摘要
背景:有研究报道,单纯纳米管改性的钛种植体表面可促进体外成骨细胞的黏附、增殖、分化,胶原涂层也具有增强成骨细胞黏附和体内骨结合的作用。目的:在纯钛表面制备TiO_2纳米管结构表层并结合Ⅰ型胶原,观察改性后的纯钛表面对体外成骨细胞黏附和体内骨结合的影响。方法:采用阳极氧化处理在纯钛表面制备TiO_2纳米管涂层,并结合Ⅰ型胶原,采用扫描电镜观察、接触角测定对纯钛(纯钛组)、TiO_2纳米管(纳米管组)和TiO_2纳米管结合Ⅰ型胶原(胶原/纳米管组)3种表面进行表征。将MC3T3-E1小鼠前成骨细胞株分别接种于3组材料上,培养4h后,利用扫描电镜观察细胞黏附形态,倒置荧光显微镜观察细胞黏附计数,激光共聚焦显微镜观察细胞骨架与黏附斑蛋白表达,实时荧光定量PCR分析黏附斑蛋白与护骨素基因表达。将3组试样分别植入SD大鼠(中国医学科学院放射医学研究所实验动物中心提供)胫骨内,4周后取胫骨标本,分别进行生物力学推出实验与组织学观察。结果与结论:①扫描电镜显示,纯钛表面仅可见机械打磨的划痕;纳米管组表面形成了可控、均一的垂直排列的纳米管状结构,管径约70 nm;胶原/纳米管组表面纳米管结构周围可见胶原附着,封闭了部分管口;②胶原/纳米管组的亲水性大于纳米管组、纯钛组(P<0.05);③与纯钛组、纳米管组比较,胶原/纳米管组成骨细胞黏附数目增加(P <0.05),细胞伸展完全,骨架结构明显,黏附斑蛋白表达强度高(P <0.05),黏附斑蛋白和护骨素基因表达升高(P <0.05);④动物体内植入实验显示,胶原/纳米管组最大推出力显著高于纯钛组、纳米管组(P<0.05);苏木精-伊红染色显示,纯钛组种植体周围骨质较少,可见较多的纤维结缔组织;纳米管组种植体周围新生骨较多,纤维结缔组织较少;胶原/纳米管组种植体周围形成结构致密的新生骨,仅残留菲薄的纤维结缔组织;⑤结果表明,纯钛表面TiO_2纳米管结合Ⅰ型胶原的新型改性方法,可有效增强体外成骨细胞的黏附,促进体内骨结合。
BACKGROUND: Simple nanotube surface modification of titanium implant has been shown to promote adhesion, proliferation and differentiation of osteoblasts. Collagen coating can promote osteoblast adhesion and osseointegration in vivo. OBJECTIVE: To observe the effects of type I collagen combined titanium dioxide nanotube composite coating modified titanium surface on osteoblast adhesion in vitro and osseointegration in vivo. METHODS: The titanium dioxide nanotube was fabricated on the pure titanium surface, then type I collagen was combined with the nanotube structure to form composite coating. Scanning electron microscope observation was used to characterize the surface topography of the pure titanium, titanium dioxide nanotube and type I collagen combined titanium dioxide nanotube surfaces. Contact angle test was employed to evaluate the hydrophilicity of different samples. MC3T3-E1 murine preosteoblasts were seeded on the three kinds of materials for 4 hours. Cell adhesion morphology was examined by scanning electron microscope. Adherent cell counting was detected under inverted fluorescence microscope. Expression of actin cytoskeleton and vinculin was observed under laser scanning confocal microscope. The gene expression of vinculin and osteoprotegerin mRNA was detected by real-time quantitative PCR. The three kinds of samples were implanted into the tibia of Sprague-Dawley rats (provided by Laboratory Animal Center, Institute of Radiation Medicine, Chinese Academy of Medical Sciences), and tibia samples were removed after 4 weeks of implantation for biological push-out test and histological observation. RESULTS AND CONCLUSION:(1) Scanning electron microscope: There was mechanical scratch on the pure titanium surface. There was controllable, and uniform vertical arrangement of nanotubular structures with a diameter of approximately 70 nm on the titanium dioxide nanotube surface, and collagen adhered surrounding the nanotubular structures on the type I collagen combined titanium dioxide nanotube substrate, and partial tubule orifices were closed.(2) The hydrophicility of type I collagen combined titanium dioxide nanotube was significantly larger than those of the other two materials (P < 0.05).(3) Compared with the pure titanium and titanium dioxide nanotube surfaces, the type I collagen combined titanium dioxide nanotube substrate displayed increased adherent cell number, much well-organized cytoskeleton, enhanced immunofluorescence intensity of vinculin protein staining, and increased expression levels of vinculin and osteoprotegerin mRNA levels (all P < 0.05).(4) In vivo test revealed that the maximum push-out force in the type I collagen combined titanium dioxide nanotube group was significantly higher than that in the pure titanium and titanium dioxide nanotube groups (P < 0.05). Hematoxylin-eosin staining results showed that there were few bones, but many fibrous connective tissue surrounding the implant in the pure titanium group;there were more newly-born bones, and less fibrous connective tissue surrounding the implant in the titanium dioxide nanotube group;there were dense newly-born bones, and few thin fibrous connective tissue surrounding the implant in the type I collagen combined titanium dioxide nanotube group.(5) These results indicate that type I collagen combined titanium dioxide nanotube surface can facilitate osteoblast cell adhesion and promote osseointegration in vivo.
作者
李莺
尤亚鹏
李宝娥
宋云嘉
马奥博
陈博
韩文
李长义
Li Ying;You Yapeng;Li Baoe;Song Yunjia;Ma Aobo;Chen Bo;Han Wen;Li Changyi(Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, China;School of Materials Science and Engineering, Hebei University of Technology, Tianjin 300130, China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第14期2169-2176,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81870809
31470920)
项目负责人:李长义
国家自然科学基金(81500886)
项目负责人:李莺
天津市自然科学基金(16JCYBJC28700)
项目负责人:李莺
河北省自然科学基金(E2017202032)
项目参与者:李莺~~
