Effects of titanium-based nanotube films on osteoblast behavior in vitro Miruna-Silvia Stan,1 Indira Memet,1 Cornel Fratila,2 Elzbieta Krasicka-Cydzik,3 Ioan Roman,4 Anca Dinischiotu1 1

Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Bucharest, 91-95 Splaiul Independentei, Bucharest 050095, Romania 2 Research and Development National Institute for Nonferrous and Rare Metals, 102 Biruintei Blvd, Pantelimon 077145, Romania 3  ra 65-417, Poland Department of Biomedical Engineering, University of Zielona Gora, ul. Licealna 9, Zielona Go 4 METAV Research & Development, 31 C.A. Rosetti, Bucharest 020011, Romania Received 3 October 2013; revised 16 December 2013; accepted 19 February 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35148 Abstract: One of the major research interests of nanomedicine is the designing of harmless and biocompatible medical devices. To improve the features of Ti surface, TiO2 based nanotube (TNT) films (50 nm diameter) achieved by anodic oxidation and thermal treatment were grown on titanium and on Ti6Al4V and Ti6Al7Nb alloys. Their in vitro toxicity and biocompatibility were investigated using G292 osteoblast cell line. The LDH release after 24 and 48 h of exposure demonstrated that TNT layers were not cytotoxic. The cell growth on TNT films deposited on titanium and Ti6Al4V was significantly increased compared with Ti6Al7Nb. F-actin staining showed a better organized actin cytoskeleton in osteoblasts grown on these two samples, which provide the best conditions for osteoblast attachment and spreading. Analysis of

GSH distribution revealed a higher nuclear level in the samples with TNTs compared with Ti plate without nanotubes, indicating an active proliferation. Thus, nuclear glutathione levels can be used as a useful biomarker for biocompatibility assessment. Our results suggest that the substrate for TNTs can have a significant impact on cell morphology and fate. In conclusion, the TNT/Ti and TNT/Ti6Al4V were toxicity-free and can provide a proper nanostructure for a positive cell C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: response. V 00A:000–000, 2014.

Key Words: TiO2 based nanotube, osteoblast, cytotoxicity, biocompatibility, glutathione

How to cite this article: Stan M-S, Memet I, Fratila C, Krasicka-Cydzik E, Roman I, Dinischiotu A. 2014. Effects of titaniumbased nanotube films on osteoblast behavior in vitro. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

Titanium is a currently used biomaterial for oral implantology and orthopedics due to its outstanding physico-chemical properties and inertia in biological environments.1 Having an extremely low toxicity and being tolerated by bone and soft tissue, Ti-based implants are used in cardiovascular, orthopedic, and dental surgery, but also in reconstructive and plastic surgery.2–4 In the last years, the research has been directed on the addition of titanium surface functional groups able to induce and accelerate the deposition of bone (osteoblasts adhesion and nucleation of hydroxyapatite crystals). One method is the bioactivation of titanium surface by making nanostructured surfaces consisting of TiO2 nanotubes. Titanium dioxide can be prepared in the form of tubes with diameter in nanometers and lengths ranging from several nanometers to micrometers. This type of nanostructure can be achieved on titanium by its electrochemical–anodic oxidation in fluoride-containing electrolytes. The

growth of nanotubes occurs as a result of two simultaneous processes: the anodic oxidation of the surface followed by the local dissolution of the growing titanium dioxide by fluoride ions.5,6 The in vitro assessment of biomaterials’ toxicity is the first step in biocompatibility studies, and is usually performed using immortalized cell lines. It is not necessarily a qualitative analysis, including also tests such as MTT or neutral red which are essential quantitative methods and are widely used particularly in indirect assessment. Toxicity involves impaired cellular homeostasis, which further disturbs cellular functions and leads to a variety of biochemical changes. Cell adhesion is an important parameter in the evaluation of implant materials and to establish if they are suitable as medical devices.7 Typical characteristics of cellular alterations caused by toxic materials include cell membrane perforation, shrinking of the nucleus, cytoplasm fragmentation, formation of granulations, rounding up, and

Correspondence to: A. Dinischiotu; e-mail: [email protected] or [email protected] Contract grant sponsor: MNT-ERA.NET Transnational Call 2010, TNTBIOSENS project

