Bi-HA biphasic coatings on Titanium: Fabrication, characterization, antibacterial and biological activity

Colloids and Surfaces B: Biointerfaces 189 (2020) 110813

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Zn-HA/Bi-HA biphasic coatings on Titanium: Fabrication, characterization, antibacterial and biological activity

T

Qing Bia, Xian Songa, Yujia Chena, Yaping Zhenga, Ping Yina,*, Ting Leib,* a b

Centre of Stomatology, Xiangya Hospital, Central South University, Changsha, 410008, China State Key Laboratory of Powder Metallurgy, Central South University, Changsha, 410083, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Sol-gel method Zn-substituted Bi-substituted Antibacterial Biomedical

Hydroxyapatite (HA) coatings have been of important as biocompatible coatings for dental and bone tissue engineering application. However, the poor antibacterial performance and weak biological activity of HA coatings limited their clinical applications. As a strategy to improve the antibacterial performance and biological activity of HA, Zinc and bismuth ions were incorporated into HA lattice by substituting Ca2+ ions, respectively, and thus zinc substituted hydroxyapatite/bismuth substituted hydroxyapatite (Zn-HA/Bi-HA) biphasic coatings on titanium plates with various ratios were fabricated via sol-gel and dip-coating processes. The purity of the ZnHA and Bi-HA phase was confirmed by X-ray diffraction (XRD) test. The biphasic coatings showed slower dissolution rate than pure HA coating. Furthermore, the Zn-HA/Bi-HA coatings reveal good biomineralization activity in simulated body fluid (SBF) by forming regular spherical apatite agglomerates. Moreover, the biphasic Zn-HA/Bi-HA coatings exhibited that improved antimicrobial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as compared to pure HA coatings. The CCK-8 assays demonstrate Zn-HA/BiHA coatings showed no toxicity to MG63 cells, and the Zn-HA/Bi-HA2 (Zn-HA:Bi-HA=64:1) coating is more effective to enhance the proliferation of MG63 cells compared to other coatings. This finding suggests Zn-HA/BiHA biphasic coatings are promising candidates for biomedical applications.

1. Introduction Hydroxyapatite has been utilized as coating material for medical metal implants for about 40 years. Because it is very similar to the inorganic components of human teeth and bones and has excellent biocompatibility, which can promote the growth of bone tissue [1–3]. HA coating on titanium is widely used in orthopedics and dentistry as a way to improve the surface properties of implant materials. However, HA has drawbacks such as poor antibacterial properties [4] and weak biological activity, which restrict its clinical application. Additionally, it has been found that bone tissue infections at surgical sites are associated with gram-positive and gram-negative bacteria [5–7]. Infections may occur in several stages of the implantation process [8]. Antibiotics should be used in a sterile environment for a short period of time before clinical surgery begins. Accordingly, implant materials with antibacterial activity have drawn much attention in clinical treatments. The structure of HA exhibits flexibility in accommodating different ions such as monovalent ions (Na+, K+), divalent ions (Sr2+, Mg2+) and trivalent ions (Fe3+, Al3+). These ion substitutions affect the properties of the compounds such as adsorption of protein [9], ion exchangers

⁎

[10]. Some studies have been reported on the biological activity of Cu, Mg, Sr and Zn ions doped in HA lattice [11–13]. Accordingly, ionic substitution in HA provides a strategy to improve its antibacterial property and biological performance. Bismuth has been associated with medicine for more than a century [14]. Bismuth compounds, are clinically used in combination with antibiotics to treat Helicobacter pylori infection [15–17], Some studies demonstrated Bismuth doped calcium phosphate as antibacterial agent can significantly inhibit the growth of bacteria [18]. Zinc is essential for the growth and development of all species, as well as for protein synthesis and cell proliferation [19]. Zinc oxide is commonly used as a bandage ingredient in wound healing. which protects and relieves the skin around inflamed ulcers [20]. Zinc could inhibit osteoclast differentiation and promote activity of osteoblast [21,22], and thus further promote the early formation of bone tissue. Zinc deficiency has been reported to reduce bone mineral density and increase the risk of osteoporosis [23,24]. Zinc is also usually used as an antibacterial agent against infection [25]. Cytotoxicity-free is a critical factor for biological materials. High concentrations of zinc and bismuth could not only kill bacteria but also

Corresponding authors. E-mail addresses: [email protected] (P. Yin), [email protected] (T. Lei).

https://doi.org/10.1016/j.colsurfb.2020.110813 Received 3 November 2019; Received in revised form 10 January 2020; Accepted 18 January 2020 Available online 25 January 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved.

