Article pubs.acs.org/journal/abseba
Inflammatory Bone Resorption and Antiosteosarcoma Potentials of Zinc Ion Sustained Release ZnO Chips: Friend or Foe? Sik-Won Choi,†,⊥ Won Jin Choi,‡,⊥ Eun Hye Kim,† Seong-Hee Moon,†,§ Sang-Joon Park,∥ Jeong-O Lee,*,‡ and Seong Hwan Kim*,† †
Laboratory of Translational Therapeutics, Pharmacology Research Center, Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea ‡ Advanced Materials Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea § Department of Biology, Chungnam National University, Daejeon 305-764, Republic of Korea ∥ Department of Histology, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea S Supporting Information *
ABSTRACT: Multifunctional zinc oxide (ZnO) has been generated as nanoparticles or nanorods and applied to various medical purposes since it exhibits several biological actions including anticancer activity. Especially, due to antibacterial activity and effects on bone regeneration, ZnO is widely used in implants and scaffolds in the orthopedic and dental fields. However, concerns over side effects have been raised recently in the clinical use of ZnO, and it is necessary to assess the safety of ZnO regarding its inflammatory potential in the bone environment. This made us hypothesize that the inflammatory activity of zinc ions released from ZnO NPs could be harmful to induce bone resorption but that their cytotoxicity would be beneficial to kill osteosarcoma. To clarify this hypothesis, in the present work, the effects of ZnO on bone matrix and abnormal bone environments were investigated quantitatively using ZnO chips, filter paper, or glass slides coated with thin films of ZnO grown via atomic layer deposition (ALD). ALD-grown ZnO thin films exhibit thickness with atomic precision, which enables the quantitative analysis of the effects of ZnO. In vivo application of ZnO chips to mouse calvarial bone induced bone resorption, presumably due to the activation of osteoclasts by zinc ion-induced TNF-α release. However, in vitro application of ZnO chips to osteosarcoma cells induced caspase-dependent apoptosis and oxidative stress. Taken together, the results showed two sides of ZnO as our hypothesis. Therefore, careful design and multiple evaluations for the safety and efficacy of ZnO materials are necessary for its successful clinical application. KEYWORDS: ZnO, zinc ions, bone, cancer, inflammatory bone resorption, biomaterials
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INTRODUCTION The fast evolution of biomaterials has led to their debut on the clinical stage for the functional replacement and repair of human tissues and organs.1−4 Multifunctional zinc oxide (ZnO) has been generated as nanoparticles (NPs) or nanorods and applied to various medical purposes, including biosensing, imaging, drug delivery, and clinical implants.5−7 The properties of biomaterials, such as biocompatibility and bioactivity, have been the primary focus for generating precisely tuned biomaterials, and as the clinical use of ZnO-related biomaterials has increased, concerns about their potential toxicity in humans have been raised.8 The cytotoxic and apoptotic activity of zinc ions (at >5 μg/ mL) released from ZnO NPs was demonstrated recently in vitro,9,10 and their inflammatory activity was shown by their potential to increase the production of inflammatory cytokines, such as tumor necrosis factor (TNF)-α.11−13 Especially in bone, ZnO-induced TNF-α may induce resorption of the bone matrix © XXXX American Chemical Society
by increasing the mobilization of bone marrow osteoclast precursors and their differentiation into functionally active mature osteoclasts,14−16 but this hypothesis has not been clarified. Therefore, in this study, we generated and implanted zinc ion sustained release ZnO chips into bone and evaluated whether they could induce inflammatory bone resorption. In addition, the biological potential of the ZnO chips in abnormal bone environments, such as osteosarcoma, was evaluated.
