钛表面透明质酸微图形调控血管内膜形成的研究
发布时间：2018-09-08 07:07
【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。
[Abstract]:Surface endothelialization of cardiovascular implants is considered to be the main way to prevent thrombosis and intimal hyperplasia after implantation. Therefore, it is very important and meaningful to improve the quality of surface endothelialization of these biomaterials. However, the endothelium formed by a single cell on the surface of this material has some problems, such as insufficient anticoagulant function and easy large-scale exfoliation. In order to solve this problem, more research has turned to how to achieve endothelialization in vivo, but on the surface of the material endothelial cell environment and fluid-like shear force ring. Therefore, based on the construction and improvement of endothelial cell environment and fluid-like shear stress environment on the surface of materials, the construction of bionic intima on the surface of materials was studied.
Titanium (Ti) with good biocompatibility was used as the substrate material to simulate the stretching mechanical effect of fluid shear on endothelial cells (ECs) by using hyaluronic acid (HA) strip micrographs on Ti surface. Two co-culture models of vascular endothelial cells and smooth muscle cells, namely, ordered co-culture of ECs and SMCs (SMCs-HAa-ECs) by hydrolyzing HA with hyaluronidase (HAa) and ordered co-culture of ECs and SMCs (SMCs-ColIV-ECs) by shielding the impedance effect of HA with collagen type IV (Col IV), were constructed. Biomimetic vascular intima on metal surface was constructed and its biological function was evaluated. Firstly, P10/40 (HA stripe width 10 micron; naked alkali-activated Ti stripe width 40 micron); P25/25 (HA stripe width 25 micron; naked alkali-activated Ti stripe width 25 micron); P40/10 (HA stripe width 40 micron; naked alkali-activated Ti stripe width 40 micron); naked alkali-activated Ti stripe width 40 micron; naked alkali-activated Ti width 25 micron strip The effects of three micrographic sizes on the morphology, proliferation, secretion of functional factors and anticoagulant function of endothelial cells were studied. The most suitable micrographic sizes for the physiological function of umbilical vein endothelial cells were selected. Secondly, the co-existence of SMCs-HAa-ECs and SMCs-ColIV-ECs was constructed. By comparing the morphology, quantity, secretion function of anticoagulant factors, anticoagulant function and shear stress function of endothelial cells in the two models, a co-culture model more suitable for bionic intima of Ti basilar vessels was screened. Finally, based on the further screening, the initial planting density of smooth muscle cells was optimized and Ti was achieved. The surface vascular bionic intima was preliminarily constructed and its function was evaluated. The hyaluronic acid micrograph on Ti surface was analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle analysis, Fourier transform infrared spectroscopy (FTIR), enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and so on. The morphology, quantity and secretory function of endothelial cells were evaluated by enzyme linked immunosorbent assay (ELISA) and immunofluorescence staining. Especially the morphology and behavior of endothelial cells and smooth muscle cells in co-culture system were observed by double staining. The morphological index of endothelial cells was calculated by ImageJ. The anticoagulant function of endothelial cells was evaluated by platelet adhesion and activation test, activated partial thrombin time (APTT) and plasma prothrombin time (PT) test. The shear force of human aortic blood flow was simulated in a flow chamber. The shear force function was evaluated.
Another related work of this paper is to construct a hyaluronic acid micrograph and a cross-secreted surface of ECM on Ti substrate by combining hyaluronic acid micrograph on Ti surface and acellular technique. Fibrinogen degeneration, endothelial progenitor cell adhesion and anticoagulant function, and smooth muscle fineness were performed on the surface. Biocompatibility evaluation of cell adhesion and macrophage adherence.
The main results are as follows:
1. Hyaluronic acid micrographs were prepared on the surface of Ti substrate. The two-dimensional micrographs had good structural and functional stability. Among the three dimensions of P10/40, P25/25 and P40/10, P25/25 was more suitable for the physiological function of endothelial cells, including morphological bionics, secretion of functional factors and anticoagulant function.
2. The hemocompatibility of hyaluronic acid micrographs is not ideal, but the morphology of endothelial cells and the synthesis of extracellular matrix can be controlled by micrographs, and the graphically distributed extracellular matrix can be obtained by acellular technique. Preliminary biological evaluation results show that the surface of the extracellular matrix has a good inhibition of fibers. Proteogen denaturation promotes adhesion, orderly distribution and anticoagulation of EPCs, inhibits adhesion of smooth muscle cells, promotes phenotypic contraction of smooth muscle cells, and inhibits macrophage adhesion.