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2.4. Characterization of Bi-HA powders and coatings

lead to cytotoxicity on cell [26–29], so it is important to control Zinc and Bismuth ions at suitable low concentrations to ensure cytotoxicityfree of coatings. Previously, we have prepared the biphasic FHA/Sr-HA coatings and their biological and solubility properties have been investigated [30,31]. In the present work, new biphasic coatings consisting of Zn-HA as the matrix phase and the Bi-HA as the soluble phase were synthesized on Titanium plates via sol-gel and dip-coating processes. In such a constructed biphasic structure, the synergistic action of Zinc and Bismuth ions which could improve the antibacterial property and biological activity of the coatings.

In order to test the morphology of Bi HA powder and coating, a field-emission scanning electron microscope (Quanta FEG250 SEM) equipped with Energy Dispersive Spectrometer was used. The phase composition of the Bi-HA powders and all coatings were analyzed by Xray diffractometry (XRD:D/MAX-255) with Cu-Kα1 radiation. The tube voltage and the tube electric current of XRD were 40 KW and 250 mA respectively. 2.5. Adhesion strength of the coatings

2. Experimental Determination of the bonding strength between coating and substrate by micro scanning scratch tester (Shimadzu, SST-101). Using a spherical Rockwell C diamond tip with a diameter of 10 μ m, with a load from 0 to 30N. The tip scanned the coating at a speed of 2 μ m / s, with an amplitude of 50 μ m, perpendicular to the scratch direction. The load on which the coating completely peels off the substrate is called the "upper critical load". Which was used as one of the measurement methods of adhesion strength between coating and substrate. Five samples in each group were tested, and standard deviation was determined.

2.1. Materials Commercially pure titanium plates (99.9 % purity, Kermel Co, China) of 14 mm in diameter and 4 mm in thickness were used as the substrate materials. Chemical agents include Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O, Sigma-Aldrich, AR), bismuth nitrate pentahydrate (Bi(NO3)3.5H2O, Sigma-Aldrich, AR), Zinc nitrate hexahydrate (Zn(NO3)2.6H2O, Sigma-Aldrich, AR), phosphorous pentoxide (P2O5, Merck, GR), Diammonium hydrogen phosphate ((NH)2HPO4, Merk, GR).

2.6. Solubility of the coatings 2.2. Preparation of Bi-HA powders and Zn-HA sol-gel Aqueous tris(hydroxymethyl) aminomethane ((HOCH2)3CNH2, Tris) solution was applied to evaluate the solubility of the coatings. The cumulative concentration of ions dissolved from the coating can be utilized to measure the dissolution rate of the coating. 0.05 mol/l Tris solution was prepared in deionized water, and its pH value was adjusted to 7.25 by adding 1 mol / L hydrochloric acid. These samples were immersed in Tris solution at 37 ± 0.5 °C in a ration of 20 ml/cm2. After soaking for 7 days, 5 ml solution was extracted to measure Ca, P, Zn, Bi concentration by an inductively coupled plasma emission spectrometer (ICP-OES, JOBYVON 70 plus). Soaking each sample in triplicate.

