【作者】 任娜；

【导师】 刘宏；

【作者基本信息】 山东大学 ， 材料物理与化学， 2013， 博士

【摘要】 生物医用材料,是用于取代、修复活组织的天然或人造材料。生物医用材料与延长人类寿命和提高生活质量息息相关,因而生物材料领域越来越受到重视。近年来,材料学和生物学的研究进展为生物医用材料的发展奠定了基础。而随着全球人口老龄化进程的加速、人类对健康和长寿的追求、运动创伤的增多、疑难病患者的增加和需要替换组织的病人年龄降低等现象都对生物材料提出了非常大的需求。目前,不仅植入手术增加,植入后的修复手术也随之增加。修复手术给患者造成了疼痛,并且造价高成功率低,这就对生物材料提出了更高的要求。生物医用钛材料由于其良好的力学性能、低弹性模量、易加工性、抗腐蚀性和生物相容性被广泛应用作人工骨、人工关节、骨钉、牙种植体等硬组织替代材料和心脏瓣膜、血管支架等血管材料。但是,钛及其合金本身存在着生物惰性的缺点,使其植入体内与周围组织的结合仅仅是简单的机械锁合,不能形成良好的键合作用。因而钛植入体往往被纤维组织所包裹而与宿主组织隔离开来,长此以往会引起血栓从而导致植入体的失败。目前主要的解决方法是在植入体表面形成磷灰石,形成骨性结合。但这种方法也存在着致命的缺点就是与钛基体的结合力弱,容易造成脱落。而其他物理方法包括离子注入,热喷涂等方法也存在着造价昂贵的问题。另外,在外科手术中,细菌感染是临床上面临的棘手问题。全面的消毒和严格的无菌操作条例等措施下,术后感染经常发生。严重的感染会延长住院时间、增加患者的病痛和经济负担等一系列问题。临床上主要的解决途径是抗生素注射的方法,而抗生素抗菌具有特异性,需要长期注射而且会引发抗药细菌株。植入体表面微纳米结构通过对络氨酸激酶活性,胞内信号通路的调节来控制转录活性和基因表达影响细胞的贴壁、形态、排列、粘附、迁移、增殖、分化和骨架组装；钙镁离子在骨代谢中扮演着重要角色；氧化石墨烯和还原氧化石墨烯对蛋白和生长因子的富集作用可以调控细胞分化；纳米银具备有效的抑制多种细菌、真菌和病毒的特性。基于以上问题,本论文提出在钛表面原位构建纳米结构活化钛表面；通过载入促进细胞成骨分化的离子Ca2+和Mg2+定向调控干细胞命运；而氧化石墨烯和还原氧化石墨烯组装在具有钛酸钠纳米结构的钛表面构建复合纳米材料改善生物学功能；在钛酸钠纳米结构上载入Ag+并还原得到Ag纳米颗粒原位制备兼备抑菌和生物相容性的钛植入体材料,具体包括如下工作：1.钛表面纳米结构的构建及其生物学性能。利用碱-水热的方法通过调节反应条件在医用金属钛表面原位构建了纳米网络结构和纳米管结构的单斜钛酸钠Na2Ti307;通过静滴法测量了超纯水在三组样品,包括抛光的纯钛片、钛酸钠网络结构和纳米管结构上的接触角,并对三组样品表面的亲疏水性能比较,探讨了表面纳米结构对前成骨细胞(MC3T3-E1)行为的影响；细胞增殖、细胞骨架染色和碱性磷酸酶活性测试得到的结果可以看出：钛酸钠纳米网络结构能够促进细胞的贴壁、铺展、迁移和增殖,特别是在成骨诱导因子的协同作用下更加明显。而纳米管结构的钛酸钠与之相比,更能促进细胞的成骨分化；钛酸钠纳米网络结构的构建为后续工作奠定了基础。2.钛表面构建不同离子的钛酸盐纳米结构,调控干细胞的命运。通过简单的水热技术和随后的离子交换过程制备了Na+-钛酸盐,Mg2+-钛酸盐和Ca2+-钛酸盐等不同离子的钛酸盐纳米结构。钛片浸没在MgCl2和CaCl2溶液中后NazTi3O7纳米结构中的Na+被完全取代。这种方法改变了用于细胞培养的钛植入体表面的化学环境,如钛酸盐纳米结构中无机阳离子的种类和数量,从而调控间充质干细胞的命运,包括贴壁,增殖和分化。与Na+-钛酸盐的样品相比,Mg2+-钛酸盐和Ca2+-钛酸盐的钛酸盐纳米结构样品均能促进细胞的增殖和成骨分化。但是过量的离子会抑制细胞活动。Ca2+-钛酸盐比Mg2+-钛酸盐在促进细胞分化方面更具有优势。3.在含钛酸钠纳米结构的钛表面组装氧化石墨烯(GO)和还原氧化石墨烯(rGO)进行表面改性并研究其生物学性能。构建氧化石墨烯和还原氧化石墨烯功能化的钛表面材料"GO-Ti"和’rGO-Ti"。具体方法是：首先通过碱-水热处理得到了利于细胞活动的钛酸钠的纳米网络结构；而结构中所含的Ti-O可以与偶联剂3-氨丙基三乙氧基硅烷(APTES)结合，然后组装GO得到’GO-Ti";随后通过联氨原位还原"GO-Ti"表面的GO得到"rGO-Ti"。通过Raman, XPS和SEM确定了功能化的钛表面成分分别为氧化石墨烯(GO)和还原氧化石墨烯(rGO)；另外也证实了钛表面组装GO和rGO后改变了接触角,从而影响了材料表面的生物学功能；蛋白吸附实验和细胞实验证实了GO和rGO的功能化使得钛表面对蛋白分子和细胞生长因子具有明显的富集作用,特别是GO中的含氧基团的辅助作用,使其具备了调控钛表面的生物学功能和细胞的增殖和分化能力；在钛表面组装石墨烯的工作具有开创性,而且有效的调控了细胞行为,包括贴壁、铺展、增殖和分化等。4.钛表面原位构建具有抑菌性能和生物相容性的钛酸／银纳米颗粒／钛酸的三明治纳米结构。在Ti植入体表面成功原位构建了钛酸-银纳米颗粒-钛酸的三明治纳米结构。首先通过碱水热反应在Ti片表面合成钛酸钠纳米结构；接着在硝酸银溶液中Ti片表面上钛酸钠纳米结构中的Na+离子被溶液中的Ag+离子取代；最后在葡萄糖溶液中Ag+被还原为Ag纳米颗粒。三明治结构上的银纳米颗粒镶嵌在钛酸盐纳米结构的片层结构之间,尺寸和含量随着硝酸银溶液和葡萄糖浓度的增加而增加。与未被葡萄糖还原的样品中Ag+离子的爆发式释放相比,还原后得到的三明治结构中对Ag+的释放起到了缓释作用,实现了稳定的持续释放过程,因而提高了抑菌效果。确定了在0.01mmol/L的硝酸银溶液中离子交换的样品为最优选择具有高达99.99%的抑菌率和良好的生物相容性。目前的研究工作证实了钛酸-银纳米颗粒-钛酸的三明治纳米结构具有抑菌性能和细胞相容性的双重功能。更多还原

【Abstract】 Biomedical materials are artificial or natural materials, used to replace or revise the lost or diseased biological structure to restore their function. They are closely related with the improvement of the quality of life and longevity of human being. Therefore, this field has drawn more and more attentions. Recently, the progress of material and biological science promote the development of biomaterial. Due to the acceleration of the aging process, the pursuit of human health and longevity, the increase of physical trauma and patients suffering complicate diseases, the demand for biomaterials is tremendous. At present, the increasing number of the repair and replacement surgeries cause pains to the patients, and the increase of the cost. Moreover, the successful rate of the surgery is rather low. Therefore, higher requirements for biological materials are expected.Biomedical metallic titanium (Ti) is widely used for artificial bone, artificial