ti-60 group和Zn-Ti-80 group之间没有统计学意义。
4.体内试验
经过组织学的前期处理后,进行硬组织磨片,特殊三色染色,得出的染色后结果分析表明;经过锌离子注入沉积改性后的钛片组(Zn-Ti)周围骨融合与骨再生情况明显优于纯钛组(cp-Ti)。
结论:
通过以上实验数据表面,四级钛表面纳米锌离子注入沉积改性技术可以提高种植体的生物相容性及抗菌性,这对于今后我们研发具有自主知识产权的新型表面改性技术的种植体意义重大,并且发掘其潜在的临床应用价值。
Background and Purpose
Implant surface microtopography is an important factor influencing implant osseointegration and its long-term survival. The titanium itself does not have anti-bacterial properties; peri-implant inflammation is one of the major factors leading to implant failure. Almost all implanting materials show no or little effectiveness of bone induction. The available treatment methods of implant surface have their own weakness and shortcoming. The implanting material with good biocompatibility and antimicrobial properties has not yet been reported. The ideal implant surface topography remains undefined. The purpose of the present study is to modify the implant surface using nano-zinc ion deposition for increasing of the adaptability of implant denture and improving the long-term implant success rate.
Materials and Methods
The fourth-generation plasma immersion ion implantation deposition device were used to generated the ion implantation and deposition. The device was designed by the national Laboratory of Advanced Welding Production Technology,a famous institute of the Harbin Institute of Technology. Experiments were performed at the Beijing Institute of Stomatology, School of Stomatology, Capital Medical University.
Evaluation of the titanium surface properties
Plasma immersion ion implantation and deposition technologies, electron microscopy (SEM), laser scanning confocal microscope (CO-FOCAL), x-ray diffraction instrument (XRD), X ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) were used to analyze of the surface properties and composition.
Antimicrobial test
The morphological observation of colonies and assessment of the results, colony count, the influence of colony adhesion and colony morphology were used to evaluate the antimicrobial effect of modified implant surface.
Biological safety
Osteoblast recovery and cultivation, cytotoxicity test (MTT), cell adhesion and proliferation experiments, cell adhesion and proliferation experiments, acridine orange staining, PI flow cytometry, formation of osteoblast adhesion plaque, formation and expression of extracellular matrix of osteoblast cells were used to assess the biological safety. Immunofluorescent assay of the formation of collagen type I, real time-PCR detection of mRNA expression of collagen type I, and Western blotting were performed to detect the expression of collagen type I.
In vivo study
Miniature pigs were used for this in vivo study as a large animal model. Titanium Plate (Zn-Ti; experimental group) and cp-Ti (the control group) were implanted into miniature pig jaws; samples were taken at different time points (30d; 60d; 90d) for histological and scanning electron microscopy observation.
Results
1. Evaluation of the titanium surface properties
(1) X ray photoelectron spectroscopy
The full range of X ray photoelectron spectroscopy indicated that the main difference of zinc PIIID (plasma immersion ion implantation and deposition) on the disc was the appearance of the signals of zinc and oxygen. At the same time, the relative atomic concentration of zinc increased gradually with the ion- implanting time, highest concentration of zinc was found in Zn-Ti-80 min (d) group compared with that of other groups.
(2) Scanning electron microscope (SEM)
Tiny minor fissures were existed in the surfaces cp-Ti, Zn-Ti-20 min and Zn-Ti-40 min, which presented a smooth shape. However, A good many of homogeneously sporadic distributed.
micropores on Zn-Ti-60 min and Zn-Ti-80 min surfaces. Especially, surfaces of Zn-Ti-80 min appeared the nano- scale space, which indicated impacting into the basal material on its surface.Diameter about 60-100nm, as the depth of implantation and deposition , diameter about 200nm; the smooth surface about grade 9, roughness 0.4um.
