纯钛表面TiN/Ag多层复合结构制备及生物学性能的评价
|
摘要
背景:在钛种植体表面建立一种能促进细胞早期黏附增殖、分化且无细胞毒性抑菌效果明显的界面至关重要。目的:在纯钛种植体材料表面制备Ti N/Ag多层复合结构涂层,分析其抗菌性能及对MC3T3-E1细胞生物学行为的影响。方法:采用酸蚀喷砂技术及多弧离子镀技术在光滑纯钛表面制备Ti N/Ag多层复合结构。取生长状态良好的MC3T3-E1细胞,分别接种于光滑钛片、酸蚀喷砂钛片及载Ti N/Ag多层复合结构钛片表面,接种后24 h,激光共聚焦显微镜观察细胞黏附及细胞活化情况,扫描电镜观察细胞生长形态;接种后24,48,72 h,CCK-8法检测细胞增殖及毒性分级。将金黄色葡萄球菌菌液分别滴加于光滑钛片、酸蚀喷砂钛片及载Ti N/Ag多层复合结构钛片表面,16 h后,激光共聚焦显微镜观察细菌生长情况。结果与结论:(1)激光共聚焦显微镜显示,光滑组细胞呈两极或三极的纺锤状结构,F-肌动蛋白和丝状伪足表达量较少;酸蚀喷砂组与载银涂层组细胞分散附着排布,方向不同,并形成大量丝状伪足,相互交织融合在一起,细胞铺展较充分,F-肌动蛋白明显高表达,肌动蛋白纤维较粗;(2)扫描电镜显示,光滑组细胞黏附与铺展不够充分;酸蚀喷砂组、载银涂层组细胞铺展黏附良好,铺展大,细胞状态较好;(3)CCK-8实验显示接种72 h后,载银涂层组细胞生长良好,增殖明显,细胞毒性为1级;(4)激光共聚焦显微镜显示,光滑组和酸蚀喷砂组钛片表面金黄色葡萄球菌生长良好,载银涂层组钛片表面细菌出现大面积死亡;(5)结果表明,Ti N/Ag多层复合结构涂层具有良好的细胞相容性及抗菌性能。
BACKGROUND: It is of vital importance to fabricate an interface on the titanium implant surface which can promote early cell adhesion, proliferation, and differentiation, and exert better antibacterial effects with no cytotoxicity. OBJECTIVE: To prepare a Ti N/Ag composite coating on the surface of pure titanium implant, and to explore its antibacterial properties and effects on MC3 T3-E1 biobehaviors. METHODS: Acid etching blasting and multi-arc ion plating were adopted to prepare Ti N/Ag composite coating on the smooth surface of pure titanium. Then, MC3 T3-E1 cells that grew well were inoculated onto pure titanium plate, sandblasted and acid-etched titanium plate, and Ti N/Ag-coated titanium plate. Twenty-four hours later, cell adhesion and viability were observed under confocal laser scanning microscope, and cell morphology was observed under scanning electron microscope. Cell counting kit-8 was used to detect cell proliferation and cytotoxicity at 24, 48, 72 hours after inoculation. In addition, Staphylococus aureus solution was dropped onto the smooth titanium plated, acid-etched and sandblasted titanium and Ti N/Ag-coated titanium plate, and the growth of bacteria was observed by the laser confocal scanning microscope at 16 hours. RESULTS AND CONCLUSION: Under the confocal laser scanning microscope, spindle cells with bipolar or three poles were observed on the smooth titanium surface, and there was less F-actin and filopodia expression; cells on the Ti N/Ag-coated titanium surface and sandblasted and acid-etched titanium surface were scattered with a large amount of interconnected filopodia that were fully stretched and adhered to the titanium surface, highly expressed F-actin was detected, and actin fibers were thickened. Under the scanning electron microscope, the cells on the smooth titanium surface were not fully adhered and stretched, and those on the Ti N/Ag-coated titanium surface or the sandblasted and acid-etched titanium surface exhibited better adhesion and extension. Findings from the cell counting kit-8 showed that after 72 hours of inoculation, the cells on the smooth titanium surface grew well, with cytotoxicity level 1. In addition, Staphylococus aureus grew well on the smooth titanium surface under the confocal laser scanning microscope, while a large amount of Staphylococus aureus died on the Ti N/Ag-coated titanium surface or on the sandblasted and acid-etched titanium surface. These findings indicated that Ti N/Ag coating has good biocompatibility and antibacterial properties.
