Flame sprayed zinc doped hydroxyapatite coating with antibacterial and biocompatible properties

Author’s Accepted Manuscript Flame sprayed zinc doped hydroxyapatite coating with antibacterial and biocompatible properties Yung-Chin Yang, Chien-Chung Chen, Jhong-Bo Wang, Yen-Ching Wang, Feng-Huei Lin www.elsevier.com/locate/ceri

PII: DOI: Reference:

S0272-8842(17)31158-6 http://dx.doi.org/10.1016/j.ceramint.2017.05.318 CERI15478

To appear in: Ceramics International Cite this article as: Yung-Chin Yang, Chien-Chung Chen, Jhong-Bo Wang, YenChing Wang and Feng-Huei Lin, Flame sprayed zinc doped hydroxyapatite coating with antibacterial and biocompatible properties, Ceramics International, http://dx.doi.org/10.1016/j.ceramint.2017.05.318 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Flame sprayed zinc doped hydroxyapatite coating with antibacterial and biocompatible properties

Yung-Chin Yanga,*, Chien-Chung Chenb, Jhong-Bo Wanga,

Yen-Ching Wangc, Feng-Huei Lind

a

Department of Material and Mineral Resources Engineering, National Taipei University of Technology, Taipei, Taiwan

b

Graduate Institute of Biomedical Materials & Tissue Engineering, Taipei Medical University, Taipei, Taiwan

c

Materials Analysis Technology Incorporated (MA-TEK)

d

Department of Biomedical Engineering, National Taiwan University, Taipei, Taiwan

* Corresponding author: Tel.:+886-2-27712171 ext. 2762; fax:+886-2-27317185; E-mail: [email protected] (Yung-Chin Yang)

Abstract

Hydroxyapatite (HA) has good biocompatibility, high strength and low fracture toughness. It is often used as the main material for artificial bones in bioceramics because of the similar composition. In this study, we prepared zinc-doped hydroxyapatite by the co-precipitation method to produce artificial bone material has antibacterial properties, for use in post joint surgery to inhibit bacterial infection or proliferation and ultimately the incidence of osteomyelitis. The co-precipitation method prepared hydroxyapatite with different concentrations of zinc chloride. The experimental results revealed that after sintering, it has some new phases generated of ZnO and CaZn2(PO4)2 when we add zinc chloride that concentration reaches 0.15 M. The minimum inhibition zone revealed the antibacterial ability of Zinc-HA increased when the concentration of zinc chloride increased from 0.0125 M to 0.15 M. The granulated powder of zinc-doped hydroxyapatite was coated on the titanium substrate by the flame-spraying method. The Zinc-HA coating was found with good biocompatibility and antibacterial ability in the biological and antibacterial test.

Keywords: A: Powders: chemical preparation, D: Apatite, E: Biomedical applications

1

1. Introduction

Hydroxyapatite (HA) is the main inorganic component in the human skeleton which accounts for 60-70% of the total bone content. It has been reported that the synthesized hydroxyapatite has good biocompatibility and has been used clinically orthopedic and dental aspects, such as bone filling [1]; and hydroxyapatite approved alloy used for artificial joints [2]. Other widely used metallic materials such as titanium alloy and stainless steel have shown to have good mechanical properties, and used in bone repair, artificial tooth root and artificial joint implant. However, they have poor biocompatibility and often resulted in loosening or failure of the implant after a long duration. If the hydroxyapatite is applied to the surface of metal dental or orthopedic implants, the new bone can form a chemical bond between the hydroxyapatite and increase the affinity between the implant and the organism. Thus it would lead to the increase in stability of the implant at the initial implantation [3,4].

Zinc ions are found in all tissues of the human body, and most of the zinc is stored in the skeleton (0.012-0.025 wt. %). In many biochemical reactions, it plays many roles such as activation of enzymes, nuclei acid metabolism, protein synthesis, maintaining the structure and function of the cell membrane and the metabolism of hormones and so on. In addition, zinc intercalation of hydroxyapatite can promote the

2

deposition of biological apatite and inhibit osteoclast bone resorption; and stimulate new bone formation. Therefore, the addition of zinc phosphate and hydroxyapatite are of great interest to many researchers [5-7]. As biomedical materials would be directly used in the body, the pre-sterilization and preservation of antibacterial properties are very important. The antibacterial properties of biomedical materials can be extended to its application, and antibacterial properties also prevents infection.