Bismuth-substituted hydroxyapatite (Bi-HA) with 15 mol% Ca2+ replaced by Bi3+ is prepared by precipitation method, and Zinc-substituted hydroxyapatite (Zn-HA) with 1 mol% Ca2+ replaced by Zn2+ is prepared by sol-gel method, respectively. To prepare 15 % Bi-HA powders. Ca(NO3)2.4H2O and (NH)2HPO4 was dissolved in deionized water forming 1 mol/L solution, respectively. The pH of (NH)2HPO4 solution was maintained at 12 by adding NH4OH. Bi(NO3)3.5H2O was dissolved in 1 mol/L mixed solution of double distilled water and concentrated nitric acid (H2O: HNO3 = 2:1). The molar ratio of calcium ions to bismuth ions is maintained to be 85:15. The molar ratio of (Ca + Bi)/P is 1.67. (NH)2HPO4 was added dropwise into the mixed solution of Ca(NO3)2.4H2O and Bi(NO3)3.5H2O to form suspension. Which was stirred at 70 °C using a magnetic stirrer for 2 h, then the white precipitate was filtered and washed three times with deionized water, dried at 80 °C for 24 h and calcined at 700OC in muffle furnace for 2 h. To prepare 1 % Zn-HA, Ca(NO3)2.4H2O and (Zn(NO3)2.6H2O was dissolved into ethanol to form 4 mol/L solution, respectively, and P2O5 was dissolved into anhydrous ethanol to form 2 mol/L solution. The molar ratio of Ca(NO3)2.4H2O and Zn(NO3)2.6H2O was maintained to be 100:1. The molar ratio of (Ca + Zn)/P = 1.67. P2O5 solution was added dropwise to the mix solution of Ca(NO3)2.4H2O and Zn (NO3)2.6H2O. The solution was stirred at 60 °C for 30 min using a magnetic stirrer, stopped stirring and kept heating at 70OC for 90 min., Zn-HA sol was obtained by aging at room temperature for 24 h.

To evaluate the bio-mineralization of the coatings, in vitro tests were performed in simulated body fluid(SBF) (containing NaCl 8.0 g/L, KCl 0.4 g/L, CaCl2 0.14 g/L, NaHCO3 0.35 g/L, D-C6H6O6 0.35 g/L, MgSO4.7H2O 0.2 g/L, KH2PO4 0.1 g/L and Na2HPO4.12H2O 0.06 g/L) in accordance with ASTMG31-72. The pH value of solution was adjusted to 7.25 at 37 ± 0.5 °C with 1 mol/L HCL and Tris solution. The samples were immersed in the solution for 7 days. The solution should be replaced every day to avoid forming precipitation. After 7 days, the samples were dried at 80 °C for 6 h. The sample surface morphology after immersion was observed by field emission scanning electron microscopy.

2.3. Preparation of biphasic Zn-HA/Bi-HA coatings on Titanium plates

2.8. Antimicrobial activity

The titanium surface was polished with 80−1500 grit stiff paper coated with powdered emery, and cleaned with distilled water and acetone in an ultrasonic cleaner for 30 min. The purified samples were soaked in 8 mol/L NaOH solution for 12 h, washed with acetone for 30 min, washed with distilled water for 20 min, then dried at 80 °C for 8 h. The Bi-HA powder was added into Zn-HA / Bi-HA sol at 96:1, 64:1, 32:1 M ratio, and dispersed by ultrasonic for 30 min to form Colloidal mixture sol. The titanium plates were immersed in colloidal sol, extracted at a rate of 8 cm / min and dried at 80 °C for 24 h, calcined in a furnace at 700OC for 30 min. The as-prepared biphasic coatings were denoted as Zn-HA/Bi-HA1, Zn-HA/Bi-HA2 Zn-HA/Bi-HA3, respectively. As a control, pure HA and Zn-HA coatings were fabricated by the same process.

The antibacterial activity of the coating samples was determined by Escherichia coli (ATCC25922) and Staphylococcus aureus (CMCC26003). Before bacterial culture, samples are autoclaved at 121 °C for 20 min. To prepare bacterial stock solution, the bacterial stock solution is made by the continuous oscillation of bacteria in Luria Bertani (LB) medium at room temperature for overnight growth. The original bacterial solution was diluted to the final concentration of 105 CFU / ml in physiological saline, and 100 μl bacterial solution with the concentration of 105 CFU / ml was inoculated on each sample and cultured at 37℃. After 24 h, 1 ml physiological saline was dipped on the samples, then the samples were shaken continuously for 10 min. After that, 100 μl bacterial suspension was extracted from the bacterial culture plate and inoculated into the standard agar medium, and culturing