BACKGROUND: It is of vital importance to fabricate an interface on the titanium implant surface which can promote early cell adhesion, proliferation, and differentiation, and exert better antibacterial effects with no cytotoxicity. OBJECTIVE: To prepare a Ti N/Ag composite coating on the surface of pure titanium implant, and to explore its antibacterial properties and effects on MC3 T3-E1 biobehaviors. METHODS: Acid etching blasting and multi-arc ion plating were adopted to prepare Ti N/Ag composite coating on the smooth surface of pure titanium. Then, MC3 T3-E1 cells that grew well were inoculated onto pure titanium plate, sandblasted and acid-etched titanium plate, and Ti N/Ag-coated titanium plate. Twenty-four hours later, cell adhesion and viability were observed under confocal laser scanning microscope, and cell morphology was observed under scanning electron microscope. Cell counting kit-8 was used to detect cell proliferation and cytotoxicity at 24, 48, 72 hours after inoculation. In addition, Staphylococus aureus solution was dropped onto the smooth titanium plated, acid-etched and sandblasted titanium and Ti N/Ag-coated titanium plate, and the growth of bacteria was observed by the laser confocal scanning microscope at 16 hours. RESULTS AND CONCLUSION: Under the confocal laser scanning microscope, spindle cells with bipolar or three poles were observed on the smooth titanium surface, and there was less F-actin and filopodia expression; cells on the Ti N/Ag-coated titanium surface and sandblasted and acid-etched titanium surface were scattered with a large amount of interconnected filopodia that were fully stretched and adhered to the titanium surface, highly expressed F-actin was detected, and actin fibers were thickened. Under the scanning electron microscope, the cells on the smooth titanium surface were not fully adhered and stretched, and those on the Ti N/Ag-coated titanium surface or the sandblasted and acid-etched titanium surface exhibited better adhesion and extension. Findings from the cell counting kit-8 showed that after 72 hours of inoculation, the cells on the smooth titanium surface grew well, with cytotoxicity level 1. In addition, Staphylococus aureus grew well on the smooth titanium surface under the confocal laser scanning microscope, while a large amount of Staphylococus aureus died on the Ti N/Ag-coated titanium surface or on the sandblasted and acid-etched titanium surface. These findings indicated that Ti N/Ag coating has good biocompatibility and antibacterial properties.
引文
[1]杨晓丰,刘帆,刘奕,等.医用钛材料表面改性研究进展[J].临床口腔医学杂志,2016,32(9):569-571.
[2]焦艳军,王珏,潘福勤,等.纯钛种植体的2种表面处理对细菌黏附能力的影响[J].实用口腔医学杂志,2009,25(2):166-169.
[3]Besinis A,Hadi SD,Le H.R,et al.Antibacterial activity and biofilm inhibition by surface modified titanium alloy medical implants following application of silver,titanium dioxide and hydroxyapatite nanocoatings.Nanotoxicology.2017;11(3):327-338.
[4]Zhao L,Chu PK,Zhang Y,et al.Antibacterial coatings on titanium implants.J Biomed Mater Res B Appl Biomater.2009;91(1):470-480.
[5]Vimbela GV,Ngo SM,Fraze C,et al.Antibacterial properties and toxicity from metallic nanomaterials.Int J Nanomedicine.2017;12:3941-3965.
[6]Rossi MC,Bezerra FJB,Silva RA,et al.Titanium-released from dental implant enhances pre-osteoblast adhesion by ROSmodulating crucial intracellular pathways.J Biomed Mater Res A.2017;105(11):2968-2976.
[7]Fernandes GV,Cavagis AD,Ferreira CV,et al.Osteoblast adhesion dynamics:a possible role for ROS and LMW-PTP.J Cell Biochem.2014;115(6):1063-1069.
[8]Arita S,Suzuki,M,Kazama-Koide M,et al.Shear bond strengths of tooth coating materials including the experimental materials contained various amounts of multi-ion releasing fillers and their effects for preventing dentin demineralization.Odontology.2017.doi:10.1007/s10266-016-0290-1.[Epub ahead of print]
[9]才学敏,刘桐,唐慧琴,等.离子束辅助沉积Ti N_Ag多层膜的抗菌性和抗腐蚀性[J].核技术,2007,30(12):1028-1032.
[10]党超群,白雪冰,李金龙,等.Ti Si N/Ag纳米多层涂层的抗菌及摩擦学性能研究[J].摩擦学学报,2017,37(1):1-10.
