鈦表面透明質酸微圖形調控血管內膜形成的研究
發(fā)布時間：2018-09-08 07:07
【摘要】：心血管植入材料表面內皮化被認為是防止植入后血栓和內膜增生的主要途徑,因此提高這類生物材料的表面內皮化質量是非常重要和有意義的。目前,體外研究證明在生物材料表面覆蓋完整的內皮層具有一定的抑制血栓形成和平滑肌細胞增生的作用。但是,這種材料表面單一細胞形成的內膜植入體內后,存在抗凝血功能不足和容易大面積脫落等問題。為了解決這一問題,更多的研究轉向如何實現(xiàn)體內內皮化,但對材料表面內皮細胞周細胞環(huán)境和仿流體剪切力環(huán)境的構建與改善的研究也不可缺少�；诖�,本文在構建與改善材料表面內皮細胞周細胞環(huán)境和仿流體剪切力環(huán)境的基礎上,進行了材料表面仿生內膜構建的相關研究。 本文采用具有較好生物相容性的鈦(Ti)作為基底材料,利用Ti表面透明質酸(HA)條帶微圖形模擬體內流體剪切力對內皮細胞(ECs)的拉伸力學作用；利用微圖形對平滑肌細胞(SMCs)的限制作用為內皮細胞提供仿生的平滑肌周細胞環(huán)境；同時構建了利用透明質酸酶(HAa)水解HA以實現(xiàn)ECs和SMCs的有序共培養(yǎng)(SMCs-HAa-ECs)和利用四型膠原(ColⅣ)屏蔽HA的阻抗效果以實現(xiàn)ECs和SMCs的有序共培養(yǎng)(SMCs-ColIV-ECs)兩種血管內皮細胞和平滑肌細胞的共培養(yǎng)模型,并在此基礎上初步實現(xiàn)了Ti金屬表面血管仿生內膜構建,同時對該內膜的生物學功能進行了評價。首先,研究了P10/40(HA條紋寬度10μm；裸露的堿活化Ti條紋寬度40μm)、P25/25(HA條紋寬度25μm；裸露的堿活化Ti條紋寬度25μm)、P40/10(HA條紋寬度40μm；裸露的堿活化Ti條紋寬度10μm)三種微圖形尺寸對內皮細胞的形態(tài)、增殖、功能性因子分泌和抗凝血功能的影響,篩選出最適合臍靜脈內皮細胞生理功能發(fā)揮的微圖形尺寸。其次,在此基礎上構建了"SMCs-HAa-ECs"和"SMCs-ColIV-ECs"兩種血管內皮細胞和平滑肌細胞的共培養(yǎng)模型。通過對比兩個模型中內皮細胞形態(tài)、數(shù)量、抗凝血因子分泌功能、抗凝血功能以及抗切應力功能,篩選出更適合Ti基底血管仿生內膜的共培養(yǎng)模型。最后,在前一步篩選的基礎上通過對平滑肌細胞初始種植密度的優(yōu)化,實現(xiàn)了Ti表面血管仿生內膜的初步構建,并對構建的仿生內膜進行了功能評價。綜合利用掃描電子顯微鏡(SEM)、原子力顯微鏡(AFM)、水接觸角分析、傅立葉變換紅外光譜(FTIR)、酶聯(lián)免疫吸附實驗(ELISA)、免疫熒光染色分析等方法對Ti表面透明質酸微圖形的物化性能和穩(wěn)定性進行了表征。運用酶聯(lián)免疫吸附實驗(ELISA)和免疫熒光染色分析方法對內皮細胞的形態(tài)、數(shù)量和分泌功能進行了評價,尤其是運用雙染方法觀察共培養(yǎng)體系中內皮細胞和平滑肌細胞的形態(tài)和行為。運用細胞形態(tài)測量和計算軟件(ImageJ)統(tǒng)計出內皮細胞的形態(tài)指數(shù)。通過血小板粘附和激活實驗、活化部分凝血酶時間(APTT)、血漿凝血酶原時間(PT)實驗對內皮細胞抗凝血功能進行了評價。在流動腔裝置內模擬人體主動脈血流剪切力對材料表面內皮細胞的抗剪切力功能進行了評價。 本文的另一個相關工作是結合Ti表面透明質酸微圖形對內皮細胞的調控和脫細胞技術在Ti基底上構建了透明質酸微圖形和內皮細胞外基質交錯分泌的表面,并對該表面進行了纖維蛋白原變性、內皮祖細胞粘附與抗凝血功能、平滑肌細胞粘附和巨噬細胞粘附等生物相容性評價。 全文主要結果如下： 1、透明質酸微圖形制備到Ti基底表面,該二維微圖形具備較好的結構穩(wěn)定性和功能穩(wěn)定性。P10/40、P25/25、P40/10三個尺寸的微圖形中P25/25更適合內皮細胞生理功能的發(fā)揮,包括形態(tài)仿生、功能性因子的分泌及抗凝血功能等。 2、透明質酸微圖形表面本身的血液相容性并不理想,但是通過微圖形調控內皮細胞形態(tài)和細胞外基質的合成,再結合脫細胞技術可獲得圖形化分布的內皮細胞外基質。初步生物學評價結果顯示,該細胞外基質表面具有較好的抑制纖維蛋白原變性功能、促內皮祖細胞粘附、有序分布和抗凝血功能,抑制平滑肌細胞粘附和促進平滑肌細胞表型收縮功能,以及抑制巨噬細胞粘附的功能。 3、通過透明質酸酶的加入可改變透明質酸阻抗細胞粘附的作用,實現(xiàn)了"SMCs-HAa-ECs"共培養(yǎng)體系的構建；利用ColⅣ的引入屏蔽透明質酸對內皮細胞的阻抗作用,實現(xiàn)了"SMCs-ColIV-ECs"共培養(yǎng)體系的構建；通過兩套模型中內皮細胞抗凝血因子的分泌、抗凝血功能、抑制平滑肌細胞增生功能和抗流體剪切力功能的綜合比較,發(fā)現(xiàn)"SMCs-ColIV-ECs"共培養(yǎng)模型具有更大的生物學優(yōu)勢。 4.在"SMCs-ColIV-ECs"共培養(yǎng)模型的基礎上,利用內皮細胞和平滑肌細胞的初始種植密度差(1×105cells/ml:2.5×104cells/ml)實現(xiàn)了Ti基底表面血管仿生內膜的初步構建,初步的生物學和力學評價結果顯示,該仿生內膜與材料表面單獨培養(yǎng)內皮細胞相比,具有形成血管仿生內膜較快、抗凝血因子分泌多、抗流體剪切力能力強等優(yōu)勢。為后續(xù)無機材料表面仿生內膜的優(yōu)化提供了重要的實驗基礎。
[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.
【學位授予單位】：西南交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R318.08
本文編號：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.
【學位授予單位】：西南交通大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R318.08
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