2.7. Mineralization of the coatings in simulated body fluid

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smaller Zn2+ ions substitute Ca2+ ions results in the HA lattice shrinkage. In contrast, the replacement of Ca2+ ion by Bi3+ions with a larger ionic radius (0.108 nm) results in the HA lattice expansion. The crystallinity of hydroxyapatite changes with the addition of zinc or bismuth ions, which is in agreement with previous reports [32–34]. The result suggested that Zinc ions and bismuth ions have been successfully substituted Ca2+ ions in the hydroxyapatite lattice respectively. Fig. 2(c) shows the XRD analysis of pure HA and biphasic Zn-HA/Bi-HA coatings derived from colloidal sols after heat treatment of each coating at 700 °C for 30 min. It is worthy to note that the content of Bi-HA in biphasic coating increased from 1/96 to 1/32 mol fraction, and all coatings still exhibit the identical XRD patterns of Zn-HA with no presence of impurity phase. Which suggested that a small amount of Bi-HA nano powders added into colloidal sol has no effect on lattice parameters.

for 24 h at 37 °C. 2.9. In vitro biocompatibility All samples were sterilized by autoclaving at 121 °C for 20 min. In this study Human osteosarcoma MG-63 cells (ATCC, Rockville, MD) were used to evaluate the osteoblastic cell response on coating surfaces. The cells were seeded onto the samples in 24-well plates at a density of 104 cells/ml. The base medium for this cell was Dulbeccos modified Eagle’ medium (DMEM, GIBCO) supplemented with 10 % fetal bovine serum (FBS,GIBCO), and 1 % penicilin/streptomycin (GIBCO). After 24 h of culture, the samples to be tested were taken from the culture. All sample for SEM observation were fixed with 2.5 % paraformaldehyde in 0.1 M cacodylate buffer for 24 h at 4 °C. The fixed samples were dehydrated in an ethanol series (30 %,50 %, 70 %,80 %,90 %,100 %), then followed by a hexamethyldisilane (HMDS) drying procedure. The cell morphology was observed by SEM after gold coating the surface.

3.3. Surface morphology and adhesion strength of the coatings The SEM morphology of the coating observed in the SEM photo is as shown in Fig. 3. The surface morphology of pure HA and Zn HA coating is relatively smooth with a few cracks, as shown in Fig. 3(a, b). Compared with pure HA coating, more cracks were observed on the surface of all biphasic coatings and the roughness of the coating is slightly higher than that of HA coating. These cracks may be caused by shrinkage of the coating during 700 °C drying. From the figure we can observe that gradually increasing the amount of Bi-HA leads the coatings to be rougher, as indicated by the arrow, more aggregates may be due to the thickening effect of adding Bi-HA nano particles. From a close observation, the main component of these coatings are nano apatite particles.

2.10. CCK-8 assays Cell proliferation was tested by Cell Counting Kit-8 (CCK-8). These cells were inoculated on the surface of the sterilization coatings of the 24 well plate and cultured at a density of 1 × 104 cells / ml for 1, 3 and 5 days. During each incubation period, the samples were removed from the old plate and transferred to the new 24 well plate. Phosphate buffered saline (PBS) was used to wash the surface of the sample three times to remove the nonadherent dead cells, then the mixture of 500 μl DMEM and 50 μl CCK-8 were added into each pore and cultured in 37 ℃ carbon dioxide incubator for 3 h. 100 μl of the mixed solution was extracted from the plate holes of each sample and transferred to the 96well plate, which was read by the plate reader at 450 nm. The viability of osteoblasts can be indirectly reflected by the calculation of OD value of each specimen. One-way ANOVA was used for statistical analysis and p < 0.05 was considered statistically significant. Four duplicate samples were used in CCK-8 analysis to ensure the repeatability. The data were expressed as mean ± standard deviation.