joints, bone screws, dental implants as well as heart valves, vascular stents and other vascular materials, because of their good mechanical property, high corrosion resistance, low elasticity modulus, biocompatibility and simple processing. However, the bionert property of titanium makes it difficult to bind with surrounding tissues and the fibrous tissue would appear during the usage period, which cause thrombus and failure of implants. The mainly available way to resolve the above problems is to form apatite coatings on the surface integrated with host tissues. But a fatal disadvantage is the weak bonding strength between the coatings and the titanium substrate, which makes it easy to fall off. For other physical methods, such as ion implantation, thermal spraying and so on, the cost increased. Another complicate issue for the application of titanium implant is the potential bacterial infection. With the comprehensive disinfection and strictly aseptic operation, the postoperative infection is still a frequent occurrence. Serious infections will prolong hospitalization time, increase the patient’s pains, financial burden and a series of troublesome issues. The main solution for infection is to inject clinically antibiotic, which possess antimicrobial specificity, but this will lead to drug-resistant strains of bacteria after a long-term injection.It’s known that the surface micro-/nanostructure influences cell activity, including cell adhesion, spread, proliferation, differentiation; calcium and magnesium cations play a vital role in bone metabolism; the preconcentration effects of graphene oxide and graphene regulate cell growth and differentiation; silver nanoparticles possess the property of inhibiting bacteria, fungi and viruses, which could be applied in preparing antibacterial implants.Based on the above problems, this paper proposed four topics as below:1. In situ construction of sodium titanate nanostructure on titanium and the biological researchUsing alkali-hydrothermal method, two kinds of sodium titanate nanostructure, nanonetwork and nanotube were constructed through the reaction between metallic titanium and concentrated NaOH; SEM, EDX, HRTEM and Raman confirmed both the sodium titanate nanostructures are monoclinic crystal Na2Ti3O7; the formed nanostructure improved the hydrophilicity of the surface, which is an important factor for affecting cell activity; cell experiments, including cell proliferation, cytoskeleton staining and alkaline phosphatase (ALP) activity demonstrated that sodium titanate nano-network structure promoted cell proliferation and sodium tianate nanotubes and enhanced ALP (the marker of osteogenesis) activity; moreover, the preparation of sodium titanate activates the titanium surface and which is helpful for the following research work.2. Nanostructured titanate with different metal ions on surface of metallic titanium for regulating the fate of rat bone marrow mesenchymal stem cellsNa+-titanate, Mg2+-titanate and Ca2+-titanate nanostructure can be synthesized by a facile method based on a hydrothermal technique and followed by an ion-substitution process. By immersing titanium foil with Na2Ti3O7nanostructured on the surface in MgCl2and CaCl2solution, Na+in titanate can be totally substituted by Mg2+or Ca2+ions. This treatment modified the chemical environment on surface of titanium implants for cell culture, i.e. category and quantity of inorganic cations in titanate