钛表面透明质酸微图形调控血管内膜形成的研究
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摘要
心血管植入材料表面内皮化被认为是防止植入后血栓和内膜增生的主要途径,因此提高这类生物材料的表面内皮化质量是非常重要和有意义的。目前,体外研究证明在生物材料表面覆盖完整的内皮层具有一定的抑制血栓形成和平滑肌细胞增生的作用。但是,这种材料表面单一细胞形成的内膜植入体内后,存在抗凝血功能不足和容易大面积脱落等问题。为了解决这一问题,更多的研究转向如何实现体内内皮化,但对材料表面内皮细胞周细胞环境和仿流体剪切力环境的构建与改善的研究也不可缺少。基于此,本文在构建与改善材料表面内皮细胞周细胞环境和仿流体剪切力环境的基础上,进行了材料表面仿生内膜构建的相关研究。
本文采用具有较好生物相容性的钛(Ti)作为基底材料,利用Ti表面透明质酸(HA)条带微图形模拟体内流体剪切力对内皮细胞(ECs)的拉伸力学作用；利用微图形对平滑肌细胞(SMCs)的限制作用为内皮细胞提供仿生的平滑肌周细胞环境；同时构建了利用透明质酸酶(HAa)水解HA以实现ECs和SMCs的有序共培养(SMCs-HAa-ECs)和利用四型胶原(ColⅣ)屏蔽HA的阻抗效果以实现ECs和SMCs的有序共培养(SMCs-ColIV-ECs)两种血管内皮细胞和平滑肌细胞的共培养模型,并在此基础上初步实现了Ti金属表面血管仿生内膜构建,同时对该内膜的生物学功能进行了评价。首先,研究了P10/40(HA条纹宽度10μm；裸露的碱活化Ti条纹宽度40μm)、P25/25(HA条纹宽度25μm；裸露的碱活化Ti条纹宽度25μm)、P40/10(HA条纹宽度40μm；裸露的碱活化Ti条纹宽度10μm)三种微图形尺寸对内皮细胞的形态、增殖、功能性因子分泌和抗凝血功能的影响,筛选出最适合脐静脉内皮细胞生理功能发挥的微图形尺寸。其次,在此基础上构建了"SMCs-HAa-ECs"和"SMCs-ColIV-ECs"两种血管内皮细胞和平滑肌细胞的共培养模型。通过对比两个模型中内皮细胞形态、数量、抗凝血因子分泌功能、抗凝血功能以及抗切应力功能,筛选出更适合Ti基底血管仿生内膜的共培养模型。最后,在前一步筛选的基础上通过对平滑肌细胞初始种植密度的优化,实现了Ti表面血管仿生内膜的初步构建,并对构建的仿生内膜进行了功能评价。综合利用扫描电子显微镜(SEM)、原子力显微镜(AFM)、水接触角分析、傅立叶变换红外光谱(FTIR)、酶联免疫吸附实验(ELISA)、免疫荧光染色分析等方法对Ti表面透明质酸微图形的物化性能和稳定性进行了表征。运用酶联免疫吸附实验(ELISA)和免疫荧光染色分析方法对内皮细胞的形态、数量和分泌功能进行了评价,尤其是运用双染方法观察共培养体系中内皮细胞和平滑肌细胞的形态和行为。运用细胞形态测量和计算软件(ImageJ)统计出内皮细胞的形态指数。通过血小板粘附和激活实验、活化部分凝血酶时间(APTT)、血浆凝血酶原时间(PT)实验对内皮细胞抗凝血功能进行了评价。在流动腔装置内模拟人体主动脉血流剪切力对材料表面内皮细胞的抗剪切力功能进行了评价。
本文的另一个相关工作是结合Ti表面透明质酸微图形对内皮细胞的调控和脱细胞技术在Ti基底上构建了透明质酸微图形和内皮细胞外基质交错分泌的表面,并对该表面进行了纤维蛋白原变性、内皮祖细胞粘附与抗凝血功能、平滑肌细胞粘附和巨噬细胞粘附等生物相容性评价。
全文主要结果如下：
1、透明质酸微图形制备到Ti基底表面,该二维微图形具备较好的结构稳定性和功能稳定性。P10/40、P25/25、P40/10三个尺寸的微图形中P25/25更适合内皮细胞生理功能的发挥,包括形态仿生、功能性因子的分泌及抗凝血功能等。
2、透明质酸微图形表面本身的血液相容性并不理想,但是通过微图形调控内皮细胞形态和细胞外基质的合成,再结合脱细胞技术可获得图形化分布的内皮细胞外基质。初步生物学评价结果显示,该细胞外基质表面具有较好的抑制纤维蛋白原变性功能、促内皮祖细胞粘附、有序分布和抗凝血功能,抑制平滑肌细胞粘附和促进平滑肌细胞表型收缩功能,以及抑制巨噬细胞粘附的功能。
3、通过透明质酸酶的加入可改变透明质酸阻抗细胞粘附的作用,实现了"SMCs-HAa-ECs"共培养体系的构建；利用ColⅣ的引入屏蔽透明质酸对内皮细胞的阻抗作用,实现了"SMCs-ColIV-ECs"共培养体系的构建；通过两套模型中内皮细胞抗凝血因子的分泌、抗凝血功能、抑制平滑肌细胞增生功能和抗流体剪切力功能的综合比较,发现"SMCs-ColIV-ECs"共培养模型具有更大的生物学优势。
4.在"SMCs-ColIV-ECs"共培养模型的基础上,利用内皮细胞和平滑肌细胞的初始种植密度差(1×105cells/ml:2.5×104cells/ml)实现了Ti基底表面血管仿生内膜的初步构建,初步的生物学和力学评价结果显示,该仿生内膜与材料表面单独培养内皮细胞相比,具有形成血管仿生内膜较快、抗凝血因子分泌多、抗流体剪切力能力强等优势。为后续无机材料表面仿生内膜的优化提供了重要的实验基础。
Surface endothelialization was considered to be the main way to resolve thrombosis and hyperplasia after the cardiovascular devices implanted. Therefore, improving the endothelial surface quality of such biomaterials is very important and meaningful. Currently, in vitro studies have shown that complete endothelium coverage of the biomaterial surface has a certain inhibition effect on thrombosis and smooth muscle cell proliferation. However, monolayer formed by this single cell on the surface was found lots of problems after implantation, such as anti-coagulation deficiencies and easily falling off in a large area. To solve these problems, more research turned to in vivo endothelialization, while the studies on construction and improvement of the endothelial cells pericytes environment and imitation of fluid shear stress environment on the materials surface were also important. Thus, in this work research on building biomimetic pericytes environment of endothelial cells and in vivo fluid shear stress environment to contribute to biomimetic intimal building on the materials surface was performed.