3.4. Dissolution behavior of coatings Tris solution contains one cation H + and one anion Cl - without other ionic components, so as to avoid the interference of chemical reactions of other ions [35]. The solubility of the coating in Tris solution is shown in Fig. 4. After immersion for 7 days, ICP was used to monitor the cumulative concentration of Ca2+, PO43−, Zn2+, Bi3+ ions from the coatings. As is shown in Fig. 4, pure HA shows the fastest dissolution rate, while the dissolution rate of pure Zn HA coating is the lowest among all coatings. Interestingly, the Zn2+ ions concentration released from biphasic coating increased with the increase of Bi-HA content, while the Ca2+, PO43− ions concentrations are suppressed. It has been demonstrated that the presence of zinc ions can reduce the dissolution rate of apatite [36]. Along with the dissolution of calcium, phosphate ions and zinc ions into Tris solution, the precipitation of a hopeite-like phase on the coating surface occurred according to reaction (1), which suppressed the further release of calcium and phosphate ions from the apatite [37]. Accordingly, the release of calcium and phosphate ions concentration decreases along with the increase of zinc ion concentration in the solution.

3. Results and discussion 3.1. Surface morphology and element analysis of Bi-HA powders Fig. 1(a) shows that the of Bi-HA powders is nanosized spherical and rod-like particles with a size of about 50−80 nm. This nano-sized structure is benefit for its well dispersion in colloid sol for biphasic coating preparation. The inset EDS analysis revealed the Bi-HA is consisted of 62.11 at% oxygen, 14.29 at% phosphorus, 20.23 at% calcium and 3.36 at% bismuth elements. 3.2. XRD analysis of HA, Zn-HA, Bi-HA and biphasic coatings The structure and phase composition of HA, Zn-HA, Bi-HA were investigated by XRD analysis. As could be seen in Fig. 2(a), the XRD pattern of HA exhibits high intensity peaks, which are well matched with the standard JCPDS card (JCPDS 09-0432) for HA. Notably, the XRD patterns of Zn-HA and Bi-HA showed the same characteristic diffraction peak of HA while no other impurities are observed. Additionally, the diffraction peaks of Zn-HA become wider and less intensive when the doped Zn content is 1at.%. while the diffraction peaks of 15 at% Bi-HA become narrower and more intensive. Moreover, the high-intensity three diffraction peaks of (002), (102) and (210) planes between 24 and 30 degrees are magnified as shown in Fig. 2(b). It is observed that (002) peak of Zn-HA significantly shifted to higher 2theta angle in comparison to the HA sample. while the (002) peak of BiHA apparently shifted to lower 2-theta angle. Given that the ionic radius of Zn2+ (0.074 nm) is smaller than that of Ca2+ ions (0.099 nm), a

3Zn2+ +2H2PO4− +4H2O → Zn3(PO4)2. 4H2O + 4H+

(1)

3.5. Bio-mineralization In vitro bio-mineralization of coating in SBF solution is contribute to simulate the process of bone deposition in vivo [38,39]. Fig. 5 shows the surface morphologies of coatings after soaking in SBF solution for 7 days. The surface morphology of the coating shows that there is a new layer formed on the surface. close observation indicated irregular agglomerates of apatite deposited on the pure HA coating surface, while regular spherical agglomerates of apatite were observed on the surface of pure Zn-HA and biphasic coatings, which are about 1−10 μm in diameter. The depositions on the biphasic coating surfaces, especially 3

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Fig. 1. Scanning electron microscope (SEM) images(a) and Energy Dispersive Spectrometer (EDS) analysis (b) of Bi-HA nano powders. The scale bar of image is 500 nm. Oxygen, phosphorus, calcium and bismuth elements were present in EDS analysis.

phenomenon is attributed to initially the less negative surface charge of the coatings due to a poverty in surface hydroxyl and phosphate groups after sintered at high temperature (700 °C). The doping of zinc and bismuth in hydroxyapatite results in the deposition of spherical aggregates of apatite with different morphologies and sizes on the surface of the coatings, indicating the biphasic coatings of Zn-HA/Bi-HA possess excellent biomineralization capability.

on Zn-HA/Bi-HA3 coating (Fig. 5e1) appear to be more regular and circular in shape than that on Zn-HA coating (Fig. 5b1), likely the addition of Bi-HA affects the nucleation of apatite deposited on the coating surface. The effect of zinc on the nucleation of apatite on the coating surface is reported [40]. As we all know, the nucleation and growth of apatite begin from immersion, and the volume of apatite will gradually increase with the increase of immersion time [41]. The formation and shape of apatite on the coating surface are controlled by the specific surface area, structure and variety of ions. It is demonstrated that there are a lot of negative surface charges on the surface of apatite coating, which can interact with the positive ions (Ca2 +) in the solution to form calcium rich Amorphous calcium phosphate (ACP). The coating surface shows positive charge, and then interact with negative phosphate ions (OH−, PO43-) in solution to form calcium-poor ACP, the above processes are stacked layer by layer due to the change of charge. which is stable by crystallization into bone-like apatite [42]. This