Commercially, nano-silver particles have been widely used with great success. However, with the degreadation of the HA, nano-silver particles with low toxicity are released. In the present study, zinc-doped HA is replaced by zinc in the calcium in HA, zinc ions are slowly released and have antibacterial properties when HA degrades. Compared with the addition of nano-silver nanoparticles, zinc ions are non-toxic and more stable than the nano-silver. In addition, the price of HA doped with zinc is lower than that of adding nano silver.

In this study, zinc-doped HA (ZHA) was synthesized by the co-precipitation method. The ZHA coating was deposited on the Ti-6Al-4V alloy substrate by the flame spraying method. This study is aimed to synthesize zinc-doped hydroxyapatite coating into good physical properties and antibacterial ability.

3

2. Materials and Methods

2.1. Synthesize HA powder by the co-precipitation method

In

this

experiment,

we

prepared

hydroxyapatite

powder

by

using

co-precipitation method. Chemical equation as eq. 1:

10 Ca (OH) 2 + 6H3PO4 → Ca 10(PO4)6(OH) 2 + 18H2O

(1)

0.15 M Phosphoric acid (H3PO4, Showa, 16163150, Japan) titrated to 0.25 M Calcium hydroxide (Ca(OH) 2, Sigma, 31219, USA). Ca(OH) 2 provided Ca ions, while H3PO4 provided P ions. The pH was adjusted to 9.0 with ammonia aqueous. Whole blending process was stirred for 4 hours of 70°C~90°C. After completion of the reaction, the solution was left alone for 24 hours without heating. The upper clear solution was removed, and the remaining slurry was dried in an oven. Finally, the grinding powder is the experimental powder.

Zinc chloride with different concentrations was added into Zinc-doped hydroxyapatite in the 0.25M Ca(OH)2 solution. The concentrations of Zinc chloride (ZnCl2, Alfa Aesar, A16281, USA) solution were 0.0125M, 0.025M, 0.05M, 0.1M, and 0.15M. In this study, we have attempted to find the best concentration for this process formula.

4

2.2. Flame spraying technique

Hydroxyapatite powder particles prepared by co-precipitation method were very small and easy to agglomerate. In order to facilitate spraying, the powder should be in granulation form. The granulation step was: 9% Polyvinyl alcohol (PVA, Sigma, P1763, USA) was added to the powder and mixed uniformly, and then dried in an oven. After drying, the massive hydroxyapatite was screened with a set of sieves. The upper to lower layers of the sieve were 80, 120, 200 mesh, and the powder in the middle layer was 120 mesh sieve (the diameter was about 120 m) which is original materials for spraying.

Before coating on Ti-6Al-4V alloy substrate, sand blasting was carried out to improve the substrate surface roughness (Ra = 6 m) which would inproved the coating and adhesive strength. The HA coating was prepared by flame spraying (CastoDyn DS 8000), using a fuel atmosphere of acetylene and oxygen, respectively. The process use of neutral flame or carbon flame spraying, and the working distance was kept at 10 cm or 20 cm. XRD, SEM, roughness and porosity measurement were carried out on the basis of four spray parameters results.

5

2.3. Analysis of Material Properties

The surfaces of HA coatings were coated with gold and the surface morphology was examined by secondary electron image (SEI) of SEM (S-4700, Hitachi, Japan). For the measurement of thickness and observation of cross-sectional microstructure, the specimen was cross-sectioned with a low-speed diamond saw and mounted by the epoxy resin. The specimen was then grounded, polished, coated with carbon, and examined using backscattering electron image (BEI) of SEM. The cross-sectional photographs were transferred to a computer image file for quantitative analysis of the volumetric porosity content using a computer image analyzer. To avoid inducing extra pores or cracks, the specimens for cross-sectional views were ground and polished carefully. The structure of the HACs was examined by X-ray diffractometer (Rigaku Geigerflex D/Max-VB, Japan). In this study, the component properties of powder are also examined by XRD at different sintering temperature.