In this paper, titanium (Ti) with good biocompatibility was chosen as the base materials. Tensile role on the endothelial cells by in vivo fluid shear stress was simulated by micro-strip graphic of hyaluronic acid (HA) on the Ti surface. Bionic smooth muscle cells pericytes environment was provided for the endothelial cells through the limitation of HA micro-strip on the smooth muscle cells. We built two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs". respectively. On this basis, bionic vascular intimal on Ti metal surface was initial built, and the biological function of the endometrium was evaluated. At first, the effects of three kinds of micro-pattern size, P10/40, P25/25, P40/10, on endothelial cell morphology, proliferation, functional cytokine secretion and anticoagulant function were studied. The micro-pattern size which was the most suitable for physiological functions of human umbilical vein endothelial cells was filtered out. Secondly, two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs" were built on this basis. By comparing the two models on endothelial cell morphology, the number of anti-coagulation factor secretion, anti-coagulation and anti-shear stress function, the model which was more suitable for culturing bionic vascular intima on Ti substrate was filtered out. Finally, on the basis of the previous step, the initial planting density of smooth muscle cells was optimized, and ultimately the construction of blood vessels bionic initial on Ti surface was realized, and the biological function of the endometrium was evaluated. Utilizing scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle analysis, Fourier transform infrared spectroscopy (FTIR), enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis methods to characterize physical and chemical properties and stability of the hyaluronic acid micro-graphics on Ti surface. We use enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining methods to evaluate endothelial cell morphology, number and secretory function, in particular using double staining to observe endothelial cells and smooth muscle cell morphology and behavior in the co-culture system. We use cell morphology measurement and calculation software (ImageJ) statisticed endothelial cell morphology index. By platelet adhesion and activation experiments and clotting time (APTT, PT) test, endothelial cells anticoagulation property was evaluated. In the flow chamber device, the human aortic flow shear stress was simulated to evaluate anti-shear-strength function of endothelial cells on the surface.
