携载蛋白质的氧化钛纳米管表面成骨细胞行为

通过阳极氧化技术,在光滑钛表面制备一层管径约100nm的纳米管,并选用小牛血清白蛋白（BSA）和纤维连接蛋白（FN）以及两种蛋白的混合溶液（BF）进行蛋白吸附试验。将吸附蛋白后的试样进行细胞培养,所用细胞为MC-3T3E1成骨细胞株,结果表明,较光滑钛吸附蛋白前后的纳米管试样显示出更好的成骨活性,同时,吸附单组分纤维连接蛋白的纳米管试样能更好地促进成骨细胞的黏附和生长,具有更高的生物活性。     

利用MicroBCA和QCM对钛薄膜表面固定纤连蛋白的量化研究与对比

采用物理化学稳定性较好的石英玻璃为基底,用非平衡磁控溅射技术于其表面沉积生物相容性良好的钛薄膜,然后在钛薄膜表面共价固定纤连蛋白。采用傅立叶红外光谱(FT-IR)和X射线光电子能谱(XPS)对各步处理后的材料表面特征进行检测和分析。主要研究了用MicroBCA检测法对样品表面固定的纤连蛋白的定量表征的可行性,并将该结果与用石英晶体微天平(QCM)方法检测获得的纤连蛋白量做了对比和深入分析。结果显示,纤连蛋白可以成功固定到钛薄膜表面,MicroBCA检测法和QCM法的检测结果具有一致性,样品表面纤连蛋白的固定量大约在767～789ng/cm2。     

胶原/HA复合涂层多孔钛的细胞响应行为

为提高医用多孔钛的表面活性及生物相容性,以纤维烧结多孔钛为载体,采用碱热处理+模拟体液等化学方法进行羟基磷灰石(HA)沉积,随后进行水溶性胶原涂覆,制备具有三维贯通孔结构的胶原/HA复合涂层多孔钛。研究了胶原/HA复合涂层的表面形貌及化学成分,探讨复合涂层的形成机理;进行体外细胞毒性实验并分析了多孔钛表面细胞响应行为。结果表明,胶原/HA复合涂层在钛纤维表面均匀附着,胶原的填充连接可修复HA涂层表面的微裂纹,有利于小鼠前成骨细胞的黏附、增殖及分化,促进细胞的孔内跨纤维生长。胶原/HA复合涂层可以得到更好的细胞响应,对于促进早期骨与植入体的固定有很大的潜力。     

酸碱辅以热处理对钛表面生物活性的影响

利用先酸后碱复合处理钛表面,部分样品加热到450和700℃进行热处理,在钛表面获得不同形貌和不同晶型的TiO2薄膜。将样品浸泡于模拟体液中,观察表面HA沉积情况,并进行体外细胞毒性实验以探讨样品的生物相容性。结果表明,酸碱复合处理样品在450℃热处理后表现出最好的生物活性,主要归因于钛表面纳米级花蕊网状结构形貌。酸碱复合处理样品在700℃热处理后表现出最差的生物活性,这是因为此法处理后的样品表面呈现纳米棒晶体结构,纳米棒直径和长度均大于450℃热处理的纳米丝形貌的钛及未热处理钛,而且纳米棒紧密结合程度也高于450℃热处理及未热处理的钛,正是由于这样紧密的较大尺寸晶体结构造成其生物活性低于450℃热处理及未热处理的钛的情况。     

碱热处理对钛表面生物活性的影响

利用碱热法处理钛表面,部分样品加热到450和700℃进行热处理,获得不同形貌和不同晶型的TiO2薄膜。实验得出,未经热处理的样品表面以非晶态钛凝胶层为主,450℃热处理样品主要以锐钛矿相TiO2存在,700℃热处理样品主要以金红石相TiO2出现。700℃热处理后的样品表面分布有大量50nm左右的纳米颗粒,这些纳米粒子的尺寸效应能较好地诱导HA在样品表面沉积形成,提高细胞的黏附和增殖,因此表现出较高的生物活性。450℃热处理后的样品表面相对于未处理样品有更多的HA沉积,是因为表面层的晶型起了主要作用,即锐钛矿TiO2晶体比无定型凝胶态TiO2更利于诱导HA沉积。但相对于未经任何处理的空白样品,仅用NaOH处理后的样品表面较利于HA沉积,可能是碱处理后钛表面羟基增加,进而提高了其诱导HA沉积的能力。     