2.4. Analysis of antibacterial ability

Qualitative Antibacterial assessment method was determined by the size of the inhibition zone. In this method, the bacteria concentration and the size of the round specimen were immobilized (diameter: 10 mm; thickness: 2 mm). The radius of the 6

inhibition zone around the test piece was compared. The larger radius of the inhibition zone, the better the antibacterial effect. The experimental process is as follows: The plate of medium was first solidified, and the bacteria were put on the solidified medium of Escherichia coli DH5α (E. coli) to isolate a single colony. The selected strains were placed in liquid medium and incubated at 37°C for 16 h to 18 h. Then the broth was homogeneously mixed with the solid medium (Former broth: solid medium = 1：15), and poured into dishes. The specimen was positioned in the center after the medium solidified. Finally, the inhibition zone of the test specimen was observed and the antibacterial effect of the test specimen was compared.

Quantitative antibacterial method is based on JIS Z2801 ： 2000 of the antibacterial testing specifications. The concentration 1~5×105 CFU/ml of bacteria was inoculated on the test specimen for a specific time (0.5 h, 1 h, 2 h and 3 h), and the bacterial suspension shook with the 10ml Phosphate buffered saline (PBS, Gibco, 21300058) for 5 min as a stock solution. After serial dilution, the broth was incubated in a solid plate incubator at 37°C for 16h to 18 h. Finally, colony forming units (CFU) was counted from the culture dishes to determine the antibacterial ability. The inhibition rate is calculated by the Eq. 2：

Inhibitory rate (%) = [(A-B)/A] × 100%

7

(2)

A：The number of colonies in the control group

( CFU/ml )

Ｂ：The number of colonies in the sample group

( CFU/ml )

2.5. Biocompatibility Test

The WST-1 cell activity assay was performed in accordance to ISO 10993-5: Cytotoxicity test. The preparation method comprises the following steps: ZHA powder was sterilized and immersed in medium (Dulbecco's Modified Eagle Medium, DMEM, Gibco, 12648010) for one day, and the pH value was adjusted to about 7.0. Cells were placed on 96 well cell culture plates for one day with Trypsin-EDTA (Hyclone) for two days prior to the experiment. One day before the experiment, 200 μl of the material extract was placed in a 96-well microtiter plate. Then the background group and the control group was set.200 μl of the cell culture solution was added to the background group. At this time medium solution contained 1% fetal bovine serum (FBS, Hyclone) and 1% antibiotic antimycotic solution (Sigma, A5955). Cytotoxicity tests were performed to measure cell light absorbance (O.D. value, absorbance proportional to cell viability) by the ELISA reader (Tecan, Trading AG, Switzerland, Wavelength = 450 nm).

8

The Lactate dehydrogenase (LDH, Clontech, 630117) cytotoxicity test procedure was done with WST-1. During the addition of the agent to the cell culture plate, equal volume of LDH (50 μl / well) was added to another 96-well microtiter plate in 50 μl form 200 μl of cell culture medium. Then the absorbance was measured by ELISA reader (wave length = 490 nm), and the highest toxicity value was determined by the Total lysis group.

3. Results and Discussion

3.1. Characterization

Hydroxyapatite was prepared by co-precipitation method, and it was then doped by different zinc concentration. XRD analysis as shown in Fig.1 showed the hydroxyapatite powder can be successfully prepared by co-precipitation method of the reaction temperature of 70 ~ 90 ° C and pH value of 9.0. When the concentration of zinc chloride was increased from Zn1-HA (0.0125 M) to Zn5-HA (0.15 M), the phase of the hydroxyapatite gradually changed into the Scholzite phase. This was due to the reaction process of zinc oxide (ZnO) generation, and the amount of zinc oxide increased with the

9

concentration of zinc chloride added. Ca[Zn(OH)3]2．2H2O was synthesized by the reaction of zinc oxide with calcium hydroxide (Ca(OH)2). This intermediate reacts with phosphoric acid (H3PO4) to form the phosphosilicate (CaZn2 (PO4)2．2H2O), the final product. The reaction equations are shown in Eqs. 3 and 4：

Ca(OH)2 + 2ZnO + 4H2O → Ca[Zn(OH)3]2．2H2O

Ca[Zn(OH)3]2．2H2O + 2H3PO4 →CaZn2 (PO4)2．2H2O + 6H2O

(3)

(4)

ZHA powder synthesized by co-precipitation method is used in flame spraying process, so the material must be investigated for the reaction temperature. ZHA was calcined at 560°C, most temperature of coating and substrate is lower than 500°C under thermal spraying. The phase identification results are shown in Fig. 2. It is found that the zinc chloride concentration of 0.05 M (Zn3-HA) still have hydroxyapatite phase in this sintering temperature. When the concentration is higher than 0.05 M, the zinc oxide and calcium/zinc phosphate occur with hydroxyapatite phase. When the concentration reaches 0.15M (Zn5-HA), ZnO and CaZn2(PO4)2 becomes the main phase. The reaction is shown in eq. 5:

CaZn2 (PO4)2 ．2H2O

CaZn2 (PO4)2．H2O

10

CaZn2 (PO4)2

(5)

The experimental results show that Zn3-HA (below 0.05M) can be stabilized in high temperature with HA and it can be applied to the process of flame spraying.

