Cost and Time Effective Nanolithography of Reusable Millimeter Size Bone Tissue Replicas for Induced MSCs Differentiation

Cells sense the physical environment at both the nano- and micro-scale. Specifically, cells interact with the surrounding via formation of focal adhesions, with integrins acting as a link between the ECM and the cytoskeleton. Several studies have shown that substrate topography, stiffness, and ligand type and density control these interactions, which determine reorganization of the cytoskeleton, and changes in cell and nuclear morphology, ultimately affecting chromosomal structure, gene expression, and cell behavior[1,2]. For these reasons, the ability to replicate the microenvironment of biological tissues creates unique biomedical possibilities for stem cell applications. Current fabrication methods are limited by either the control on feature size and shape, or by the throughput and size of the replicas.<br/>Here we report a method to study the synergistic effect of the substrate topography, stiffness, and surface chemistry on the cell-surface interactions. This remains challenging due to the limitations of conventional nanofabrication methods in terms of costs, resolution, control of geometry and size in x-y-z (e.g. impossibility to fabricate complex 3D nano-structures), and choice of materials (e.g., materials with tunable chemistry and stiffness). We introduced a novel platform that combines thermal scanning probe lithography (tSPL)[2] with innovative methodologies for the low-cost and high-throughput nanofabrication of large area quasi-3D bone tissue replicas with high fidelity, sub-15 nm lateral precision, and sub-2 nm vertical resolution. This bio-tSPL platform [4] features a biocompatible polymer resist that withstands multiple cell culture cycles and can be patterned and chemically activated [5] allowing the reuse of the replicas, further decreasing costs and fabrication times. Moreover, we modulated the mechanical properties of this resist in order to study and improve cell-surface adhesion in acqueous solutions.<br/>The achieved level of time and cost-effectiveness, as well as the cell compatibility of the replicas, make bio-tSPL a promising platform to produce tissue-mimetic replicas to study stem cell-tissue microenvironment interactions, test drugs, and ultimately harness the regenerative capacity of stem cells and tissues for biomedical applications.<br/>[1] H. Donnelly, M. J. Dalby, M. Salmeron-Sanchez, P. E. Sweeten, <i>Nanomed-Nanotechnol</i> <b>2018</b>, <i>14</i>, 2455.<br/>[2] M. M. Stevens,J. H. George, <i>Science</i> <b>2005</b>, <i>310</i>, 1135<br/>[3] R. Szoszkiewicz, T. Okada, S. C. Jones, T. D. Li, W. P. King, S. R. Marder, E. Riedo, <i>Nano Lett.</i> <b>2007</b>, <i>7</i>, 1064<br/>[4] X. Liu, A. Zanut, M. Sladkova-Faure, L. Xie, M. Weck, X. Zheng, E. Riedo, and G. M. de Peppo. <i>Adv. Func. </i><i>Mat </i><b>2021, </b>31, 2008662<br/>[5] Liu, X. Y.<i> et al.</i> Sub-10 nm Resolution Patterning of Pockets for Enzyme Immobilization with Independent Density and Quasi-3D Topography Control. <i>Acs Appl Mater Inter</i> <b>11</b>, 41780-41790