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
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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。
As hard tissue implants, pure titanium is widely used due to its advantages inbiocompatibility, mechanical properties, corrosion resistance, and light weight.However, the application of pure titanium is limited in clinic because of its lowbiological property, which leads to the poor interface of the implant-bone tissue. Inthis research, the clickable ECM-MAO film, which could improve the biologicalproperties of the pure titanium hard tissue implant, was biomimetic constructed onthe implant surface by using micro-arc oxidation, decellularization and click reaction.
In the ECM-MAO film, the MAO film is mainly formed by anatase, rutile andapatite. This MAO film could load the allen sodium phosphate (AL) and control therelease of AL up to50h. On the other hand, it could also load the antimicrobial drugPAMAM Dendrimer stably up to30days in PBS solution. The MAO film withPAMAM Dendrimer could exhibit the antimicrobial activity to bacteria P. aeruginosaand S. Aureus while keeping excellent biocompatibility and bioactivity to HBMSC(Human Bone Mesenchymal Stem Cells). The ECM film in the ECM-MAO film isconstructed from the decellularized film model. The decellularized model is form bythe extracellular matrix (ECM) secreted by HUVEC (Human Umbilical VeinEndothelial Cells), which could improve the adhesion, proliferation and angiogenesisof HUVEC in vitro compared to the control groups. The ECM-MAO film which isbiomimetic constructed from this model and formed by ECM secreted by HUVEC orHBMSC shows better biological behavior than MAO film. It could improveHBMSC’s proliferation and expression of Col-I and OPN in vitro.
For ECM-MAO film, the high biocompatibility of the chemistry method is required toprotect the bioactive molecular in ECM while integrating bioactive molecular on it.Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is one kind of the click reactions withhigh biocompatibility. After researched the effect of reactive condition on the reactive rateand biocompatibility, we use CuAAC to stain the live cell and bacteria with dye BN, CN andthe new dye BCN designed by us. After reaction, the cell and bacteria could exhibitfluorescence while keeping alive. At the same time, compared to BN and CN, the BCN could exhibit two kinds of fluorescence. This click reaction supports a kind of method to integratebioactive molecular on the clickable ECM-MAO film.
Finally, we import the alkyl group into the ECM film to construct the clickable ECMmodel. After integrated QK with click reaction, the model could improve the angiogenesis ofHUVEC evidently. The clickable ECM-MAO biomimetic film is biomimetic constructedfrom this model, which could show better biological behavior for improving the expression ofosteogenic gene of HBMSC after integrated with QK by click reaction. In addition, comparedthe film integrated QK peptide in air, the film integrated QK peptide in nitrogen has bettereffect on improving the expression of osteogenic gene of HBMSC at the first day.
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。
As hard tissue implants, pure titanium is widely used due to its advantages inbiocompatibility, mechanical properties, corrosion resistance, and light weight.However, the application of pure titanium is limited in clinic because of its lowbiological property, which leads to the poor interface of the implant-bone tissue. Inthis research, the clickable ECM-MAO film, which could improve the biologicalproperties of the pure titanium hard tissue implant, was biomimetic constructed onthe implant surface by using micro-arc oxidation, decellularization and click reaction.
In the ECM-MAO film, the MAO film is mainly formed by anatase, rutile andapatite. This MAO film could load the allen sodium phosphate (AL) and control therelease of AL up to50h. On the other hand, it could also load the antimicrobial drugPAMAM Dendrimer stably up to30days in PBS solution. The MAO film withPAMAM Dendrimer could exhibit the antimicrobial activity to bacteria P. aeruginosaand S. Aureus while keeping excellent biocompatibility and bioactivity to HBMSC(Human Bone Mesenchymal Stem Cells). The ECM film in the ECM-MAO film isconstructed from the decellularized film model. The decellularized model is form bythe extracellular matrix (ECM) secreted by HUVEC (Human Umbilical VeinEndothelial Cells), which could improve the adhesion, proliferation and angiogenesisof HUVEC in vitro compared to the control groups. The ECM-MAO film which isbiomimetic constructed from this model and formed by ECM secreted by HUVEC orHBMSC shows better biological behavior than MAO film. It could improveHBMSC’s proliferation and expression of Col-I and OPN in vitro.
For ECM-MAO film, the high biocompatibility of the chemistry method is required toprotect the bioactive molecular in ECM while integrating bioactive molecular on it.Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is one kind of the click reactions withhigh biocompatibility. After researched the effect of reactive condition on the reactive rateand biocompatibility, we use CuAAC to stain the live cell and bacteria with dye BN, CN andthe new dye BCN designed by us. After reaction, the cell and bacteria could exhibitfluorescence while keeping alive. At the same time, compared to BN and CN, the BCN could exhibit two kinds of fluorescence. This click reaction supports a kind of method to integratebioactive molecular on the clickable ECM-MAO film.
Finally, we import the alkyl group into the ECM film to construct the clickable ECMmodel. After integrated QK with click reaction, the model could improve the angiogenesis ofHUVEC evidently. The clickable ECM-MAO biomimetic film is biomimetic constructedfrom this model, which could show better biological behavior for improving the expression ofosteogenic gene of HBMSC after integrated with QK by click reaction. In addition, comparedthe film integrated QK peptide in air, the film integrated QK peptide in nitrogen has bettereffect on improving the expression of osteogenic gene of HBMSC at the first day.
引文
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