3.2. Microstructures of the Zinc-doped HA powder

Hydroxyapatite synthesized by co-precipitation method and the surface morphology of Zn-doped hydroxyapatite powder with different concentrations were shown in Fig. 3. The morphology of the grains is fine and needle-like. It is found that when the zinc doping concentration was increased and the population of needle-like grain increased. The grain size was about 50 nm ~ 200 nm.

The elemental identification of hydroxyapatite and different concentrations of Zn-doped hydroxyapatite powder was carried out by energy dispersive spectroscopy (EDS). Powder Ca, P, Zn atomic percentage semi-quantitative the results is shown in Table 1. EDS data showed that the higher the amount of zinc chloride added, the higher the concentration of Zn doped in HA. When the Zn content increases, Ca content decreases, P content also decreases slightly and the Ca/P ratio was also decreased. Because of the addition of ZnCl2 concentration, Zn ions will replace the Ca ions to form zinc-doped hydroxyapatite.

11

3.3. Antibacterial properties of zinc-doped HA

Qualitative and antibacterial analysis of the powder was carried out by the inhibition zone method. Bacteria concentration and specimen size were fixed, and the scope of coverage of inhibition zone was observed. It is found that the greater the scope of their antimicrobial performance is better. The results are shown in Fig. 4. Antimicrobial activity is increased with the increase of zinc doping concentration. No calcination and three calcination-treated powders (320°C, 440°C and 560°C) show this behavior. In Fig. 4, it is also found that the antibacterial properties of Zn4-HA and Zn5-HA specimen increases significantly. The reason was presumably due to the obvious formation of zinc oxide phase. However, the HA phase in Zn4-HA and Zn5-HA was replaced by zinc phosphate and zinc oxide phases. In this study, Zn3-HA (0.05 M) was chosen as the raw material for the preparation of flame sprayed zinc-doped hydroxyapatite coatings.

12

3.4. Analysis of coating properties by flame spraying

In this study, commercial HA powder was sprayed by flame spraying. Four flame spray parameters were used to prepare the HA coating and investigate the effect of the spray parameters on the coating properties. XRD pattern is shown in Fig. 5. Reducing the O2/C2H2 supply ratio and shortening the working distance significantly enhance the (00,2n) (n=1,2,3…..) diffraction peak intensity. It is shown that the formation of hexagonal HA is mainly on the (00l) plane. The crystal lattice grows along the c-axis, and there was anisotropic crystallization observed. From the cross-sectional views of Fig. 6, it can be observed that the working distance of 20 cm powders melting is likely good for adhesion. It is observed that the film crack of the working distance of 20 cm is significantly reduced compared to the working distance of 10 cm. It is presumed that a longer working distance would help to improve the strength of the film, and it is found that using carbon flame spray exhibits less holes than the neutral flame in layers. Studies also found that the carbon flame is easier to make the powder attached to the substrate.

Based on the above results, ZHA coating was prepared by using Zn3-HA powder with carbonization flame and 20cm spraying distance. The XRD of flame sprayed ZHA coating was shown in Fig. 7. Ca3 (PO4)2 (TCP) phase appears at high

13

temperature, and it is found that the TCP phase becomes more obvious as the spraying time increases. This is due to the phase change of hydroxyapatite that cause damage to OH- at elevated temperatures. The chemical equation is shown in Eq. 6:

Ca10 (PO4)6(OH) 2

3Ca3 (PO4)2 +CaO+H2O

(6)

The microstructure observation of the ZHA coating is shown in Fig. 8. ZHA coating is found to have many small holes formed near the pore network melting structure (Fig. 8(a)). Fig. 8(b) shows the cross-sectional structure of the ZHA coating. It is found that there are many holes in the interior of the coating stacked during the spraying, which is consistent with Fig. 8(a). ZHA was a porous structure of the melting method coated on the substrate. Thus it results in a large number of pores inside the film. ZHA coating porosity is about 26%, and commercial HA (Sulzer HA) coating porosity is 16.1%. The porosity of homemade ZHA coating is much larger than that of commercial HA coating. It was shown that zinc doped

hydroxyapatite

has a large number of pores in the coating due to its melt-coating in a porous manner. This is useful for bone tissue growth, and the increased specific surface area results in the antibacterial properties.