nanostructure, which can tune protein adsorption and the fate of mesenchymal stem cells, including adhesion, proliferation and differentiation. Both Mg2+and Ca2+in titanate nanostructure can enhance proliferation and osteogenic differentiation of BMSCs, compared with Na+-titanate nanostructures. Excessive Mg2+and Ca2+in titanate can inhibit cell proliferation and differentiation of BMSCs, yet, after some cations (Mg2+, Ca2+) release into osteogenic inductive medium during cell culture, osteoblastic differentiation could be enhanced. Certainly, Ca2+-titanate has more advantages than Mg2+-titanate in cell differentiation. This facile method of introducing Mg2+and Ca2+into titanate by ion- substitution would be promising in titanium implant design and manufacture.3. A graphene oxide and reduced graphene oxide functionalized Ti implant for improving biological propertyBased on the great significance of graphene and its derivatives in biological applications, graphene oxide (GO) and reduced graphene oxide (rGO) were firstly successfully assembled on titanium foils with sodium titanate nanostructure on the surface to obtain the composite nanostructures. The typical process is through alkylation method using coupling agent to bind GO and sodium titanate nanostructure and then GO was in situ reduce on the surface by hydrazine. Raman, XPS and SEM confirmed the structure and composition of GO and rGO on titanium surface. The modification altered the hydrophilicity of the surface and thus influenced the biological function. Then the protein adsorption and cell experiments demonstrated that functionalized samples, especially GO with a large scale of oxygen-containing groups can concentrate protein and growth factors which are associated with cell activities and promote cell proliferation and differentiation. Because of the great enhancement in cell growth, as well as the hot topic of graphene, the titanium material functionalized with graphene will be promising in the biomedical future.4. In situ construction of a titanate-silver nanoparticle-titanate sandwich nanostructure on a metallic titanium surface for bacteriostatic and biocompatible implantsThe sodium titanate nanonetwork film was first synthesized on the Ti foil surface by the alkaline hydrothermal method. Subsequently, the silver ions substituted for the sodium ions in the layer-structured sodium titanate by immersing sodium titanate into the AgNO3solution. Finally, Ag+ions were reduced to the Ag nanoparticles (AgNPs) in the glucose solution. As a result, a titanate-silver nanoparticle-titanate sandwich nanostructure was successfully in situ constructed on the titanium surface. The size and the amount of AgNPs in the sandwich nanostructure increased with an increase in the concentrations of the silver nitrate and the glucose. Compared with Ag+-titanate samples without reduction, this sandwich nanostructure showed a steady and prolonged release manner of Ag+ions, and thereby improved the bacteriostatic efficacy. An optimal concentration of AgNO3solution for ion-substitution was0.01mM and its bacteriostatic rate was determined as high as99.99%and also it possessed good cytocompatibility. The present work demonstrated a titanate-silver nanoparticle-titanate sandwich nanostructure that has dual functions of antibacterial activity and cytocompatibility. Therefore, the Ti substrate with such a sandwich nanostructure on the surface is a promising implantable biomaterial.更多还原