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Synthesis, characterization, antimicrobial activity and mechanism of a novel hydroxyapatite whisker/nano zinc oxide biomaterial
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015001 (http://iopscience.iop.org/1748-605X/10/1/015001) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 132.203.227.61 This content was downloaded on 29/12/2014 at 07:28
Please note that terms and conditions apply.
IOP Publishing
10.1088/1748-6041/10/1/015001
Biomed. Mater. 10 (2015) 015001
Paper
received
14 September 2014
Synthesis, characterization, antimicrobial activity and mechanism of a novel hydroxyapatite whisker/nano zinc oxide biomaterial
accep ted for publication
22 October 2014 published
22 December 2014
Jian Yu1,2, Wenyun Zhang1,5, Yang Li1,2, Gang Wang1, Lidou Yang1, Jianfeng Jin3, Qinghua Chen4 and Minghua Huang4 1
Department of Stomatology, Kunming General Hospital of Chengdu Military Region, #212 Daguan Road, Xishan District, Kunming, Yunnan Province, People’s Republic of China 2 Clinical College of Kunming Medical University, Kunming, People’s Republic of China 3 Kunming Municipal Stomatological Hospital, Kunming, People’s Republic of China 4 Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming, People’s Republic of China 5 Author to whom correspondence should be addressed. E-mail: [email protected] Keywords: hydroxyapatite whisker, zinc oxide, antibacterial mechanism, biomaterial
Abstract Postoperative infections remain a risk factor that leads to failures in oral and maxillofacial artificial bone transplantation. This study aimed to synthesize and evaluate a novel hydroxyapatite whisker (HAPw) / nano zinc oxide (n-ZnO) antimicrobial bone restorative biomaterial. A scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) and x-ray diffraction (XRD) were employed to characterize and analyze the material. Antibacterial capabilities against Staphylococcus aureus, Escherichia coli, Candida albicans and Streptococcus mutans were determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), and kinetic growth inhibition assays were performed under darkness and simulated solar irradiation. The mode of antibiotic action was observed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). The MIC and MBC were 0.078–1.250 mg ml−1 and 0.156–2.500 mg ml−1, respectively. The inhibitory function on the growth of the microorganisms was achieved even under darkness, with gram-positive bacteria found to be more sensitive than gram-negative, and enhanced antimicrobial activity was exhibited under simulated solar excitation compared to darkness. TEM and CLSM images revealed a certain level of bacterial cell membrane destruction after treatment with 1 mg ml−1 of the material for 12 h, causing the leakage of intracellular contents and bacteria death. These results suggest favorable antibiotic properties and a probable mechanism of the biomaterial for the first time, and further studies are needed to determine its potential application as a postoperative anti-inflammation method in bone transplantation.
1. Introduction Postoperative infections remain a severe risk of artificial bone transplantation in oral and maxillofacial regions, which leads to failures and psychological trauma for patients [1–3]. An increasing number of experiments involving hard tissue substitutes loaded with antibiotics were investigated to provide antimicrobial efficacy against relevant pathogens [4–6]. Nevertheless, the presence of certain disadvantages associated with the composite constrains its application. It was reported that bone cement as a drug carrier had limited drug release and imperfect biodegradation, and might bring about necrosis between the surface of the cement and the surrounding tissue [7, 8] as well as dose-dependent © 2015 IOP Publishing Ltd
cytotoxicity from the additives [9]. Therefore, employing bone repair materials possessing excellent biological and mechanical performance incorporated with appropriate antibacterial agents was imperative for clinical work. Hydroxyapatite (HAP), a pervasively used bioceramic, constitutes the major component of skeleton and teeth in human body and is supposed to have enormous potential on account of its favorable biocompatibility and osteoinductive activity [10, 11]. HAP whisker (HAPw) is a type of monocrystal fiber with high purity and modulus, principally utilized for reinforcing the biomechanical properties of HAP [12, 13]. Despite being a promising artificial bone substitute, the independent antibiotic capability of HAPw is scarcely
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
investigated. Acknowledged as a representative of inorganic antimicrobial biomaterials, zinc oxide (ZnO) has been given great significance in recent years because of the superiority of its broad antibacterial spectrum, biosecurity, durability and stability when compared to organic antimicrobial biomaterials [14–16]. Furthermore, continuous studies with respect to ZnO nanostructures were carried out to indicate the probable antibiotic mechanism and photocatalysis of ZnO [17–19]. However, the exact antimicrobial mechanism of ZnO nanoparticles against a wide range of microorganisms is still an extensive field of research that is generally unexplored. In this paper, our objective is to prepare a novel biomaterial that couples the ideal mechanical and biological functions of HAPw with the superior antibacterial ability of ZnO. A sol–gel technology was applied to synthesize a hydroxyapatite whisker/nano zinc oxide (HAPw/n-ZnO) biocomposite in which nanometer-sized ZnO were fused on the surface of HAPw. The properties of the composite were characterized and analyzed using SEM, EDS and XRD. Both gram-positive and gram-negative bacteria were considered for antibacterial assays, including determination of MIC, MBC and kinetic growth inhibition under darkness and simulated solar irradiation. The mode of antibiotic action on the tested microorganisms was evaluated using TEM and CLSM. These results were employed to discuss the antimicrobial activity and possible mechanism of HAPw/n-ZnO biomaterial for the first time.