14

3.5. Antibacterial and biocompatibility of ZHA coating

After spraying the ZHA coating, a quantitative antibacterial analysis was carried out for four specific times (0.5 h, 1 h, 2 h, 3 h) with reference to the antibacterial test specification of JIS Z2801: 2000. The growth condition of the colony was shown in Fig. 9, and the antibacterial effect was shown in Table 2. It was found that the antibacterial effect was enhanced with the time of contact of the test specimen with the bacterial solution, and the antibacterial rate reached 99.9% after 3 h. This was due to the sustained release of Zn ions from the specimen.

The 3T3 cells were cultured in a 96 well of a powder extract to test and compare whether the material was helpful for cell growth and the absorbance value would be proportional to the number of viable cells. WST-1 cell activity test results were analyzed by an ELISA reader at a wavelength of 450 nm as shown in Fig. 10. Cells in commercial HA, homemade HA, ZHA are compared with the control group. The cell growth number was shown increased, proving that the synthesis of the powder for the cell activity was helpful in this study. In addition, the LDH test measured the number of dead cells. The number of dead cells was higher with the measured value. The enzyme was analyzed at 490 nm using an enzyme-linked immunosorbent assay (ELISA reader). As shown in Fig. 11, It was found that

15

commercial HA, homemade HA, ZHA of the LDH values were much lower than the total lysis group. This represents that the powder synthesized does not produce substances that affect cell death in this study. Therefore, the material does not have toxicity for the cells, and exhibits good biocompatibility.

4. Conclusions

In this study, Zinc-doped hydroxyapatite has been synthesized successfully by the co-precipitation method, and the grain size was about 50 nm to 200 nm. Zinc doping concentration of 0.05M (Zn3-HA) exhibited

good antibacterial and also

avoided the production of calcium zinc phosphate phase while maintaining the stable phase of hydroxyapatite. 0.05 M zinc-doped HA was prepared on a Ti-6Al-4V alloy for ZHA coating by the flame spraying. In the qualitative and quantitative antibacterial tests, show significant inhibition zone. Because of the persistent release of Zn ions, the antibacterial effect was enhanced with the time of contact of the test specimen with the bacteria solution, and the antibacterial rate reaches 99.9% after 3 h. ZHA coating was tested by the WST-1 cell activity assay and LDH cytotoxicity assay. The number of cells grew normally and not considered toxic to the cells. It reveals the good biocompatibility of ZHA coating.

16

Conflict of Interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Acknowledgement

The first and second authors contribute equally to this work. Authors would like to thank the financial support from National Taipei University of Technology - Taipei Medical University Joint Research Program (NTUT-TMD Joint Research Program: NTUT-TMU-100-13).

17

References

[1] M. Jarcho, Calcium phosphate ceramics as hard tissue prosthetics, Clin. Orthop. Rel. Res., 157 (1981) 259–278. [2] P.M. Pilliar, H.U. Cameron, A.G. Binnington, J.A. Szvek, Bone ingrowth and stress shielding with a porous surface coated fracture fixation plate, J. Biomed. Mater. Res., 13 (1979) 799–810. [3] J.B. Park, R.S. Lakes, Biomaterials an introduction, Plenum Press, New York, 1992. [4] J.B. Park, Biomaterials Science and Engineering, Plenum Press, New York, 1985. [5] A. Ito, K. Ojima, H. Naito, N. Ichinose, T. Tateishi, Preparation, solubility, and cytocompatibility od zinc-releasing calcium phosphate ceramics, Journal of Biomedical Materials Research, 50 (2000) 178–183. [6] H. Kawamura, A. Ito, S. Miyakawa, P. Layrolle, K. Ojima, N. Ichinose, T. Tateishi, Stimulatory effect of zinc-releasing calcium phosphate implane on bone formation in rabbit femora, Journal of Biomedical Materials Research, 50 (2000) 184–190. [7] B.S. Moonga, D.W. Dempster, Zinc is a potent inhibitor of osteocalstic bone resorption in vitro, Journal of Bone and Mineral Research, 10 (1995) 453–457. [8] A.H. Melcher, Summary of biological considerations, Journal of Dental Education, 52 (1988) 812–814 [9] L.L. Hench, Bioceramics: from concept to clinic, Journal of American Ceramic

