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Kaori Iwanami‐Kadowaki, [Tetsuo Uchikoshi](https://orcid.org/0000-0003-3847-4781), Masayoshi Uezono, [Masanori Kikuchi](https://orcid.org/0000-0002-9451-8147), Keiji Moriyama

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This is the peer reviewed version of the following article: Development of novel bone-like nanocomposite coating of hydroxyapatite/collagen on titanium by modified electrophoretic deposition, which has been published in final form at https://doi.org/10.1002/jbm.a.37182. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.[In Copyright](http://rightsstatements.org/vocab/InC/1.0/)

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[Development of novel bone‐like nanocomposite coating of hydroxyapatite/collagen on titanium by modified electrophoretic deposition](https://mdr.nims.go.jp/datasets/afe8e6c6-8a25-466f-a66a-912712575d20)

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Development of novel bone-like nanocomposite coating of hydroxyapatite/collagen on titanium by modified electrophoretic depositionKaori IWANAMI-KADOWAKI 1,2, Tetsuo UCHIKOSHI3, Masayoshi UEZONO1, Masanori KIKUCHI2, Keiji MORIYAMA11 Department of Maxillofacial Orthognathics, Graduate School of Tokyo Medical and Dental University, Tokyo 113-8510, Japan2 Bioceramics Group, National Institute for Materials Science, Ibaraki 305-0047, Japan 3 Materials Processing Unit, National Institute for Materials Science, Ibaraki 305-0047, JapanCorresponding authorMasanori Kikuchi、Ph.D.KIKUCHI.Masanori@nims.go.jpAbstractElectrophoretic deposition (EPD) is a simple, rapid, and inexpensive technique to accomplish uniform coatings with controlled thicknesses. The EPD using binders that do not require a thermal degreasing process, which also eliminates the polymer components of the composite, are required for coating polymer-ceramic composites. This study demonstrated the application of a modified EPD technique utilizing Mg2+ ions to coat a bone-like hydroxyapatite/collagen nanocomposite (HAp/Col) on a titanium (Ti) substrate. The coating thickness was successfully controlled by varying the applied voltage and/or the treatment time. The adhesive strength of the modified EPD coating, evaluated by the tape test, showed class 0 (coating was not peeled off) and drastically increased in comparison to that of the non-Mg2+ EPD coating, class 5 (coating was completely peeled off). The MG63 cells on the HAp/Col-coated Ti demonstrated similar proliferation to and superior alkaline phosphatase activity to that on the bare Ti. Thus, the HAp/Col-coated Ti is expected to facilitate the surrounding bone formation than the bare-Ti. The results of the study indicated the HAp/Col-coated Ti prepared by the modified EPD is effective for applications in novel instruments such as subperiosteal temporary anchorage devices (TADs) which strongly requires rapid osseointegration at the bone-implant surface.Key Words: Hydroxyapatite/collagen, electrophoretic deposition, coating, orthodontic device, implantIntroductionTitanium (Ti) and its alloys are utilized in various medical and dental applications including as implant materials. Surface treatments of Ti-based materials had been being investigated to achieve rapid fixation to bone, which is also useful to temporary anchorage devices (TADs) (1) to expand the boundary of tooth movement in orthodontic treatment. Two types of TADs are generally used: miniscrews with diameters less than 2.0 mm and miniplates coupled with fixation screws. The selection of the TADs type depends on the treatment objectives and the anatomical characteristics of the placement site. Bone fixation of these TADs is achieved by the drilling into craniofacial bone tissues; therefore, there are risks of trauma to the periodontal ligaments, dental roots, tooth germs, nerve tissues, blood vessels, and maxillary sinuses that are encapsulated in the bone tissues. Bone-bonding subperiosteal devices, the other type of TADs that developed to avoid drilling, are limited in clinical application due to requirement of longer fixation period, 3–4 months, by osseointegration to achieve sufficient adhesion.Previous studies demonstrated that a dip-coated bone-like