用MicroBCA方法定量检测硅烷化表面固定的纤维连接蛋白

使用microBCA方法（micro-bicinchoninicacid protein assay）,定量研究了人纤维连接蛋白（fi-bronectin,Fn）在硅烷化石英玻璃表面实现共价固定的情况。首先对石英玻璃进行化学清洗,使表面的羟基得以充分暴露。再利用硅烷偶联剂（APTE）将氨基基团引入,并通过EDC/NHS催化体系把人纤维连接蛋白以酰胺键的形式共价固定于该表面。最后用Mi-croBCA方法对该表面固定化的生物分子（Fn）进行量化表征。应用水接触角、X射线光电子能谱（X-rayphotoelectron spectroscopy,XPS）、原子力显微镜（atomic force microscope,AFM）等方法对各步处理效果进行跟踪分析。结果表明Fn在硅烷化石英玻璃表面实现了固定,一定范围内,硅烷化表面的Fn固定量随所使用Fn孵化液浓度的增加而增大。为生物材料表面改性与修饰研究中生物大分子的量化表征提供了一种新方法。     

超亲水多孔TiO_2薄膜的制备及其超亲水机理研究

以钛酸四丁酯为钛前体,聚乙二醇(PEG)为成孔剂,采用溶胶-凝胶法制备多孔TiO2薄膜。结果表明,该薄膜具有多孔表面特征,表面粗糙不平,在避光的条件下与水接触角小于5°。当煅烧温度为450℃以及煅烧时间为2h时,得到的样品超亲水性能为最佳。同时,还探讨了薄膜超亲水性能的形成机理,薄膜的超亲水特性是由光催化物质以及粗糙表面共同作用的,并且表观接触角是服从Cassie-Baxter模型。     

TiO_2薄膜表面构建催化活性层改善抗血小板黏附性能

一氧化氮(nitric oxide,NO)是心血管系统的信号分子,具有抗凝血抗增生多种生物学功能。在TiO2薄膜表面固定生物分子胱胺、硒代胱胺,构建催化活性层,催化供体释放NO,从而使改性后的材料表面具有抗凝效应。构建分为两个步骤,首先通过膦酸单分子层自组装固定十二烷基膦酸,再借助光化学方法在材料表面固定4-叠氮基苯甲酸分子使表面羧基官能团化;随后,用EDC/NHS/MES体系活化表面羧基,进而固定胱胺、硒代胱胺。傅里叶红外吸收光谱(Fourier transform infrared spectroscopy,FTIR)与X光电子能谱(X-ray photoelectron spectroscopy,XPS)结果显示各分子均在TiO2薄膜表面成功固定。体外催化实验结果证实了催化活性层的催化活性。该改性层可原位催化内源性一氧化氮供体释放出NO,改善材料表面抗血小板粘附性能,其在体外血小板黏附实验中得到证实。改善了TiO2薄膜的生物相容性,为无机材料的生物活化提供了一种新途径。     

钛材料表面固定多聚赖氨酸-肝素纳米颗粒以改善血液相容性的研究

通过将多聚赖氨酸(PLL)-肝素纳米颗粒固定在多巴胺涂覆的钛表面,以改善其血液相容性。利用zeta电位仪及甲苯胺蓝法检测纳米颗粒的粒径及成分,通过傅立叶变换红外光谱(FT-IR)、X射线光电子能谱(XPS)及水接触角等对颗粒固定前后表面理化性质的变化进行表征。通过体外血小板粘附实验、肝素释放及活化部分凝血活酶时间(APTT)检测对改性样品的血液相容性进行评价。结果表明,PLL-肝素纳米颗粒成功固定在多巴胺沉积的钛表面,纳米颗粒的固定有效降低钛材料表面血小板的粘附行为,大大提高了血液相容性。     

钛表面铜离子注入对细菌和细胞行为的影响

医用钛及其合金被广泛用作骨组织替换材料,但缺乏抗菌性,易导致细菌感染。铜具有良好的抗菌性能,将其引入到钛表面,可改善医用钛的抗菌性能;然而铜含量过高对细胞具有毒性。因此,需要调节铜的含量,实现铜的抗菌性能和细胞相容性之间的平衡。本研究采用等离子体浸没离子注入技术对医用钛进行表面改性,获得表面含铜量不同的样品,并研究改性钛表面对细菌和细胞行为的影响。结果表明,钛表面含铜量较低的样品能够促进大鼠骨髓间充质干细胞(rBMSCs)和人脐静脉内皮细胞(HUVECs)的增殖,但对大肠杆菌和金黄色葡萄球菌没有抑制能力;随着离子注入时间的延长,钛表面含铜量较高的样品抗菌能力显著提高,同时也未产生明显细胞毒性。因此,通过控制钛表面的铜含量,可以获得兼具良好抗菌性能和生物相容性的钛植入材料。     

Structure, morphology and fibroblasts adhesion of surface-porous titanium via anodic oxidation

Surface-porous titanium samples were prepared by anodic oxidation in H₂SO₄, H₃PO₄ and CH₃COOH electrolytes under various electrochemical conditions. X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) were employed to characterize the structure, morphology and chemical composition of the surface layer, respectively. Closer analysis on the effect of the electrochemical conditions on pore configuration was involved. It can be indicated that porous titania was formed on the surface layer, and the pore configuration was influenced by electrolyte composition and crystal structure of the titania. The fibroblast cells experiment showed that anodic oxidation of titanium surface could promote fibroblast adhesion on Ti substrate. The results suggested that anodic oxidation of Ti in CH₃COOH was suitable to obtain surface-porous titanium oxides layers, which might be beneficial for better soft tissue ingrowths.     