3.6. Antibacterial activity Peri implant inflammation caused by bacterial infection is the main cause of implant failure. Therefore, endowing implants with antibacterial activity is an effective way to reduce the incidence of inflammation, so as to promote the success probability of clinical surgery. The gram-negative bacteria E. coli and gram-positive bacteria S. aureus were usually utilized to evaluate the antibacterial effect of samples. The

Fig. 2. X-ray diffractometry (XRD) patterns of HA, Zn-HA, Bi-HA and biphasic coatings. The structure and phase composition of HA, Zn-HA, Bi-HA Fig. 2(a). the highintensity three diffraction peaks of planes between 24 and 30 degrees were magnified Fig. 2(b). The phase composition of powders scraped from the HA and biphasic coatings Fig. 2(c). 4

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Fig. 3. Scanning electron microscope (SEM) images of HA (a, a1), Zn-HA (b, b1), Zn-HA/Bi-HA1(c, c1), Zn-HA/Bi-HA2 (d, d1), and Zn-HA/BiHA3 (e, e1) coatings. (a–e)Scale bars represent 5 μm, and (a1-e1) are higher magnification(500 nm) of the images.

the control. The result of present study demonstrated that the biphasic coating containing higher content Bi-HA exhibited higher bactericidal effect on E. coli and S. aureus bacteria. The antibacterial ability of coatings may be due to the toxicity of zinc and bismuth ions to bacteria. Zinc ion and bismuth ion have different antibacterial mechanisms. Compared to single ion antibacterial mechanism, A variety of different antibacterial mechanisms working at the same time can better inhibit the growth of bacteria. The mechanisms of bactericidal effect of Zn, Bi ions are as follows: firstly, the Zinc ions released from coating, then bind to the protein and inactivates it; secondly, Zinc Ions interact with the cell wall of bacteria, resulting in changes in the structure and permeability of the cell wall; finally, these ions interact with the nucleic acid of bacteria to prevent them from migrating elsewhere [43]. In addition, as for the antibacterial mechanism of bismuth ion, which can form a precipitation membrane on the surface of bacteria, then directly inhibit the growth of bacteria, and inhibit protease, lipase, phospholipase and other enzymes secreted by bacteria [44]. In summary, the interaction of zinc and bismuth ions with bacteria can inhibit the growth and maturation of bacteria, fix bacteria and reduce the formation of bacterial membrane on the surface of biomedical devices. Regarding the lower release rate of Bi3+ ions from biphasic coating, it is reasonable to suggest that Zn2+ ions released from biphasic coating play an important role in antibacterial activity for E. coli and S. aureus bacteria. This conclusion is supported by the higher dissolution rate of Zn-HA/BiHA2 ad Zn-HA/BiHA3 coating as demonstrated by ICP analysis. It is worthy to note that Other researchers' experiments show that Zn-HA or Bi-HA possess excellent antibacterial effect [45,46]. Accordingly, the above result indicates that the doping of zinc and bismuth ions in hydroxyapatite lattice and their synergistic effect improved the antibacterial ability of coatings.

Fig. 4. Ionic concentration of Ca, P, Zn, and Bi released from HA, Zn-HA and biphasic costings. in Tris solution after 7 days soaking. Results were expressed as the means ± standard of Soaking each sample in triplicate.

