Stable ZnO-doped hydroxyapatite nanocoating for anti-infection and osteogenic on titanium

Journal Pre-proof Stable ZnO-doped Hydroxyapatite Nanocoating for Anti-infection and Osteogenic on Titanium Bakri Mamat, Naiyin Zhang, Ling Yan, Jianghong Luo, Chaoming Xie, Yingbo Wang, Chuang Ma, Tingjun Ye

PII:

S0927-7765(19)30875-6

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110731

Reference:

COLSUB 110731

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

7 August 2019

Revised Date:

30 November 2019

Accepted Date:

13 December 2019

Please cite this article as: Mamat B, Zhang N, Yan L, Luo J, Xie C, Wang Y, Ma C, Ye T, Stable ZnO-doped Hydroxyapatite Nanocoating for Anti-infection and Osteogenic on Titanium, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110731

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Stable ZnO-doped Hydroxyapatite Nanocoating for Anti-infection and Osteogenic on Titanium

Bakri Mamata1, Naiyin Zhangbc1, Ling Yana, Jianghong Luoa, Chaoming Xied, Yingbo Wanga,* [email protected], Chuang Mad,* [email protected], Tingjun Yeb,* [email protected]

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College of Chemical Engineering, Xinjiang Normal University, 102Xinyi Road,

Urumqi 830054, Xinjiang, P. R. China. b

Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases,

Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao

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Tong University School of Medicine, 197 Ruijin 2nd Road, Shanghai 200025, P. R. China. c

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College of Life Information Science and Instrument Engineering, Hangzhou Dianzi

University, Xiasha Higher Education Zone, Hangzhou, Zhejiang 310018, P. R. China. Department of Orthopedics Center, the First Affiliated Hospital of Xinjiang Medical

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University, 393 Xinyi Road, Urumqi 830054, P. R. China

These authors contributed equally.

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1

*

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Corresponding Author

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Graphical Abstract

The preparation of HA/PPy/ZnO nanocomposite coating on titanium substrate

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with bioactive, physiological stable, antibacterial, angiogenic and

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osteoinductive properties.

HIghlights

Pulse electrochemical deposition method for preparing polypyrrole.

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PPy as a dual regulator of hydroxyapatite and ZnO nanoparticles.

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Nano-coating with anti-infection, angiogenic and osteoinductivity.

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Reduced the release rate of Ca2+ and Zn2+ from the nano-coating.

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Abstract: Titanium and titanium alloys have been widely used in orthopedics and related fields. However, their clinical applications are limited due to the lack of anti-

infection, osteoinductivity and angiogenic ability. In the present study, we utilized pulse electrochemical deposition method to prepare polypyrrole (PPy) by the in-situ oxidative polymerization of pyrrole (Py), and through the coordination and doping of ions, the function of PPy as a dual regulator of hydroxyapatite nanoparticles (HA-NPs) and zinc oxide nanoparticles (ZnO-NPs) was achieved. Bioactivity test showed that the composite coating could induce the formation of apatite, and the apatite was in a neat arrangement preferentially grew along the (002) crystal plane, indicating good bioactivity. The release test showed that the dual regulation effect of PPy coordination and doping reduced the release rate of Ca2+ and Zn2+ from the composite coating.

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Antibacterial tests showed that the composite coating against Escherichia coli and Staphylococcus aureus. Besides, bone marrow-derived mesenchymal stem cells

(BMSCs) exhibited good adhesion, proliferation and differentiation on the composite

coating, and fluorescence staining experiments demonstrated good osteoinductivity of the composite coating. In this study, a multifunctional composite coating with anti-

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infection, angiogenic and osteoinductivity was successfully constructed on the

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titanium surface via pulse electrochemical deposition method.

Keywords: Titanium; Osteoinductivity; Anti-infection; Angiogenic; Electrochemical

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deposition

1. Introduction As an essential trace element in organisms, Zinc (Zn) participates in the catalytic activation reaction of more than 300 enzymes in organisms.[1] Human body contains about 2~3 g of Zn, 57% of which is distributed in skeletal muscle and 29% in bone. The main function of Zn is to promote bone growth, bone mineralization and bone formation in tissue culture.[2, 3] D. Liu et. al[4] confirmed from experiments that Zn is an important component of early growth factor 3 (Erg3) in the DNA segment transcriptional activator of vascular endothelial growth factor (VEGF). VEGF is stimulated by Erg3 to promote the proliferation and migration of vascular endothelial

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cells (VEC), and the formation of blood vessels. Metal oxide nanoparticles (MO-NPs) are known to have a broad antibacterial spectrum of Gram-positive and Gram-

negative strains, and are the candidates expected to address antibiotic resistance issues.[5] The antibacterial activity of zinc oxide nanoparticles (ZnO-NPs) has