C 2014 WILEY PERIODICALS, INC. V

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detachment of the cells from the support.8,9 Cell attachment to the material tested involves the production of extracellular matrix proteins and reorganization of cytoskeleton proteins to stabilize the cell-material interface. Morphological characteristics specific to cell adhesion involve the aspect of filopodia, or that of lamellipodia.10 Among the most common intracellular changes induced by the action of a foreign element are the variations of the major cellular antioxidant levels–glutathione (GSH). It is considered to be essential for the survival of mammalian cells, being involved in protection against oxidative damage and detoxification of reactive oxygen species (ROS), reactive nitrogen species and end products of lipid peroxidation.11,12 Also, nuclear GSH is involved in maintaining the redox homeostasis and thus, it plays a decisive role in cellular proliferation.13 The usage of titanium dioxide nanotube films in biomedical applications requires the investigation of the stability and toxicity of such materials. To assess the biocompatibility of nanotube films grown on titanium and Ti6Al4V and Ti6Al7Nb alloys, the degree of in vitro toxicity was evaluated, and the cell morphology, adhesion, and spreading on the surface of nanotubes were also established. Further, the measurement of the nuclear and cytoplasmic GSH levels was performed to assess the influence of TiO2 based nanotube (TNT) films on cell proliferation. In addition, the biological response generated following exposure to the nanotubes was compared with a simple titanium plate. MATERIALS AND METHODS

TiO2-based nanotube synthesis The metallic substrates of 10 3 10 mm surface and 0.5 mm thickness were Ti plates (Ti 5 base; Al 5 0.30; Cd 5 0.003; Cr 5 0.010; Cu 5 0.020; Fe 5 0.040; Mg 5 0.05, Mn 5 0.005; Mo 5 0.005; Ni 5 0.009, Pb 5 0.40; Sb 5 0.020; Si 5 0.05; Zn 5 0.005), Ti6Al4V alloy plates (Ti 5 base; N 5 0.0051; C 5 0.030; Al 5 5.53; V 5 3.90; Fe 5 0.13; Si 5 0.05 2 0.1; Ni 5 0.01 2 0.05; Cr 5 0.005 2 0.01; Co < 0.005; Cu ffi 0.001; Pb < 0.005) and Ti6Al7Nb alloy plates (Ti 5 base; C < 0.08; N < 0.05; Fe < 0.25; H < 0.009; O < 0.20; Ta < 0.5; Al 5 5.5 2 -6.5; Nb 5 6.5 2 -7.5). Glycerol-based electrolyte (90% glycerol, 9.3% H2O, 0.7% NH4F, pro analysis reagents) was used due to its high viscosity which influences the diffusion of ionic species and the nanotube formation kinetics and morphology.14 Prior to anodization, all metallic plates were initially polished employing emery paper in successive grits of 320, 400, 600, and finally with diamond paste, ultrasonic degreased in acetone followed by rinsing with deionized water, and drying in hot air stream. Anodization was performed in a standard two-electrode bath with circular platinum mesh cathode and MLW DC power source, of 150 V and 10 A. The anodization temperature was fixed to laboratory temperature of 25 C. The resulting TiO2 nanostructures were rinsed and subsequently annealed in a VULCAN 3–350 furnace, in air, at 550 C for 1 h. The samples were sterilized by autoclaving at 120 C for 30 min before use in biological experiments. An identically