Another relevant work of this paper is a combination of regulation of HA micro-pattern on endothelial cells and decellularization technology, which can build an orderly distribution of endothelial extracellular matrix surface on Ti substrate surface. The initial blood compatibility evaluation, evaluation of rapid endothelialization, smooth muscle cell culture evaluation and evaluation of macrophage cultures for the extracellular matrix surface were performed.
Full results are as follows:
1. Hyaluronic acid micro-graph on Ti substrate surface has good stability. P25/25better suits to play a physiological function of endothelial cells among P10/40, P25/25and P40/10three micro-graphs, including imitation situ morphological, functional cytokine secretion and anticoagulant function.
2. Hyaluronic micropattern surface itself does not have ideal blood compatibility. Micro-pattern can regulate endothelial cell morphology and extracellular matrix synthesis. Combining decellularization technology, a patterned distribution of endothelial extracellular matrix can be achieved. This extracellular matrix surface has good blood compatibility, rapid endothelialization function, certain inhibitions of smooth muscle over proliferation and inhibition of macrophage adhesion by preliminary biological evaluation.
3. Because hyaluronidase can change the property of hyaluronic acid that inhibit cell adhesion, we take advantage of this to achieve a "SMCs-HAa-ECs" co-culture system construction; We take advantage of ColIV shield impedance function of hyaluronic acid on endothelial cell, to achieve the "SMCs-ColIV-ECs" co-culture system construction; Comparing two models on anti-clotting factor secretion, anti-clotting function, inhibition of smooth muscle function and anti fluid shear stress of endothelial cells,"SMCs-ColIV-ECs" co-culture model is considered to have a greater physiological advantage。
4. On the basis of "SMCs-ColIV-ECs" co-culture model, initial construction of bionic vascular intima has been realized by the initial planting density difference of endothelial cells and smooth muscle cells (1×105cells/ml:2.5×104cells/ml). After initial of the biological and mechanical evaluation, the biomimetic membrane surface compared with endothelial cells cultured alone, has the advantages of growing fast, anti-coagulation factor secretion and better ability to resist shear stress and so on。 This work provides important experimental basis for subsequent optimization of biomimetic membrane on inorganic material surface.