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received great attention in the past decades. Sevinc et al. [6] reported that the

bactericidal concentration against S. aureus was 80 μg·mL-1 ~ 1.2 mg·mL-1 when the size of ZnO-NPs was in the range of 8 ~ 125 nm. Reddy et al. [7] reported that the

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complete inhibitory concentration against E. coli was 280 μg·mL-1 when the size of ZnO-NPs was 13 nm, while the complete inhibitory concentration against S. aureus

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was 80 μg·mL-1. Atsuo Ito et al. [8] prepared ZnO/β-TCP/HA composite ceramics using high temperatures, and their study has found that MC3T3-E1 osteoblasts proliferated significantly when Zn content was 0.6 ~ 1.2 wt%. ZnO-NPs (7-8nm) is

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toxic to human periodontal ligament fibroblast and mouse dermal fibroblast cells when its concentration exceeds 0.05-0.1 mg·mL-1 [9]. It is reported that ZnO is safe to

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organisms when the concentration of ZnO is below 50 mg·mL-1, and the serum Zn level in normal people is 0.88 ± 0.6 μg·mL-1 [10]. The antibacterial mechanism of ZnO is mainly the production of reactive oxygen species (ROS), which is highly

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reactive, able to destroy the integrity of bacterial cell membranes and cause bacterial death.[11] Besides, Zn2+ ions released from ZnO are attracted by the negatively charged polysaccharide layer of cell membrane, disturb the charge balance of the membrane and cause bacterial death.[12] Materials contain ZnO mostly have antibacterial property. Sevinc et al. [6] reported that the bactericidal concentration against S. aureus was 80 μg·mL-1 ~ 1.2 mg·mL-1 when the size of ZnO-NPs was in the range of 8 ~ 125 nm. Reddy et al. [7]

reported that the complete inhibitory concentration against E. coli was 280 μg·mL-1 when the size of ZnO-NPs was 13 nm, while the complete inhibitory concentration against S. aureus was 80 μg·mL-1. Memarzadeh et al. [13] prepared ZnO/HA composite coating using Electrohydrodynamic atomisation (EHDA) method. The coating had significant antibacterial properties against S. aureus, and the cell morphology of hMSCs was normal on the coating surface. This composite coating can be used in the preparation of antibacterial and biocompatible bone implant. Laurenti et al. [14] prepared ZnO-NPs and ZnO films by hydrothermal method, both of which can promote the growth of MC3T3-E1 osteoblasts and osseointegration.

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However, ZnO has shortcomings. H. Elham et. al. [15] synthesized ZnO-NPs using flame pyrolysis method. In their experiments, ZnO-NPs and hydroxyapatite (HA) was coated on the surface of titanium (Ti) substrate following heat-treatment to prepare

HA/ZnO nanocomposite coating layer. However, the HA/ZnO nanocomposite coating showed aggregations of ZnO-NPs, which would reduce the cytocompatibility of

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materials and cause cytotoxicity.[16] N. Sharma et. al.[17] prepared Ag-NPs and ZnO-NPs composites using co-precipitation method, but the bioactivity of the

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material was not ideal. J. Zhang et. al.[18] synthesized ZnO-NPs using coprecipitation method and Ag-loaded ZnO-NPs at high temperature. The size of the

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particles increased from 200 nm for ZnO-NPs to 1 μm for Ag-loaded ZnO-NPs. However, the Ag-loaded ZnO-NPs showed aggregation, which corresponded for potential toxicity.[19] Therefore, the development of multifunctional ZnO composite

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coatings with bioactivity, biocompatibility and osteoinductivity is an urgent need. Currently, the most commonly used methods for the preparation of ZnO composite coating are mixed heating, sol-gel and in-situ cationic polymerization. J.

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Sawa et. al.[20] mixed ZnO powder with polyvinyl alcohol (PVA) following heat treatment to prepare amorphous carbon coated ZnO-NPs (ZnOCC), but the composite

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coating prepared with this method showed chunks of crystals and aggregates of nanoparticles. J. Yu et. al.[21] synthesized HA/ZnO powder with sol-gel method and the composite coating exhibited crystal chunks with width of 1μm. A. Bhowmick et. al.[22] prepared composite material composed of organic montmorillonite-loaded chitosan (CTS) and HA-ZnO via in situ cationic polymerization. However, the composite material was not homogeneous and aggregates were shown. Monalisha et. al.[23] synthesized ZnO-HA composite material with chemical deposition method but nanoparticle aggregations also existed. All the methods mentioned above presented

unfavorable particle aggregates or agglomerates in the composites. This issue can be addressed by pulse electrochemical deposition method. Pulse electrochemical deposition is an effective way of controlling the reaction rate, due to the continuously repeated process of "power on - power off - power on" in the electrolytic cell when the pulse signal is applied. The chemical reaction on the electrode is intermittent, which is favorable for the diffusion and reduction of concentration polarization, greatly increase deposition efficiency and achieve better deposition results.[24] In this study, polypyrrole (PPy) is used as a regulator to achieve homogeneous dispersion of HA-NPs and ZnO-NPs on the surface of Ti substrate. PPy is a kind of