hydroxyapatite/collagen nanocomposite (HAp/Col) (2) on Ti is three times faster osseointegration than a nano-HAp-coated Ti and a strong candidates for the fabrication of subperiosteal devices(3). Although dip coating is a simple and inexpensive technique, it is difficult to apply for clinical devices because it has problems in utilization for complicated shapes, control of coating thickness and obtainment of sufficient adhesive strength between the coating and the substrate. Since collagen in the HAp/Col decomposes or denatures by heating, electrophoretic deposition (EPD) using organic polymer binders which requires combustion degrease(4) is not suitable. However, the deposition without binders are simply compression molded and will easily peel off when the dry coating is rubbed. EPD using binders that do not require a thermal degreasing process are required for coating polymer-ceramic composites.A recent report demonstrated that adhesive strength of an Eu2+ coating on an indium tin oxide/glass substrate was improved by a modified EPD technique using Mg2+ as an inorganic binder (5). The modified EPD is available to apply the HAp/Col coating, because Mg2+ is a constituent of bone, is acceptable in high concentrations of up to 84 mg/L in the human blood plasma, and is reported to promote bone formation in an optimal concentration range.In this study, the HAp/Col coating layers were formed by the modified EPD technique at various conditions to solve problems in utilization for complicated shapes, control of coating thickness and obtainment of sufficient adhesive strength between the coating and the substrate with Mg2+, and their physicochemical and cytocompatible properties were investigated.Materials & methods2.1.1 Preparation and characterization of the HAp/Col powderThe HAp/Col was synthesized by a simultaneous titration method with the same reagents used in the literatures (2),(6) and crushed into powder. Inorganic phases in the HAp/Col powder were identified by the powder X-ray diffraction (XRD, RINT-Ultima III, Rigaku Corporation, Tokyo, Japan) from 20–60 ° using CuKα radiation at a scanning rate of 2 °/min and an acceleration voltage and excitation current of 40 kV and 40 mA.2.1.2 Preparation and analysis of the HAp/Col suspensionThe colloidal suspensions of HAp/Col were prepared via a slight modification of a previously reported mechanism (5) to suit the HAp/Col. The following materials were utilized for the preparation: 1 g of the HAp/Col powder, 100 mL of 2-propanol ((CH3)2CHOH, Nacalai Tesque, Inc., Kyoto, Japan), 2 mL of glycerol (C3H5(OH)3, Kanto Chemical Co., Inc., Tokyo, Japan), 1–50 mg of Mg(NO3)2•6H2O (Kanto Chemical Co., Inc.) as a particle charge modifier (positive), and 2 mL of distilled water. C3H5(OH)3 acted as the dispersion medium. These materials were sequentially added to a polypropylene container as in the list order and dispersed by stirring with ultrasonication (GSD 600AT, Sonic Technology Co., Ltd., Saitama, Japan) for 10 min in ice water to obtain a homogeneous mixture. The HAp/Col colloidal suspension was obtained by the subsequent wet ball milling using ZrO2 balls (φ 1.0 mm) at 4 °C for 24 h and ultrasonication for 10 min in ice water. The zeta potential (n = 5) of the HAp/Col in suspension was measured using a zeta potential analyzer (Zetasizer Nano Z, Malvern Instruments, Malvern, UK). Sedimentation of particles in the suspension was determined by measuring the settled particle height, H, in the suspensions using the conventional fixed-point observations, and the sedimentation rate, H/H0, where H0 is sedimentation height at time 0, was calculated. The optimal Mg amounts was determined from the aforementioned measurements and applied in the following experiments.2.1.3 Coating of the HAp/Col by modified EPDCommercially pure Ti plates (Grade 2, Kobe Steel, Ltd., Hyogo, Japan) were cut into sheets of 20×50×0.5 mm3 and passivated according to the ASTM F86. A stainless-steel sheet (SUS-316L, Nilaco Corporation, Tokyo, Japan) was cut into the same size as that of the Ti sheets and cleaned with acetone and pure water. The modified EPD was performed using the Ti sheet and the stainless-steel sheet as the cathodic substrate and the anodic counter