表面梯度化材料对内皮细胞黏附与迁移影响的研究
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摘要
为探讨表面梯度化材料对内皮细胞的黏附与迁移行为的影响,我们通过不同方式体外构建材料表面I型胶原蛋白梯度(蛋白密度梯度和图案化不同曲率和弧度梯度的蛋白基底),来研究内皮细胞在梯度表面的黏附状态和运动趋化性。实验一通过连续碱水解聚乳酸膜制备形成羧基密度,并共价偶联胶原蛋白形成相应的蛋白密度梯度。接触角测量仪和激光扫描共聚焦显微镜分别表征了- COOH密度梯度和胶原蛋白密度梯度,通过荧光显微镜可观测到明显的胶原蛋白梯度,以上结果证实了聚乳酸薄膜上通过连续碱水解方法制备胶原蛋白梯度的可行性。在低密度和中间密度的胶原蛋白梯度区域培养的内皮细胞表现出较强的沿着梯度方向的运动性(净迁移,趋化指数,迁移率和细胞轨迹证实),然而,内皮细胞在高浓度蛋白密度区域的运动与胶原蛋白梯度反向。结果表明,细胞运动受胶原蛋白梯度趋向,但必须是适宜的利于细胞黏附的蛋白密度。实验二基于仿生学原理,根据人体内不同种类和不同口径(从最细小的毛细血管到大动脉)的血管厚度和弯曲度不同设计出一系列弧度梯度(20μm,60μm,100μm弧度宽度),并运用微接触印刷技术制备出不同曲率半径的胶原蛋白基底,扫描电子显微镜和光学显微镜检验PDMS印章表面平整度,激光共聚焦显微镜检验基底蛋白黏附情况和均匀度,细胞骨架蛋白肌动蛋白和粘着斑蛋白染色观测细胞粘附状态,活细胞工作站对细胞6h运动情况进行录像,运用相关软件测定20,60,100μm弧度宽度梯度之间或自身不同曲率半径之间的细胞迁移速度和细胞迁移总位移与净位移比值。结果表明细胞的黏附状态与弧度宽度成正比;设计的曲率半径越小,迁移速度越快,运动性越强;在不同弧度宽度的材料表面细胞运动能力如下:100μm>20μm>60μm。本研究分析了内皮细胞在不同形式的胶原蛋白梯度上的一系列运动迁移参数,获得了内皮细胞黏附、运动迁移的最适宜的蛋白梯度基底,为进一步研究内皮细胞化学诱导运动性提供依据,为植入体在植入宿主后的微循环血管新生中的脉管重塑和血管再生提供新的思路,为组织工程血管支架材料的内皮化提供理论依据。
To investigate the effect of surface gradient biomaterials on endothelial cells adhesion and motility, we developed two novel approachs for the fabrication of type I collagen gradient (collagen density gradient and the gradient of micro-radian width with various curvatures) onto substrate in this study. The first experiment involved sequential alkali hydrolysis of PDLLA films to produce–COOH density gradient along substrates, followed by covalently immobilizing collagen onto hydrolyzed PDLLA films. This treatment resulted in a surface-density gradient of collagen onto PDLLA surfaces. Contact angle measurement and confocal laser scanning microscopy were employed to characterize the–COOH gradient and collagen gradient, respectively. The collagen gradient onto PDLLA films was clearly visible by fluorescence microscopy observation. All results confirmed the feasibility of the fabrication of collagen gradient onto PDLLA films via alkali hydrolysis approach. Endothelial cells cultured on the low surface-density and moderate surface-density of collagen gradient areas displayed a strong motility (net displacement, chemotactic index, and migration rate, cell trajectories) tendency in parallel to the gradient. However, endothelial cells grown on the high surface-density of collagen gradient areas demonstrated a reverse response of motility to collagen gradient clues. The result suggests that cell motility is regulated by collagen gradient, however, with appropriate surface-density. The second experiment involves the micropatterning technology. Based on the principle of bionics, we designed some new patterns (the width of the micro-radian pattern is 20μm, 60μm, 100μm respectively) which similar to different blood vessels (from capillary to artery). From the patterns we can obtain a collagen substrate microenvironment with various curvatures. Scanning electron microscopy was employed to characterize the quality of PMDS stamp. Confocal laser scanning microscopy (CLSM) was utilized to characterize the absorption of FITC conjugated collagen solution. The staining of F-action and vinculin showed cells’adhesion on the substrates surfaces. Live Cell Imaging System was used to take cell’s migration image recording of 6h. Software was used to analyze migration rate and cell’s migration distance / net displacement between 20, 60 and 100μm on the protein substrates. The result suggests that Cell adhesion status is proportional to the width of the curvature, the smaller radius of curvatures,the faster of the migration rate. Meanwhile, it can make the cell motility strengthen. Cell movement ability on different width of the micropattern is: 100μm>20μm >60μm.