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conductive and biocompatible polymer that can be prepared by electrochemical method.[25] PPy nanoparticles (PPy-NPs) can synthesized by addition polymerization of pyrrole monomers (Py) on the α and β sites under electrochemical oxidation.[26] Anions such as PO43- could be doped in the main chain of PPy to neutralize its

electrical performance in the electrochemical oxidative polymerization.[27] PO43-

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doped PPy-NPs can be used as a template for HA nucleation and growth. Besides, the PPy molecule has an amine group that is widely used to adsorb metal ions such as

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Cr4+ [28], Co3+ [29], and Zn2+[30, 31], and form complex molecules with them. This indicates that PPy can be used as a stabilizer for ZnO-NPs to prevent its

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agglomeration. PPy-based regulator has the ability of enabling the uniform distribution of molecules. However, the current research is all based on the coordination of amine groups in the PPy molecule with metal ions, which is difficult

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to achieve co-deposition of two or more nano-coatings. This study is based on a pulse electrochemical method, in which PPy is used as a dual regulator of HA-NPs and ZnO-NPs. In the antibacterial system containing ZnO-NPs, a bioactive HA/ZnO

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nanocomposite coating was constructed on the surface of Ti through the coordination and doping of PPy with anions and cations. The nanocomposite coating was PPy-

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regulated, physiologically stable, antibacterial, osteoinductive, and able to promote angiogenesis. We constructed the nanocomposite coatings by controlling Py polymerization conditions such as pulse potential, Py monomer concentration and the concentration of Zn2+ and Ca2+. Firstly, Py monomer lost electrons and became a cationic radical on the surface of the electrode under the action of an electric field, which was subsequently combined with another monomer to form a Py dimer. The steps were repeated and the chain growth continued, leading the formation of a PPy macromolecular chain. In this process, PO43- in the electrolyte was doped into the

anion structure of the PPy macromolecular chain, and formed HA with Ca2+ and OHdue to electrostatic forces. Meanwhile, amine groups in the PPy chain were coordinated with Zn2+, producing a homogeneously distributed nanocomposite coating. The preparation of HA/PPy/ZnO nanocomposite coating, antibacterial test and the mechanism of promoting VEC growth is shown in Scheme 1.

2. Experimental Section 2.1. Materials and Instruments

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Ca(NO3)2·4H2O (AR) and Zn(NO3)2·6H2O were from Tianjin Shengao Chemical Reagent Co., Ltd., China. Py (98%) was from Sigma-Aldrich, US. Pure titanium was from Baoji INT Medical Titanium Co., Ltd., China.

X-ray powder diffractometer (XRD, D2-PHASER, 40kV, BRUKER, Germany)

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was used to characterize the crystal phase of the surface nanocomposite coatings on Ti. The thin film XRD (TF-XRD) test was conducted with X-ray sources of Cu-Kα

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(λ=1.5418), scanning rate of 0.5º·s-1, and scanning range of 20~60°. Scanning electron microscope (SEM, SU8010, HITACHI, 5.0 kV accelerating voltage, Japan)

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with energy dispersive spectroscope (EDS) was used to observe the surface microstructure and analyze elemental composition and distribution of materials, respectively. Atomic absorption spectrophotometer (AAS, Z2000, HITACHI, Japan)

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was used to perform quantitative analysis of material ion release. Thermogravimetric analyzer (TGA, STA449F3, NETZSCH) was used to quantify material weight losses and reveal its composition. Cell incubator (PBH-9082, Yiheng Technology, China)

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was used to perform bioactivity test and physiological stability test.

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2.2. Preparation of nanocomposite coatings A solution with Ca and P ratio of n(Ca)/n(P) = 1.67 (the concentration of Ca2+

and PO43- was 5.0 mmol·L-1 and 3.0mmol·L-1, respectively) was prepared. Then Zn(NO3)2 solution with various concentrations and Py solution with concentration of 2 ml·L-1 were prepared with deionized water, and the pH of the electrolytes were adjusted to 5.0 with ammonia. Titanium (10 mm×10 mm×1 mm), platinum and saturated calomel electrode was used as the working electrode, auxiliary electrode, and reference electrode, respectively. The three-electrode system was placed in the

electrolyte, and the sample was prepared by controlled electrodeposition on the titanium working electrode. The whole process was conducted for 2h at room temperature, with the pulse width of 100 seconds. The samples were collected after preparation, washed with deionized water and air dried. 2.3. Bioactivity test Samples was placed in a fast mineralization fluid (SCPS, composition is shown in Table S1) for 10 days and SCPS was changed every 24 hours. After the mineralization process, samples were air dried, washed with deionized water, and air

