鈦表面透明質(zhì)酸微圖形調(diào)控血管內(nèi)膜形成的研究
發(fā)布時(shí)間：2018-09-08 07:07
【摘要】：心血管植入材料表面內(nèi)皮化被認(rèn)為是防止植入后血栓和內(nèi)膜增生的主要途徑,因此提高這類生物材料的表面內(nèi)皮化質(zhì)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(nèi)皮層具有一定的抑制血栓形成和平滑肌細(xì)胞增生的作用。但是,這種材料表面單一細(xì)胞形成的內(nèi)膜植入體內(nèi)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(zhuǎn)向如何實(shí)現(xiàn)體內(nèi)內(nèi)皮化,但對(duì)材料表面內(nèi)皮細(xì)胞周細(xì)胞環(huán)境和仿流體剪切力環(huán)境的構(gòu)建與改善的研究也不可缺少�；诖�,本文在構(gòu)建與改善材料表面內(nèi)皮細(xì)胞周細(xì)胞環(huán)境和仿流體剪切力環(huán)境的基礎(chǔ)上,進(jìn)行了材料表面仿生內(nèi)膜構(gòu)建的相關(guān)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(zhì)酸(HA)條帶微圖形模擬體內(nèi)流體剪切力對(duì)內(nèi)皮細(xì)胞(ECs)的拉伸力學(xué)作用；利用微圖形對(duì)平滑肌細(xì)胞(SMCs)的限制作用為內(nèi)皮細(xì)胞提供仿生的平滑肌周細(xì)胞環(huán)境；同時(shí)構(gòu)建了利用透明質(zhì)酸酶(HAa)水解HA以實(shí)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(shí)現(xiàn)ECs和SMCs的有序共培養(yǎng)(SMCs-ColIV-ECs)兩種血管內(nèi)皮細(xì)胞和平滑肌細(xì)胞的共培養(yǎng)模型,并在此基礎(chǔ)上初步實(shí)現(xiàn)了Ti金屬表面血管仿生內(nèi)膜構(gòu)建,同時(shí)對(duì)該內(nèi)膜的生物學(xué)功能進(jìn)行了評(píng)價(jià)。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(duì)內(nèi)皮細(xì)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(nèi)皮細(xì)胞生理功能發(fā)揮的微圖形尺寸。其次,在此基礎(chǔ)上構(gòu)建了"SMCs-HAa-ECs"和"SMCs-ColIV-ECs"兩種血管內(nèi)皮細(xì)胞和平滑肌細(xì)胞的共培養(yǎng)模型。通過對(duì)比兩個(gè)模型中內(nèi)皮細(xì)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(yīng)力功能,篩選出更適合Ti基底血管仿生內(nèi)膜的共培養(yǎng)模型。最后,在前一步篩選的基礎(chǔ)上通過對(duì)平滑肌細(xì)胞初始種植密度的優(yōu)化,實(shí)現(xiàn)了Ti表面血管仿生內(nèi)膜的初步構(gòu)建,并對(duì)構(gòu)建的仿生內(nèi)膜進(jìn)行了功能評(píng)價(jià)。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(shí)驗(yàn)(ELISA)、免疫熒光染色分析等方法對(duì)Ti表面透明質(zhì)酸微圖形的物化性能和穩(wěn)定性進(jìn)行了表征。運(yùn)用酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)和免疫熒光染色分析方法對(duì)內(nèi)皮細(xì)胞的形態(tài)、數(shù)量和分泌功能進(jìn)行了評(píng)價(jià),尤其是運(yùn)用雙染方法觀察共培養(yǎng)體系中內(nèi)皮細(xì)胞和平滑肌細(xì)胞的形態(tài)和行為。運(yùn)用細(xì)胞形態(tài)測(cè)量和計(jì)算軟件(ImageJ)統(tǒng)計(jì)出內(nèi)皮細(xì)胞的形態(tài)指數(shù)。