18

Society, 74 (1991) 1487–1510. [10] C.A. Vacanti, J.P. Vacanti, Bone and cartilage reconstruction of Tissue Engineering, R.G. Landes Co, New York, 1997. [11] G. Klaas de , Bioceramics of calcium phosphate, CRC Press Inc, Florida, 1983. [12] S.K. Yen, C.M. Lin, Cathodic reactions of electrolytic hydroxyapatite coating on pure titanium, Material chemistry and phys, 77 (2003) 70–76. [13] C.F. Feng, K.A. Khor, E.J. Liu, P. Cheang, Phase transformations in plasma sprayed hydroxyapatite coatings, Scripta Material, 42 (2000) 103–109. [14] Y.

Hu,

X.

Miao,

Comparson

of

hydroxyapatite

ceramics

and

hydroxyapatite/borosilicate glass composites prepared by slip casting, Ceramic international, 30 (2004) 1787–1791. [15] M.R. Saeri, A. Afshar, M. Ghorbani, N. Ehsani, C.C. Sorrell, The wet precipitation process of hydroxyapatite, Materials Letters, 57 (2003) 4064–4069. [16] J.M. Jarcho, C.H. Bolen, M.B. Thomas, J. Bobick, J.F. Kay, R.H. Doremus, Hydroxylapatite synthesis and characterization in dense polycrystalline form, Journal of Materials Science, 11 (1976) 2027–2035. [17] J. Zhou, X. Zhang, J. Chen, S. Zeng, G. Klaas de, High temperature characteristics of synthetic hydroxyapatite, Journal of Materials Science-Materials in Medicine, 4

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(1993) 83–85. [18] M.J. Root, Inhibition of the amorphous calcium phosphate transformation reaction by polyphosphonates and metal ions, Calcified Tissue International, 47 (1990) 112– 116. [19] Y. Okamoto, S. Hidaka, Studies on calcium phosphate precipitation : Effects of metal ions used in dental materials, Journal of Biomedical Materials Research, 28 (1994) 1403–1410. [20] A. Bigi, E. Foresti, M. Gandolfi, M. Gazzano, N. Rovri, Inhibiting effect of Zinc on hydroxyapatite crystallization, Journal of Inorganic Biochemistry, 58 (1995) 49–58. [21] N. Kanzaki, K. Onuma, G. Treboux, S. Tsutsumi, A. Ito, Inhibitory effect of magnesium and zinc on crystallization kinetics of hydroxyapatite (0001) face, Journal of Physical Chemistry B, 104 (2000) 4189–4194.

20

List of Table Table 1 Compositions of zinc-doped HA powder by the EDS analysis (at%) Table 2 Antibacterial ability of the ZHA coating

List of Figures Fig. 1. XRD patterns of Zinc-doped HA powders Fig. 2. XRD pattern of ZHA powder calcined at 560°C Fig. 3. Surface morphology of the HA powder (a)HA, (b)Zn1-HA, (c) Zn2-HA, (d) Zn3-HA, (e) Zn4-HA, (f) Zn5-HA Fig. 4. The inhibition zone after inhibition test of the ZHA powder specimens Fig. 5. XRD patterns of commercial HA coating prepared by flame spraying Fig. 6. Surface morphology of the HA coating by flame spraying (a) neutral flame & SD=10cm, (b) carbon flame & SD=10cm, (c) neutral flame & SD=20cm, (d) carbon flame & SD=20cm Fig. 8. Cross-sectional morphology of commercial HA coating prepared by flame spraying (a) neutral flame & SD=10cm, (b) carbon flame & SD=10cm, (c) neutral flame & SD=20cm, (d) carbon flame & SD=20cm Fig. 7. XRD pattern of the flame sprayed ZHA (Zn3-HA) coating Fig. 8. Microstructures of the flame sprayed ZHA (Zn3-HA) coating, (a) morphology, (b) cross-sectional view. Fig. 9. Quantitative colony of the flame sprayed ZHA coating Fig. 10. WST-1 cell activity test Fig. 11. LDH cytotoxicity test