[11]黄美东,李云珂,王萌萌,等.多弧离子镀沉积Ti Al N_Ti N多层膜的结构与性能[J].天津师范大学学报(自然科学版),2015,35(1):26-29.
[12]姜雪峰,刘清才,王海波.多弧离子镀技术及其应用[J].重庆大学学报(自然科学版),2006,29(10):55-57,68.
[13]杜娟,姜焕焕,莫嘉骥,等.钛表面形貌和亲水性表面对成骨细胞增殖分化的影响[J].中国口腔颌面外科杂志,2012,10(3):182-187.
[14]Wu P,Gao H,Sun J,et al.Biosorptive dehydration of tert-butyl alcohol using a starch-based adsorbent:characterization and thermodynamics.Bioresour Technol.2012;107:437-443.
[15]Tomankova K,Horakova J,Harvanova M,et al.Cytotoxicity,cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.Food Chem Toxicol.2015;82:106-115.
[16]Abbas HK,Yoshizawa T,Shier WT.Cytotoxicity and phytotoxicity of trichothecene mycotoxins produced by Fusarium spp.Toxicon.2013;74:68-75.
[17]Miao X,Wang D,Xu L,et al.The response of human osteoblasts,epithelial cells,fibroblasts,macrophages and oral bacteria to nanostructured titanium surfaces:a systematic study.Int JNanomedicine.2017;12:1415-1430.
[18]Qin H,Cao H,Zhao Y,et al.In vitro and in vivo anti-biofilm effects of silver nanoparticles immobilized on titanium.Biomaterials.2014;35(33):9114-9125.
[19]Ye L.Current dental implant design and its clinical importance.Hua Xi Kou Qiang Yi Xue Za Zhi.2017;35(1):18-28.
[20]邵磊,赵宝红.钛种植体骨结合界面组织学研究进展[J].中国实用口腔科杂志,2014,7(7):440-445.
[21]Ma Z,Li M,Liu R,et al.In vitro study on an antibacterial Ti-5Cu alloy for medical application.J Mater Sci Mater Med.2016;27(5):91.
[22]Vahabzadeh S,Roy M,Bandyopadhyay A,et al.Phase stability and biological property evaluation of plasma sprayed hydroxyapatite coatings for orthopedic and dental applications.Acta Biomater.2015;17:47-55.
[23]莫尊理,胡惹惹,王雅雯,等.抗菌材料及其抗菌机理[J].材料导报,2014,28(1):50-52,90.
[24]Qin H,Zhu C,An Z,et al.Silver nanoparticles promote osteogenic differentiation of human urine-derived stem cells at noncytotoxic concentrations.Int J Nanomedicine.2014;9:2469-2478.
[25]Roy M,Pompella A,Kubacki J,et al.Photofunctionalization of dental zirconia oxide:Surface modification to improve bio-integration preserving crystal stability.Colloids Surf B Biointerfaces.2017;156:194-202.
[26]Okazaki Y,Doi K,Oki Y,et al.Enhanced Osseointegration of a Modified Titanium Implant with Bound Phospho-Threonine:APreliminary In Vivo Study.J Funct Biomater.2017;8(2).pii:E16.doi:10.3390/jfb8020016.
[27]Meng HW,Chien EY,Chien HH.Dental implant bioactive surface modifications and their effects on osseointegration:a review.Biomark Res.2016;4:24.
[28]胡敏,刘莹,赖珍荃,等.磁控溅射Ti N薄膜的工艺及电学性能研究[J].功能材料,2009,40(2):222-225.
[29]袁建鹏.钛合金表面多弧离子镀Ti Al N薄膜微观组织结构及性能研究[J].热喷涂技术,2012,4(3):84-88.
[30]赵时璐,张钧,刘常升.多弧离子镀(Ti,Al,Zr,Cr)N多组元氮化物膜的研究[J].真空科学与技术学报,2009,29(6):707-711.
[31]Qiao S,Cao H,Zhao X,et al.Ag-plasma modification enhances bone apposition around titanium dental implants:an animal study in Labrador dogs.Int J Nanomedicine.2015;10:653-664.
[32]汤京龙,王硕,刘丽,等.纳米银颗粒的细胞毒性作用及机制初探[J].北京生物医学工程,2013,32(5):485-489.
[33]刘泉,黄文,熊颖铭,等.纳米银改性钛片细胞生物毒性实验研究[J].现代口腔医学杂志,2014,28(4):214-217.
[34]Dong F,Mohd Zaidi NF,Valsami-Jones E,et al.Time-resolved toxicity study reveals the dynamic interactions between uncoated silver nanoparticles and bacteria.Nanotoxicology.2017;11(5):637-646.