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EXPERIMENTAL SECTION
Fabrication and Microscopic Imaging of ZnO Chips. ZnO chips were fabricated by coating circular Whatman grade 3MM Chr cellulose chromatography paper (thickness, 0.34 mm; Whatman, Maidstone, UK; cat. no. 3030−917; for in vivo transplantation) or glass Received: September 15, 2015 Accepted: February 25, 2016
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DOI: 10.1021/acsbiomaterials.5b00395 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
day, and then the cell viability was measured using Cell Counting Kit 8 (CCK-8, Dojindo Molecular Technologies, MD, USA) according to the manufacturer’s protocol. Cell culture media were discarded for the removal of detached cells before treating with the CCK-8 reagent. As shown in the product description, CCK-8 is one of the tetrazolium salt-based colorimetric assays for the determination of cell viability. Tetrazolium salt is reduced by dehydrogenase in live cells to give a yellow formazan dye, which is soluble in cell culture media. The amount of formazan dye is directly proportional to the number of living cells. Absorbance of the formazan dye was measured at 450 nm using a Wallac EnVision microplate reader (PerkinElmer, MA, USA). Real-Time PCR. TNF-α mRNA expression was evaluated by realtime PCR. Briefly, RAW264.7 cells were plated in a 6-well plate at a density of 1 × 106 cells/well and cultured for 1 day. Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, CA, USA) and reverse transcription and PCR carried out using the Omniscript RT kit (Qiagen, CA, USA) and Brilliant SYBR Green Master Mix (Stratagene, CA, USA) according to the manufacturers’ protocols. First-strand cDNA was synthesized from 1 μg of total RNA and SYBR green-based real-time PCR performed in the Stratagene Mx3000 Real-Time PCR system. GAPDH was used as the internal control. The primer sequences used in this study were TNF-α forward, 5′- GTC TAC TTT GGA GTC ATT GC-3′; TNF-α reverse, 5′- CTC TGA GGA GTA GAC AAT AA-3′; GAPDH forward, 5′- AAC TTT GGC ATT GTG GAA GG-3′; and GAPDH reverse, 5′- ACA CAT TGG GGG TAG GAA CA-3′. Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA kit for mouse TNF-α was purchased from KOMA Biotech (Seoul, Korea). RAW264.7 cells were plated in triplicate in a 96-well plate at a density of 5 × 104 cells/well and cultured for 1 day. Cells were incubated with media containing zinc ions for 1 day, and then the amount of TNF-α in the culture media was quantified using a Wallac EnVision microplate reader (PerkinElmer, MA, USA). The standard curve of TNF-α was generated by the provided recombinant mouse TNF-α according to the manufacturer’s protocol. Western Blotting. Protein extraction was carried out on ice. Cells were lysed in RIPA buffer (Elpis Biotech, Korea; 50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, and 1 mM phenylmethanesulfonylfluoride). After centrifugation at 18,000g for 10 min at 4 °C, the protein content of the supernatant was quantified using RC DC Protein Assay Kit (Bio-Rad, CA). Proteins were mixed with 5× sample buffer (Elpis Biotech; 0.375 M Tris-HCl [pH 6.8], 5% sodium dodecyl sulfate, 5% β-mercaptoethanol, 50% glycerol, and 0.05% bromophenol blue), denatured by boiling at 100 °C for 5 min, loaded onto a 10% sodium dodecyl sulfate polyacrylamide gel, separated via electrophoresis, and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, NJ, USA). The membrane was preincubated with 5% skim milk in TBST (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween-20) at room temperature for 1 h, and then probed with the indicated primary antibody. After incubation overnight at 4 °C, the membrane was washed three times for 30 min with TBST and then incubated for 2 h at room temperature with the secondary antibody. After washing with TBST, the membrane was developed using Pierce SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Chemical Co., IL, USA) in a LAS-3000 Luminescent Image Analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan). Antibodies against phosphorylated (p)-Elk and actin and the horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (TX, USA). All other antibodies were obtained