纯钛表面微米坑复合纳米管形貌对骨髓间充质干细胞增殖及成骨分化的影响
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摘要
背景:近年来对钛种植体材料表面微米纳米的仿生设计成为了生物材料领域的研究热点。目的:观察纯钛试件表面微米坑复合纳米管形貌对骨髓间充质干细胞增殖和成骨分化的影响。方法:采用酸蚀阳极氧化方法,在纯钛表面构建微米坑复合纳米管形貌,作为实验组,以抛光处理的光滑钛试样为对照组,采用场发射扫描电子显微镜观察两组试样表面形貌。将骨髓间充质干细胞接种于两组钛试样表面,常规培养7 d内,MTT法检测细胞增殖能力;成骨诱导培养3,7,14 d后,检测碱性磷酸酶活性;成骨诱导培养7,14,21 d,RT-qPCR和Western blot检测成骨标志的mRNA及蛋白表达。结果与结论:(1)对照组试样表面无微米及纳米级结构,实验组试样表面可见为凹凸不平的微米坑形貌与均匀分布的纳米管阵列;(2)随着培养时间的增长,两组试样表面的骨髓间充质干细胞数量逐渐增加,实验组培养3,5,7 d的细胞数量明显多于对照组(P<0.05);(3)实验组成骨诱导培养7,14 d的碱性磷酸酶活性高于对照组(P<0.05);(4)实验组成骨诱导培养7,14,21 d的骨桥蛋白、骨特异性转录因子、骨钙素的mRNA及蛋白表达均高于对照组;(5)结果表明,钛表面微米坑复合纳米管形貌有利于骨髓间充干细胞的增殖与成骨分化。
BACKGROUND: In recent years, the biomimetic design of titanium implant surface at micron-nano level has become a research hotspot in the biomaterial research. OBJECTIVE: To observe the effect of acid etching and anodic oxidation on the surface morphology of pure titanium and the proliferation and osteogenesis of bone marrow mesenchymal stem cells(BMSCs). METHODS: The nanotubes with micron roughness were prepared on the titanium surface by acid etching and anodizing method. The surface morphology of nanotubes with micron roughness was used as experimental group and smooth titanium sample treated by polishing as control group. BMSCs were seeded on the surface of titanium samples and observed using field emission scanning electron microscope. Proliferation of the cells was detected by MTT assay within 7 days of conventional culture. Alkaline phosphatase activity was measured At 3, 7, 14 days after osteogenic induction. Osteogenic marker genes and proteins were detected using RT-qPCR and western blot at 7, 14, 21 days after osteogenic induction. RESULTS AND CONCLUSION: The control group had no micron and nano structure surface of the samples, while uneven surface, micron-sized holes, and uniformly distributed nanotube arrays were visible in the experimental group. With the culture time, the number of BMSCs on the sample surface in the two groups was gradually increased, and the cell number in the experimental group was significantly higher than that in the control group at 3, 5, 7 days after culture(P < 0.05). The alkaline phosphatase activity in the experimental group was also higher than that in the control group at 7 and 14 days after osteogenic induction(P < 0.05). Compared with the control group, the expression levels of osteopontin, bone-specific transcription factors, osteocalcin mRNAs and proteins were increased in the experimental group at 7, 14, 21 days after culture. These results suggest that the acid etching and anodic oxidation of the titanium surface of nanotubes can promote BMSCs proliferation and osteogenic differentiation.