通過血小板粘附和激活實(shí)驗(yàn)、活化部分凝血酶時(shí)間(APTT)、血漿凝血酶原時(shí)間(PT)實(shí)驗(yàn)對(duì)內(nèi)皮細(xì)胞抗凝血功能進(jìn)行了評(píng)價(jià)。在流動(dòng)腔裝置內(nèi)模擬人體主動(dòng)脈血流剪切力對(duì)材料表面內(nèi)皮細(xì)胞的抗剪切力功能進(jìn)行了評(píng)價(jià)。 本文的另一個(gè)相關(guān)工作是結(jié)合Ti表面透明質(zhì)酸微圖形對(duì)內(nèi)皮細(xì)胞的調(diào)控和脫細(xì)胞技術(shù)在Ti基底上構(gòu)建了透明質(zhì)酸微圖形和內(nèi)皮細(xì)胞外基質(zhì)交錯(cuò)分泌的表面,并對(duì)該表面進(jìn)行了纖維蛋白原變性、內(nèi)皮祖細(xì)胞粘附與抗凝血功能、平滑肌細(xì)胞粘附和巨噬細(xì)胞粘附等生物相容性評(píng)價(jià)。 全文主要結(jié)果如下： 1、透明質(zhì)酸微圖形制備到Ti基底表面,該二維微圖形具備較好的結(jié)構(gòu)穩(wěn)定性和功能穩(wěn)定性。P10/40、P25/25、P40/10三個(gè)尺寸的微圖形中P25/25更適合內(nèi)皮細(xì)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(zhì)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(diào)控內(nèi)皮細(xì)胞形態(tài)和細(xì)胞外基質(zhì)的合成,再結(jié)合脫細(xì)胞技術(shù)可獲得圖形化分布的內(nèi)皮細(xì)胞外基質(zhì)。初步生物學(xué)評(píng)價(jià)結(jié)果顯示,該細(xì)胞外基質(zhì)表面具有較好的抑制纖維蛋白原變性功能、促內(nèi)皮祖細(xì)胞粘附、有序分布和抗凝血功能,抑制平滑肌細(xì)胞粘附和促進(jìn)平滑肌細(xì)胞表型收縮功能,以及抑制巨噬細(xì)胞粘附的功能。 3、通過透明質(zhì)酸酶的加入可改變透明質(zhì)酸阻抗細(xì)胞粘附的作用,實(shí)現(xiàn)了"SMCs-HAa-ECs"共培養(yǎng)體系的構(gòu)建；利用ColⅣ的引入屏蔽透明質(zhì)酸對(duì)內(nèi)皮細(xì)胞的阻抗作用,實(shí)現(xiàn)了"SMCs-ColIV-ECs"共培養(yǎng)體系的構(gòu)建；通過兩套模型中內(nèi)皮細(xì)胞抗凝血因子的分泌、抗凝血功能、抑制平滑肌細(xì)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(xué)優(yōu)勢(shì)。 4.在"SMCs-ColIV-ECs"共培養(yǎng)模型的基礎(chǔ)上,利用內(nèi)皮細(xì)胞和平滑肌細(xì)胞的初始種植密度差(1×105cells/ml:2.5×104cells/ml)實(shí)現(xiàn)了Ti基底表面血管仿生內(nèi)膜的初步構(gòu)建,初步的生物學(xué)和力學(xué)評(píng)價(jià)結(jié)果顯示,該仿生內(nèi)膜與材料表面單獨(dú)培養(yǎng)內(nèi)皮細(xì)胞相比,具有形成血管仿生內(nèi)膜較快、抗凝血因子分泌多、抗流體剪切力能力強(qiáng)等優(yōu)勢(shì)。為后續(xù)無機(jī)材料表面仿生內(nèi)膜的優(yōu)化提供了重要的實(shí)驗(yàn)基礎(chǔ)。
[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(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
本文編號(hào)：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(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R318.08
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