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sized flat Ti plate (without TNT film) was used as a control after being chemically cleaned with acetone and isopropyl alcohol, dried, and autoclaved. TNT surface analysis The nanostructural films were investigated as such, without any preparation (TiO2 is a semiconductor) by scanning electron microscopy (SEM, QUANTA INSPECT F) and by high resolution transmission electron microscopy (HRTEM, TECNAI F30 G2) with a line resolution of 1 Å. Cell culture and exposure to TNT films G292 osteoblastic cells American Type Culture Collection (ATCC CRL-1423), originally isolated from human osteosarcoma, were purchased from the ATCC (Manassas, VA). The cells were cultured in McCoy’s 5a medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 lg/mL streptomycin, in a humidified atmosphere with 5% CO2 at 37 C. The culture medium was changed every 2 days until cells reached confluence. Then, the cells were trypsinized using 0.25% trypsin – 0.03% EDTA (Sigma-Aldrich) and were seeded onto TiO2 nanotube substrates, Ti samples without nanotubes and directly on tissue culture polystyrene (control cells) in a six-well plate at a density of 2 3 104 cells/ well, for 24 or 48 h. Four wells were used per treatment for each sample. Lactate dehydrogenase (LDH) release assay LDH leakage into the culture medium as a result of plasma membrane lysis was evaluated with a cytotoxicity detection kit (TOX-7, Sigma-Aldrich) according to the manufacturer’s protocol. The LDH activity was determined spectrophotometrically after 30 min of incubation at 25 C of 50 lL of culture medium and 100 lL of the reaction mixture, by measuring the oxidation of NADH at 490 nm in the presence of pyruvate, according to the manufacturer’s kit instructions. F-actin staining G292 cells were seeded onto six-well plates (15 3 104 cells/ well) in the presence of TiO2 nanotube samples or Ti plates. After 24 and 48 h of incubation, the cells were fixed in 4% paraformaldehyde for 20 min at 4 C and permeabilized with 0.5% Triton X-100 for 1 h. After three washes with PBS, the cells were incubated for 40 min in PBS containing 20 mg/mL phalloidin–FITC (Sigma-Aldrich). The nuclei were counterstained with 2 mg/mL DAPI (Invitrogen). The fluorescence imaging was performed using an Olympus IX71 fluorescence microscope. Cell counting The number of cells attached to the surface of each sample was established by counting the blue nuclei, which were identified by DAPI staining. Ten fields per sample captured at 1003 magnification with a DAPI filter were randomly selected and used for nuclei counting with Cell Counter plugin from ImageJ 1.48 software. The mean value of these measurements was used to estimate the overall count for each sample.

EFFECTS OF TNT FILMS ON OSTEOBLAST BEHAVIOR IN VITRO

ORIGINAL ARTICLE

GSH fluorescence detection The osteoblasts were cultivated onto six-well plates (104 cells/well) on the surface of TiO2 nanotube or Ti polished samples. After 24 and 48 h of incubation, the medium was removed, and the cells were stained to detect GSH with 5 lM CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA; Molecular Probes from Invitrogen) in cell culture medium without FBS for 30 min at 37 C and 5% CO2. After washing with prewarmed media, cells were left to rest for 30 min at 37 C and 5% CO2 in the medium to allow the hydrolysis of CMFDA to the fluorescent 5-chloromethylfluorescein (CMF) by intracellular esterases and conjugation with GSH preferentially by GSH S-transferase (GST) or the diffusion of the unconjugated dye. The CMFDA specificity for detection of both nuclear and cytoplasmic GSH detection is 95%, which proves it is a useful tool in the characterization of GSH distribution between the nucleus and cytoplasm in animal cells.13,15 The nuclei were counterstained with 2 mg/mL Hoechst 33342 (Invitrogen). The cells attached to the titanium surfaces were observed using an Olympus IX71 fluorescence microscope. The simultaneous detection of CMF fluorescence and nuclear staining in the same cells was done using a custom DAPI/FITC dual band fluorescence filter (Ex. 340–380/450–490 nm, Em. 435–485/520–560 nm). Quantification of fluorescence area The fluorescence tracking was performed using CMF and Hoechst images taken at 2003 magnification, which were transformed into grayscale images. The area encompassing the nucleus and the entire cellular area excluding the nucleus (i.e., cytoplasm) were drawn according to the area marked with Hoechst 33342. Nucleus/cytoplasm ratio for GSH in every cell analyzed was obtained by dividing the mean of green CMF fluorescence of the nuclear area by the mean of CMF fluorescence in the cytoplasm area. The area drawing and fluorescence quantification were performed for 50 cells per condition in three independent experiments using ImageJ 1.48 software. Only completely displayed cells in the field of view were selected from 20 images randomly captured and the average fluorescence intensity of these cells was used as an overall estimation for each sample. Statistical evaluation All results were expressed as mean values 6 SD of three independent experiments. The data were analyzed for statistical significance using GraphPad Prism software (Version 6; GraphPad Software, La Jolla, CA) by one-way ANOVA followed by a post hoc Bonferroni test. A P value of