Acid etching and anodic oxidation of titanium surface of nanotubes promotes the proliferation and osteogenesis of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0997

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Dental Implants, Titanium, Bone Marrow, Mesenchymal Stem Cells, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Ma Yan, Zhang Hua, Shang Jian-ping, Jiang Qi. Acid etching and anodic oxidation of titanium surface of nanotubes promotes the proliferation and osteogenesis of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4614-4619.

Figures/Tables 2

2.1 骨髓间充质干细胞的培养与鉴定原代细胞形状多呈长梭形、多角形，24 h后可见少量细胞贴壁，8 d左右长满瓶底，呈纺锤形，细胞排列呈漩涡状、辐射状；传代后，细胞贴壁及生长迅速，1 d左右可完全贴壁，五六天铺满瓶底，细胞形态较均一，呈漩涡状、辐射状或鱼群样排列，见图1。流式细胞仪检测结果显示，第3代骨髓间充质干细胞表面CD44、CD29、CD90均呈阳性表达，其阳性表达率分别为96.5%，94.6%及98.2%；细胞表面CD14、CD34、CD45均呈阴性表达，符合骨髓间充质干细胞的生物学特性。 2.2 钛试样表面细胞增殖检测结果随着培养时间的增长，实验组与对照组钛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)，提示在酸蚀阳极氧化处理钛表面接种的骨髓间充质干细胞增殖迅速，见图2。 2.3 钛试样表面细胞碱性磷酸酶活性检测结果实验组与对照组成骨诱导培养3 d的碱性磷酸酶活性比较差异无显著性意义(P > 0.05)，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)，见表2，说明酸蚀阳极氧化处理的钛表面可促进骨髓间充质干细胞分泌碱性磷酸酶。 2.4 钛试样表面细胞碱性磷酸酶染色与茜素红染色结果碱性磷酸酶染色：成骨诱导培养第7天，两组钛试样表面细胞胞浆内可见被染成红色的颗粒状碱性磷酸酶，说明经诱导后细胞表达碱性磷酸酶，并且实验组胞浆阳性颗粒数多于对照组，见图3。茜素红染色：成骨诱导第21天，两组钛试样表面细胞胞外基质中可见大小不等的聚集团状结节，实验组钙结节数量及面积多于对照组，见图3。 2.5 钛试样表面细胞成骨标志基因的表达见图4。骨桥蛋白基因表达：两组均可见骨桥蛋白基因表达，骨桥蛋白基因表达在成骨诱导培养第14天达高峰，实验组成骨诱导培养7，14，21 d的骨桥蛋白基因表达均高于对照组(P < 0.05)。骨特异性转录因子基因表达：两组均可见骨特异性转录因子基因表达，并且随着诱导时间的延长，骨特异性转录因子基因表达逐渐升高，实验组成骨诱导培养不同时间点的骨特异性转录因子基因表达均高于对照组，但仅在成骨诱导7，14 d时差异有显著性意义(P < 0.05)。骨钙素基因表达：两组均可见骨钙素基因表达，骨钙素基因在成骨诱导培养的第21天时达高峰，实验组成骨诱导培养不同时间点的骨钙素基因表达均高于对照组，但仅在成骨诱导培养14，21 d时差异有显著性意义(P < 0.05)。 2.6 钛试样表面细胞成骨标志蛋白的表达两组钛试样表面细胞的成骨标志蛋白表达与基因检测结果一致，实验组诱导培养7，14，21 d的骨钙素蛋白、骨特异性转录因子及骨桥蛋白表达条带均强于对照组，见图5。"

References

[1] Tsutsumi K, Horiuchi T, Hongo K. Mechanical evaluation of cerebral aneurysm clip scissoring phenomenon: comparison of titanium alloy and cobalt alloy. J Mater Sci Mater Med. 2017;28(10):159.

[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.TiO2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 TiO2 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 of γ-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. PLoS 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] Özdal-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] McKee 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 integrin β1 for directed migration induced by osteopontin in vitro. In Vitro Cell Dev Biol Anim. 2011;47(3):241-250.