title: Improved in vitro angiogenic behavior on anodized titanium dioxide nanotubes.
abstract: BACKGROUND
  Neovascularization over dental implants is an imperative requisite to achieve successful osseointegration onto implanted materials. The aim of this study was to investigate the effects on in vitro angiogenesis of anodized 70 nm diameter TiO2nanotubes (NTs) on Ti6Al4V alloy synthesized and disinfected by means of a novel, facile, antibacterial and cost-effective method using super oxidized water (SOW). We also evaluated the role of the surface roughness and chemical composition of materials of materials on angiogenesis.
  
  
  METHODS
  The Ti6Al4V alloy and a commercially pure Ti were anodized using a solution constituted by SOW and fluoride as electrolyte. An acid-etched Ti6Al4V was evaluated to compare the effect of micro-surface roughness. Mirror-polished materials were used as control. Morphology, roughness, chemistry and wettability were assessed by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy, atomic force microscopy, energy dispersive X-ray spectroscopy (EDX) and using a professional digital camera. Bovine coronary artery endothelial cells (BCAECs) were seeded over the experimental surfaces for several incubation times. Cellular adhesion, proliferation and monolayer formation were evaluated by means of SEM. BCAEC viability, actin stress fibers and vinculin cellular organization, as well as the angiogenic receptors vascular endothelial growth factor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) were measured using fluorescence microscopy.
  
  
  RESULTS
  The anodization process significantly increased the roughness, wettability and thickness of the oxidized coating. EDX analysis demonstrated an increased oxygen (O) and decreased carbon (C) content on the NTs of both materials. Endothelial behavior was solidly supported and improved by the NTs (without significant differences between Ti and alloy), showing that endothelial viability, adhesion, proliferation, actin arrangement with vinculin expression and monolayer development were evidently stimulated on the nanostructured surface, also leading to increased activation of VEGFR2 and eNOS on Ti6Al4V-NTs compared to the control Ti6Al4V alloy. Although the rougher alloy promoted BCAECs viability and proliferation, filopodia formation was poor.
  
  
  CONCLUSIONS
  The in vitro results suggest that 70 nm diameter NTs manufactured by anodization and cleaned using SOW promotes in vitro endothelial activity, which may improve in vivo angiogenesis supporting a faster clinical osseointegration process.
authors.affiliation: Department of Biomaterials and Tissue Engineering, Faculty of Dentistry Mexicali, Autonomous University of Baja California (UABC), Ave. Zotoluca and Chinampas St., 21040, Mexicali, Baja California, Mexico.|Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California (UABC), Blvd. Benito Juarez and Normal St., 21280, Mexicali, Baja California, Mexico.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Beltrán-Partida
authors.affiliation: Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California (UABC), Blvd. Benito Juarez and Normal St., 21280, Mexicali, Baja California, Mexico. benval@uabc.edu.mx.
authors.forename: Benjamín
authors.initials: B
authors.lastname: Valdéz-Salas
authors.affiliation: Department of Biomedical Innovation, Center for Scientific Research and Higher Education of Ensenada (CICESE), Ensenada, Baja California, Mexico.
authors.forename: Aldo
authors.initials: A
authors.lastname: Moreno-Ulloa
authors.affiliation: Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California (UABC), Blvd. Benito Juarez and Normal St., 21280, Mexicali, Baja California, Mexico.
authors.forename: Alan
authors.initials: A
authors.lastname: Escamilla
authors.affiliation: Department of Corrosion and Materials, Engineering Institute, Autonomous University of Baja California (UABC), Blvd. Benito Juarez and Normal St., 21280, Mexicali, Baja California, Mexico.
authors.forename: Mario A
authors.initials: MA
authors.lastname: Curiel
authors.affiliation: Laboratory of Basic Sciences, Faculty of Stomatology, Autonomous University of San Luis Potosi (UASLP), San Luis Potosí, Mexico.
authors.forename: Raúl
authors.initials: R
authors.lastname: Rosales-Ibáñez
authors.affiliation: School of Medicine, University of California San Diego (UCSD), 9500 Gilman Dr, La Jolla, CA, 92093, USA.
authors.forename: Francisco
authors.initials: F
authors.lastname: Villarreal
authors.affiliation: National Centre for Metallurgical Research (CENIM), CSIC, Ave. Gregorio del Amo 8, 28040, Madrid, Spain.
authors.forename: David M
authors.initials: DM
authors.lastname: Bastidas
authors.affiliation: National Centre for Metallurgical Research (CENIM), CSIC, Ave. Gregorio del Amo 8, 28040, Madrid, Spain.
authors.forename: José M
authors.initials: JM
authors.lastname: Bastidas
mesh_terms: D000497:Alloys; D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D049109:Cell Proliferation; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D015921:Dental Implants; D042783:Endothelial Cells / Q000187:drug effects; D018625:Microscopy, Atomic Force; D008855:Microscopy, Electron, Scanning; D043942:Nanotubes / Q000737:chemistry*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010316:Particle Size; D013052:Spectrometry, X-Ray Emission; D013499:Surface Properties; D014025:Titanium / Q000737:chemistry*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D017155:Wettability
chemical_list: D000497:Alloys; D043925:Angiogenesis Inducing Agents; D015921:Dental Implants; C031462:titanium alloy (TiAl6V4); C009495:titanium dioxide; D014025:Titanium; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Anodization; Dental implants; Endothelialization; Nanotubes; Super oxidized water; Titanium