BACKGROUND: In recent years, the biomimetic design of titanium implant surface at micron-nano level has become a research hotspot in the biomaterial research. OBJECTIVE: To observe the effect of acid etching and anodic oxidation on the surface morphology of pure titanium and the proliferation and osteogenesis of bone marrow mesenchymal stem cells(BMSCs). METHODS: The nanotubes with micron roughness were prepared on the titanium surface by acid etching and anodizing method. The surface morphology of nanotubes with micron roughness was used as experimental group and smooth titanium sample treated by polishing as control group. BMSCs were seeded on the surface of titanium samples and observed using field emission scanning electron microscope. Proliferation of the cells was detected by MTT assay within 7 days of conventional culture. Alkaline phosphatase activity was measured At 3, 7, 14 days after osteogenic induction. Osteogenic marker genes and proteins were detected using RT-qPCR and western blot at 7, 14, 21 days after osteogenic induction. RESULTS AND CONCLUSION: The control group had no micron and nano structure surface of the samples, while uneven surface, micron-sized holes, and uniformly distributed nanotube arrays were visible in the experimental group. With the culture time, the number of BMSCs on the sample surface in the two groups was gradually increased, and the cell number in the experimental group was significantly higher than that in the control group at 3, 5, 7 days after culture(P < 0.05). The alkaline phosphatase activity in the experimental group was also higher than that in the control group at 7 and 14 days after osteogenic induction(P < 0.05). Compared with the control group, the expression levels of osteopontin, bone-specific transcription factors, osteocalcin mRNAs and proteins were increased in the experimental group at 7, 14, 21 days after culture. These results suggest that the acid etching and anodic oxidation of the titanium surface of nanotubes can promote BMSCs proliferation and osteogenic differentiation.
引文
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[8]王婷婷,王丽娜,范震.钛种植体阳极氧化的研究[J].口腔颌面外科杂志,2016,26(4):290-294.
[9]朱晟译.钛种植体表面纳米改性方法及研究进展[J].中国口腔种植学杂志,2011,16(3):180-182,187.
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[13]付晓龙,李莺,李宝娥,等.纯钛表面阳极氧化增强成骨细胞生物活性及护骨素基因的表达[J].中国组织工程研究,2014,18(39):6240-6245.
[14]陈岩,赵文婷,咏梅,等.正畸微种植体表面阳极氧化处理对周围骨组织的影响[J].中华口腔正畸学杂志,2012,19(3):149-151.
[15]Huang B,Guang M,Ye J,et al.Effect of Increasing Doses ofγ-Radiation on Bone Marrow Stromal Cells Grown on Smooth and Rough Titanium Surfaces.Stem Cells Int.2015;2015:359416.
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[19]Wang H,Jiang Z,Zhang J,et al.Enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells on titanium substrates by inhibiting Notch3.Arch Oral Biol.2017;80:34-40.
[20]Bressel TAB,de Queiroz JDF,Gomes Moreira SM,et al.Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.Stem Cell Res Ther.2017;8(1):269.
[21]Arpornmaeklong P,Pripatnanont P,Chookiatsiri C,et al.Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.Int J Oral Maxillofac Implants.2017;32(1):e35-e46.
[22]?zdal-Kurt F,Tu?lu I,Vatansever HS,et al.The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts.Biotech Histochem.2016;91(6):412-422.
[23]Zhang W,Cao H,Zhang X,et al.A strontium-incorporated nanoporous titanium implant surface for rapid osseointegration.Nanoscale.2016;8(9):5291-5301.
[24]Li G,Cao H,Zhang W,et al.Enhanced Osseointegration of Hierarchical Micro/Nanotopographic Titanium Fabricated by Microarc Oxidation and Electrochemical Treatment.ACS Appl Mater Interfaces.2016;8(6):3840-3852.
[25]刘娜,陈丽丽.骨质疏松状态下钛种植体周围骨形成的动态观察[J].全科医学临床与教育,2017,15(2):129-131,143.
[26]孙鑫,徐金标,魏军水.热处理提高钛种植体表面成骨细胞活性的实验研究[J].健康研究,2017,37(4):393-395,399.
[27]施育才,严洪海.不同粗糙表面的纯钛种植体表面的体外细胞培养评价[J].中国口腔种植学杂志,2010,15(2):55-58,62.
[28]张君红,姜海行,覃山羽,等.全骨髓细胞贴壁法分离与培养大鼠骨髓间充质干细胞的多向分化能力[J].中国组织工程研究,2012,16(36):6685-6689.
[29]王婧,顾新华.种植体基台表面性状及其对软组织附着的影响[J].口腔医学,2017,37(10):942-945.
[30]Nakatsu Y,Nakagawa F,Higashi S,et al.Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.Pharmacol Rep.2018;70(1):29-36.
[31]Nemoto A,Chosa N,Kyakumoto S,et al.Water-soluble factors eluated from surface pre-reacted glass-ionomer filler promote osteoblastic differentiation of human mesenchymal stem cells.Mol Med Rep.2018;17(3):3448-3454.