antibacterial property of pure HA, Zn-HA and three biphasic HA coatings against the E. coli were presented in Fig. 6(a1-a6). The results showed that the number of E. coli colonies in the nutrient agar medium of Zn-HA coated samples was significantly lower than that of pure hydroxyapatite samples. Moreover, the number and size of E. coli colonies in the nutritional agar medium of Zn-HA/Bi-HA1 sample are significantly reduced compared to Zn-HA sample. Fig. 6(a3-a4) indicate the colony numbers changed from six to four, and the size of the single colony reduced significantly. No colonies of E. coli in the nutritional agar medium for Zn-HA/BiHA2 and Zn-HA/BiHA3 coatings were observed, indicating that Zn-HA/BiHA2 and Zn-HA/BiHA3 could effectively kill E. coli colonies. Fig. 6(b1-b6) shows the antimicrobial activity against S. aureus bacteria and represents number of colonies for pure hydroxyapatite (HA), Zn-HA and biphasic Zn-HA/Bi-HA. The S. aureus bacteria were obviously inhibited by Zn-HA/BiHA2 and Zn-HA/BiHA3 as indicated in Fig. 6(b5, b6). Zn-HA/Bi-HA3 coating exhibits the highest antibacterial activity followed by Zn-HA/BiHA2 compared to

3.7. Biocompatibility of biphasic coatings SEM micrographs of HA, Zn-HA, Zn-HA/Bi-HA1 Zn-HA/Bi-HA2 and Zn-HA/Bi-HA3 coatings after 24 h culture (Fig. 7) showed the morphological characteristics of MG63 cells. The cells attached to the coating surface in the shape of long fusiform. The dysfunctional cells 5

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Fig. 5. Scanning electron microscope (SEM) images of HA (a, a1), Zn-HA (b, b1), Zn-HA/Bi-HA1(c, c1), Zn-HA/Bi-HA2(d, d1), and Zn-HA/Bi-HA3(e, e1) coatings after soaking in simulated body fluid (SBF) for 7 days. (a–e)Scale bars represent 50 μm, and (a1-e1) are higher magnification(10 μm) of images.

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Fig. 6. Optical photos of plate counting antibacterial(Escherichia coli and Staphylococcus aureus) test of control (a1, b1), HA (a2, b2), Zn-HA (a3, b3), Zn-HA/BiHA1(a4, b4), Zn-HA/Bi-HA2(a5, b5), and Zn-HA/Bi-HA3(a6, b6) coatings. After 24 h of culture on the coatings, the bacteria were transferred to the agar medium and cultured in 37 °C incubator for 24 h. The number and size of colonies were compared with the blank control.

Calcium, phosphorus, zinc, bismuth ions, whose concentrations are non-toxic effect on cells.

are always spherical, and no dysfunctional cells could be observed. The cell morphology observation demonstrated a good cell viability and a high biological affinity of biphasic coatings, which implies that the concentration of calcium, phosphorus, zinc and bismuth in the biphasic coating is beneficial to the growth of cells on its surface. It is well known that Calcium and phosphorus are necessary elements for cell growth. The biphasic coatings with different Bi-HA content in Zn-HA matrix phase provides the possibility of controlling the release of

3.8. CCK-8 assays The results of the CCK-8 assay for MG63 cells cultured on the coating for 1, 3 and 5 days are shown in Fig. 7B. The test results show that OD value increases significantly with time. When the cells were

Fig. 7. A)Scanning electron microscope (SEM) images of coating surface upon cell cultivation on HA(a), Zn-HA(b), Zn-HA/Bi-HA1(c), Zn-HA/Bi-HA2(d), and Zn-HA/ Bi-HA3(e) coatings. fixed with 2.5 % paraformaldehyde in 0.1 M cacodylate buffer for 24 h at 4 °C. The fixed samples were dehydrated in ethanol, followed by a hexamethyldisilane (HMDS) drying procedure. Gold spray treatment of coating surface. Scale bar is indicating 50 μm in each image. B)CCK-8 assays of MG63 cells cultured on HA, Zn-HA, Zn-HA/Bi-HA1, Zn-HA/Bi-HA2, and Zn-HA/Bi-HA3 coatings for 1, 3, 5 day. Four duplicate samples were used in CCK-8 analysis, the data were expressed as means ± standard deviation. statistical difference between HA and biphasic coatings were significant at *p < 0.05 after 3 days. 7

