In order to gain insight into how interfacial effects influence cell responses, chemically modified anodized TiO2 nanotubes (ATNs) were used to simultaneously investigate the effects of nanoscale substrate structure and angstrom-scale chemicals on cell morphological change and cell growth. Two small chemicals were used to modify the ATNs, namely, 3-aminopropyltrimethoxysilane (APTMS) and 3-mercaptopropyltrimethoxysilane (MPTMS), resulting in APTMS-modified ATNs (APTMS-ATNs) and MPTMS-modified ATNs (MPTMS-ATNs), respectively. In our in vitro observation of NIH/3T3 fibroblasts, cells thrived on both unmodified and modified ATNs. Quantitative analyses of cell numbers exhibited that APTMS-ATNs effectively facilitated cell proliferation and directed cell orientation owing to full cell-substrate contact caused by positively charged amino groups (-NH3(+)) on the surface. In addition, scanning electron microscopy and fluorescence images showed different cell morphologies on APTMS-ATNs and MPTMS-ATNs. APTMS-ATNs resulted in flat spreading of fibroblasts, while MPTMS-ATNs resulted in fibroblasts with a three-dimensional solid shape and clear contours. The results indicate that the synergistic effects of nanotube surface topology and small chemical modification and, to a lesser extent, surface hydrophilicity, alter the interfacial interactions between cells and substrates, significantly affecting cell morphology, attachment, and growth. Using ATNs with different interfacial effects from various small chemicals, orientation of cells into various patterns can be achieved and investigation of cell fates, such as proliferation or stem cell differentiation, can be performed for future advanced medical or biological applications
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