TiO2 nanotubes were prepared on a Ti disc surface by an anodizing method in a two-electrode (distance between the two electrodes is 7 cm) electrochemical cell with platinum foil as the counter electrode at a constant anodic potential of 25 V and current density of 20 V, in a 1 M H3PO4 (Merck, Whitehouse Station, NJ, USA) and 0.3 wt.% HF (Merck) aqueous solution with 100-rpm magnetic agitation at 20°C. The Ti disc specimen was commercially pure titanium grade IV. The specimen was cleaned ultrasonically in ethanol for 10 min and chemically polished in a 10 vol.% HF and 60 vol.% H2O2 solution for 3 min. All electrolytes were prepared from reagent-grade chemicals and deionized water. Heat treatment of TiO2 nanotubes was carried out for 3 h at 350°C in air. The morphology of the TiO2 nanotubes was observed by field emission scanning electron microscopy (FE-SEM; JSM 6700F, Jeol Co., Akishima-shi, Japan), and their crystal structure was analyzed by wide-angle X-ray diffraction (WAXD, PANalytical’s X’PertPro, Almelo, The Netherlands).
Immobilization of PDA on a nt-TiO2 disc
The immobilization of PDA on the TiO2 nanotube (nt-TiO2) disc was carried out in three steps. First, the carboxyl group (−COOH) was introduced to the nt-TiO2 disc surface by a reaction of aminopropyl triethoxysilane (APTES; Sigma-Aldrich, St. Louis, MO, USA) with l-glutamic acid γ-benzyl ester (Sigma-Aldrich) followed by alkaline hydrolysis. Subsequently, PDA was immobilized on the carboxyl groups of the nt-TiO2 disc surface using water-soluble carbodiimide (WSC). Briefly, a nt-TiO2 disc (1 × 1 cm2) was immersed in an APTES-water solution (1:9) and sonicated for 30 min. The disc was then heated to 95°C for 2 h with gentle stirring. The silanized nt-TiO2 disc was washed with water in an ultrasonic cleaner and dried under reduced pressure and room temperature to produce a primary amine-coupled TiO2 nanotube disc (nt-TiO2-A). The nt-TiO2-A was then immersed in a beaker containing aqueous solution of l-glutamic acid γ-benzyl ester (23.93 mg in 100 ml water) and WSC solution (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.5 g, 0.25 wt.%; Sigma-Aldrich) and N-hydroxysuccinimide (0.5 g, 0.25 wt.%; Sigma-Aldrich) dissolved in 20 ml water) and stirred gently for 5 h at 4°C followed by alkaline hydrolysis to obtain the carboxyl functional TiO2 nanotube disc (nt-TiO2-G). The nt-TiO2-G was immersed in a solution of pamidronic acid disodium salt hydrate (10−4 M, 100 ml; Sigma-Aldrich) and WSC and stirred gently for 12 h at 4°C to obtain a PDA-immobilized nt-TiO2 disc (nt-TiO2-P; Figure ). The nt-TiO2-P was then washed in distilled water and dried. The chemical composition of the nt-TiO2-P surface was analyzed by electron spectroscopy for chemical analysis (ESCA, ESCA LAB VIG Microtech, East Grinstead, UK) using Mg Kα radiation at 1,253.6 eV and a 150-W power mode at the anode.
Schematic diagram showing the PDA-immobilized TiO2 nanotubes.
Osteoblastic cell culture
To examine the interaction of the surface-modified and unmodified TiO2 discs (Ti, nt-TiO2, and nt-TiO2-P) with osteoblasts (MC3T3-E1), the circular TiO2 discs were fitted to a 24-well culture dish and immersed in a Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco, Invitrogen, Carlsbad, CA, USA). Subsequently, 1 mL of the MC3T3-E1 cell solution (3 × 104 cells/mL) was added to the TiO2 disc surfaces and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 4 h, 2 days, 3 days, and 4 days. After incubation, the supernatant was removed and the TiO2 discs were washed twice with phosphate-buffered silane (PBS; Gibco) and fixed in a 4% formaldehyde aqueous solution for 15 min. The samples were then dehydrated, dried in a critical-point drier, and sputter-coated with gold. The surface morphology of the TiO2 disc was observed by FE-SEM.