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dried again for XRD analysis and SEM characterization to evaluate crystal phase change, morphology change and bioactivity of the samples. 2.3. Physiological stability

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Immerse the composite coating in 40 mL phosphate buffered saline (PBS) at a constant temperature of 37 °C for 0.5 to 10 days, and observe the release profile of

Ca2+ and Zn2+. Each experimental group has 3 parallel samples placed in a centrifuge

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tube and each centrifuge tube is added with 40 mL PBS solution. At six time points (0.5, 1, 3, 5, 7, 10 d), the release solution in the centrifuge tube was removed, and new

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PBS solution was added in the centrifuge tube. AAS (Table S2) was used to determine the Ca2+ and Zn2+ in the release solution. The concentration of Ca2+ and Zn2+ in the release solution was calculated according to the standard curve (Fig. S1) to

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investigate the physiological stability of the composite coating.

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2.4. Antibacterial property test

The antibacterial property of the nanocomposite coatings was test with

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Escherichia coli and Staphylococcus aureus, and the results were analyzed qualitatively and quantitatively. Spread plate method was used for the qualitative analysis, while film adhering method was used for the quantitative analysis. Three groups of samples were studied for the antibacterial test including HA, HA/ZnO and HA/PPy/ZnO, and triplicate samples were tested for each group (n = 3). For the qualitative analysis, strains of Escherichia coli and Staphylococcus aureus were inoculated on MH agar medium, and activated at 37 °C for 12 hours. Then appropriate quantity of strains was acquired from the MH agar medium with an

inoculating loop and placed in liquid medium following 12 hours of culture to obtain bacterial suspension of each strain. The bacterial suspensions were then formulated into new suspensions with volume of 10 mL and concentration of 1.5×108 cell·mL-1 by the addition of physiological saline. The new suspensions were then incubated in a shaker at 37 °C and 200 r·min-1 for 24 hours before a volume of 100 μL was taken out from each, seeded on a plate and cultured at 37 °C for another 24 hours. The growth condition of the two strains was observed and photographed. For the quantitative analysis, the antibacterial rate of the sample was tested by surface adhering method. Briefly, both strains were inoculated on MH agar medium

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and incubated at 37 °C for 12 hours. This process was repeated three times to obtain purified colonies. Then single colonies of both strains were selected and inoculated in liquid media to culture at 37 °C and 200 r·min-1 in a shaker for 12 hours before being prepared into suspensions with concentration of 3.0×107 cell·mL-1 by the addition of

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PBS. The suspensions of the two strains were then diluted 103 times for the following tests. Samples to be tested were placed on a glass slide in a Petri dish, and sterile water was added to cover the bottom of the Petri dish in order to prevent bacteria

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suspension from evaporation. 50 μL of the bacteria suspension was dropped on the sample surface and cultured at 37 °C for 12 hours. The bacterial suspension was then

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transferred to 500 μL of PBS solution, shaken well to make homogeneous suspension, and 50 μL of the suspension was taken out and seeded on a plate. Bacteria were cultured on the plate for 12 hours and the number of colonies was counted. The

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antibacterial efficiency of the samples can be represented by calculating the antibacterial rate, and the calculation method is shown in equation (1), 𝐶𝐶𝐶𝐺−𝐶𝐶𝐸𝐺 𝐶𝐶𝐶𝐺

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Antibacterial rate =

× 100%

(1)

where CCCG is the colony count of the control group and CCEG is the colony count

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of the experimental group. 2.5. In vitro cytocompatibility test In vitro test of bone mesenchymal stem cells (BMSCs). BMSCs was extracted from the bone marrow of newborn SD rats (10 days old). The experiment protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Xinjiang Medical University. All the procedures were in

accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The experiment was divided into three groups: HA, HA/ZnO, and HA/PPy/ZnO, with 7 parallel samples in each group (n = 7). BMSCs were seeded on the surface of the samples at a concentration of 5.0×104 cell·mL-1 and cultured in 3.0 mL α-MEM at 37 °C in a CO2 incubator. (1) After 1, 3, 5, and 7 days of culture, the cells were fixed and dehydrated, and the morphology of the cells was observed. (2) After 1, 3, 5, and 7 days of culture, cell viability was also examined using CCK-8. Specifically, 100 μL of cell suspension was transferred to a 96-well plate and 10 μL of CCK-8 solution was added and the plate was incubated at 37 °C for 3 hours. The

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proliferation of the cells was quantified by measuring the absorbance at 450 nm by a microplate reader. (3) After 7 days of culture, cells were stained with FITC. Briefly, cells were fixed with paraformaldehyde, treated with Tritonx-100, and placed in

serum for 20 minutes. Finally, cells were stained with FITC at 37 °C for 1.5 hours

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followed by DAPI staining for 5 minutes.