21

Table 1 Compositions of zinc-doped HA powder by the EDS analysis (at%) Ca

P

Zn

Ca / P

HA

60.92

39.08

x

1.56

Zn1-HA

55.24

41.28

3.48

1.34

Zn2-HA

52.74

41.18

6.08

1.28

Zn3-HA

50.36

41.06

8.58

1.23

Zn4-HA

48.18

40.18

11.08

1.21

Zn5-HA

42.05

37.28

20.67

1.13

Table 2 Antibacterial ability of the ZHA coating 0.5h

1h

2h

3h

5x105

2.6x106

6.2x106

8.8x106

ZHA ( CFU/ml )

1x105

7.5x104

2.5x104

200

Inhibition rate (%)

80.0

97.1

99.6

99.9

Control ( CFU/ml )

22







(004)

  

CaZn2(PO4)2 . 2H2O

(222) (132) (213)





(212) (310)





(202) (512)



(211) (420) (112)

(020) (002) (220) (221) (102) (210)

(311)

Relative intensity (Arbitrary units) 20



 Ca10(PO4)6(OH)2

 

 Zn5-HA Zn4-HA Zn3-HA Zn2-HA Zn1-HA HA

24

28

32

36

40

44

48

52

2(degree)

Fig. 1.

XRD patterns of Zinc-doped HA powders

23

56

60

20

(002)

　 Ca10(PO4)6(OH)2





　 ZnO CaZn2(PO4)2













(213)







(222) (102)





(310)

(002) (121)



(100) (211) (113)

(101)



(111) (020)

Relative intensity (Arbitrary units)





Zn5-HA Zn4-HA Zn3-HA Zn2-HA Zn1-HA HA

25

30

35

40

45

50

55

2(degree)

Fig. 2.

XRD pattern of ZHA powder calcined at 560°C.

24

60

Fig. 3. Surface morphology of the HA powder (a) HA, (b) Zn1-HA, (c) Zn2-HA, (d) Zn3-HA, (e) Zn4-HA, (f) Zn5-HA

25

Initial

320°C

440°C

560°C

HA

Zn1-HA

Zn2-HA

Zn3-HA

Zn4-HA

Zn5-HA

Fig. 4.

The inhibition zone after inhibition test of the ZHA powder specimens

26

(004)

(213)

(222)

(212) (310)

(211) (112) (300) (202)

(102) (210)

(002)

Relative intensity (Arbitrary units) 20

Carburizing flame Stand-off distance = 10 cm

Neutral flame Stand-off distance = 10 cm Carburizing flame Stand-off distance = 20 cm Neutral flame Stand-off distance = 20 cm PDF#09-4320 HA

25

30

35

40

45

50

55

60

2(degree)

Fig. 5.

XRD patterns of commercial HA coating prepared by flame spraying

27

Fig. 6. Cross-sectional morphology of commercial HA coating prepared by flame spraying (a) neutral flame & SD=10cm, (b) carbon flame & SD=10cm, (c) neutral flame & SD=20cm, (d) carbon flame & SD=20cm

28

(170) (211) (122)

 Ca3(PO4)2 　  ZnO CaZn2(PO4)2

20

25



30

35

(212) (222) (222) (213)



(131)





(310)



 

(101)



(210) (122)



(002)





(132)



　  Ca10(PO4)6(OH)2

(113) (043) (080)

(202)



(150)

Relative intensity (Arbitrary units)







40

45



50

55

60

2(degree)

Fig. 7.

XRD pattern of the flame sprayed ZHA (Zn3-HA) coating

29

Fig. 8. Microstructures of the flame sprayed ZHA (Zn3-HA) coating, (a) morphology, (b) cross-sectional view.

30

Control

ZHA

0.5h

1h

2h

3h

Fig. 9. Quantitative colony of the flame sprayed ZHA coating

31

0.6 DAY1

450 nm ( O. D. )

0.5

DAY3

0.4

0.3

0.2

0.1

0.0

Control

Commercial HA

Fig. 10.

HA

WST-1 cell activity test

32

ZHA

0.7 DAY1

490 nm ( O. D. )

0.6

DAY3

0.5 0.4 0.3 0.2 0.1 0.0

Fig. 11. LDH cytotoxicity test

33