3. The co-culture system of SMCs-HAa-ECs was constructed by the addition of hyaluronidase, which could change the effect of hyaluronic acid on cell adhesion, and the co-culture system of SMCs-ColIV-ECs was constructed by using Col IV to shield the effect of hyaluronic acid on endothelial cells. Comparing the secretion, anticoagulation, inhibition of smooth muscle cell proliferation and anti-fluid shear stress, we found that the SMCs-ColIV-ECs co-culture model had more biological advantages.
4. On the basis of SMCs-ColIV-ECs co-culture model, the bionic intima of blood vessels on Ti substrate surface was constructed by using the difference of initial planting density between endothelial cells and smooth muscle cells (1 *105 cells/ml: 2.5 *104 cells/ml). The preliminary biological and mechanical evaluation results showed that the bionic intima was cultured separately from the surface of the material. Compared with other biomimetic intima, it has the advantages of faster formation of vascular biomimetic intima, more secretion of anticoagulant factors, and stronger ability to resist fluid shear stress.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
本文编号：2229747
[Abstract]:Surface endothelialization of cardiovascular implants is considered to be the main way to prevent thrombosis and intimal hyperplasia after implantation. Therefore, it is very important and meaningful to improve the quality of surface endothelialization of these biomaterials. However, the endothelium formed by a single cell on the surface of this material has some problems, such as insufficient anticoagulant function and easy large-scale exfoliation. In order to solve this problem, more research has turned to how to achieve endothelialization in vivo, but on the surface of the material endothelial cell environment and fluid-like shear force ring. Therefore, based on the construction and improvement of endothelial cell environment and fluid-like shear stress environment on the surface of materials, the construction of bionic intima on the surface of materials was studied.
Titanium (Ti) with good biocompatibility was used as the substrate material to simulate the stretching mechanical effect of fluid shear on endothelial cells (ECs) by using hyaluronic acid (HA) strip micrographs on Ti surface. Two co-culture models of vascular endothelial cells and smooth muscle cells, namely, ordered co-culture of ECs and SMCs (SMCs-HAa-ECs) by hydrolyzing HA with hyaluronidase (HAa) and ordered co-culture of ECs and SMCs (SMCs-ColIV-ECs) by shielding the impedance effect of HA with collagen type IV (Col IV), were constructed. Biomimetic vascular intima on metal surface was constructed and its biological function was evaluated. Firstly, P10/40 (HA stripe width 10 micron; naked alkali-activated Ti stripe width 40 micron); P25/25 (HA stripe width 25 micron; naked alkali-activated Ti stripe width 25 micron); P40/10 (HA stripe width 40 micron; naked alkali-activated Ti stripe width 40 micron); naked alkali-activated Ti stripe width 40 micron; naked alkali-activated Ti width 25 micron strip The effects of three micrographic sizes on the morphology, proliferation, secretion of functional factors and anticoagulant function of endothelial cells were studied. The most suitable micrographic sizes for the physiological function of umbilical vein endothelial cells were selected. Secondly, the co-existence of SMCs-HAa-ECs and SMCs-ColIV-ECs was constructed. By comparing the morphology, quantity, secretion function of anticoagulant factors, anticoagulant function and shear stress function of endothelial cells in the two models, a co-culture model more suitable for bionic intima of Ti basilar vessels was screened. Finally, based on the further screening, the initial planting density of smooth muscle cells was optimized and Ti was achieved. The surface vascular bionic intima was preliminarily constructed and its function was evaluated. The hyaluronic acid micrograph on Ti surface was analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM), water contact angle analysis, Fourier transform infrared spectroscopy (FTIR), enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and so on. The morphology, quantity and secretory function of endothelial cells were evaluated by enzyme linked immunosorbent assay (ELISA) and immunofluorescence staining. Especially the morphology and behavior of endothelial cells and smooth muscle cells in co-culture system were observed by double staining. The morphological index of endothelial cells was calculated by ImageJ. The anticoagulant function of endothelial cells was evaluated by platelet adhesion and activation test, activated partial thrombin time (APTT) and plasma prothrombin time (PT) test. The shear force of human aortic blood flow was simulated in a flow chamber. The shear force function was evaluated.