In vitro test of VECs. VECs were extracted from the kidneys of newborn SD rats (3 days old). The experiment protocol was approved by the IACUC of the First

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Affiliated Hospital of Xinjiang Medical University. All the procedures were in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of

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Laboratory Animals. The experiment was divided into three groups: HA, HA/ZnO, and HA/PPy/ZnO, with 7 parallel samples in each group (n = 7). VECs were seeded on the surface of these samples at a concentration of 1.5×104 cell·mL-1 and cultured at

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37 °C in 3.0 mL DMEM in a CO2 incubator. (1) After 1, 3, 5, and 7 days of culture, the cells were fixed and dehydrated, and the morphology of the cells was observed. (2)

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After 1, 3, 5, and 7 days of culture, cell viability was also examined using CCK-8. Specifically, 100 μL of cell suspension was transferred to a 96-well plate and 10 μL of CCK-8 solution was added and incubated at 37 °C for 3 hours. The proliferation of

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the cells was quantified by measuring the absorbance at 450 nm by a microplate reader.

2.6. Statistical analysis All data are presented as mean ± standard deviation (SD) and a One Sample t Test was used for statistical analysis (assuming unequal variance). The difference between two set of data is considered statistically significant when p < 0.05.

3. Results and Discussions 3.1. Preparation of HA/PPy/ZnO composite coating During the preparation of the composite coating, the concentration of the electrolyte component and the redox potential had great influence on the composition and morphology of the composite coating. In our study, the effects of Py concentration, Zn2+ concentration and redox potential on the composite coating were investigated. Besides, the bioactivity, physiological stability, antibacterial property

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and cytocompatibility of the composite coatings were analyzed.

The effect of Py concentration on composite coating: When Py concentration was

0.01 mol·L-1, majority of the coating showed nano-needle shape (Fig. 1a), and the

reason was that the amount of PPy formed by electrochemical polymerization was too

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little to function as an effective template when Py concentration was in a low value. When the Py concentration increased to 0.03 mol·L-1, the coating was composed of

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relatively uniform spherical-shaped nanoparticles (Fig. 1b). The average diameter of the nanoparticles in the composite is 141.4 nm (Fig. S2a), And this was due to the

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oxidation to reduction reaction change near the cathode when the oxidation potential of 1.0 V decreased to the reduction potential of -2.5 V. At the meantime, PPy doped with PO43- was gathered near the cathode and the reduction potential was favorable

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for the nucleation growth of HA. Moreover, there was electrostatic interaction between PPy macromolecules, effectively preventing HA-NPs from agglomeration. When Py concentration increased to 0.05 mol·L-1, the coating was a nanorod network

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with uneven size distribution (Fig. 1c). This phenomenon revealed that when Py concentration was too high, the excess Py molecules would oxidize and polymerize

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into PPy macromolecules under the action of electric field before reaching the electrode microreaction region, and would not function as an effective template. Therefore, Py concentration for the effective production of PPy macromolecules using electrochemical polymerization method should be in an appropriate range. Its average thickness is 8.43 μm, 8.29 μm, 7.68 μm (Fig. S3). As can be seen from Fig. 1d, the diffraction peaks of ZnO and HA coincide at 31.7°, and the diffraction peaks of the HA (112) and (300) crystal planes appear at 32.3° and 32.9°, which is consistent with the standard card of ZnO (# 03-0888) and HA (# 74-0566). According to the

calculated crystallinity of Jade5 software, the crystallinity of the composite coating is 72%, 48%, and 68%, and it is higher at 0.01 mol·L-1, 0.05 mol·L-1 compared to 0.03 mol·L-1. According to literature report [32], the crystallinity is low and the diffraction peak is weak. Therefore, the diffraction peak of Ti substrate at 39° is relatively weak at 0.03 mol·L-1. The diffraction peak of HA did not show significant changes when the concentration of Py was increased, revealing the minimal effect of Py concentration on the crystallization of the substance. The effect of Zn2+ concentration on composite coating: In the electrochemical deposition process, Zn2+ in the electrolyte was reduced to obtain Zn the element. Zn

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was more active and able to be further oxidized to obtain ZnO that could improve antibacterial property of the coating. [33] When the concentration of Zn2+ was 0.3

mmol·L-1 and 0.6 mmol·L-1, the microstructure of the coating was in nano-spherical shape and nanorod shape, respectively (Fig. 1 f~g). After the addition of Zn2+ to the