本文采用具有较好生物相容性的钛(Ti)作为基底材料,利用Ti表面透明质酸(HA)条带微图形模拟体内流体剪切力对内皮细胞(ECs)的拉伸力学作用；利用微图形对平滑肌细胞(SMCs)的限制作用为内皮细胞提供仿生的平滑肌周细胞环境；同时构建了利用透明质酸酶(HAa)水解HA以实现ECs和SMCs的有序共培养(SMCs-HAa-ECs)和利用四型胶原(ColⅣ)屏蔽HA的阻抗效果以实现ECs和SMCs的有序共培养(SMCs-ColIV-ECs)两种血管内皮细胞和平滑肌细胞的共培养模型,并在此基础上初步实现了Ti金属表面血管仿生内膜构建,同时对该内膜的生物学功能进行了评价。首先,研究了P10/40(HA条纹宽度10μm；裸露的碱活化Ti条纹宽度40μm)、P25/25(HA条纹宽度25μm；裸露的碱活化Ti条纹宽度25μm)、P40/10(HA条纹宽度40μm；裸露的碱活化Ti条纹宽度10μm)三种微图形尺寸对内皮细胞的形态、增殖、功能性因子分泌和抗凝血功能的影响,筛选出最适合脐静脉内皮细胞生理功能发挥的微图形尺寸。其次,在此基础上构建了"SMCs-HAa-ECs"和"SMCs-ColIV-ECs"两种血管内皮细胞和平滑肌细胞的共培养模型。通过对比两个模型中内皮细胞形态、数量、抗凝血因子分泌功能、抗凝血功能以及抗切应力功能,筛选出更适合Ti基底血管仿生内膜的共培养模型。最后,在前一步筛选的基础上通过对平滑肌细胞初始种植密度的优化,实现了Ti表面血管仿生内膜的初步构建,并对构建的仿生内膜进行了功能评价。综合利用扫描电子显微镜(SEM)、原子力显微镜(AFM)、水接触角分析、傅立叶变换红外光谱(FTIR)、酶联免疫吸附实验(ELISA)、免疫荧光染色分析等方法对Ti表面透明质酸微图形的物化性能和稳定性进行了表征。运用酶联免疫吸附实验(ELISA)和免疫荧光染色分析方法对内皮细胞的形态、数量和分泌功能进行了评价,尤其是运用双染方法观察共培养体系中内皮细胞和平滑肌细胞的形态和行为。运用细胞形态测量和计算软件(ImageJ)统计出内皮细胞的形态指数。通过血小板粘附和激活实验、活化部分凝血酶时间(APTT)、血浆凝血酶原时间(PT)实验对内皮细胞抗凝血功能进行了评价。在流动腔装置内模拟人体主动脉血流剪切力对材料表面内皮细胞的抗剪切力功能进行了评价。
本文的另一个相关工作是结合Ti表面透明质酸微图形对内皮细胞的调控和脱细胞技术在Ti基底上构建了透明质酸微图形和内皮细胞外基质交错分泌的表面,并对该表面进行了纤维蛋白原变性、内皮祖细胞粘附与抗凝血功能、平滑肌细胞粘附和巨噬细胞粘附等生物相容性评价。
全文主要结果如下：
1、透明质酸微图形制备到Ti基底表面,该二维微图形具备较好的结构稳定性和功能稳定性。P10/40、P25/25、P40/10三个尺寸的微图形中P25/25更适合内皮细胞生理功能的发挥,包括形态仿生、功能性因子的分泌及抗凝血功能等。
2、透明质酸微图形表面本身的血液相容性并不理想,但是通过微图形调控内皮细胞形态和细胞外基质的合成,再结合脱细胞技术可获得图形化分布的内皮细胞外基质。初步生物学评价结果显示,该细胞外基质表面具有较好的抑制纤维蛋白原变性功能、促内皮祖细胞粘附、有序分布和抗凝血功能,抑制平滑肌细胞粘附和促进平滑肌细胞表型收缩功能,以及抑制巨噬细胞粘附的功能。
3、通过透明质酸酶的加入可改变透明质酸阻抗细胞粘附的作用,实现了"SMCs-HAa-ECs"共培养体系的构建；利用ColⅣ的引入屏蔽透明质酸对内皮细胞的阻抗作用,实现了"SMCs-ColIV-ECs"共培养体系的构建；通过两套模型中内皮细胞抗凝血因子的分泌、抗凝血功能、抑制平滑肌细胞增生功能和抗流体剪切力功能的综合比较,发现"SMCs-ColIV-ECs"共培养模型具有更大的生物学优势。
4.在"SMCs-ColIV-ECs"共培养模型的基础上,利用内皮细胞和平滑肌细胞的初始种植密度差(1×105cells/ml:2.5×104cells/ml)实现了Ti基底表面血管仿生内膜的初步构建,初步的生物学和力学评价结果显示,该仿生内膜与材料表面单独培养内皮细胞相比,具有形成血管仿生内膜较快、抗凝血因子分泌多、抗流体剪切力能力强等优势。为后续无机材料表面仿生内膜的优化提供了重要的实验基础。
Surface endothelialization was considered to be the main way to resolve thrombosis and hyperplasia after the cardiovascular devices implanted. Therefore, improving the endothelial surface quality of such biomaterials is very important and meaningful. Currently, in vitro studies have shown that complete endothelium coverage of the biomaterial surface has a certain inhibition effect on thrombosis and smooth muscle cell proliferation. However, monolayer formed by this single cell on the surface was found lots of problems after implantation, such as anti-coagulation deficiencies and easily falling off in a large area. To solve these problems, more research turned to in vivo endothelialization, while the studies on construction and improvement of the endothelial cells pericytes environment and imitation of fluid shear stress environment on the materials surface were also important. Thus, in this work research on building biomimetic pericytes environment of endothelial cells and in vivo fluid shear stress environment to contribute to biomimetic intimal building on the materials surface was performed.