Enhanced endothelialization guided by fibronectin functionalized plasma polymerized acrylic acid film

The plasma polymerized acrylic acid (PPAA) films with high carboxyl concentration were fabricated by low temperature plasma polymerization technology. Vacuum thermal treatment was used to modify the PPAA films. The stability of the PPAA film was evidenced by scanning electron microscopy (SEM). The result of toluidine blue-O (TBO) method showed that the –COOH concentration on the surface of the PPAA films decreased from 20.73 nM to17.90 nM after vacuum thermal treatment. Fibronectin (Fn) was successfully covalently immobilized onto the modified surface PPAA via a covalent bond reacted with carboxyl groups. Diffuse reflectance Fourier infrared transform spectroscopy (DRFTIR) and X-ray photoelectron spectroscopy (XPS) were utilized to characterize the layer-by-layer PPAA surface modification. From the evaluation of immunostaining for actin and Cell Counting Kit-8 (CCK-8), the surface of Fn-immobilized thermal treated PPAA films could be used to enhance the adhesion and proliferation of human umbilical vein endothelial cells (HUVECs). Especially, the exposure of RGD domain existing in the immobilized Fn was ascertained by ELISA, which was one of main improvement factors of HUVEC's adhesion and proliferation. This study demonstrated a promising surface modification used for vascular devices.     

有机磷酸酯改性钛表面的蛋白质吸附行为

通过磷酸三乙酯对钛表面进行改性,使表面含有机磷并且具有一定的疏水性。考察了不同初始浓度、温度和pH值条件下,小牛血清白蛋白（BSA）在钛表面的吸附行为,通过紫外分光光度计表征了BSA吸附的动力学特征。研究发现,在与含磷钛表面接触的前40 min内,BSA发生迅速吸附,随后逐渐达到动态平衡。根据Langmiur理论计算获得吸附等温线和最大吸附量。通过红外光谱和X射线光电子能谱分析表明BSA化学吸附到表面。对BSA在改性钛表面的吸附机制进行了初步的分析。      

Osseointegration evaluation of laser-deposited titanium dioxide nanoparticles on commercially pure titanium dental implants

The nanotechnology field plays an important role in the improvement of dental implant surfaces. However, the different techniques used to coat these implants with nanostructured materials can differently affect cells, biomolecules and even ions at the nano scale level. The aim of this study is to evaluate and compare the structural, biomechanical and histological characterization of nano titania films produced by either modified laser or dip coating techniques on commercially pure titanium implant fixtures. Grade II commercially pure titanium rectangular samples measuring 35?×?12?×?0.25?mm length, width and thickness, respectively were coated with titania films using a modified laser deposition technique as the experimental group, while the control group was dip-coated with titania film. The crystallinity, surface roughness, histological feature, microstructures and removal torque values were investigated and compared between the groups. Compared with dip coating technique, the modified laser technique provided a higher quality thin coating film, with improved surface roughness values. For in vivo examinations, forty coated screw-designed dental implants were inserted into the tibia of 20 white New Zealand rabbits’ bone. Biomechanical and histological evaluations were performed after 2 and 4 weeks of implantation. The histological findings showed a variation in the bone response around coated implants done with different coating techniques and different healing intervals. Modified laser-coated samples revealed a significant improvement in structure, surface roughness values, bone integration and bond strength at the bone-implant interface than dip-coated samples. Thus, this technique can be an alternative for coating titanium dental implants.     

Immobilization of the cell-adhesive peptide Arg–Gly–Asp–Cys (RGDC) on titanium surfaces by covalent chemical attachment

Surface modification of acid-pretreated titanium with 3-aminopropyltriethoxylsilane (APTES) in dry toluene resulted in covalently bonded siloxane films with surface coverage that was relatively controllable by regulating the reaction conditions. A hetero-bifunctional cross-linker, N-succinimidyl-3-maleimidopropionate (SMP), reacted with the terminal amino groups, forming the exposed maleimide groups. Finally, a model cell-binding peptide, Arg–Gly–Asp–Cys (RGDC), was immobilized on the surface through covalent addition of the cysteine thiol groups to the maleimide groups. X-ray photoelectron spectroscopy, radiolabelling techniques, and ellipsometry were used to quantify and characterize the modified surfaces.     