electrode, respectively at an electric field between the electrodes of 20 to 60 V/cm for 2–6 min. The Ti plate was extracted after the EPD, rinsed by dipping in 2-propanol, and finally dried at room temperature. The HAp/Col coating by the EPD without addition of Mg(NO3)2•6H2O were also prepared as a conventional EPD coating.2.2 Physicochemical properties of the HAp/Col coatingThe HAp/Col coating and the pretreated Ti surfaces were observed by the naked eye and with a scanning electron microscope (SEM, JSM-5610, Jeol Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV after platinum spatter coating at 30 nm (ESC-101, Elionix Inc., Tokyo, Japan).  The coating thickness (n = 5) was measured using a constant-pressure thickness gauge (J type PG-01, JIS K6732, Teclock Co., Ltd., Nagano, Japan). The surface roughness of the coated and bare Ti substrate (n = 5) was determined by shape-measurement laser microscopy (VK9-9700G, Keyence Corporation, Osaka, Japan). The inorganic crystalline phases in the coating layer were identified by the powder XRD. The elemental distribution of calcium, carbon, magnesium, and titanium in the HAp/Col coated surface was analyzed with an energy dispersive spectroscope (EDS, JED-2300, Jeol Ltd.) equipped with the SEM at 20 kV. The adhesive strength of the coating was evaluated by the tape test according to the ASTM D3359 to be classified in 6 classes as class 0 (no peels) to class 5 (complete peel).2.3 Cell seedingThe Ti specimens (10×10× 0.5 mm3) with the HAp/Col coating thicknesses of 5, 10, and 20 µm were dehydrothermally cross-linked and sterilized with ethylene oxide gas. The same size uncoated Ti plate was used as a control. A 0.5-mm-thick silicone rubber sheet (SR-50, Tigers Polymer Corporation, Osaka, Japan) was placed at the bottom of each well of 24-well plates (Falcon®, Becton, Dickinson and Company, New Jersey, USA) to restrict cell attachment only to the specimen. The specimen was then placed on the silicone rubber sheet.Twenty thousand human osteoblast-like cell lines (MG63) in 1 mL of Dulbecco’s minimum essential medium (Sigma Aldrich, Missouri, USA) supplemented with 10 % fetal bovine serum (Sigma Aldrich) and 1 % penicillin/streptomycin (Gibco®, Life technologies, Japan) were seeded on the specimen and cultured for 1, 3, 5, and 7 d in an incubator (HeracellTM 150i, Thermo Scientific, Japan) at a relative humidity of 95 % under a 5 % CO2 atmosphere. The medium was refreshed every 2 days.2.4 Viability/cytocompatibility & alkaline phosphatase assayThe cell viability was evaluated by the live/dead cell staining assay using a double staining kit (Cellstain®, Dojindo Laboratories, Kumamoto, Japan) and observed with a fluorescence microscope (BX51, Olympus Corp., Tokyo, Japan) using 490 nm and 545 nm filters. Cell morphology was observed at the same time. The area occupied by the cell attachment area was measured with the use of the image analysis software (ImageJ, NIH, Bethesda, MD, USA). The measured area was divided by number of cells and calculated. The cell proliferation on the specimens was estimated by measuring t the total DNA amounts (n = 5) extracted from the MG63 cells on the specimen measured at 1, 3, 5, and 7 days after seeding. The cells were harvested with the specimen and washed with phosphate-buffered saline (Sigma Aldrich), and were detached from the specimen using 500 µL of a detaching solution, composed of 500 µL of ethylenediamine tetraacetic acid (EDTA, Sigma Aldrich), 1.3 mg/mL of collagenase (Wako Pure Chemical, Co., Osaka, Japan), and 0.25 % trypsin (Lonza, Basel, Switzerland). The cells were analyzed total DNA quantitative using previous reports methods to inhibit adsorption of DNA on the HAp nanocrystals in the HAp/Col(7).The osteogenic activity of the MG63 cells was evaluated by the alkaline phosphatase (ALP) activity assay by the conventional p-nitrophenol phosphate (Sigma Aldrich) method (n = 5) using the same cell lysates for the DNA assay. The amount of p-nitrophenol (pNP, Sigma Aldrich) was calculated from the light absorbance at 405 nm using a standard curve that was prepared from the serial dilution of pNP. The ALP activity was normalized against the amount of the total DNA in each sample.3 Statistical analysis.All the