2. Materials and methods 2.1. Preparation of the materials HAPw was provided by Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Yunnan, China. Zinc acetate [(CH 3COO) 2Zn·2H 2O], polyethylene glycol 6000 (PEG-6000), anhydrous ethanol, aqueous ammonia and glacial acetic acid were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd, Tianjin, China. The HAPw/n-ZnO biomaterial was synthesized using a sol–gel technique. First, zinc acetate and PEG-6000 powders were dissolved into anhydrous ethanol solution then bathed in water for 2 h at 70 °C in clarified liquid obtained with a rotary evaporator, which was the precursor product [Zn5(OH)8(Ac)2·2H2O] of nanometer zinc oxide. Subsequently, HAPw (after drying for 24 h at 80 °C), with a weight ratio of 2:1 of zinc acetate and PEG-6000, was dissolved into anhydrous ethanol solution to obtain a dispersion liquid by means of ultrasonic oscillating and magnetic stirring. Third, with magnetic stirring at room temperature, the clarified liquid was titrated slowly into the dispersion liquid with a constant flow pump, while simultaneously applying aqueous ammonia or glacial acetic acid to maintain PH = 6.4. After titration, the mixed solution was transferred to the rotary evaporator and bathed in 2
water for 6 h at 70 °C, followed by ultrasonic dispersion for 20 s and heating to 78–80 °C to be dehydrated, then proceeding with thorough desiccation in a thermostatic drying oven. Finally, the obtained mixture was heated to 800 °C at 2 °C per min and sintered for 1 h at 800 °C. After being cooled naturally to an ambient temperature, the composite powders were obtained. 2.2. Characterization of the composite The surface microstructure of HAPw and the composite powder were observed using a scanning electron microscope (SEM, FEI Quanta 200, USA), and the contents of the relevant elements in the composite were spot analyzed with energy dispersive spectroscopy (EDS). To determine the components and structure of the composite powder, x-ray diffraction (XRD) analysis was performed with an x-ray diffractometer (D/max2200, Rigaku Corporation, Japan), which measured Cu-Kα (λ = 0.15406 Å) radiation in the 2θ range from 0° to 90° with a scanning speed of 4° per min at 36 kV and 30 mA. 2.3. Microorganisms and culture conditions The following microorganisms were used in this experiment. Gram-negative: Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231) and Streptococcus mutans (ATCC 25175). Gram-positive: Escherichia coli (ATCC 8739). These bacterial strains were obtained from the Department of Microbiology and Immunology, Kunming Medical University, Yunnan, China. S. aureus and E. coli were grown in nutrient agar medium and C. albicans and S. mutans were grown in sabouraud dextrose agar (SDA) medium and tryptone peptone yeast (TPY) agar medium, respectively. S. aureus, E. coli and C. albicans were aerobic cultured at 37 °C for 24–48 h, while S. mutans was under anaerobic condition (80% N2, 10% H2 and 10% CO2). All microorganisms were maintained by subculture at 4 °C in a refrigerator. The media used in the research were all purchased from Qingdao Hope Bio-Technology Co., Ltd, Shandong, China. 2.4. Antimicrobial assay of the HAPw/n-ZnO biocomposite The antimicrobial activity of the HAPw/n-ZnO biocomposite was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To evaluate the MIC, HAPw and commercial n-ZnO (20–50 nm in diame ter, Kunming Nuob ei Biotechnology Co., Ltd, Yunnan, China) were used for comparison. The three samples were suspended in sterile distilled water and prepared for suspension at the required concentrations of 20–0.039 mg ml−1 prior to the experiment, using the method of two-fold serial dilutions [20]. Bacterial suspensions with a turbidity equivalent to a 0.5 McFarland standard were prepared and then further diluted to 1:10 twice to obtain a final concentration of 1.5 × 106 colony-forming units
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
(CFU) ml−1. We added 1 ml of suspensions of three samples with each concentration into separate sterile test tubes containing 0.9 ml of the corresponding broth medium inoculated with 0.1 ml of each bacterial suspension, then allowed it to culture overnight at 37 °C. The tubes were checked for visible turbidity after 24–48 h of culturing for the tested microorganisms and were referenced by positive and negative growth control tubes. The positive control tube was free of the three samples and the negative control tube did not contain bacterial inoculum. The MIC was the lowest concentration in each sample, and no visible growth of microorganism could be detected after incubation with regard to the controls [21]. To avoid the possibility of misinterpretations owing to any turbidity of insoluble samples, the MBC was determined by application of 0.1 ml from each test tube with no turbidity and incubation onto respective agar plates at 37 °C for 48 h. The MBC was regarded as the lowest concentration of each sample that produced no colony growth of microorganisms on the plate [21]. All experiments were performed in triplicate and protected from light.