Surface modification of a novel porous titanium dioxide/glass composite

A novel technique is used to produce an open porous titanium dioxide/glass composite, named Ecopore, with promising structural and biological properties for the development as a bone graft. This study aims at a fast and lasting integration of the new material by means of biochemical surface modification. Surface etching of Ecopore, aminosilanization and covalent coupling of the cellular attachment mediator fibronectin was employed as modification strategy. In a comparison of different etching procedures, alkaline etching led to the highest density of amino functions after subsequent aminosilanization. Fibronectin was immobilized using a bifunctional aminoreactive PEG‐linker. This protein coating improved the attachment of human osteoblast‐like cells (HOB) on non‐porous Ecopore as displayed by vital staining. XTT metabolism assays indicated an enhanced HOB growth in the initial phase of cultivation on fibronectin‐coated versus non‐coated specimens. In a first feasibility study, cultivation of HOB on coated porous Ecopore cylinders with a median pore size diameter of 130 μm showed that cellular growth was uniform and dense on the external surface of the specimen, but was sparse in the interior pore system. Ecopore batches with larger pores will be modified and investigated in vitro and in vivo in the next step of the study.     

Surface functionalization of materials to initiate auto‐biocompatibilization in vivo

Cellular growth and differentiation in contact with artificial materials is controlled by exchange of information between solid and components of the extracellular matrix. Usually cells are connected with the extracellular matrix via adhesive proteins, f. i. fibronectin or laminin. Fibronectin is a large glycoprotein consisting of a dimer of two subunits which are connected via two disulfide‐bonds at their carboxy terminal ends. Each subunit contains binding sites for heparin, collagen and the cell itself. Fibronectin is bound via sequences of type III to cellular integrins. The cell‐specifity of the implant‐surface can be realized by physical, chemical or biological functionalization. The RGD type III‐sequence arg‐gly‐asp of fibronectin can be connected to the implant surface via metal organic spacers. Already experimentally performed was the reaction between short‐chain III sequences with titanium alcoxides. The metal‐organic compound carries the sequence as a functional group. The coupling to the metal(oxide)‐surface takes place with a sol‐gel‐process via hydrolysis and condensation of the alkoxide groups. In appropriate preparatory work mono amino acids, polycarbocylic acids and other short chain biological effective molecules have already been bound to titanium. The free functionalities: ‐NH₂, ‐COOH, ‐SO₃ were prooved by FT‐IR‐ or surface enhanced raman spectroscopy (SERS). The provided surfaces show differently strong mineralization after incubation in simulated body fluids containing calcium phosphates; a sterilization in an autoclave does not modify the chemical composition and the morphology of the surface modification. The goal for implants with contact to hard tissue is to prepare a surface with free functionalities which adsorb preferently adhesive proteins from the host extracellular matrix as an auto‐biocompatibilization.     

Immobilized-OPG-Fc on a titanium surface inhibits RANKL-dependent osteoclast differentiation in vitro

The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-κB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37°C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.     

Surface modification of titanium by hydrothermal treatment in Mg-containing solution and early osteoblast responses

Surface modification on titanium was carried out in order to improve its bioactivity. Pure titanium was hydrothermally treated in distilled water and 0.1 M MgCl₂ solutions at 200°C for 24 h. Surface morphology, roughness, wettability and chemical composition were characterized before and after treatment. Bovine serum albumin was used as model to study protein adsorption. MC3T3-E1 cells were cultured and initial cell attachment, morphology, proliferation were evaluated. After hydrothermal treatment, nano-sized precipitations were observed and samples showed superhydrophilicity. Magnesium (Mg) was immobilized into titanium surface by hydrothermal treatment. Protein adsorption was significantly increased on Mg-containing samples. Cell attachment was improved and cell spreading was enhanced on Mg-containing samples compared with untreated or those treated in distilled water. Increased early cellular attachment on the MgTi surface resulted in subsequent increase of number of proliferated cells. Hydrothermal treatment in MgCl₂ solution was expected to be an effective method to fabricate titanium implant with good bioactivity.     

Study on double-glow plasma niobium surface alloying of pure titanium

A niobium modified layer on a pure titanium surface was obtained by means of a double-glow plasma surface alloying technique. Microstructure and phases resident in the alloy layer were analyzed. The processing parameters and effects of cathode sputtering before the diffusion process were also studied. The results show that the surface niobium content in the modified layer is similar to that in the Ti-45Nb alloy, and decreases gradually from the surface into the underlying substrate. The oxidation behavior of the modified pure titanium at 900 °C was noticeably improved after the niobium alloying process. Characterization was performed using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The oxidation mechanism is also discussed.