Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films Lubica Grausova 1 , Alexander Kromka 2 , Zuzana Burdikova 1 , Adam Eckhardt 1 , Bohuslav Rezek 2 , Jiri Vacik 3 , Ken Haenen 4 , Vera Lisa 1 , Lucie Bacakova 1 * 1 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 2 Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 3 Nuclear Physics Institute, Academy of Sciences of the Czech Republic, Prague, Czech Republic, 4 Institute for Materials Research (IMO), Hasselt University & Division IMOMEC, IMEC vzw, Diepenbeek, Belgium Abstract Intrinsic nanocrystalline diamond (NCD) films have been proven to be promising substrates for the adhesion, growth and osteogenic differentiation of bone-derived cells. To understand the role of various degrees of doping (semiconducting to metallic-like), the NCD films were deposited on silicon substrates by a microwave plasma-enhanced CVD process and their boron doping was achieved by adding trimethylboron to the CH 4 :H 2 gas mixture, the B:C ratio was 133, 1000 and 6700 ppm. The room temperature electrical resistivity of the films decreased from .10 MV (undoped films) to 55 kV, 0.6 kV, and 0.3 kV (doped films with 133, 1000 and 6700 ppm of B, respectively). The increase in the number of human osteoblast-like MG 63 cells in 7-day-old cultures on NCD films was most apparent on the NCD films doped with 133 and 1000 ppm of B (153,000614,000 and 152,000610,000 cells/cm 2 , respectively, compared to 113,000610,000 cells/cm 2 on undoped NCD films). As measured by ELISA per mg of total protein, the cells on NCD with 133 and 1000 ppm of B also contained the highest concentrations of collagen I and alkaline phosphatase, respectively. On the NCD films with 6700 ppm of B, the cells contained the highest concentration of focal adhesion protein vinculin, and the highest amount of collagen I was adsorbed. The concentration of osteocalcin also increased with increasing level of B doping. The cell viability on all tested NCD films was almost 100%. Measurements of the concentration of ICAM-1, i.e. an immunoglobuline adhesion molecule binding inflammatory cells, suggested that the cells on the NCD films did not undergo significant immune activation. Thus, the potential of NCD films for bone tissue regeneration can be further enhanced and tailored by B doping and that B doping up to metallic-like levels is not detrimental for cells. Citation: Grausova L, Kromka A, Burdikova Z, Eckhardt A, Rezek B, et al. (2011) Enhanced Growth and Osteogenic Differentiation of Human Osteoblast-Like Cells on Boron-Doped Nanocrystalline Diamond Thin Films. PLoS ONE 6(6): e20943. doi:10.1371/journal.pone.0020943 Editor: Meni Wanunu, University of Pennsylvania, United States of America Received November 30, 2010; Accepted May 16, 2011; Published June 10, 2011 Copyright: ß 2011 Grausova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was financially supported by the Academy of Sciences of the Czech Republic (grants No. KAN400480701, KAN400100701 and IAAX00100902) and by the Grant Agency of the Czech Republic (P108/11/0794). In Belgium, the work was supported by Research Programs G.0068.07, G.0430.07 and G.0555.10N of the Research Foundation - Flanders (FWO), the Methusalem ‘‘NANO network’’, and by IAP-P6/42 project ‘Quantum Effects in Clusters and Nanowires’. The funders had no role in the study design, data collection and analysis, the decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lucy@biomed.cas.cz Introduction Nanostructured materials, i.e. materials characterized by dimensions of less than 100 nanometres, are promising for a wide range of advanced technologies, including nanomedicine. In this novel interdisciplinary scientific field, nanostructured materials are developed or applied as carriers for targeted drug and gene delivery, as tracers for bioimaging, tools for nanoscale surgery, components of nanoelectronic biosensors, and also as cell carriers for tissue engineering, i.e. for constructing bioartificial replace- ments for irreversibly damaged tissues and organs. Artificial materials currently used for constructing body implants, characterized by microscale topography of the cell- material interface, often do not evoke proper cellular responses needed for integrating them with the surrounding tissue and for tissue regeneration. The cells typically studied on these materials, i.e. anchorage-dependent mammalian cells of various tissues and organs, including the bone, adhere with spreading across usually tens of mm (for a review, see [1]). Thus, irregularities on the same scale can hamper appropriate spreading of the cells. The cells have to bridge the irregularities or to spread only in a limited space in the grooves among the prominences, which reduces the cell- substratum contact area, proliferation activity, viability and functioning of the cells, e.g., the activity of alkaline phosphatase in osteoblasts, necessary for bone tissue mineralization [2–5]. On the other hand, nanostructured materials imitate the nanoscale architecture of natural tissue components, such as extracellular matrix (ECM) and cell membrane with cell adhesion receptors. In addition, the ECM molecules, such as vitronectin, fibronectin, collagen or laminin, which mediate the cell adhesion on artificial materials, and are adsorbed spontaneously to the material surfaces, are attached in advantageous geometrical conformations, which enable good contact between specific bioactive sites in the ECM molecules (e.g., oligopeptidic ligands for cell adhesion receptors) and cell adhesion receptors (e.g., integrins) [6–8]. Thus, nanostructured materials are usually PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20943