软刻蚀方法制备细胞模板及内皮/平滑肌细胞的协同培养
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摘要
生物材料表面内膜功能及完整性的恢复是解决心血管系统植入装置血栓与栓塞并发症、提高远期的管腔通畅率的最佳方法之一。借鉴仿生表面工程的设计理念,本文采用微转移模塑方法在NaOH活化的钛(TiOH)表面成功制备出了透明质酸(HA)微图形,采用扫描电子显微镜(SEM)、傅里叶红外变换光谱分析(FTIR)、接触角测量仪、表面轮廓仪和电子能谱仪(EDX)对制备HA微图形及其稳定性进行了表征,进而研究了HA的抗细胞粘附功能,并利用酶联免疫吸附实验(Elisa)和细胞粘附实验研究了透明质酸酶(HAa)对HA的酶解作用,以及纤维连接蛋白(FN)和Ⅳ型胶原蛋白(CollagenⅣ)关闭HA的抗蛋白、抗细胞粘附功能。在此基础上,作为细胞模板用于研究对血小板、内皮细胞、平滑肌细胞粘附的影响,以及在细胞模板上血小板/内皮细胞、内皮/平滑肌细胞联合培养的研究。
SEM、FTIR和接触角测量结果表明：碱活化后钛(Ti)表面形貌和粗糙度发生改变,出现-OH伸缩振动峰,其亲水程度明显增加；SEM和表面轮廓仪检测结果表明：采用浇铸法制备出沟、脊分明、表面平整的PDMS弹性印章；SEM和EDX检测结果表明：采用软刻蚀(微转移模塑)的方法在TiOH表面获得了轮廓清晰的HA条纹微图形,但HA在脊上呈不均匀的分布；XPS结果表明,涂覆HA的TiOH样品经过磷酸缓冲液(PBS)浸泡24h后仍能够稳定吸附在TiOH样品表面,HA微图形样品经过PBS浸泡后图形结构仍清晰可见。采用三种方法关闭HA抗蛋白、细胞粘附的功能,细胞粘附实验表明,采用HAa酶解、CollagenⅣ和FN组装的方法都可以有效地关闭HA的阻抗细胞粘附功能。CCK-8结果表明这三种处理方法对平滑肌细胞的增殖能力的影响不大。采用CollagenⅣ组装方法时内皮细胞粘附率要高于其它两种方法。
单种细胞的培养结果表明,HA微图形可以有效地将血小板、内皮、平滑肌胞三种细胞限制在微图形样品表面的粘附区域。当图形脊/沟尺寸为20μm/30μm,与细胞大小相当时,可以有效地引导内皮细胞、平滑肌细胞细胞按照图形的方向伸长,呈有序的细胞阵列分布；HA微图形对内皮细胞的接触引导行为的影响非常显著,与平板样品相比, HA微图形表面培养的内皮细胞形态指数更接近体内生理条件下的内皮细胞的形态指数。在HA微图形表面可以得到内皮细胞/血小板和内皮/平滑肌细胞有序分布的协同培养体系。两种细胞的协同培养结果表明,血小板图案并不能影响内皮细胞粘附的形态,而图案化的平滑肌细胞的更细长形态,可以调控内皮细胞的长轴取向。但长时间的协同培养会引起平滑肌细胞跨过内皮细胞呈交错、多层生长的结构。
以上研究结果表明,HAa酶解、CollagenⅣ和FN组装可以有效地关闭HA阻抗蛋白、细胞粘附的功能。细胞模板可以很好地调控细胞粘附的区域和细胞的铺展形态,利用该细胞模板获得了短期的内皮/平滑肌细胞联合培养体系。
The recovery of intimal function and integrity on biomaterials surface is one of the best ways to prohibit the thrombosis and embolism complications of cardiovascular implanted devices and improve long-term lumen patency. In this thesis, Microtransfer Molding was used to fabricate the hyaluronic acid (HA) micropattern (Cells template) on the activated titanium (TiOH) surface, which was based on the design concept of biomimetic surface engineering. The HA micropatterns were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), contact angle measur-ement, surface profiler and energy dispersive X-ray spectroscopy (EDX). Furthermore, the stability and anti-adhesion function of HA were investigated. The zymohy- drolysis of hyaluronidase, collagen and fibronectin (FN) self-assembly were used to switch the anti-adhesion function of HA which was evaluated enzyme-linked immunosorbent assay (Elisa) and cell adhesion experiments. The adhesion behaviors of platelet, endothelial cells and smooth muscle cells on the cells template were studied, respectively, and the co-culture of endothelial cells /platelet and endothelial cells/ smooth muscle cells on the cells template were also investigated.