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Declaration of Competing Interest

inoculated on the coating for one day, the OD 450 value of the spectrophotometer was about 0.23. On the first day, there was no significant difference in cell growth between HA and biphasic coatings. After 3 or 5 days of incubation, OD450 value was significantly enhanced for the biphasic coatings. Compared with the pure HA coating, the synergistic effect of ions released from the biphasic coatings is beneficial to cell survival and proliferation. The CCK-8 assay observation demonstrates that biphasic coatings are noncytotoxic, and could stimulate cell proliferation compared to pure HA coatings. Meanwhile, the OD value of Zn-HA/Bi-HA2 coating is relatively high after 5 days of culture, which shows it has better proliferation ability compared with other coatings. Implying that the surface properties of Zn- HA/Bi-HA2 coating and the concentration of various ions released from the coating can better promote cell proliferation. There may be an optimal concentration of calcium, phosphorus, zinc and bismuth ions for cell proliferation. Zinc containing calcium phosphate has been demonstrated to promote the formation of new bone [47]. FHA coating combined with zinc ion can enhance the activity of osteoblasts [48]. Prasad et al. have shown that zinc is involved in the synthesis of DNA and some important proteins [49]. However, the excessive concentration of zinc ions has toxic effects to cells, thus inhibiting the activity of some protease [50]. The actual mechanism in which how Bi ions interacts with the cell is unclear and need to be investigated further. Additionally, Cell proliferation was significantly enhanced after 3 and 5 days of cell culture compared with 1 day, indicating that the promotion of various ions on cell proliferation was a long-term process. The biological activity of the coatings can be adjusted by altering the phase composition of the coating and the proportion of different ions. In general, Bi-HA nanoparticles were dispersed in Zn-HA sol colloidal matrix that considered to be an effective way to promote osteoblast coating interaction, so as to promote cell proliferation.

None. Acknowledgment This work was supported by innovative research and development project of Hunan Province (Grant No.2019412). References [1] M. Yoshinari, Y. Oda, T. Inoue, K. Matsuzaka, M. Shimono, Bone response to calcium phosphate-coated and bisphosphonate-immobilized titanium implants, Biomaterials 23 (2002) 2879–2885, https://doi.org/10.1016/s0142-9612(01) 00415-x. [2] T. Hayakawa, M. Yoshinari, H. Kiba, H. Yamamoto, K. Nemoto, J. Jansen, Trabecular bone response to surface roughened and calcium phosphate (Ca-P) coated titanium implants, Biomaterials 23 (2002) 1025–1031, https://doi.org/10. 1016/s0142-9612(01)00214-9. [3] G. Darimont, R. Cloots, E. Heinen, L. Seidel, R. Legrand, In vivo behaviour of hydroxyapatite coatings on titanium implants: a quantitative study in the rabbit, Biomaterials 23 (2002) 2569–2575, https://doi.org/10.1016/s0142-9612(01) 00392-1. [4] B. 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4. Conclusion In the present work, low concentration Zn and Bi ions substituted HA were synthesized via sol-gel process. The biphasic Zn-HA/Bi-HA coatings on titanium plates were fabricated successfully by using Zn-HA as the matrix phase and Bi-HA as the soluble phase. Adding nano Bi-HA powder to Zn-HA matrix can adjust the phase composition of the twophase coating. The presence of a small amount of Bi-HA in the biphasic coatings had minor effect on the phase of the coatings. It is found that the biphasic coatings have lower solubility than pure HA coating. Immersion test of biphasic coating samples in SBF solution reveals their good biomineralization capacity. Zn-HA/Bi-HA2 and Zn-HA/Bi-HA3 biphasic coatings show satisfied antibacterial activity against E. coli and S. aureus bacteria. Zn-HA/Bi-HA2 biphasic coating exhibits the most positive effect on osteoblast proliferation. In summary, the development of antibacterial and bioactive function of biphasic Zn-HA/BiHA coatings makes them potential in bone tissue engineering. Credit author statement I have made substantial contributions to the conception or design of the work, analysis, or interpretation of data for the work. I have drafted the work or revised it critically for important content, and I have approved the final version to the published, and I agree to be accountable for all aspects of the work in ensuing that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons who have made substantial contributions to the work reported in the manuscript, including those who provided editing and writing assistance but who are not authors, are named in the acknowledgments section of the manuscript and have given their written permission to be named. Authors: Qing Bi, Xian Song, Yujia Chen, YapingZheng, Ping Yin, Ting Lei 8

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