Original Article Hydrogenated Amorphous Carbon Nanopatterned Film Designs Drive Human Bone Marrow Mesenchymal Stem Cell Cytoskeleton Architecture Sabata Martino, Ph.D., 1 Francesco D’Angelo, Ph.D., 1 Ilaria Armentano, Ph.D., 2 Roberto Tiribuzi, Ph.D., 1 Manuela Pennacchi, Ph.D., 1,2 Mariaserena Dottori, Ph.D., 2,3 Samantha Mattioli, Ph.D., 1,2 Auro Caraffa, M.D., 4 Giuliano Giorgio Cerulli, M.D., 4 Jose ` Maria Kenny, Ph.D., 2 and Aldo Orlacchio, Ph.D. 1 The interaction between stem cells and biomaterials with nanoscale topography represents a main route in the roadmap for tissue engineering–based strategies. In this study, we explored the interface between human bone marrow–derived mesenchymal stem cells (hBM-MSCs) and hydrogenated amorphous carbon (a-C:H) film designed with uniform, groove, or grid nanopatterns. In either case, hBM-MSCs preserved growth rate and multi-differentiation properties, suggesting that the films were biocompatible and suitable for stem cell culture. hBM-MSCs responded to different nanopattern designs with specific changes of microtubule organization. In particular, the grid pattern induced a square-localized distribution of a-tubulin=actin fibers, whereas the groove pattern exerted a more dynamic effect, associated with microtubule alignment and elongation. Introduction A n important step in regenerative medicine consists of the appropriate interaction between stem cells and biomaterials to recreate the original tissue architecture. A biocompatible material is needed to provide structural support for stem cells that promotes cell adhesion and pro- liferation as well as secretion of extracellular molecules for stimulation of new tissue formation. Thus, the design criteria of materials have become one of the most discussed topics in biomaterial research. The effects of micro-topography and, more recently, the effect of nano-topography on cell biology represent a particularly critical issue. 1–7 Amorphous carbon is a useful biomaterial for tissue engi- neering because of its chemical–physical properties (e.g., chemically inert, wear- and corrosion-resistance, bio- and hemo-compatibility, high electrical resistivity, infrared trans- parency, low surface roughness, and high refractive index). 8 Recently, hydrogenated amorphous carbon (a-C:H) coatings have been used as a protective layer for artificial implants that have contact with blood (e.g., mechanical heart valves, arterial grafts and stents) and orthopedic implants. 9–11 Selection of a specific cell type able to actively respond to the biomaterial is another critical issue for regenerative med- icine. In this regard, human bone marrow–derived mesen- chymal stem cells (hBM-MSCs), because of their easy access from bone marrow, extensive expansion capacity in vitro, and capability to differentiate into multiple cell lineages, are a potential source for developing in vivo and in vitro applica- tions in regenerative medicine. 12–14 In this article, we investigate how biomaterial surface design could be a critical factor for controlling interactions between cells and materials and their orientations. To this aim, we investigated the behavior of hBM-MSCs cultured on a-C:H films produced using radiofrequency plasma-enhanced chemical vapor deposition in three differ- ent configurations: uniform, grooved, and grid patterned. Our work demonstrates that uniform and nanopatterned a-C:H are biocompatible materials for hBM-MSC culture and that a-C:H nanogrooves induce human stem cell alignment and elongation. Our results could be useful in optimizing biomedical de- vices in which cell adhesion and orientation are required properties. Materials and Methods Film preparation and characterization Uniform and patterned a-C:H films were prepared ac- cording to the radiofrequency plasma-enhanced chemical 1 Department of Experimental Medicine and Biochemical Science, Section of Biochemistry and Molecular Biology, University of Perugia, Perugia, Italy. 2 Materials Engineering Center, Resarch Unit, Interuniversity Consortium on Materials Science and Technology, Nanocompositi e Ibridi Polimerici Multifunzionali, University of Perugia, Terni, Italy. 3 National Institute of Biostructures and Biosystems, Materials Engineering Center, University of Perugia, Perugia, Italy. 4 Department of Orthopedics and Traumatology, University of Perugia, Perugia, Italy. TISSUE ENGINEERING: Part A Volume 15, Number 10, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089=ten.tea.2008.0552 3139