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HA/PPy coating, the diameter of the spherical-shaped nanoparticles became smaller. Agglomeration appeared with the increase of Zn2+ concentration (0.6 mmol·L-1),

which leads to the increase of particle size (Fig. S2b). This was because that the PPy-

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Zn chelate formed by PPy and Zn2+ had a slower moving rate to the working electrode, thus reduced the reduction rate of Zn on the electrode surface. At the meantime, the

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electrostatic repulsion between the PPy macromolecules prevented the agglomeration of Zn-NPs. Zn was oxidized to ZnO by oxidation potential (Fig. S4) and ZnO was uniformly dispersed in the coating. According to the XRD data analysis (Fig. 1h), the

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number of diffraction peaks and peak intensity of HA composite coating increased along with the addition of Zn2+. However, as the concentration of Zn2+ continued to

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increase, peak intensity of HA composite coating was weakened, This is because the radius of Zn2+ (0.074 nm) is smaller than the radius of Ca2+ (0.099 nm) so Zn2+ can replace Ca2+ in HA lattice, resulting in a decrease in the lattice parameters of HA

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(Table S3) and affecting the crystallinity of HA [34]. When the Zn2+ concentration was increased from 0.3 mmol·L-1 to 0.6 mmol·L-1, the crystallinity of the composite coating decreased from 48% to 40%. The effect of redox potential on composite coating: The pulse potential also had a great effect on the PPy-controlled composite coating. The results showed that the composite coating was in uneven sized nano-spherical shape when the pulse potential was 1.0V/-2.0V (Fig. 1i). The as prepared coating was HA/PPy because Zn-NPs

would not form until the reduction potential reach - 2.4 V. When the pulse potential increased to 1.0V/-2.5V, the coating was still in nano-spherical shape while the size of the nanoparticles became uniform (Fig. 1j). However, when the pulse potential continued to increase to 1.0V/-3.0V, the coating was consistently in nano-spherical shape but the particle size became smaller and agglomeration occurred (Fig. 1k). The average diameters of the nanoparticles are 275 nm, 243 nm, and 105 nm (Fig. S2c). XRD analysis (Fig. 11) showed that the intensity of HA diffraction peak was strengthened as the pulse potential increased, indicating better crystallization. From the analysis above, the optimized experiment conditions were achieved

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with Py concentration of 0.03 mol·L-1, Zn2+ concentration of 0.3 mmol·L-1, and pulse potential of 1.0V/-2.5V. TGA results of the HA/PPy/ZnO composite coating prepared under these conditions (Fig. 1m) showed uniformly distributed PPy content of 22.3%, which contained elements such as Ca, P and Zn (Fig. 1n, p). Zn content in the

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composite coating was 2.1%, and the ratio of calcium to phosphorus was Ca/P = 1.78

(Table S4). The increase of Ca/P ratio was due to the deposition reaction of Zn2+ with PO43- that produced a precipitate of Zn3(PO4)2, which reduced the proportion of

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phosphate. In the infrared spectrum of HA/PPy/ZnO composite coating (Fig. 1o), the absorption peaks of C=C, C-H, and C-C bonds in PPy were observed at 1637 cm-1,

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1425 cm-1, and 973 cm-1, respectively. While the peak observed at 1066 cm-1 was the characteristic absorption peak of PO43- group of HA. In summary, PPy acted as a dual

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regulator to evenly distribute HA-NPs and ZnO-NPs in the coating.

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3.2. Bioactivity and physiological stability of nano-coating The bioactivity analysis of the HA/PPy/ZnO composite coating showed that the

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microscopic morphology of the composite coating before mineralization was nanospherical particles (Fig. 2a). After mineralization, the surface of the composite coating was deposited with a layer of flaky substance that was laid in a neat row and grew in the same direction. XRD showed that the deposited material was HA (Fig. 2b) and the number of diffraction peaks after mineralization was significantly increased. Besides, significant preferential growth occurred at the (002) crystal plane that was represented by the peak at 25.8°, that is, along the Z axis of HA. This result was consistent with the observation from SEM images. It has been reported that the mineralization of the

material in the simulated body fluid was accomplished by the self-assembled monolayers (SAMs).[35] In another word, the surface of the material reacted with the PO43-, OH- and Ca2+ in the solution repeatedly and attracted each other under the action of electrostatic force, enabling the formation of calcium phosphate initial nucleus and oriented growth to HA. In this study, the phenomenon that HA exhibited a significant preferential growth at 25.8° was probably caused by the higher concentration of Ca2+ and PO43- in the SCPS solution. This study has shown that HA/PPy/ZnO composite coatings had good bioactivity. Ca2+ can stimulate the osteogenesis of BMSCs, while the release of Zn2+ is a