data were statistically analyzed using the Wilcoxon rank sum test by the “R” software (version R 4.0.2 for Mac OS X, http://www.r-project.org/).Results 4.1 Characterization of the HAp/Col suspensionThe results of the zeta potential measurements for the HAp/Col in the suspension are summarized in Fig. 1. The addition of Mg(NO3)2•6H2O significantly increases the zeta potential of HAp/Col (p < 0.01). The suspension, prepared with the addition of 25 mg of Mg(NO3)2•6H2O, exhibited the highest positive zeta potential and lowest sedimentation rate, i.e., most stable dispersion (Fig. 2); therefore, the suspension prepared using 25 mg of Mg(NO3)2•6H2O was optimal for the modified EPD.4.2 Physicochemical properties of coating layerFigure 3 shows the macroscopic photographs of the surfaces of the Ti substrates after the EPD. The visual observations confirmed the uniform coating of a white substance, which was presumed to be HAp/Col, on the Ti substrate. The qualitative degree of whiteness of the treated region increased with the increase in the treatment time and/or the applied voltage. The SEM observations (Fig. 4) revealed that the surface of the coating formed at 20 V for 2 min was smoother and flatter than that of the coatings formed under different conditions. Furthermore, the quantity of cracks and that of islands separated by cracks on the coating increased with the increaseing in the treatment time and/or the applied voltage. Increasing in island thickness appeared at cracks with the increase in the treatment time and/or the applied voltage was agreed with increasing in the coating thickness, measured with the thickness gauge, with the increase in the deposition time and/or the applied voltage (Fig. 5). Further, the surface roughness (Ra) of the coatings was increased with the increase in the coating thickness (Fig. 6).The XRD analysis (data not shown) for formed coatings demonstrated that inorganic phase of the coating layer was identified as low-crystalline HAp without any detectable differences between before and after the coating. Further, no Mg compounds were detected. The EDS analysis (data not shown) confirmed the uniform distribution of the elements in the HAp/Col and small amount of Mg2+ in the coating layer.The tape test results are summarized in Fig. 7. The adhesive strength of the conventional and modified EPD-coated HAp/Col was designated as class 5 and class 0, respectively. Crack formation in the coating layer showed no influences on the adhesive strength between the HAp/Col coating and the Ti substrate for the modified-EPD coated HAp/Col.4.3 Cell culture testFigure 8 shows the live/dead staining images of the cells. The HAp/Col coating exhibited the presence of multiple green-fluorescent, live, cells and a few red-fluorescent, dead, cells (indicated by arrowheads). This distribution was similar to that in the control Ti specimen. Therefore, the viability of the cells on the HAp/Col coating was identical to that of the cells on the control Ti specimen. Spread area of MG63 on Ti (2482 ± 753 µm2) without coating is larger than that with coating (1062 ± 456 µm2), and these showed significant differences (p < 0.01). Former was flat-spread and latter was round.Figure 9 shows that the increase in the amount of the total DNA, the index of the number of cells, followed an approximately identical trend for all the test and control groups. However, the following slight but significant differences were found between the 20 µm-thick layer and the control. The cells on the 20 µm-thick coating showed significantly small number than that of the control at 1 day’s culture, but cell number relation was inverted at 7 days’ culture.The ALP activity of MG63 cells was increased with the increase in the culture period and that for the 20 µm-thick coating was significantly higher than that for the control at all periods as shown in Fig. 10. Other coating layers also exhibit higher ALP activities than the control.DiscussionsMg(NO3)2•6H2O was completely dissociated in solution(5) to yield Mg2+. Mg2+ was attracted to the HAp/Col particles; thus, the zeta potential of the suspension increased with the increase in the amount of Mg(NO3)2•6H2O from 0 to 25 mg. When the quantity of Mg(NO3)2•6H2O