Another related work of this paper is to construct a hyaluronic acid micrograph and a cross-secreted surface of ECM on Ti substrate by combining hyaluronic acid micrograph on Ti surface and acellular technique. Fibrinogen degeneration, endothelial progenitor cell adhesion and anticoagulant function, and smooth muscle fineness were performed on the surface. Biocompatibility evaluation of cell adhesion and macrophage adherence.
The main results are as follows:
1. Hyaluronic acid micrographs were prepared on the surface of Ti substrate. The two-dimensional micrographs had good structural and functional stability. Among the three dimensions of P10/40, P25/25 and P40/10, P25/25 was more suitable for the physiological function of endothelial cells, including morphological bionics, secretion of functional factors and anticoagulant function.
2. The hemocompatibility of hyaluronic acid micrographs is not ideal, but the morphology of endothelial cells and the synthesis of extracellular matrix can be controlled by micrographs, and the graphically distributed extracellular matrix can be obtained by acellular technique. Preliminary biological evaluation results show that the surface of the extracellular matrix has a good inhibition of fibers. Proteogen denaturation promotes adhesion, orderly distribution and anticoagulation of EPCs, inhibits adhesion of smooth muscle cells, promotes phenotypic contraction of smooth muscle cells, and inhibits macrophage adhesion.
3. The co-culture system of SMCs-HAa-ECs was constructed by the addition of hyaluronidase, which could change the effect of hyaluronic acid on cell adhesion, and the co-culture system of SMCs-ColIV-ECs was constructed by using Col IV to shield the effect of hyaluronic acid on endothelial cells. Comparing the secretion, anticoagulation, inhibition of smooth muscle cell proliferation and anti-fluid shear stress, we found that the SMCs-ColIV-ECs co-culture model had more biological advantages.
4. On the basis of SMCs-ColIV-ECs co-culture model, the bionic intima of blood vessels on Ti substrate surface was constructed by using the difference of initial planting density between endothelial cells and smooth muscle cells (1 *105 cells/ml: 2.5 *104 cells/ml). The preliminary biological and mechanical evaluation results showed that the bionic intima was cultured separately from the surface of the material. Compared with other biomimetic intima, it has the advantages of faster formation of vascular biomimetic intima, more secretion of anticoagulant factors, and stronger ability to resist fluid shear stress.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
【参考文献】
相关期刊论文 前10条
1 于学军,何作云,王晓燕,高凌云,牟娇,杨生平,董琼兰;血管内皮-平滑肌细胞双层联合培养模型的改进[J];第三军医大学学报;2004年06期
2 陈江;郭翔;杨苹;王锦标;郑楠;孙红;黄楠;;微图形Ti-O薄膜亲水性对细胞行为的影响[J];功能材料;2010年10期
3 廖玉珍;杨苹;王进;黄楠;;内皮细胞在钛金属表面透明质酸微图形上的粘附行为[J];功能材料;2010年10期
4 李晖;心室辅助装置的内皮化[J];国外医学.生物医学工程分册;2003年04期
5 陈双红,姜宗来,张炎,陈槐卿;内皮细胞和平滑肌细胞联合种植研究[J];国外医学.生物医学工程分册;1999年02期
6 姜宗来,陈双红,张炎,张传森,李玉泉,丛兴忠;与平滑肌细胞联合培养的内皮细胞的形态学和生长增殖[J];解剖学报;2000年03期
7 韩林,张宝仁,覃开荣,朱家麟,柳兆荣;不同切应力下的内皮细胞条件培养基对平滑肌细胞增殖和胶原合成的影响[J];中国动脉硬化杂志;2000年03期
8 陈佳龙;李全利;陈俊英;黄楠;;心血管植入材料体内内皮化研究进展[J];生物医学工程学杂志;2009年06期
9 赵宏伟;王贵学;戴传云;;血管内支架和支架内皮化[J];生物技术通讯;2005年06期
10 李晖;黄焕雷;肖学钧;林秋雄;成安衡;;气动左心辅助循环泵-罗叶泵内皮化的实验研究[J];中国体外循环杂志;2011年02期
相关博士学位论文 前1条
1 石锦霞;软刻蚀技术在高分子科学中的应用[D];中国科学技术大学;2008年
，本文编号：2229747
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2229747.html