2. Antimicrobial test
The zinc implanted titanium could affect adherence and the form of S.mutans group. The adherence amount of bacteria in Zn-Ti-60 min and -80 min group were significantly less than those in cp-Ti group, Zn-Ti-20 min and -40 min group. The similar results were found in the volume of bacterium on modified surface during the ion-implanted and deposition procedure, e.g. few bacterial colony in Zn-Ti-80min.
3. Biological safety
(1) MG-63 cells were of irregular polygon shapes
SEM analysis revealed that Zn-Ti In vitro the morphologies of MG-63 cells were influenced ,which were revealed through the analysid of SEM.
An increased cell-to-substrate contact ratio was found in the spreading and flattening of cells on the Zn-Ti discs,which was faster than the ratio of those grew on the cp-Ti discs.The density of zinc ion improved the cells activities, such as proliferation, differentiation and externalization. Furthermore, the implanting time was contributed with the density of cells, which on the cp-Ti had far less and shorter fibrillar extensions.
(2) The number of cells which were observed in different times.No statistical differences were found after 6 hours.However, the number of cells attached on Zn-Ti surfaces increased rapidly compared with the cp-Ti surfaces after 24 hours. The trend was more significantly with the time extended. But no significant difference were found between the Zn-Ti-60 group and the Zn-Ti-80 group.
4. In vivo study
The grinded bone-implant slices were obtained after histological pretreatment. Bone integration and osteogenesis with bone regenerative materials were clearly detected. Much better bone regeneration was found in experimental groups (Zn-Ti) compared with the control groups (cp-Ti).
Conclusion
Our data demonstrated that implant modification using the nano-zinc implantation and deposition technologies can improve biocompatibility and antimicrobial properties of implants, which may have potential wide clinical application for dental implantation.
种植体的表面微形貌是影响种植体及种植体长期存活的重要因素。由于钛自身不具备抗菌性能,而种植体周围炎是导致种植义齿失败的主要原因之一。
几乎所有常用的牙科种植材料都没有诱导成骨的效用,或者诱导成骨的能力很弱,不能达到理想的骨结合效果。
现有的种植体表面处理方法存在着各自的弱点与不足,同时具有良好的生物相容性和抗菌性能的种植材料国内外尚未见报道。
理想的种植体表面形貌尚未明确且表面处理技术均来自于国外。
意义:扩大种植义齿适应症、提高种植义齿的长期成功率、减轻患者的经济负担、提高病人的生存质量。
材料和方法
1.全方位离子注入与沉积装置是哈尔滨工业大学现代焊接生产技术国家重点实验室自主研发的第四代等离子体浸没离子注入沉积装置;
2.首都医科大学北京口腔医学院研究所进行预实验且已完成部分实验;
3.首都医科大学和中科院化学研究所具备先进的仪器设备,可进行改性后表面形貌的检测,并对此项研究提供相关的技术指导;
4.首都医科大学附属北京口腔医院研究所设备先进,是全国最具权威的菌库基地之一,技术成熟,能支持并完成本实验的微生物部分及细胞分子水平的实验;
5.动物模型选用小型猪,这也是首医王松灵教授领导的课题小组的一大特色,掌握一定的技术和方法;
纯钛表面处理方法及其特性的研究:
应用全方位离子注入与沉积技术,通过扫描电镜(SEM)、激光共聚焦显微镜(CO-FOCAL)、x线衍射仪(XRD)、X线光电子能谱分析(XPS)、透射电镜(TEM)等分析表面特性及成分。
生物安全性研究:
成骨细胞的复苏和培养、细胞毒性试验(MTT)、细胞粘附和增殖实验、扫描电镜观察(SEM)、丫啶橙染色法、PI流式细胞仪分析、成骨细胞粘着斑的形成、成骨细胞细胞外基质的形成和表达、免疫荧光法检测I型胶原的形成、Realtime-PCR检测I型胶原mRNA的表达、蛋白印迹法(Western Blotting)检测I型胶原蛋白的表达。动物体内对比研究
建立动物模型,将实验组鈦片(Zn-Ti)与对照组(cp-Ti)分别植入小型猪颌骨内,不同时间(30d;60d;90d)采集标本,进行组织形态学及扫描电镜观察。
结果:
1.纯钛表面处理方法及其特性的评价
(1) X线光电子能谱分析
X线光电子能谱分析表明,经过PIIID(全方位等离子体浸没离子注入与沉积)改性后的钛片表面新出现了锌和氧元素的信号。同时,随着锌离子注入沉积时间的延长,锌含量逐渐增加,在Zn-Ti-80 min组时,锌含量最高。
(2)扫描电镜(SEM)
通过扫描电镜(SEM)可见在纯钛组(cp-Ti),材料表面有少量的微小裂隙,锌离子注入沉积组,Zn-Ti-20 min和Zn-Ti-40 min组,改性后材料表面整体呈现云雾状的光滑表面。在锌离子注入沉积后的Zn-Ti-60 min and Zn-Ti-80 min组的钛片上表面呈现出均匀的“蜂窝状”纳米级样结构,直径约60-100nm,深度随着注入沉积时间的延长而增加,到80min组时深度约为200nm;该表面光滑9级,粗糙度0.4um。
2.抑菌实验
锌离子注入沉积钛表面可以减少变形链球菌的粘附,间接的提升其有抑菌功能。变形链球菌定植在钛表面的数量及菌体形态显示;在Zn-Ti-60 min和Zn-Ti-80 min组时变形链球菌的数量远远少于Zn-Ti-20 min和Zn-Ti-40 min组。Zn-Ti-80 min组的钛片表面几乎没有菌体附着,只能看见少数残存的菌落碎片。
3.生物安全评价
(1) MG-63细胞形态变化
在体外实验中,经过全方位离子注入沉积改性后的锌、钛离子表面对MG - 63细胞的形貌的影响,通过扫描电镜分析表明;在纯钛组表面细胞数量及伸展均不足,改性后的钛片组表面细胞数量远远比纯钛组密度高,且伸展充分,Zn-Ti-80 min组最明显。
(2)在6小时时间点上,各组间(纯钛组与改性组)比较分析,细胞增殖数量经统计学分析p>0.05,无统计学意义;在24小时时间点上,改性后的各组与纯钛组经统计学分析p<0.05,有统计学意义;但是Zn-Ti-60 group和Zn-Ti-80 group之间没有统计学意义。
4.体内试验
经过组织学的前期处理后,进行硬组织磨片,特殊三色染色,得出的染色后结果分析表明;经过锌离子注入沉积改性后的钛片组(Zn-Ti)周围骨融合与骨再生情况明显优于纯钛组(cp-Ti)。
结论:
通过以上实验数据表面,四级钛表面纳米锌离子注入沉积改性技术可以提高种植体的生物相容性及抗菌性,这对于今后我们研发具有自主知识产权的新型表面改性技术的种植体意义重大,并且发掘其潜在的临床应用价值。
Background and Purpose
Implant surface microtopography is an important factor influencing implant osseointegration and its long-term survival. The titanium itself does not have anti-bacterial properties; peri-implant inflammation is one of the major factors leading to implant failure. Almost all implanting materials show no or little effectiveness of bone induction. The available treatment methods of implant surface have their own weakness and shortcoming. The implanting material with good biocompatibility and antimicrobial properties has not yet been reported. The ideal implant surface topography remains undefined. The purpose of the present study is to modify the implant surface using nano-zinc ion deposition for increasing of the adaptability of implant denture and improving the long-term implant success rate.
Materials and Methods
The fourth-generation plasma immersion ion implantation deposition device were used to generated the ion implantation and deposition. The device was designed by the national Laboratory of Advanced Welding Production Technology,a famous institute of the Harbin Institute of Technology. Experiments were performed at the Beijing Institute of Stomatology, School of Stomatology, Capital Medical University.