from Cell Signaling Technology (MA, USA). Actin was used as a loading control. Serum Response Element (SRE) Reporter Luciferase Assay. The plasmid for the SRE reporter luciferase activity assay was constructed by inserting two copies of the SRE sequence (CCATATTAGG) into the pGL3 basic vector (Promega, WI, USA). Using Lipofectamine 2000 (Invitrogen Life Technologies), 2 × SRE luciferase (200 ng) plus pGL4-renilla (20 ng; Promega) as the control plasmid were cotransfected into HEK293T cells plated at a density of 5 × 104 cells/well on a 48-well plate in triplicate and incubated for 1 day. After 48 h, the transfected cells were lysed with
slides (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany; for in vitro assays) with ZnO using an atomic layer deposition (ALD) technique, in order to precisely control the thickness of ZnO. ALD is a unique technique normally in use in the semiconductor industry, which ensures precision control of the film thickness. Briefly, a single cycle of ALD is composed of a pulse of diethyl zinc (DEZ, Sigma-Aldrich, MO, USA) followed by a purge process, which results in the formation of a Zn-terminated substrate (glass or filter papers). Then, a subsequent pulse of H2O (deionized water) follows, to attach O atoms to Zn-terminated substrates, to form a layer of ZnO.17 The DEZ−purge−water−purge cycles were repeated to form ZnO thin films with desired thickness. Because of the selflimited nature of ALD, atomic scale control of film thickness is possible, which is directly connected to the precise amount of released Zn ions. As reported by Choudhury et al., a single cycle of the ALD process produces ∼200 ng/cm2 ZnO; the total amount of ZnO on 35 mm-diameter glass slides with 400-cycled ZnO is approximately ∼0.8 mg.18 ALD was performed in the Lucida D-100 chamber, with DEZ− purge−water−purge cycles controlled at 0.5 s−10 s−0.1 s−30 s. The deposition temperature was fixed at 150 °C. As reported in the previous work, ALD-grown ZnO shows atomically smooth surfaces with roughness below ∼1 nm.17 Prior to microscopic imaging, ZnO chips (fabricated on filter papers) were coated with Pt for 120 s for better imaging. SEM images were taken with Mira 3 LMU FEG SEM (Tescan, Czech Republic), with an accelerating voltage of 20 kV. For EDS analysis, a Quantax 200 energy dispersive X-ray spectrometer (Bruker, MA, USA) was used with an energy resolution smaller than 127 eV. In Vivo Mouse Calvarial Bone Assay. In vivo mouse calvarial bone assays were carried out according to the Ethics Guidelines of the Korea Research Institute of Chemical Technology (Protocol ID No. 7D-M5). The protocol was approved by the Institutional Committee (Approval No. 2014-7D-04-05). Five-week-old male ICR mice (Damul Science Co., Korea) were maintained in a room illuminated daily from 07:00 to 19:00 (a 12:12-h light/dark cycle) under controlled temperature (23 ± 1 °C) and ventilation (10−12 times per hour). Humidity was maintained at 55 ± 5%. The mice had free access to a standard animal diet and tap water. After anesthetizing the mice with an intraperitoneal injection of 1.2% avertin (2,2,2-tribromoethanol, Sigma-Aldrich; 700 μL/mice), the sagittal incision on the scalp was made to implant the ZnO (100 or 500 cycle)-deposited papers on the calvarium (n = 3 mice per group) and sutured as shown in Supporting Information, Figure S1. ZnO-uncoated papers were implanted in the control group. Two weeks after implantation, the calvariae were harvested, fixed in 4% paraformaldehyde, decalcified in 12% EDTA, embedded in paraffin, and sectioned. For histological examination, 3 sections per mouse were stained with hematoxylin and eosin (H&E), and the representative image was presented in the result. Preparation of ZnO Extracts and Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) Analysis. After incubating ZnO-coated glass chips with 5 mL of cell culture media in a 6-well plate for 2 days, media (called “ZnO extracts” in the following) were collected and used for cell-based assays. Before treating cells with ZnO extracts, the concentrations of zinc ion in ZnO extracts were analyzed using iCAP 6500 (Thermo Scientific, MA, USA). Since the standard deviation was