To examine the cytotoxic effects of PDA, after 2 days of culture, the osteoblast cells were suspended in PBS with a cell density of 1 × 105 to 1 × 106 cells/mL. Subsequently, 200 μL of a cell suspension was mixed with a 100-μL assay solution (10 μL calcein-AM solution (1 mM in DMSO) and 5 μL propidium iodide (1.5 mM in H2O) was mixed with 5 mL PBS) and incubated for 15 min at 37°C. The cells were then examined by fluorescence microscopy (Axioplan 2, Carl Zeiss, Oberkochen, Germany) with 490-nm excitation for the simultaneous monitoring of viable and dead cells.
The proliferation of osteoblasts on the Ti, nt-TiO2 and nt-TiO2-P discs was determined by a 3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, MC3T3-E1 osteoblasts were seeded at a concentration of 3 × 104 cells/mL on the Ti, nt-TiO2, and nt-TiO2-P disc surfaces, which fitted in a 24-well plate, and cell proliferation was monitored after 2 and 3 days of incubation. A MTT solution (50 μL, 5 mg/mL in PBS) was added to each well and incubated in a humidified atmosphere containing 5% CO2 at 37°C for 4 h. After removing the medium, the converted dye was dissolved in acidic isopropanol (0.04 N HCl-isopropanol) and kept for 30 min in the dark at room temperature. From each sample, the medium (100 μL) was taken, transferred to a 96-well plate, and subjected to ultraviolet measurements for the converted dye at a wavelength of 570 nm on a kinetic microplate reader (ELx800, Bio-Tek® Instruments, Inc., Highland Park, VT, USA).
The calcium deposition of MC3T3-E1 cells cultured was studied by Alizarin Red S staining. The cells were cultured for 15 days on Ti, nt-TiO2, and nt-TiO2-P discs under the same condition as described earlier. After incubation, the cells were washed with PBS, fixed in 10% formaldehyde for 30 min, and then triple washed with distilled water for 10 min. The samples were then treated with Alizarin Red S stain solution (1 mL) and incubated for 20 min. After washing the sample with distilled water four times, the digital images of the stained cultures were obtained (Nikon E 4500, Shinjuku, Japan).
Differentiation of macrophage
For osteoclastic differentiation, hematopoietic stem cells (HSC, name of cell line) at a cell density of 3 × 104 cells/mL were cultivated on Ti, nt-TiO2, and nt-TiO2-P discs in DMEM containing 10% FBS, 50 ng/mL mouse recombinant receptor activator of nuclear factor kappa-B ligand (RANKL), and 50 ng/mL macrophage colony-stimulating factors from mouse (m-CSF). The culture medium was changed every 2 days.
Tartrate-resistant acid phosphatase staining and solution assays
To analyze osteoclastic differentiation, the cells after 4 days of culture in the differentiation medium were washed once with PBS and fixed with 10% formalin (50 μL, neutral buffer) at room temperature for 5 min. After fixation, cells were washed with distilled water and incubated with a substrate solution (3 mg of chromogenic substrate with 5 mL tartrate-containing buffer (pH 5.0)) for 30 min at 37°C. The cell images were obtained by fluorescence microscopy.
For immunocytochemistry, the HSCs were cultivated in a differentiation medium and fixed and immunostained after 4 days with 4′
,6-diamidino-2-phenylindole (DAPI) and (tetra-methyl rhodamine isothiocyanate)-phalloidin (TRICK), as described previously [33
]. Multinucleated cells containing more than three nuclei were considered differentiated osteoclast-like cells. The cell images were obtained by fluorescence microscopy. To confirm the viability of the differentiated macrophages on nt-TiO2
-P, the cells after 4 days of culture were stained with calcein-AM and propidium iodide, as described in the section for the osteoblastic cell culture, and examined by fluorescence microscopy.