[35]Dong F,Valsami-Jones E,Kreft JU.New,rapid method to measure dissolved silver concentration in silver nanoparticle suspensions by aggregation combined with centrifugation.J Nanopart Res.2016;18(9):259.
[36]刘玉,尹伟,史春.20-40nm银的体外生物安全性研究[J].口腔医学研究,2015,31(2):120-122.
[37]Bouallegui Y,Ben Younes R,Turki F,et al.Effect of exposure time,particle size and uptake pathways in immune cell lysosomal cytotoxicity of mussels exposed to silver nanoparticles.Drug Chem Toxicol.2017:1-6.
[38]Foldbjerg R,Dang DA,Autrup H.Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line,A549.Arch Toxicol.2011;85(7):743-750.
[39]Umeda H,Mano T,Harada K,et al.Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method.J Mater Sci Mater Med.2017;28(8):112.
[40]于卫强,徐玲,张富强.Ti O_2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响[J].口腔医学研究,2011,27(2):101-104.
[2]焦艳军,王珏,潘福勤,等.纯钛种植体的2种表面处理对细菌黏附能力的影响[J].实用口腔医学杂志,2009,25(2):166-169.
[3]Besinis A,Hadi SD,Le H.R,et al.Antibacterial activity and biofilm inhibition by surface modified titanium alloy medical implants following application of silver,titanium dioxide and hydroxyapatite nanocoatings.Nanotoxicology.2017;11(3):327-338.
[4]Zhao L,Chu PK,Zhang Y,et al.Antibacterial coatings on titanium implants.J Biomed Mater Res B Appl Biomater.2009;91(1):470-480.
[5]Vimbela GV,Ngo SM,Fraze C,et al.Antibacterial properties and toxicity from metallic nanomaterials.Int J Nanomedicine.2017;12:3941-3965.
[6]Rossi MC,Bezerra FJB,Silva RA,et al.Titanium-released from dental implant enhances pre-osteoblast adhesion by ROSmodulating crucial intracellular pathways.J Biomed Mater Res A.2017;105(11):2968-2976.
[7]Fernandes GV,Cavagis AD,Ferreira CV,et al.Osteoblast adhesion dynamics:a possible role for ROS and LMW-PTP.J Cell Biochem.2014;115(6):1063-1069.
[8]Arita S,Suzuki,M,Kazama-Koide M,et al.Shear bond strengths of tooth coating materials including the experimental materials contained various amounts of multi-ion releasing fillers and their effects for preventing dentin demineralization.Odontology.2017.doi:10.1007/s10266-016-0290-1.[Epub ahead of print]
[9]才学敏,刘桐,唐慧琴,等.离子束辅助沉积Ti N_Ag多层膜的抗菌性和抗腐蚀性[J].核技术,2007,30(12):1028-1032.
[10]党超群,白雪冰,李金龙,等.Ti Si N/Ag纳米多层涂层的抗菌及摩擦学性能研究[J].摩擦学学报,2017,37(1):1-10.
[11]黄美东,李云珂,王萌萌,等.多弧离子镀沉积Ti Al N_Ti N多层膜的结构与性能[J].天津师范大学学报(自然科学版),2015,35(1):26-29.
[12]姜雪峰,刘清才,王海波.多弧离子镀技术及其应用[J].重庆大学学报(自然科学版),2006,29(10):55-57,68.
[13]杜娟,姜焕焕,莫嘉骥,等.钛表面形貌和亲水性表面对成骨细胞增殖分化的影响[J].中国口腔颌面外科杂志,2012,10(3):182-187.
[14]Wu P,Gao H,Sun J,et al.Biosorptive dehydration of tert-butyl alcohol using a starch-based adsorbent:characterization and thermodynamics.Bioresour Technol.2012;107:437-443.
[15]Tomankova K,Horakova J,Harvanova M,et al.Cytotoxicity,cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.Food Chem Toxicol.2015;82:106-115.
[16]Abbas HK,Yoshizawa T,Shier WT.Cytotoxicity and phytotoxicity of trichothecene mycotoxins produced by Fusarium spp.Toxicon.2013;74:68-75.
[17]Miao X,Wang D,Xu L,et al.The response of human osteoblasts,epithelial cells,fibroblasts,macrophages and oral bacteria to nanostructured titanium surfaces:a systematic study.Int JNanomedicine.2017;12:1415-1430.
