纯钛硬组织植入体表面膜层的仿生构建及其生物学行为研究

发布时间：2018-03-03 16:28

本文选题：纯钛　切入点：微弧氧化　出处：《华南理工大学》2012年博士论文　论文类型：学位论文

【摘要】：纯钛硬组织植入体因具有优良的生物相容性、力学性能、耐腐蚀性和可加工性而在临床上得到广泛应用。但其表面缺乏优异的生物学性能，植入体内后无法形成牢固的植入体-骨组织结合界面。本研究以改善纯钛硬组织植入体表面生物学性能为目的，从植入体-骨组织结合界面入手，结合微弧氧化（MAO）技术、去细胞化技术和点击反应技术，创新性地在纯钛基植入体表面仿生构建了与骨组织成分相似且可利用点击反应接枝生物活性分子的细胞外基质-微弧氧化膜层（clickable ECM-MAO膜层），显著地改善了细胞在纯钛硬组织植入体表面的多项生物学行为。膜层中的MAO膜层由金红石、锐钛矿和磷灰石晶体组成。该膜层对阿伦膦酸钠具有50小时以上的控释能力。另外，载有PAMAM Dendrimer的MAO膜层在PBS缓冲液中具有30天以上的稳定性，，且在对人骨髓间充质干细胞（HBMSC）表现出优良的生物相容性和促进成骨生物活性的同时，还对临床常见的致病细菌绿脓杆菌和金黄色葡萄球菌表现出一定的抗菌性。膜层中的ECM膜层是在去细胞化膜层模型基础上构建的。去细胞化膜层模型主要成分是人血管内皮细胞（HUVEC）所分泌的细胞外基质，模型相比于对照组可以有效地促进HUVEC体外的粘附、增殖和成血管分化。在该模型基础上仿生构建的HUVEC和HBMSC两种ECM-MAO膜层相比于MAO膜层表现出更加优异的生物学性能，对HBMSC体外增殖及其成骨基因Col-I、OPN的表达具有显著的促进作用。仿生构建的膜层中含有大量细胞外基质成分，所以应用于膜层的接枝化学反应必须具有高生物相容性。铜催化叠氮-炔基Husigen环加成反应（CuAAC反应）是具有优异生物相容性的一类点击反应。本文在系统研究反应条件对反应速率和生物相容性影响规律的基础上，通过该CuAAC反应技术利用荧光染料BN和CN以及自主设计合成的荧光染料BCN标记三种活细胞并首次标记无合成缺陷的细菌。经处理的细胞和细菌不但呈现明显的荧光，而且保持了很好的活性。同时，BCN相比于BN和CN，实现了一种荧光染料双重标记细胞的效果。该技术为在仿生构建的膜层表面接枝生物活性分子的研究提供了重要的技术支持。最后，本研究创新性地利用新陈代谢方法在ECM膜层中引入炔基基团，构建了可进行点击反应的去细胞化膜层模型（clickable ECM模型）。在利用点击反应接枝多肽QK之后，模型对HUVEC体外成血管化的促进作用得到明显提升。在该模型的基础上仿生构建的clickable ECM-MAO膜层接枝多肽QK之后，对HBMSC体外成骨分化具有更好地促进作用。另外，在氮气环境中接枝QK多肽的膜层对HBMSC第一天成骨分化具有更加明显的促进作用。
[Abstract]:Pure titanium hard tissue implants have been widely used in clinic because of their excellent biocompatibility, mechanical properties, corrosion resistance and processability. In order to improve the surface biological properties of pure titanium hard tissue implants, we began with implant-bone tissue binding interface and combined with Mao (Microarc Oxidation) technique. Decellarization and click-through technology, A novel clickable ECM-MAO membrane layer, which is similar to bone tissue composition and can be grafted to bioactive molecule by click-reaction, was constructed on the surface of pure titanium based implant. Several biological behaviors on the surface of titanium hard tissue implants. The MAO layer in the film consists of rutile, anatase and apatite crystals. The film has a controlled release capacity of more than 50 hours to alendronate. In addition, the MAO film containing PAMAM Dendrimer has a stability of more than 30 days in the PBS buffer. In addition, the bone marrow mesenchymal stem cells (BMSCs) showed excellent biocompatibility and osteogenic activity. The ECM membrane in the membrane was constructed on the basis of the model of acellular membrane. The main component of the model of acellular membrane layer is the following: the main components of the model of the acellular membrane layer are as follows: (1) Antibacterial activity of Pseudomonas aeruginosa and Staphylococcus aureus (Staphylococcus aureus). The extracellular matrix secreted by human vascular endothelial cells (HUVECs), Compared with the control group, the model could effectively promote the adhesion, proliferation and angiogenesis of HUVEC in vitro. The bionic ECM-MAO and HBMSC membranes constructed based on the model showed better biological properties than those of MAO. It can promote the proliferation of HBMSC in vitro and the expression of osteogenic gene Col-Ion. The bionic membrane contains a large amount of extracellular matrix, Therefore, the graft reaction applied to the film must have high biocompatibility. Copper catalyzed azido-alkynyl Husigen cycloaddition reaction is a kind of click-through reaction with excellent biocompatibility. In this paper, the reaction is studied systematically. On the basis of the effect of conditions on reaction rate and biocompatibility, The fluorescent dyes BN and CN, as well as BCN, were used to label three kinds of living cells and to label the bacteria without synthetic defect for the first time. The treated cells and bacteria not only showed obvious fluorescence. Compared with BN and CNN, BCN has achieved the effect of double labeling with fluorescent dyes. This technique provides important technical support for the study of biologically active molecules grafted on the surface of bionic coatings. Finally, a clickable ECM model was constructed by introducing acetyl groups into the ECM membrane layer by metabolic method. The clickable ECM model was constructed after the click-reaction grafting of polypeptide QK was carried out. On the basis of this model, the clickable ECM-MAO membrane grafted polypeptide QK was constructed on the basis of this model, which could promote the osteogenic differentiation of HBMSC in vitro. The membrane layer grafted with QK peptide in nitrogen environment could promote osteogenic differentiation on the first day of HBMSC.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08

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