Influence of nanopatterend titanium surfaces on the osteogenic differentiation of human mesenchymal stem cells in combination with chemical and biological surface modifications (NanoTune)

低纳米水平各向异性、有序的表面形貌对人骨髓间充质干细胞(HMSC)行为的影响到目前为止还没有得到充分的研究和了解。然而，垂直尺寸在10纳米以下，横向尺寸在10纳米以下的形貌显示出显著影响细胞附着和成骨承诺的高潜力，特别是结合量身定制的化学和生物化学表面修饰。因此，本研究旨在研究钛表面的这种纳米结构可以显著影响蛋白质的吸附以及hMSC的黏附、迁移和成骨分化的假说，这些影响既是它们自身的，也是通过化学和生物表面修饰而放大的。低能离子轰击将被用来产生横向周期为几十纳米、高度从1 nm到5 nm的有序波纹图案。随后，硅烷化将产生量身定制的表面化学，并将使用交联来特异性地偶联两种蛋白质，玻璃化连接蛋白和纤维连接蛋白，这两种蛋白质在细胞黏附、迁移和分化中起主要作用。使用QCM-D、AFM和PM-IRRAS以及免疫细胞化学分析等手段，进一步研究未经修饰和化学多样化的表面对蛋白质的非特异性吸附能力。随后将使用已建立的黏附、迁移和分化实验来表征种植的hMSC的表面诱导的细胞反应。然后，系统地改变地形、化学和生化参数，以揭示单个表面修饰对细胞的各自影响。为此，将对hMSC的形态、细胞组成和重组、增殖和分化行为进行广泛的体外分析。因此，在这项研究中收集的关于干细胞对低纳米级底物形貌的响应的数据将有助于改进未来的设计和例如矫形和牙科植入物的可用性。

The influence of anisotropic, ordered surface topographies at the low nanometer level on the behavior of human mesenchymal stem cells (hMSC) is thus far insufficiently investigated and poorly understood. Yet topographies with vertical dimensions in the sub-10 nm range and lateral dimensions of the order few 10 nm exhibit high potential to significantly influence cellular attachment and osteogenic commitment, especially in combination with tailored chemical and biochemical surface modifications. This study therefore aims at investigating the hypothesis that such nanostructures on titanium substrates can significantly influence protein adsorption as well as hMSC adhesion, migration, and osteogenic differentiation, both by themselves and amplified with chemical and biological surface modifications. Low-energy ion bombardment will be used to generate ordered wave-like ripple patterns with lateral periodicities of few ten nanometers and heights in the range from < 1 nm to 5 nm. Subsequently, tailored surface chemistry will be generated by silanization, and crosslinking will be used to specifically couple two proteins, vitronectin and fibronectin, which play major roles in cell adhesion, migration, and differentiation. The unmodified as well as the chemically diversified surfaces will furthermore be investigated for their ability to unspecifically adsorb proteins, using QCM-D, AFM, and PM-IRRAS among others, as well as immunocytochemistry assays. Established adhesion, migration and differentiation assays will subsequently be used to characterize the surface induced cellular responses of the seeded hMSC. The topographical, chemical, and biochemical parameters will then be systematically varied to reveal the respective influences of the individual surface modifications on the cells. For this, extensive in-vitro analyses of the morphology, cellular composition and reorganization, proliferation, and differentiation behavior of the hMSC will be conducted. The data gathered within this study, regarding stem cell response to substrate topographies at the low-nanometer level, will thus be helpful to improve future design and availability of e.g. orthopaedic and dental implants.