[18]Qin H,Cao H,Zhao Y,et al.In vitro and in vivo anti-biofilm effects of silver nanoparticles immobilized on titanium.Biomaterials.2014;35(33):9114-9125.
[19]Ye L.Current dental implant design and its clinical importance.Hua Xi Kou Qiang Yi Xue Za Zhi.2017;35(1):18-28.
[20]邵磊,赵宝红.钛种植体骨结合界面组织学研究进展[J].中国实用口腔科杂志,2014,7(7):440-445.
[21]Ma Z,Li M,Liu R,et al.In vitro study on an antibacterial Ti-5Cu alloy for medical application.J Mater Sci Mater Med.2016;27(5):91.
[22]Vahabzadeh S,Roy M,Bandyopadhyay A,et al.Phase stability and biological property evaluation of plasma sprayed hydroxyapatite coatings for orthopedic and dental applications.Acta Biomater.2015;17:47-55.
[23]莫尊理,胡惹惹,王雅雯,等.抗菌材料及其抗菌机理[J].材料导报,2014,28(1):50-52,90.
[24]Qin H,Zhu C,An Z,et al.Silver nanoparticles promote osteogenic differentiation of human urine-derived stem cells at noncytotoxic concentrations.Int J Nanomedicine.2014;9:2469-2478.
[25]Roy M,Pompella A,Kubacki J,et al.Photofunctionalization of dental zirconia oxide:Surface modification to improve bio-integration preserving crystal stability.Colloids Surf B Biointerfaces.2017;156:194-202.
[26]Okazaki Y,Doi K,Oki Y,et al.Enhanced Osseointegration of a Modified Titanium Implant with Bound Phospho-Threonine:APreliminary In Vivo Study.J Funct Biomater.2017;8(2).pii:E16.doi:10.3390/jfb8020016.
[27]Meng HW,Chien EY,Chien HH.Dental implant bioactive surface modifications and their effects on osseointegration:a review.Biomark Res.2016;4:24.
[28]胡敏,刘莹,赖珍荃,等.磁控溅射Ti N薄膜的工艺及电学性能研究[J].功能材料,2009,40(2):222-225.
[29]袁建鹏.钛合金表面多弧离子镀Ti Al N薄膜微观组织结构及性能研究[J].热喷涂技术,2012,4(3):84-88.
[30]赵时璐,张钧,刘常升.多弧离子镀(Ti,Al,Zr,Cr)N多组元氮化物膜的研究[J].真空科学与技术学报,2009,29(6):707-711.
[31]Qiao S,Cao H,Zhao X,et al.Ag-plasma modification enhances bone apposition around titanium dental implants:an animal study in Labrador dogs.Int J Nanomedicine.2015;10:653-664.
[32]汤京龙,王硕,刘丽,等.纳米银颗粒的细胞毒性作用及机制初探[J].北京生物医学工程,2013,32(5):485-489.
[33]刘泉,黄文,熊颖铭,等.纳米银改性钛片细胞生物毒性实验研究[J].现代口腔医学杂志,2014,28(4):214-217.
[34]Dong F,Mohd Zaidi NF,Valsami-Jones E,et al.Time-resolved toxicity study reveals the dynamic interactions between uncoated silver nanoparticles and bacteria.Nanotoxicology.2017;11(5):637-646.
[35]Dong F,Valsami-Jones E,Kreft JU.New,rapid method to measure dissolved silver concentration in silver nanoparticle suspensions by aggregation combined with centrifugation.J Nanopart Res.2016;18(9):259.
[36]刘玉,尹伟,史春.20-40nm银的体外生物安全性研究[J].口腔医学研究,2015,31(2):120-122.
[37]Bouallegui Y,Ben Younes R,Turki F,et al.Effect of exposure time,particle size and uptake pathways in immune cell lysosomal cytotoxicity of mussels exposed to silver nanoparticles.Drug Chem Toxicol.2017:1-6.
[38]Foldbjerg R,Dang DA,Autrup H.Cytotoxicity and genotoxicity of silver nanoparticles in the human lung cancer cell line,A549.Arch Toxicol.2011;85(7):743-750.
[39]Umeda H,Mano T,Harada K,et al.Appearance of cell-adhesion factor in osteoblast proliferation and differentiation of apatite coating titanium by blast coating method.J Mater Sci Mater Med.2017;28(8):112.
[40]于卫强,徐玲,张富强.Ti O_2纳米管对MC3T3-E1前成骨细胞骨功能基因表达变化的影响[J].口腔医学研究,2011,27(2):101-104.