The results of SEM FTIR and contact angle measurement showed the changes of morphology, increases of roughness and hydrophilicity and detection of ophilic groups on the Ti surface after treatement of NaOH solution. Surface profiler and SEM results showed that polydimethyl-siloxane (PDMS) stamps with high fidelity were prepared. SEM and EDX results demonstrated that HA micropattems with HA and TiOH alternating stripes had been prepared accurately on the surface of TiOH. However, thickness of HA stripe was not uniform. XPS results demonstrated that HA micropattems were still distinct and HA adsorbed stably on the surface of TiOH after washing by phosphate buffer (PBS).
The results of cells adhesion showed that the anti-adhesion function of HA can be effectively switched by enzymatic action, collagen and FN self-assembly. The three treatments displayed weak effect on the smooth muscle cells (SMCs) proliferation, which was proven by the results of CCK-8. The self-assembly of collagen used to the change the anti-adhesion function of HA could obviously enhance the adhesion of ECs compared with other two switch methods
The results of single cell culture showed that the adhesion of platelet, endothelial cells (ECs) and SMCs were restricted by HA micropatterns. Furthermore, when the size of HA/TiOH alternating stripe was 20μm/30μm, which was comparable to the size of ECs, ECs and SMCs were elongated along the stripes. In addition, the shape index of ECs cultured on patterned surface was more similar to that of ECs grown in vivo compared with ECs on flat surfaces. The co-culture of two kinds of cells, such as ECs/platelet and ECs/ SMCs, showed that co-culture system with ordered cell distribution could be obtained on the surface of HA/TiOH alternating stripes. The pattern of platelets (which adhered on TiOH alternating stripes) did not affect the morphology of endothelial cells. However, the long axis of ECs could be regulated through SMCs alignment. But SMCs could stride over the ECs and would form staggered multi-layers structure during a long time co-colture period.
Our results indicates the anti-adhesion function of HA will be effectively changed by enzymatic action, Collagen and FN self-assembly. The cell templates can be used to regulate cell adhesion and spreading on the patterns, and construct a short-term co-culture system of ECs/SMCs.
SEM、FTIR和接触角测量结果表明：碱活化后钛(Ti)表面形貌和粗糙度发生改变,出现-OH伸缩振动峰,其亲水程度明显增加；SEM和表面轮廓仪检测结果表明：采用浇铸法制备出沟、脊分明、表面平整的PDMS弹性印章；SEM和EDX检测结果表明：采用软刻蚀(微转移模塑)的方法在TiOH表面获得了轮廓清晰的HA条纹微图形,但HA在脊上呈不均匀的分布；XPS结果表明,涂覆HA的TiOH样品经过磷酸缓冲液(PBS)浸泡24h后仍能够稳定吸附在TiOH样品表面,HA微图形样品经过PBS浸泡后图形结构仍清晰可见。采用三种方法关闭HA抗蛋白、细胞粘附的功能,细胞粘附实验表明,采用HAa酶解、CollagenⅣ和FN组装的方法都可以有效地关闭HA的阻抗细胞粘附功能。CCK-8结果表明这三种处理方法对平滑肌细胞的增殖能力的影响不大。采用CollagenⅣ组装方法时内皮细胞粘附率要高于其它两种方法。
单种细胞的培养结果表明,HA微图形可以有效地将血小板、内皮、平滑肌胞三种细胞限制在微图形样品表面的粘附区域。当图形脊/沟尺寸为20μm/30μm,与细胞大小相当时,可以有效地引导内皮细胞、平滑肌细胞细胞按照图形的方向伸长,呈有序的细胞阵列分布；HA微图形对内皮细胞的接触引导行为的影响非常显著,与平板样品相比, HA微图形表面培养的内皮细胞形态指数更接近体内生理条件下的内皮细胞的形态指数。在HA微图形表面可以得到内皮细胞/血小板和内皮/平滑肌细胞有序分布的协同培养体系。两种细胞的协同培养结果表明,血小板图案并不能影响内皮细胞粘附的形态,而图案化的平滑肌细胞的更细长形态,可以调控内皮细胞的长轴取向。但长时间的协同培养会引起平滑肌细胞跨过内皮细胞呈交错、多层生长的结构。
以上研究结果表明,HAa酶解、CollagenⅣ和FN组装可以有效地关闭HA阻抗蛋白、细胞粘附的功能。细胞模板可以很好地调控细胞粘附的区域和细胞的铺展形态,利用该细胞模板获得了短期的内皮/平滑肌细胞联合培养体系。