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crucial factor influencing the antibacterial effect of composite coating. The release data of Ca2+ and Zn2+ from composite coatings showed that Ca2+ release was 87.0%

from the HA/ZnO coating while decreased to 81.9% from the HA/PPy/ZnO coating, and Zn2+ release decreased from 64.6% for HA/ZnO coating to 36.5% for

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HA/PPy/ZnO coating. This was due to the production of chelate formed by the amino group of PPy, Ca2+ and Zn2+, regulating the deposition of Ca2+ and Zn2+, and

uniformly depositing HA-NPs and ZnO-NPs on the surface of Ti substrate. Therefore,

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long-lasting antibacterial effect.

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the HA/PPy/ZnO composite coating had good physiological stability and enabled a

3.3. Antibacterial property test of the nano-coating

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The qualitative antibacterial data of the composite coating can be seen in Fig. 3a. There were countless colonies and colony groups formed on the surface of the HA coating. However, number of colonies on the surface of HA/ZnO composite coating

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was less compared to that on the surface of HA coating, while the number of colonies on the surface of HA/PPy/ZnO composite coating was even less. Without the

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addition of regulator PPy, HA/ZnO composite coating contained a large amount of agglomeration and uneven distribution of nanoparticles (Fig. S5), which made the contact area between the coating and bacteria smaller, resulting in lower antibacterial property when compared to HA/PPy/ZnO composite coating. The reduction of colony number was induced by the addition of the modulator PPy, which enabled the uniform distribution of ZnO-NPs and improved the antibacterial ability of the HA/PPy/ZnO composite coating. In the meanwhile, the quantitative analysis showed that the antibacterial rate of HA/PPy/ZnO composite coating was 63.5% against Escherichia

coli (Fig. 3b), and 72.8% against Staphylococcus aureus (Fig. 3c). Both the quantitative and qualitative experiment results revealed that the HA/PPy/ZnO composite coating had good antibacterial effect on Escherichia coli and Staphylococcus aureus. The reason was that the polarization state inside and outside the bacteria cell membrane could be changed by the positive charge carried by Zn2+, which hindered the material transport of the cells and killed the bacteria eventually.[12] In addition, ZnO-NPs formed H2O2 and ROS during the process. H2O2 further decomposed to form OH· and the synergistic effect of OH· and ROS would destroy the polysaccharide layer of cell membrane and cause the formation of

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membrane pores. ROS subsequently entered the cell, damage the organelles and genetic substance, leading to bacterial death.[11] 3.4. Cytocompatibility test of nano-coating

Human bones are supplied with nutrients from the capillaries. Therefore, VECs

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were cultured on the surface of the composite coating to investigate its angiogenic ability. As can be seen from Fig. 4a, the cells on the surface of HA and HA/ZnO

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coatings showed edge floating phenomenon while the cells on the surface of HA/PPy/ZnO coating began to adhere when VECs were cultured for 1 day. When

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VECs were cultured for 3 to 5 days, cells on HA coating surface still exhibited edge floating while cells on the surface of HA/PPy/ZnO and HA/ZnO coating showed good adhesion and spreading. When VECs were cultured for 7 days, cells on the surface of

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HA coating had a tendency of falling off while cells on the surface of HA/ZnO and HA/PPy/ZnO coating showed further adherence and spreading. The CCK-8 results of

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VECs (Fig. 4b) demonstrated that the viability of VECs on the surface of HA/ZnO, HA/PPy/ZnO coating was higher than that on the surface of HA coating. This could be due to the stimulation effect of ZnO on the proliferation and differentiation of

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VECs.[36] VECs viability on the surface of HA/PPy/ZnO coating was higher than that on the surface of HA/ZnO coating, and this was because that PPy was able to regulate the size of ZnO to nanometer scale and enable uniform nanoparticle distribution. Studies have shown that HA/PPy/ZnO coating had good cytocompatibility with VECs. BMSCs were cultured to investigate the osteogenic capacity of HA/PPy/ZnO coatings. The CCK-8 results (Fig. 4c) showed that the viability of the cells on the

surface of HA/ZnO and HA/PPy/ZnO composite coatings was significantly higher than that on the surface of HA coating. This was because the active site on the surface of ZnO-NPs promoted protein interactions in the initial stages of osteogenesis, thereby increasing the activity of osteoblasts.[14] Besides, the viability of the cells on the surface of HA/PPy/ZnO composite coating was higher than that on the surface of HA/ZnO composite coating. The SEM results of BMSCs cultured on the surface of the coating for 7 days showed that the cells on the surface of HA and HA/ZnO coatings exhibited edge floating, while cells on the surface of HA/PPy/ZnO composite coating spread well with pseudopods protruding (Fig. 4a). This was because that ZnO-

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NPs distributed evenly in the composite coating with the addition of PPy macromolecules, which increased the surface energy of the coating and further

improved the cytocompatibility. In order to investigate the osteogenic ability of BMSCs on the surface of HA/PPy/ZnO composite coating, we performed

immunofluorescence staining (FITC), and the results positively showed that the

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composite coating could promote the differentiation of BMSCs into osteoblasts,

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indicating good cytocompatibility and osteoinductivity.

