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Surfaces, Interfaces, and Applications
High-density ZnO Nanowires as a Reversible Myogenic-Differentiation-Switch Vito Errico, Giuseppe Arrabito, Ersilia Fornetti, Claudia Fuoco, Stefano Testa, Giovanni Saggio, Stefano Rufini, Stefano Maria Cannata, Alessandro Desideri, Christian Falconi, and Cesare Gargioli ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19758 • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018
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ACS Applied Materials & Interfaces
High-density ZnO Nanowires as a Reversible Myogenic-Differentiation-Switch Vito Errico,#‡ Giuseppe Arrabito,#‡† Ersilia Fornetti,§ Claudia Fuoco,§ Stefano Testa,§ Giovanni Saggio,# Stefano Rufini,§ Stefano Cannata,§ Alessandro Desideri,§* Christian Falconi,#* Cesare Gargioli§* # § †
Department of Electronic Engineering, University of Rome Tor Vergata, Via del Politecnico 1 00133, Rome, Italy Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy Present address: Department of Physics and Chemistry, University of Palermo, Viale delle Scienze, Parco d’Orleans
II, 90128 Palermo, Italy
KEYWORDS mesoangioblasts; muscle differentiation; ZnO nanowires; tissue engineering
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ABSTRACT. Mesoangioblasts are outstanding candidates for stem cell therapy and are already being explored in clinical trials. However, a crucial challenge in regenerative medicine is the limited availability of undifferentiated myogenic progenitor cells, since growth is typically accompanied by differentiation. Here reversible myogenic-differentiation-switching during proliferation is achieved by functionalizing the
glass substrate
with high-density ZnO
nanowires.
Specifically,
mesoangioblasts grown on ZnO nanowires present a spherical viable undifferentiated cell state without lamellopodia formation during all the observation time (8 days). Consistently, the Myosin Heavy Chain, typically expressed in skeletal muscle tissue and differentiated myogenic progenitors, is completely absent. Remarkably, nanowires do not induce any damage while reversibly block differentiation, so that the differentiation capabilities are completely recovered upon cells removal from the nanowires-functionalized substrate and re-plating on standard culture glass. This is the first evidence of a reversible myogenic-differentiation switch which does not affect viability. These results can be the first step toward for the in vitro growth of a large number of undifferentiated stem/progenitor cells and therefore can represent a breakthrough for cell based therapy and tissue engineering.
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INTRODUCTION The possibility to reversibly switch off the differentiation of stem cells is a major goal of regenerative medicine.1 Some cells within the tissues of adult organisms can act as progenitor cells, capable of differentiating with different fates both in vitro and in vivo.2 Among progenitor cells, mesoangioblasts (MABs), which are multipotent progenitor cells associated with blood vessels, can differentiate into several types of mesodermal cells 3,4 and myoblasts. For this reason, MABs are extremely interesting for the therapy of primitive muscle diseases. In fact, MABs have been successfully employed to reconstruct artificially injured muscles in vivo5 or to regenerate muscles damaged by muscular dystrophy6 or myocardial infarction7 and are currently being explored in clinical trials for the Duchenne Muscular Dystrophy.8 Skeletal muscle tissue engineering may enable the repair of muscular injuries but the limited availability of myogenic progenitor cells in terms of adequate number supply and proliferative activity in adult tissue9,10 is a crucial obstacle for real applications. Indeed, different stem/progenitor myogenic cell sources have been described, characterized and employed in clinical trials, including satellite cell derived myoblast,11,12 MABs8 and muscle-derived AC133+ cells.13
Nevertheless, obtaining a sufficient number of cells for
clinical experimentation is still a hotly debated topic representing one of the major limitation for the 8