Evaluation of the titanium surface properties
Plasma immersion ion implantation and deposition technologies, electron microscopy (SEM), laser scanning confocal microscope (CO-FOCAL), x-ray diffraction instrument (XRD), X ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM) were used to analyze of the surface properties and composition.
Antimicrobial test
The morphological observation of colonies and assessment of the results, colony count, the influence of colony adhesion and colony morphology were used to evaluate the antimicrobial effect of modified implant surface.
Biological safety
Osteoblast recovery and cultivation, cytotoxicity test (MTT), cell adhesion and proliferation experiments, cell adhesion and proliferation experiments, acridine orange staining, PI flow cytometry, formation of osteoblast adhesion plaque, formation and expression of extracellular matrix of osteoblast cells were used to assess the biological safety. Immunofluorescent assay of the formation of collagen type I, real time-PCR detection of mRNA expression of collagen type I, and Western blotting were performed to detect the expression of collagen type I.
In vivo study
Miniature pigs were used for this in vivo study as a large animal model. Titanium Plate (Zn-Ti; experimental group) and cp-Ti (the control group) were implanted into miniature pig jaws; samples were taken at different time points (30d; 60d; 90d) for histological and scanning electron microscopy observation.
Results
1. Evaluation of the titanium surface properties
(1) X ray photoelectron spectroscopy
The full range of X ray photoelectron spectroscopy indicated that the main difference of zinc PIIID (plasma immersion ion implantation and deposition) on the disc was the appearance of the signals of zinc and oxygen. At the same time, the relative atomic concentration of zinc increased gradually with the ion- implanting time, highest concentration of zinc was found in Zn-Ti-80 min (d) group compared with that of other groups.
(2) Scanning electron microscope (SEM)
Tiny minor fissures were existed in the surfaces cp-Ti, Zn-Ti-20 min and Zn-Ti-40 min, which presented a smooth shape. However, A good many of homogeneously sporadic distributed.
micropores on Zn-Ti-60 min and Zn-Ti-80 min surfaces. Especially, surfaces of Zn-Ti-80 min appeared the nano- scale space, which indicated impacting into the basal material on its surface.Diameter about 60-100nm, as the depth of implantation and deposition , diameter about 200nm; the smooth surface about grade 9, roughness 0.4um.
2. Antimicrobial test
The zinc implanted titanium could affect adherence and the form of S.mutans group. The adherence amount of bacteria in Zn-Ti-60 min and -80 min group were significantly less than those in cp-Ti group, Zn-Ti-20 min and -40 min group. The similar results were found in the volume of bacterium on modified surface during the ion-implanted and deposition procedure, e.g. few bacterial colony in Zn-Ti-80min.
3. Biological safety
(1) MG-63 cells were of irregular polygon shapes
SEM analysis revealed that Zn-Ti In vitro the morphologies of MG-63 cells were influenced ,which were revealed through the analysid of SEM.
An increased cell-to-substrate contact ratio was found in the spreading and flattening of cells on the Zn-Ti discs,which was faster than the ratio of those grew on the cp-Ti discs.The density of zinc ion improved the cells activities, such as proliferation, differentiation and externalization. Furthermore, the implanting time was contributed with the density of cells, which on the cp-Ti had far less and shorter fibrillar extensions.
(2) The number of cells which were observed in different times.No statistical differences were found after 6 hours.However, the number of cells attached on Zn-Ti surfaces increased rapidly compared with the cp-Ti surfaces after 24 hours. The trend was more significantly with the time extended. But no significant difference were found between the Zn-Ti-60 group and the Zn-Ti-80 group.
4. In vivo study
The grinded bone-implant slices were obtained after histological pretreatment. Bone integration and osteogenesis with bone regenerative materials were clearly detected. Much better bone regeneration was found in experimental groups (Zn-Ti) compared with the control groups (cp-Ti).
Conclusion
Our data demonstrated that implant modification using the nano-zinc implantation and deposition technologies can improve biocompatibility and antimicrobial properties of implants, which may have potential wide clinical application for dental implantation.
引文
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