These studies analyzed a series of migration parameters about endothelial cell’s migration on various collagen protein gradient substrates, and obtained the optimal substrate for cell’s movement and adhesion. This study provides proper substrates for investigating chemical stimuli that induced cell directional motility. It is potentially important for controlled angiogenesis for implantation of tissue-engineered devices and will provide new ideas to vascular remodeling and angiogenesis.
To investigate the effect of surface gradient biomaterials on endothelial cells adhesion and motility, we developed two novel approachs for the fabrication of type I collagen gradient (collagen density gradient and the gradient of micro-radian width with various curvatures) onto substrate in this study. The first experiment involved sequential alkali hydrolysis of PDLLA films to produce–COOH density gradient along substrates, followed by covalently immobilizing collagen onto hydrolyzed PDLLA films. This treatment resulted in a surface-density gradient of collagen onto PDLLA surfaces. Contact angle measurement and confocal laser scanning microscopy were employed to characterize the–COOH gradient and collagen gradient, respectively. The collagen gradient onto PDLLA films was clearly visible by fluorescence microscopy observation. All results confirmed the feasibility of the fabrication of collagen gradient onto PDLLA films via alkali hydrolysis approach. Endothelial cells cultured on the low surface-density and moderate surface-density of collagen gradient areas displayed a strong motility (net displacement, chemotactic index, and migration rate, cell trajectories) tendency in parallel to the gradient. However, endothelial cells grown on the high surface-density of collagen gradient areas demonstrated a reverse response of motility to collagen gradient clues. The result suggests that cell motility is regulated by collagen gradient, however, with appropriate surface-density. The second experiment involves the micropatterning technology. Based on the principle of bionics, we designed some new patterns (the width of the micro-radian pattern is 20μm, 60μm, 100μm respectively) which similar to different blood vessels (from capillary to artery). From the patterns we can obtain a collagen substrate microenvironment with various curvatures. Scanning electron microscopy was employed to characterize the quality of PMDS stamp. Confocal laser scanning microscopy (CLSM) was utilized to characterize the absorption of FITC conjugated collagen solution. The staining of F-action and vinculin showed cells’adhesion on the substrates surfaces. Live Cell Imaging System was used to take cell’s migration image recording of 6h. Software was used to analyze migration rate and cell’s migration distance / net displacement between 20, 60 and 100μm on the protein substrates. The result suggests that Cell adhesion status is proportional to the width of the curvature, the smaller radius of curvatures,the faster of the migration rate. Meanwhile, it can make the cell motility strengthen. Cell movement ability on different width of the micropattern is: 100μm>20μm >60μm.
These studies analyzed a series of migration parameters about endothelial cell’s migration on various collagen protein gradient substrates, and obtained the optimal substrate for cell’s movement and adhesion. This study provides proper substrates for investigating chemical stimuli that induced cell directional motility. It is potentially important for controlled angiogenesis for implantation of tissue-engineered devices and will provide new ideas to vascular remodeling and angiogenesis.
引文
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