The recovery of intimal function and integrity on biomaterials surface is one of the best ways to prohibit the thrombosis and embolism complications of cardiovascular implanted devices and improve long-term lumen patency. In this thesis, Microtransfer Molding was used to fabricate the hyaluronic acid (HA) micropattern (Cells template) on the activated titanium (TiOH) surface, which was based on the design concept of biomimetic surface engineering. The HA micropatterns were characterized by scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), contact angle measur-ement, surface profiler and energy dispersive X-ray spectroscopy (EDX). Furthermore, the stability and anti-adhesion function of HA were investigated. The zymohy- drolysis of hyaluronidase, collagen and fibronectin (FN) self-assembly were used to switch the anti-adhesion function of HA which was evaluated enzyme-linked immunosorbent assay (Elisa) and cell adhesion experiments. The adhesion behaviors of platelet, endothelial cells and smooth muscle cells on the cells template were studied, respectively, and the co-culture of endothelial cells /platelet and endothelial cells/ smooth muscle cells on the cells template were also investigated.
The results of SEM FTIR and contact angle measurement showed the changes of morphology, increases of roughness and hydrophilicity and detection of ophilic groups on the Ti surface after treatement of NaOH solution. Surface profiler and SEM results showed that polydimethyl-siloxane (PDMS) stamps with high fidelity were prepared. SEM and EDX results demonstrated that HA micropattems with HA and TiOH alternating stripes had been prepared accurately on the surface of TiOH. However, thickness of HA stripe was not uniform. XPS results demonstrated that HA micropattems were still distinct and HA adsorbed stably on the surface of TiOH after washing by phosphate buffer (PBS).
The results of cells adhesion showed that the anti-adhesion function of HA can be effectively switched by enzymatic action, collagen and FN self-assembly. The three treatments displayed weak effect on the smooth muscle cells (SMCs) proliferation, which was proven by the results of CCK-8. The self-assembly of collagen used to the change the anti-adhesion function of HA could obviously enhance the adhesion of ECs compared with other two switch methods
The results of single cell culture showed that the adhesion of platelet, endothelial cells (ECs) and SMCs were restricted by HA micropatterns. Furthermore, when the size of HA/TiOH alternating stripe was 20μm/30μm, which was comparable to the size of ECs, ECs and SMCs were elongated along the stripes. In addition, the shape index of ECs cultured on patterned surface was more similar to that of ECs grown in vivo compared with ECs on flat surfaces. The co-culture of two kinds of cells, such as ECs/platelet and ECs/ SMCs, showed that co-culture system with ordered cell distribution could be obtained on the surface of HA/TiOH alternating stripes. The pattern of platelets (which adhered on TiOH alternating stripes) did not affect the morphology of endothelial cells. However, the long axis of ECs could be regulated through SMCs alignment. But SMCs could stride over the ECs and would form staggered multi-layers structure during a long time co-colture period.
Our results indicates the anti-adhesion function of HA will be effectively changed by enzymatic action, Collagen and FN self-assembly. The cell templates can be used to regulate cell adhesion and spreading on the patterns, and construct a short-term co-culture system of ECs/SMCs.
引文
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