successful employing of stem cells in clinic. In fact, recently it has been proposed to promote cell proliferation preserving differentiation capabilities by taking advantage of an artificial chromosome to reversibly immortalize human myogenic progenitor cells and to support cell proliferation in order to obtain an appropriate number of myogenic cells suitable for damaged/degenerated skeletal muscle tissue recovery.14 An ideal strategy to circumvent this problem would be to expand progenitor stem cells in a biocompatible matrix which can maintain the primary cells in a viable but not differentiated state without irreversibly compromising their differentiation and regenerative capabilities.15 Cellular differentiation is a complex process that dramatically changes cells size, shape, membrane potential, metabolic activity, and responsiveness to external signals. Cells adhere ACS Paragon Plus Environment
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to and spread on materials by assembling supramolecular protein complexes. However, nanoscale variations in the substrate topography can modify ligation and clustering resulting in adhesion changes. Indeed, adhesion has been shown to be crucial in modulating cellular growth and differentiation.16 Different biomaterials can be employed to facilitate myoblast growth and differentiation, such as protein-based scaffolds,17 namely polyesterurethane membranes,18 Poly Ethylenedioxy Thiophene (PEDOT)
19
or graphene.20,21 Among them, PEDOT is an outstanding
candidate for enhancing bioelectronics interfaces due to its excellent biocompatibility towards living cells and the ability to transmit and acquire electrical stimuli to the cells.22 In fact, PEDOT can enhance myocytes adhesion, proliferation and differentiation with respect to conventional metal electrode interfaces22 and it can guide epithelial cellular adhesion23 and membrane potential on glioblastoma or human dermal fibroblasts.24 PEDOT can be easily combined with various materials allowing the realization of new cellular interfaces. By combining it with ZnO nanowires and graphene, it is possible to realize flexible and low impedance electrical interfaces to neural tissues with improved signal-to-noise ratio.25 When combined with Graphene Oxide doped bacterial cellulose/Poly(3,4-ethylenedioxythiophene) nanofibers, it has been shown to mimic the structure of the native extracellular matrix (ECM) and to promote cells orientation and differentiation after electrical stimulation.26,27 Differently from PEDOT interfaces which can enhance cellular differentiation, other polymeric nanostructured surfaces have been employed as convenient tools to retain stem-cell phenotype maintaining steady stem-cell growth in a time scale of weeks. For instance, nanoscale substrates fabricated by electron beam lithography and processed into polycaprolactone polymer maintains functional Mesenchymal Stem Cells over eight weeks.28 Porous polymeric films realized by photo polymerization of hydroxyethyl methacrylate and ethylene dimethacrylate in presence of the porogens cyclohexanol and 1-decanol, promote longterm self-renewal (>3 weeks) of mouse embryonic stem cells (mESCs).29 In spite of the remarkable capability to control stem cell growth, the fabrication of polymer-based interfaces is dependent on lithographic steps and/or upon the accurate control of surface chemistry which make this approach ACS Paragon Plus Environment
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not immediately applicable to biomedicine labs. On the other hand, ZnO nanostructures can be synthesized at lower costs by wet chemical approaches30, and they have demonstrated good potentiality to gain control upon cell adhesion, survival and behavior.31,32 Moreover, ZnO quasi-1D nanostructures are especially promising because of their biocompatibility, as reported by the Food and Drug Administration (21CFR182.8991),33 piezoelectricity,34 ability to monitor the dynamic mechanical behavior of cellular traction on substrate,35 easiness to be synthesized at low temperature on cell-cultures substrates with tunable densities,36 electrochemical activity37 and easy surface functionalization.38 For these reasons, unlike ZnO nanoparticles, ZnO nanowires are already believed to be very promising for different biomedical applications.25,33,35,39–45 In fact, the biocompatibility or cytotoxicity of nanomaterials is both shape-dependent46 and cell-type dependent.47 Remarkably, ZnO nanoparticles are known to be toxic towards different kinds of cell lines.48 The mechanism of their toxicity is due to zinc release in solution due to ZnO nanoparticles dissolution process, which, in turn, is triggered especially by the downscaling of the particles to
