Immobilization of pamidronic acids on the nanotube surface of titanium discs and their interaction with bone cells
- Tae-Hyung Koo†1,
- Jyoti S Borah1,
- Zhi-Cai Xing†1,
- Sung-Mo Moon†2,
- Yongsoo Jeong†2 and
- Inn-Kyu Kang†1Email author
© Koo et al.; licensee Springer. 2013
Received: 29 January 2013
Accepted: 27 February 2013
Published: 12 March 2013
Self-assembled layers of vertically aligned titanium nanotubes were fabricated on a Ti disc by anodization. Pamidronic acids (PDAs) were then immobilized on the nanotube surface to improve osseointegration. Wide-angle X-ray diffraction, X-ray photoelectron microscopy, and scanning electron microscopy were employed to characterize the structure and morphology of the PDA-immobilized TiO2 nanotubes. The in vitro behavior of osteoblast and osteoclast cells cultured on an unmodified and surface-modified Ti disc was examined in terms of cell adhesion, proliferation, and differentiation. Osteoblast adhesion, proliferation, and differentiation were improved substantially by the topography of the TiO2 nanotubes, producing an interlocked cell structure. PDA immobilized on the TiO2 nanotube surface suppressed the viability of the osteoclasts and reduced their bone resorption activity.
The clinical success of orthopedic and dental implants depends on the interaction between the implanted surface and bone tissues and, consequently, their osseointegration . Titanium implants are used widely in orthopedic surgery and dentistry for their favorable biocompatibility and corrosion resistance [2, 3]. Surface modification of the implanted material is a critical factor for tissue acceptance and cell survival. Among three different crystalline phases of titania (anatase, rutile, and amorphous titania), anatase phase is more favorable for cell adhesion and proliferation due to lower surface contact angles and/or wettability . Several surface modification techniques, i.e., sol–gel techniques, chemical (alkali/acid) treatment, anodization, plasma spray, hydroxyapatite-coated surface, and self-assembled monolayers, have been developed and are currently used with the aim of enhancing the bioactivity of pure Ti surface [5–12].
Over the last decade, bisphosphonates (BPs) have attracted increasing attention as a surface modifier for orthopedic and dental implants. Bisphosphonates are stable pyrophosphates that prevent the loss of bone mass and are used widely to treat a range of diseases with excess bone resorption, such as bone metastasis, hypercalcemia of a malignancy, and Paget’s disease [13–16]. In orthopedic implants, the use of BP is expected to promote osteogenesis at the bone tissue/implant interface by inhibiting the activity of osteoclasts. BPs were reported to inhibit the differentiation of the osteoclast precursor and the resorptive activity of mature osteoclasts [17, 18]. Furthermore, BPs alter the morphology of osteoclasts, such as a lack of ruffled border and disruption of the actin ring, both in vitro and in vivo[19, 20]. García-Moreno et al. reported that BPs enhance the proliferation, differentiation, and bone-forming activity of osteoblasts directly . Recently, pamidronic acid, a nitrogen-containing bisphosphonate, was reported to conjugate the titanium surface and stimulate new bone formations around the implant both in vitro and in vivo, which contribute to the success of the implant technology [22, 23].
Besides chemical surface modifications, nanometric-scale surface topography and roughness of the biomaterial is also recognized as a critical factor for tissue acceptance and cell survival. Nanoscale topography affects cell adhesion and osteoblast differentiation [24–26]. It was reported that the fabrication of TiO2 nanotubes on titanium implants increased new bone formation significantly . To study the effect of the nanopore size on bone cell differentiation and proliferation, Park et al. used vertically aligned TiO2 nanotubes with six different diameters between 15 and 100 nm. They reported 15 nm to be the optimal length scale of the surface topography for cell adhesion and differentiation . TiO2 nanotubes can modulate the bone formation events at the bone-implant interface to reach a favorable molecular response and osseointegration . Immobilization of bone morphogenetic protein 2 (BMP-2) on TiO2 nanotubes stimulates both chondrogenic and osteogenic differentiation of mesenchymal stem cells (MSCs). Surface-functionalized TiO2 nanotubes with BMP-2 synergistically promoted the differentiation of MSCs [30, 31]. Furthermore, TiO2 nanotubes can control the cell fate and interfacial osteogenesis by altering their nanoscale dimensions, which have no dependency or side effects .
In this study, dual-surface modifications, i.e., nanometric-scale surface topography and chemical modification were examined to improve the osteogenesis of titanium implants. First, TiO2 nanotubes were fabricated on a Ti disc and pamidronic acid (PDA) was then immobilized on the nanotube surface. The behavior of osteoblasts and osteoclasts on the dual-surface modified and unmodified Ti disc surface were compared in terms of cell adhesion, proliferation, and differentiation to examine the potential for use in bone regeneration and tissue engineering. The motivation for the immobilization of PDA on nanotube surface was that PDA, a nitrogen-containing bisphosphonate, suppresses the osteoclast activity and improves the osseointegration of TiO2 nanotubes.
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
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 . 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 and 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.
Results and discussion
Crystal structure of TiO2 nanotubes and surface characterization of PDA-immobilized nt-TiO2
Chemical composition of nt-TiO 2 and surface-modified nt-TiO 2
Interaction of bone cells with the surface-modified TiO2 nanotubes
Adhesion, proliferation, and differentiation of osteoblasts
Differentiation of macrophages into osteoclasts and viability on nanotube surface
On the nt-TiO2 surface, differentiated osteoclasts stained with calcein-AM and propidium iodide showed a green color indicating the good viability of the cells. In contrast, along with green fluorescence, red fluorescence was also observed on the nt-TiO2-P surface, which suggests that some osteoclast cells died in contact with PDA (immobilized PDA did not show any cytotoxic effect on macrophage cells, Additional file 1: Figure S1). Osteoclasts normally destroy themselves by apoptosis, a form of cell suicide. PDA encourages osteoclasts to undergo apoptosis by binding and blocking the enzyme farnesyl diphosphate synthase in the mevalonate pathway . Thus, the viability of osteoclasts was suppressed on the nt-TiO2-P surface, leading to a decrease in bone resorption activity and an increase in osseointegration and bone maturation.
TiO2 nanotubes were successfully fabricated on Ti surface, and pamidronic acids were immobilized on the TiO2 nanotube surface. The adhesion and proliferation of osteoblasts were accelerated on the TiO2 nanotubes and pamidronic acid-conjugated TiO2 nanotubes compared to the Ti disc only. Macrophages were partially differentiated into osteoclasts by the addition of RANKL and m-CSF. The viability of osteoclasts was suppressed on the pamidronic acid-conjugated TiO2 nanotubes. This study has demonstrated that immobilization of PDA might be a promising method for the surface modification of TiO2 nanotube for use as dental and orthopedic implants. An in vivo study will be necessary to evaluate the potential of pamidronic acid-conjugated TiO2 nanotube as a therapeutic bone implant.
This study was supported by a grant (2010–0011125) and the Basic Research Laboratory Program (2011–0020264) of the Ministry of Education, Science and Technology of Korea.
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