[32]Xu FT,Li HM,Yin QS,et al.Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro.Am J Transl Res.2015;7(2):257-270.
[33]曹蓉蓉,马俊玥,李淑慧,等.TNF-α对鼠根尖乳头干细胞增殖及多向分化能力的影响[J].重庆医学,2017,46(14):1874-1877.
[34]Teng S,Liu C,Guenther D,et al.Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds.Exp Ther Med.2016;11(6):2086-2094.
[35]Mc Kee MD,Pedraza CE,Kaartinen MT.Osteopontin and wound healing in bone.Cells Tissues Organs.2011;194(2-4):313-319.
[36]史欣,张鹏飞,孙玉芬,等.骨桥蛋白影响矿化液诱导牙髓干细胞成骨分化能力的研究[J].口腔医学,2014,34(8):566-569.
[37]Zou C,Song G,Luo Q,et al.Mesenchymal stem cells require integrinβ1 for directed migration induced by osteopontin in vitro.In Vitro Cell Dev Biol Anim.2011;47(3):241-250.
[2]Francaviglia N,Maugeri R,Odierna Contino A,et al.Skull Bone Defects Reconstruction with Custom-Made Titanium Graft shaped with Electron Beam Melting Technology:Preliminary Experience in a Series of Ten Patients.Acta Neurochir Suppl.2017;124:137-141.
[3]Xing H,Wu J,Zhou L,et al.Natural teeth-retained splint based on a patient-specific 3D-printed mandible used for implant surgery and vestibuloplasty:A case report.Medicine(Baltimore).2017;96(48):e8812.
[4]Hou PJ,Ou KL,Wang CC,et al.Hybrid micro/nanostructural surface offering improved stress distribution and enhanced osseointegration properties of the biomedical titanium implant.J Mech Behav Biomed Mater.2018;79:173-180.
[5]周屹立,丁仲鹃,唐玲.钛片表面粗糙度和氧化膜对成骨细胞增殖和分化的影响[J].上海口腔医学,2013,22(3):282-286.
[6]高岩.不同波长紫外线激发微弧氧化纯钛表面光催化作用的体外生物活性研究[D].广州:南方医科大学,2012.
[7]Ma T,Ge XY,Hao KY,et al.Simple 3,4-Dihydroxy-L-Phenylalanine Surface Modification Enhances Titanium Implant Osseointegration in Ovariectomized Rats.Sci Rep.2017;7(1):17849.
[8]王婷婷,王丽娜,范震.钛种植体阳极氧化的研究[J].口腔颌面外科杂志,2016,26(4):290-294.
[9]朱晟译.钛种植体表面纳米改性方法及研究进展[J].中国口腔种植学杂志,2011,16(3):180-182,187.
[10]Li NB,Sun SJ,Bai HY,et al.Preparation of well-distributed titania nanopillar arrays on Ti6Al4V surface by induction heating for enhancing osteogenic differentiation of stem cells.Nanotechnology.2018;29(4):045101.
[11]赵旭,卢燃,陈溯.Ti O2纳米管负载BMP-2指节肽对骨髓间充质干细胞增殖与分化的影响[J].北京口腔医学,2017,25(5):241-244.
[12]Giorgi L,Dikonimos T,Giorgi R,et al.Electrochemical synthesis of self-organized Ti O2 crystalline nanotubes without annealing.Nanotechnology.2018;29(9):095604.
[13]付晓龙,李莺,李宝娥,等.纯钛表面阳极氧化增强成骨细胞生物活性及护骨素基因的表达[J].中国组织工程研究,2014,18(39):6240-6245.
[14]陈岩,赵文婷,咏梅,等.正畸微种植体表面阳极氧化处理对周围骨组织的影响[J].中华口腔正畸学杂志,2012,19(3):149-151.
[15]Huang B,Guang M,Ye J,et al.Effect of Increasing Doses ofγ-Radiation on Bone Marrow Stromal Cells Grown on Smooth and Rough Titanium Surfaces.Stem Cells Int.2015;2015:359416.
[16]Tsuchiya S,Hara K,Ikeno M,et al.Rat bone marrow stromal cell-conditioned medium promotes early osseointegration of titanium implants.Int J Oral Maxillofac Implants.2013;28(5):1360-1369.
