Sousa S R, Brás M Manuela, Moradas-Ferreira P, Barbosa M A
Laboratório de Biomateriais, Instituto de Engenharia Biomédica (INEB), Rua do Campo Alegre 823, 4150-180 Porto.
Langmuir. 2007 Jun 19;23(13):7046-54. doi: 10.1021/la062956e. Epub 2007 May 18.
In the present work we analyze the dynamics of fibronectin (FN) adsorption on two different stable titanium oxides, with varied surface roughness, and chemically similar to those used in clinical practice. The two types of titanium oxide surfaces used were TiO2 sputtered on Si (TiO2 sp) and TiO2 formed on commercially pure titanium after immersion in H2O2 (TiO2 cp). Surface characterization was previously carried out using different techniques (Sousa, S. R.; Moradas-Ferreira, P.; Melo, L. V.; Saramago, B.; Barbosa, M. A. Langmuir 2004, 20 (22), 9745-9754). Imaging and roughness analysis before and after FN adsorption used atomic force microscopy (AFM) in tapping mode, in air, and in magnetic alternating current mode, in liquid (water). FN adsorption as a function of time was followed by X-ray photoelectron spectroscopy (XPS), by radiolabeling of FN with 125I (125I-FN), and by ellipsometry. Exchangeability studies were performed using FN and HSA. AFM roughness analysis revealed that, before FN adsorption, both TiO2 surfaces exhibited a lower root-mean-square (Rq) and maximum peak with the depth of the maximum valley (Rmax) roughness in air than in water, due to TiO2 hydration. After protein adsorption, the same behavior was observed for the TiO2 sp substrate, while Rq and Rmax roughness values in air and in water were similar in the case of the TiO2 cp substrate, for the higher FN concentration used. Surface roughness was always significantly higher on the TiO2 cp surfaces. AFM led to direct visualization of adsorbed FN on both surfaces tested, indicating that after 10 min of FN incubation the TiO2 sp surface was partially covered by FN. The adsorbed protein seems to form globular aggregates or ellipsoids, and FN aggregates coalesce, forming clusters as the time of adsorption and the concentration increase. Radiolabeling of FN revealed that a rapid adsorption occurs on both surfaces and the amount adsorbed increased with time, reaching a maximum after 60 min of incubation. Time dependence is also observed for the evolution of the atomic (%) of N determined by XPS and by the increase of the thickness by ellipsometry. TiO2 cp adsorbs more FN than the TiO2 sp surfaces, after 60 min of adsorption, as shown by the radiolabeling data. FN molecules are also more strongly attached to the former surface as indicated by the exchangeability studies. The overall results provide novel evidence that FN spontaneously adsorbs as a self-assembly at TiO2 surfaces as a function of time. The aggregate structure is an intermediate feature shared by some protein fibrillar assemblies at interfaces, which is believed to promote cell adhesion and cytoskeleton organization (Pellenc, D.; Berry, H.; Gallet, O. J. Colloid Interface Sci. 2006, 298 (1), 132-144. Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113 (10), 1677-1686).
在本研究中，我们分析了纤连蛋白（FN）在两种不同的稳定二氧化钛上的吸附动力学，这两种二氧化钛具有不同的表面粗糙度，且在化学性质上与临床实践中使用的材料相似。所使用的两种二氧化钛表面分别是溅射在硅上的TiO₂（TiO₂ sp）和商业纯钛在H₂O₂中浸泡后形成的TiO₂（TiO₂ cp）。先前已使用不同技术对表面进行了表征（Sousa, S. R.; Moradas-Ferreira, P.; Melo, L. V.; Saramago, B.; Barbosa, M. A. Langmuir 2004, 20 (22), 9745 - 9754）。在FN吸附前后的成像和粗糙度分析，在空气中采用轻敲模式的原子力显微镜（AFM），在液体（水）中采用磁交流电模式。通过X射线光电子能谱（XPS）、用¹²⁵I对FN进行放射性标记（¹²⁵I - FN）以及椭偏仪跟踪FN吸附随时间的变化。使用FN和人血清白蛋白（HSA）进行可交换性研究。AFM粗糙度分析表明，在FN吸附之前，由于TiO₂水合作用，两种TiO₂表面在空气中的均方根（Rq）粗糙度和最大峰值与最大谷深（Rmax）粗糙度均低于在水中的情况。蛋白质吸附后，TiO₂ sp基底观察到相同的行为，而对于较高的FN浓度，在TiO₂ cp基底的情况下，空气中和水中的Rq和Rmax粗糙度值相似。TiO₂ cp表面的粗糙度始终显著更高。AFM直接观察到了在两种测试表面上吸附的FN，表明在FN孵育10分钟后，TiO₂ sp表面部分被FN覆盖。吸附的蛋白质似乎形成球状聚集体或椭球体，并且随着吸附时间和浓度的增加，FN聚集体合并形成簇。FN的放射性标记显示，在两种表面上都发生了快速吸附，吸附量随时间增加，孵育60分钟后达到最大值。通过XPS测定的N原子（%）的演变以及椭偏仪测量的厚度增加也观察到了时间依赖性。放射性标记数据表明，吸附60分钟后，TiO₂ cp比TiO₂ sp表面吸附更多的FN。可交换性研究表明，FN分子也更牢固地附着在前一种表面上。总体结果提供了新的证据，表明FN作为一种自组装体在TiO₂表面随时间自发吸附。聚集体结构是界面处一些蛋白质纤维状组装体共有的中间特征，据信这有助于细胞黏附和细胞骨架组织（Pellenc, D.; Berry, H.; Gallet, O. J. Colloid Interface Sci. 2006, 298 (1), 132 - 144. Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. J. Cell Sci. 2000, 113 (10), 1677 - 1686）。
