Marín-Pareja Nathalia, Cantini Marco, González-García Cristina, Salvagni Emiliano, Salmerón-Sánchez Manuel, Ginebra Maria-Pau
Biomaterials, Biomechanics, and Tissue Engineering Group, Department of Materials Science and Metallurgy, Universitat Politècnica de Catalunya. BarcelonaTech (UPC) , Av. Diagonal 647, 08028 Barcelona, Spain.
Division of Biomedical Engineering, School of Engineering, University of Glasgow , Glasgow G12 8LT, U.K.
ACS Appl Mater Interfaces. 2015 Sep 23;7(37):20667-77. doi: 10.1021/acsami.5b05420. Epub 2015 Sep 9.
Silanization has emerged in recent years as a way to obtain a stronger and more stable attachment of biomolecules to metallic substrates. However, its impact on protein conformation, a key aspect that influences cell response, has hardly been studied. In this work, we analyzed by atomic force microscopy (AFM) the distribution and conformation of type I collagen on plasma-treated surfaces before and after silanization. Subsequently, we investigated the effect of the different collagen conformations on fibroblasts adhesion and fibronectin secretion by immunofluorescence analyses. Two different organosilanes were used on plasma-treated titanium surfaces, either 3-chloropropyl-triethoxy-silane (CPTES) or 3-glycidyloxypropyl-triethoxy-silane (GPTES). The properties and amount of the adsorbed collagen were assessed by contact angle, X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and AFM. AFM studies revealed different conformations of type I collagen depending on the silane employed. Collagen was organized in fibrillar networks over very hydrophilic (plasma treated titanium) or hydrophobic (silanized with CPTES) surfaces, the latter forming little globules with a beads-on-a-string appearance, whereas over surfaces presenting an intermediate hydrophobic character (silanized with GPTES), collagen was organized into clusters with a size increasing at higher protein concentration in solution. Cell response was strongly affected by collagen conformation, especially at low collagen density. The samples exhibiting collagen organized in globular clusters (GPTES-functionalized samples) favored a faster and better fibroblast adhesion as well as better cell spreading, focal adhesions formation, and more pronounced fibronectin fibrillogenesis. In contrast, when a certain protein concentration was reached at the material surface, the effect of collagen conformation was masked, and similar fibroblast response was observed in all samples.
近年来，硅烷化已成为一种使生物分子更牢固、更稳定地附着于金属基底的方法。然而，其对蛋白质构象（影响细胞反应的一个关键方面）的影响却鲜有研究。在本研究中，我们通过原子力显微镜（AFM）分析了硅烷化前后等离子体处理表面上I型胶原蛋白的分布和构象。随后，我们通过免疫荧光分析研究了不同胶原蛋白构象对成纤维细胞黏附及纤连蛋白分泌的影响。在等离子体处理的钛表面使用了两种不同的有机硅烷，即3-氯丙基三乙氧基硅烷（CPTES）或3-缩水甘油氧基丙基三乙氧基硅烷（GPTES）。通过接触角、X射线光电子能谱、光波导光模式光谱和AFM评估吸附胶原蛋白的性质和数量。AFM研究表明，I型胶原蛋白的构象因所用硅烷而异。在非常亲水的（等离子体处理的钛）或疏水的（用CPTES硅烷化）表面上，胶原蛋白形成纤维状网络，后者形成具有串珠状外观的小球，而在具有中等疏水特性的（用GPTES硅烷化）表面上，胶原蛋白聚集成簇，且在溶液中蛋白质浓度较高时尺寸增大。细胞反应受胶原蛋白构象的强烈影响。特别是在胶原蛋白密度较低时，表现出胶原蛋白聚集成球状簇的样品（GPTES功能化样品）有利于更快、更好地促进成纤维细胞黏附，以及更好地促进细胞铺展、形成黏着斑和更明显的纤连蛋白纤维形成。相比之下，当材料表面达到一定蛋白质浓度时，胶原蛋白构象的影响被掩盖，在所有样品中观察到相似的成纤维细胞反应。