In this paper, titanium (Ti) with good biocompatibility was chosen as the base materials. Tensile role on the endothelial cells by in vivo fluid shear stress was simulated by micro-strip graphic of hyaluronic acid (HA) on the Ti surface. Bionic smooth muscle cells pericytes environment was provided for the endothelial cells through the limitation of HA micro-strip on the smooth muscle cells. We built two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs". respectively. On this basis, bionic vascular intimal on Ti metal surface was initial built, and the biological function of the endometrium was evaluated. At first, the effects of three kinds of micro-pattern size, P10/40, P25/25, P40/10, on endothelial cell morphology, proliferation, functional cytokine secretion and anticoagulant function were studied. The micro-pattern size which was the most suitable for physiological functions of human umbilical vein endothelial cells was filtered out. Secondly, two kinds of vascular endothelial cells and smooth muscle cells co-culture model named "SMCs-HAa-ECs" and "SMCs-ColIV-ECs" were built on this basis. By comparing the two models on endothelial cell morphology, the number of anti-coagulation factor secretion, anti-coagulation and anti-shear stress function, the model which was more suitable for culturing bionic vascular intima on Ti substrate was filtered out. Finally, on the basis of the previous step, the initial planting density of smooth muscle cells was optimized, and ultimately the construction of blood vessels bionic initial on Ti surface was realized, and the biological function of the endometrium was evaluated. Utilizing scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle analysis, Fourier transform infrared spectroscopy (FTIR), enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis methods to characterize physical and chemical properties and stability of the hyaluronic acid micro-graphics on Ti surface. We use enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining methods to evaluate endothelial cell morphology, number and secretory function, in particular using double staining to observe endothelial cells and smooth muscle cell morphology and behavior in the co-culture system. We use cell morphology measurement and calculation software (ImageJ) statisticed endothelial cell morphology index. By platelet adhesion and activation experiments and clotting time (APTT, PT) test, endothelial cells anticoagulation property was evaluated. In the flow chamber device, the human aortic flow shear stress was simulated to evaluate anti-shear-strength function of endothelial cells on the surface.
Another relevant work of this paper is a combination of regulation of HA micro-pattern on endothelial cells and decellularization technology, which can build an orderly distribution of endothelial extracellular matrix surface on Ti substrate surface. The initial blood compatibility evaluation, evaluation of rapid endothelialization, smooth muscle cell culture evaluation and evaluation of macrophage cultures for the extracellular matrix surface were performed.
Full results are as follows:
1. Hyaluronic acid micro-graph on Ti substrate surface has good stability. P25/25better suits to play a physiological function of endothelial cells among P10/40, P25/25and P40/10three micro-graphs, including imitation situ morphological, functional cytokine secretion and anticoagulant function.
2. Hyaluronic micropattern surface itself does not have ideal blood compatibility. Micro-pattern can regulate endothelial cell morphology and extracellular matrix synthesis. Combining decellularization technology, a patterned distribution of endothelial extracellular matrix can be achieved. This extracellular matrix surface has good blood compatibility, rapid endothelialization function, certain inhibitions of smooth muscle over proliferation and inhibition of macrophage adhesion by preliminary biological evaluation.
3. Because hyaluronidase can change the property of hyaluronic acid that inhibit cell adhesion, we take advantage of this to achieve a "SMCs-HAa-ECs" co-culture system construction; We take advantage of ColIV shield impedance function of hyaluronic acid on endothelial cell, to achieve the "SMCs-ColIV-ECs" co-culture system construction; Comparing two models on anti-clotting factor secretion, anti-clotting function, inhibition of smooth muscle function and anti fluid shear stress of endothelial cells,"SMCs-ColIV-ECs" co-culture model is considered to have a greater physiological advantage。
4. On the basis of "SMCs-ColIV-ECs" co-culture model, initial construction of bionic vascular intima has been realized by the initial planting density difference of endothelial cells and smooth muscle cells (1×105cells/ml:2.5×104cells/ml). After initial of the biological and mechanical evaluation, the biomimetic membrane surface compared with endothelial cells cultured alone, has the advantages of growing fast, anti-coagulation factor secretion and better ability to resist shear stress and so on。 This work provides important experimental basis for subsequent optimization of biomimetic membrane on inorganic material surface.
引文
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