was increased beyond 25 mg, the attraction of surplus Mg2+ to the HAp/Col particles induced the compression of the electric double layer on the particles(4), thereby increasing the Van der Waals attraction between the particles (Fig. 1). Consequently, zeta potential and the particle dispersibility decreased and affected the results of the sedimentation test (Fig. 2). Therefore, the suspension prepared with 25 mg of Mg(NO3)2•6H2O exhibited the highest dispersibility and is concluded as the optimal condition for colloidal suspension preparation.The increase in the coating thickness with the increase in the applied voltage was attributed to the strengthening of the interparticle attraction forces due to the change in pH of the suspension near the substrate up to vicinity of the isoelectric point of the particles (8). Additionally, the increase in the treatment time increases the total number of the HAp/Col particles accumulated on the substrate. This resulted in the increase in the coating thickness with the increase in the treatment time. The applied voltage and the treatment time were independent parameters, and they exerted independent positive influences on the coating thickness. Figure. 4 shows the appearance of a crack after the formation of the HAp/Col coating. The presence of this crack was induced by the shrinkage of the coating owing to the evaporation of 2-propanol and water between the HAp/Col particles. Collagen exhibits water-retention characteristics and it seems difficult to avoid cracking. Although cracks have occurred, it is considered that there is no problem in practical use because the adhesive strength is remarkably improved in Fig. 7.The reason of remarkable increases in the adhesive strength between the HAp/Col coating prepared with Mg2+ and the Ti substrate in Fig. 7 was considered as follows. When Mg2+ was absent, the bonding between the Ti substrate and the HAp/Col particles as well as that between the HAp/Col particles occurred via the Van der Waals forces and the liquid bridging forces that originated from the residual solvent. These forces resulted in weak adhesion. The addition of Mg2+ induced the formation of Mg(OH)2 hydrogels in the region near the cathode, where the pH shifted to the alkaline side owing to energization(5). The hydrogels acted as binders and formed crosslinks between the Ti substrate and the HAp/Col particles. Therefore, the addition of 25 mg of Mg(NO3)2•6H2O comprising Mg2+ resulted in strong adhesive forces between the coating and substrate. Thus, the modified EPD-coated HAp/Col exhibited a high adhesive strength.Though the conventional EPD coating of HAp/Col did not show sufficient adhesion to Ti for cell culture test, the HAp/Col itself demonstrates good cell proliferation and osteogenic activity in vitro(7),(9). Proliferation behavior of cells on the modified-EPD HAp/Col coating layers indicated the excellent biocompatibility and the absence of cytotoxicity (Fig. 8 and 9). Further, higher ALP activity of the MG63 cells on the HAp/Col coating than those on the control (Fig. 10) suggests that the HAp/Col coating by the modified EPD increased the osteogenic activity in vitro as the same as shown in previous reports(7),(9). Higher ALP activities of MG63 cells with small attachment areas shown in Fig. 8 were also consistent with the previous finding that mesenchymal stem cells (MSCs) with small attachment areas showed a high osteogenic differentiation activity(10). These results suggest that modified-EPD coating add no negative influences on the HAp/Col biological properties.The HAp/Col has been confirmed that it is resorbed by osteoclasts and promotes bone formation at the lacunae formed by the osteoclasts(2). Further, a dip-coated HAp/Col on a Ti substrate accelerates the osseointegration via the osteoclastic resorption of the HAp/Col coating(3). Since the present HAp/Col coating is much dense than the dip-coated HAp/Col, acceleration of osseointegration has to be confirmed by animal tests planned in near future. Even though, the HAp/Col coatings formed by the modified-EPD could at least be used as osteoconductive surface that gradually transit to osseointegration.To summarize, this study demonstrated the HAp/Col coating layer with controlled thickness and high adhesive strength was formed on Ti by the modified EPD using 25 mg of Mg(NO3)2•6H2O. The HAp/Col coating layer formed on Ti substrate showed excellent biological properties for MG63 similar to the original HAp/Col materials.AcknowledgementsThis study was supported by a research grant 19im0210221h0001 from AMED, Japan.References1.  