Bioactive Chemical Nanopatterns Impact Human Mesenchymal Stem Cell Fate Zhe A. Cheng, †,‡ Omar F. Zouani,* ,† Karine Glinel, ‡ Alain M. Jonas, ‡ and Marie-Christine Durrieu* ,† † Institut Europe ́ en de la Chimie et Biologie, CBMN-UMR5248, Universite ́ de Bordeaux 1, 2 Rue Robert Escarpit, 33607 Pessac, France ‡ Institut de la Matie ̀ re Condense ́ e et des Nanosciences, Universite ́ Catholique de Louvain, 1 Croix du Sud, B-1348 Louvain-la-Neuve, Belgium * S Supporting Information ABSTRACT: We present a method of preparing and characterizing nanostructured bioactive motifs using a combination of nanoimprint lithography and surface function- alization. Nanodots were fabricated on silicon surfaces and modified with a cell-adhesive RGD peptide for studies in human mesenchymal stem cell adhesion and differentiation. We report that bioactive nanostructures induce mature focal adhesions on human mesenchymal stem cells with an impact on their behavior and dynamics specifically in terms of cell spreading, cell-material contact, and cell differentiation. KEYWORDS: Nanoimprint lithography, surface functionalization, mesenchymal stem cell, focal adhesion, differentiation T he optimization of biomaterial surface interactions with biological components is important for sustaining an artificial environment capable of directing and maintaining favorable cell and tissue growth. Essentially, biomaterial surfaces should be able to function compatibly in physiological conditions by imitating the role of the in vivo extracellular matrix (ECM) with which cells and tissues naturally come in contact. In turn, a correct type of cells, an appropriate scaffold designed at different spatial scales, and a smart choice of signaling biomolecules must all be incorporated in the design of a biomaterial. 1 As in vivo cellular interactions occur on the nanoscale, surface nanopatterning has gained interest and attention as a unique means of mimicking micro and nanoenvironments in which cells and tissues thrive. 2 For instance, nanoscale topographies have a noticeable effect on the behavior of stem cells, and reports have shown the effects of nanofeature dimensions on stem cell fate. 3-8 Though numerous studies have been published on the effects of physical surface nanotopography on stem cell behavior, few are concerned with the nanoorganization of signaling molecules. We demonstrate here the impact of biochemical nano- structuring on the behavior of human mesenchymal stem cells (hMSCs), in particular the formation of mature focal adhesions (FAs) and changes in cell commitment. hMSCs are a type of adult stem cells derived from bone marrow that show promise in the regeneration of damaged tissues due to their multipotent capacities, being able to differentiate into osteoblasts, adipocytes, and chondrocytes, among other mature cell types. 9,10 The differentiation of hMSCs can be modulated by chemical and physical forces present in their ECM environment. 1,11-20 One important goal in stem cell research is thus to understand the mechanisms that direct cell differentiation into a specific cell lineage within a given nanoscale environment. A typical example of a nanoscale signaling inducing a regulation of cell behavior is provided by focal adhesions (FAs). FAs lie at the convergence of integrin clustering, signal transduction, and actin cytoskeleton organ- ization. 21,22 Cells modify FAs in response to changes in the molecular composition and physical forces present in their ECM environment. 23-31 The exact composition of a given FA will in turn regulate intracellular tension, effecting cellular behaviors such as adhesion, migration, proliferation, and differentiation. 32-36 In this study, we combined nanoimprint lithography with surface modification techniques to prepare material surfaces that are chemically patterned with nanosized bioactive features, over a total area of about 1 cm 2 . These platforms allow cellular assays to be carried out for the investigation of hMSC-material nanointeraction. Various nanofabrication techniques such as e- beam lithography, colloidal lithography, and nanoimprint lithography (NIL) were explored to produce nanopatterned surfaces. Among them, NIL offers a series of advantages related to its ease-of-processing, rapidity, and versatility. 37-45 As a template-based system, NIL operates by transferring a predefined pattern from a master mold to a material surface. Thus, surface features ranging from the microscale down to the nanoscale can be constructed with no limit on geometry. Motifs with varying shapes (circles, squares, lines), sizes, and Received: June 3, 2013 Revised: July 24, 2013 Published: July 31, 2013 Letter pubs.acs.org/NanoLett © 2013 American Chemical Society 3923 dx.doi.org/10.1021/nl4020149 | Nano Lett. 2013, 13, 3923-3929