[17]Tang Z,Xie Y,Yang F,et al.Porous tantalum coatings prepared by vacuum plasma spraying enhance bmscs osteogenic differentiation and bone regeneration in vitro and in vivo.PLo S One.2013;8(6):e66263.
[18]Colombo JS,Carley A,Fleming GJ,et al.Osteogenic potential of bone marrow stromal cells on smooth,roughened,and tricalcium phosphate-modified titanium alloy surfaces.Int J Oral Maxillofac Implants.2012;27(5):1029-1042.
[19]Wang H,Jiang Z,Zhang J,et al.Enhanced osteogenic differentiation of rat bone marrow mesenchymal stem cells on titanium substrates by inhibiting Notch3.Arch Oral Biol.2017;80:34-40.
[20]Bressel TAB,de Queiroz JDF,Gomes Moreira SM,et al.Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.Stem Cell Res Ther.2017;8(1):269.
[21]Arpornmaeklong P,Pripatnanont P,Chookiatsiri C,et al.Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.Int J Oral Maxillofac Implants.2017;32(1):e35-e46.
[22]?zdal-Kurt F,Tu?lu I,Vatansever HS,et al.The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts.Biotech Histochem.2016;91(6):412-422.
[23]Zhang W,Cao H,Zhang X,et al.A strontium-incorporated nanoporous titanium implant surface for rapid osseointegration.Nanoscale.2016;8(9):5291-5301.
[24]Li G,Cao H,Zhang W,et al.Enhanced Osseointegration of Hierarchical Micro/Nanotopographic Titanium Fabricated by Microarc Oxidation and Electrochemical Treatment.ACS Appl Mater Interfaces.2016;8(6):3840-3852.
[25]刘娜,陈丽丽.骨质疏松状态下钛种植体周围骨形成的动态观察[J].全科医学临床与教育,2017,15(2):129-131,143.
[26]孙鑫,徐金标,魏军水.热处理提高钛种植体表面成骨细胞活性的实验研究[J].健康研究,2017,37(4):393-395,399.
[27]施育才,严洪海.不同粗糙表面的纯钛种植体表面的体外细胞培养评价[J].中国口腔种植学杂志,2010,15(2):55-58,62.
[28]张君红,姜海行,覃山羽,等.全骨髓细胞贴壁法分离与培养大鼠骨髓间充质干细胞的多向分化能力[J].中国组织工程研究,2012,16(36):6685-6689.
[29]王婧,顾新华.种植体基台表面性状及其对软组织附着的影响[J].口腔医学,2017,37(10):942-945.
[30]Nakatsu Y,Nakagawa F,Higashi S,et al.Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.Pharmacol Rep.2018;70(1):29-36.
[31]Nemoto A,Chosa N,Kyakumoto S,et al.Water-soluble factors eluated from surface pre-reacted glass-ionomer filler promote osteoblastic differentiation of human mesenchymal stem cells.Mol Med Rep.2018;17(3):3448-3454.
[32]Xu FT,Li HM,Yin QS,et al.Effect of activated autologous platelet-rich plasma on proliferation and osteogenic differentiation of human adipose-derived stem cells in vitro.Am J Transl Res.2015;7(2):257-270.
[33]曹蓉蓉,马俊玥,李淑慧,等.TNF-α对鼠根尖乳头干细胞增殖及多向分化能力的影响[J].重庆医学,2017,46(14):1874-1877.
[34]Teng S,Liu C,Guenther D,et al.Influence of biomechanical and biochemical stimulation on the proliferation and differentiation of bone marrow stromal cells seeded on polyurethane scaffolds.Exp Ther Med.2016;11(6):2086-2094.
[35]Mc Kee MD,Pedraza CE,Kaartinen MT.Osteopontin and wound healing in bone.Cells Tissues Organs.2011;194(2-4):313-319.
[36]史欣,张鹏飞,孙玉芬,等.骨桥蛋白影响矿化液诱导牙髓干细胞成骨分化能力的研究[J].口腔医学,2014,34(8):566-569.
[37]Zou C,Song G,Luo Q,et al.Mesenchymal stem cells require integrinβ1 for directed migration induced by osteopontin in vitro.In Vitro Cell Dev Biol Anim.2011;47(3):241-250.
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