Dalessandri D, Salgarello S, Dalessandri M, Lazzaroni E, Piancino M, Paganelli C, Maiorana C, Santoro F. Determinants for success rates of temporary anchorage devices in orthodontics: A meta-analysis (n > 50). Eur J Orthod. 2014;36:303–13. 2.  Kikuchi M, Itoh S, Ichinose S, Shinomiya K, Tanaka J. Self-organization mechanism in a bone-like hydroxyapatite/collagen nanocomposite synthesized in vitro and its biological reaction in vivo. Biomaterials. 2001;22:1705–11. 3.  Uezono M, Takakuda K, Kikuchi M, Suzuki S, Moriyama K. Hydroxyapatite/collagen nanocomposite-coated titanium rod for achieving rapid osseointegration onto bone surface. J Biomed Mater Res - Part B. 2013;101 B:1031–8. 4.  Besra L, Liu M. A review on fundamentals and applications of electrophoretic deposition (EPD). Prog Mater Sci. 2007;52:1–61. 5.  Zhang C, Uchikoshi T, Liu L, Sakka Y, Hirosaki N. Crystalline-oriented beta-sialon:Eu2+ deposits fabricated by electrophoretic deposition (EPD) within strong magnetic field. ECS J Solid State Sci Technol. 2014;3:195–9. 6.  Kikuchi M, Ikoma T, Itoh S, Matsumoto HN, Koyama Y, Takakuda K, Shinomiya K, Tanaka J. Biomimetic synthesis of bone-like nanocomposites using the self-organization mechanism of hydroxyapatite and collagen. Compos Sci Technol. 2004;64:819–25. 7.  Yoshida T, Kikuchi M, Koyama Y, Takakuda K. Osteogenic activity of MG63 cells on bone-like hydroxyapatite/collagen nanocomposite sponges. J Mater Sci Mater Med. 2010;21:1263–72. 8.  Mishra M, Sakka Y, Uchikoshi T, Besra L. PH localization: A case study during electrophoretic deposition of ternary MAX phase carbide-Ti3SiC2. J Ceram Soc Japan. 2013;121:348–54. 9.  Kikuchi M. Osteogenic activity of MG63 cells on hydroxyapatite/collagen nanocomposite membrane. Key Eng Mater. 2007;330-332 I:313–6. 10.  Cheng K, Hirose M, Wang X, Sogo Y, Yamazaki A, Ito A. Development of an early estimation method for predicting later osteogenic differentiation activity of rat mesenchymal stromal cells from their attachment areas. Sci Technol Adv Mater. 2012;13:1–6. Figure legendsFig. 1. Changes in the zeta potential for the colloidal suspensions of HAp/Col that are prepared with different amounts of Mg(NO3)2•6H2O (n = 5). Significant differences from the control at p < 0.05 and at p < 0.01 are indicated as * and **, respectively.Fig. 2. Differences in the sedimentation rate for the colloidal suspensions of HAp/Col that are prepared with different amounts of Mg(NO3)2•6H2O. Fig. 3. Naked-eye observation of the HAp/Col coating on Ti at different treatment times and applied voltages. HAp/Col is deposited as a white layer by the modified EPD. Fig. 4. Scanning electron micrographs of the Ti substrate before the HAp/Col coating and after the HAp/Col coating at different treatment times and applied voltages. The arrowheads indicate the cracks in the coating.Fig. 5. Changes in the thickness of the coating (n = 5) as a function of the treatment time and the applied voltage.Fig. 6. Surface roughness of the HAp/Col coatings and bare Ti (n = 5). Significant differences from the control (bare Ti) at p < 0.05 and at p < 0.01 are indicated as * and **, respectively.Fig. 7. Adhesive strengths between the HAp/Col coating and the Ti substrate with and without Mg2+, as determined by the tape test according to the ASTM D3359. Fig. 8. Viability of the MG63 cells on the different-thick HAp/Col-coated Tis. The green and red fluorescence indicate the live and dead cells, respectively.Fig. 9. Total DNA amounts of the MG63 cells that are cultured on the different-thick HAp/Col coatings (n = 5). Significant differences from the control at p < 0.05 and at p < 0.01 are indicated as * and **, respectively.Fig. 10. ALP activities of the MG63 cells on the different-thick HAp/Col coatings (n = 5). Significant differences from the control at p < 0.05 and at p < 0.01 are indicated as * and **, respectively.1