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4. Conclusions

In this paper, pulse electrochemical in-situ oxidative polymerization method was used to produce PPy from Py. This method enabled the PPy dual regulation of HA-

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NPs and ZnO-NPs, successfully constructed a multifunctional titanium substrate with bioactive, physiological stable, antibacterial, angiogenic and osteoinductive

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HA/PPy/ZnO nanocomposite coating. The experimental conditions of the method were optimized to have a redox potential of 1V/-2.5V, with Py monomer concentration of 0.03 mol·L-1 and Zn2+ concentration of 3 mmol·L-1. The composite

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coating composed of uniformly distributed spherical-shaped nanoparticles. The bioactivity test showed that the composite coating could induce the formation of apatite in the simulated body fluid, and the apatite arrangement was neat and preferentially grew along the (002) crystal plane, showing good bioactivity. The ion release experiments showed that the dual regulation effect of PPy coordination and doping reduced the release rate of Ca2+ and Zn2+, which effectively improved the physiological stability of the composite coating. The antibacterial rate of

HA/PPy/ZnO composite coating against Escherichia coli and Staphylococcus aureus was 63.5% and 72.8%, respectively, indicating good antibacterial property. VECs adhered and proliferated well on the composite coating, indicating good angiogenic ability. BMSCs adhered, proliferated and differentiated well on the composite coating, and fluorescence staining experiments showed good osteoinductivity of the composite coating.

Author Contributions Section

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For this manuscript, Bakri Mamat, Naiyin Zhang, Ling Yan, Yingbo Wang, Chuang Ma and Tingjun Ye conceived and designed the experiments; Ling Yan, Jianghong Luo and Chaoming Xie analysed the data and prepared the figures; Bakri Mamat,

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Naiyin Zhang, Yingbo Wang, Chuang Ma and Tingjun Ye wrote the paper.

Conflict of interest

Acknowledgements

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The authors declare no competing financial interest.

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This work was supported by the National Natural Science Foundation of China (51662038, 81560350 and 81760397), Outstanding Youth Natural Science

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Foundation Project of Xinjiang (2017Q002), Supported by the “13th Five-Year” Plan

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for Key Discipline Chemistry, Xinjiang Normal University.

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Scheme 1. The preparation of HA/PPy/ZnO nanocomposite coating, antibacterial test

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and the mechanism of promoting vascular endothelial cell growth and angiogenesis.

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Fig. 1. Characterization of HA/PPy/ZnO composite coating. SEM micrographs of

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coating at Py concentration of (a) 0.01mol·L-1, (b) 0.03 mol·L-1 and (c) 0.05 mol·L-1; (d) XRD patterns of coatings at different Py concentrations; SEM micrographs of coating at Zn2+ concentration of (e) 0.0 mmol·L-1, f) 0.3 mol·L-1 and (g) 0.6 mol·L-1;

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(h) XRD patterns of coatings at different Zn2+ concentrations; SEM micrographs of coating at REDOX potential of i) 1.0/-2.0V, (j) 1.0/-2.5V and (k) 1.0/-3.0V; (l) XRD patterns of coatings at different REDOX potentials; (m) TGA diagram of

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HA/PPy/ZnO composite coating; (n) EDS diagram of HA/PPy/ZnO composite coating; (o) FTIR diagram of HA/PPy/ZnO composite coating; (p) EDS surface scan

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of HA/PPy/ZnO composite coating.

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Fig. 2. Biological activity and physiological stability data of HA/PPy/ZnO composite coating. (a) SEM micrographs and (b) XRD patterns of composite coating before and

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after mineralization; (c) Ca2+ and Zn2+ release profile.

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Fig. 3. Antibacterial test results of composite coatings. (a) Qualitative test of the

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composite coatings against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Quantitative analysis of the composite coatings against (b) E. coli and (c) S.

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aureus.

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Fig. 4. Cytocompatibility test results. (a) SEM micrographs of VECs and BMSCs cultured on the surface of different composite coatings; CCK-8 data plots of (b) VECs and (c) BMSCs cultured on different composite coatings for 1, 3, 5, and 7 days; (d) FITC diagram of BMSCs cultured on the surface of HA/PPy/ZnO coating for 7 days.