2.6. Statistical analysis The kinetic growth curves of the tested microorganisms were obtained using the OD values at each time point, and all the data recorded were the means of three parallel assays and were shown as mean ± SD. One way analysis of variance (ANOVA) and the least significant difference (LSD) test via SPSS 22.0 software were used to compare the bacterial growth inhibition rate of the three samples under different light conditions (α = 0.05).
2.7. Morphological observations of the microbial cells Transmission electron microscopy (TEM, JEM-1011, JEOL, Japan) was applied to examine morphological changes of four tested microbial cell treatments with the HAPw/n-ZnO composite. First, the microorganism suspensions (0.2 OD) treated with 1 mg ml−1 of HAPw/ n-ZnO composite were incubated in EP tubes overnight at 37 °C for 12 h, while S. mutans was anaerobic cultured, using the same samples untreated with the composite as blank controls. After incubation, each EP tube was centrifuged at 8000 × g for 15 min then discarded the 2.5. Kinetics of microbial growth study under supernatant. The samples were first fixed with 2.5% different light irradiation glutaraldehyde solution for 2 h then rinsed three times To examine the inhibition on the kinetics of (5 min each) with 0.1 m phosphate buffer solution microbial growth in the presence of the HAPw/ (PBS, pH = 7.2), followed by secondary fixation with n-ZnO composite under different illuminants, the 1% osmic acid for 2 h, and then rinsed three times four cultured microorganisms were suspended (5 min each) with 0.1 m PBS at room temperature. and the optical density (OD) value was adjusted to Subsequently, the specimens were dehydrated using OD595nm = 0.2 using a visible spectrophotometer (WFJ ethanol solutions (20, 50, 70, 80, 90 and 100%) and 7200, UNICO, Shanghai, China). Three samples, the acetone solution (100%), followed by infiltration and HAPw/n-ZnO composite, commercial n-ZnO and embedment of epoxy resin 618, which polymerized at HAPw, were suspended in sterile distilled water then 60 °C for 48 h. Finally, the samples were processed into concentrations of 1 mg ml−1 were obtained. Sterile 96- ultrathin sections using Leica ultramicrotome (Leica well plates (Nest Biotechnology Co., Ltd, Wuxi, Jiangsu, Ultracut R, Germany), then stained with lead citrate China) were employed in this test [22, 23]. To every 8 for 10 min and uranyl acetate for 20 min to observe the wells of the plate we added 50 μl of each sample and micrographs obtained from TEM at 100 kV. 170 μl of corresponding liquid medium containing 10 μl of each microorganism, while preparing appropriate 2.8. Confocal laser scanning microscopic analysis wells only with inoculum containing bacterium as Inspection and analysis of the overall live/dead bacteria the positive control and with liquid medium and each fluorescent proportion of the microorganisms sample as the negative control. The inoculated plates that were treated with (and without) the HAPw/ were exposed to two types of light conditions, darkness n-ZnO composite were performed using confocal and simulated solar irradiation, and were removed laser scanning microscopy (CLSM, Leica TCS SP5, from the latter after the initial 2 h. Then the plates Germany) and the fluorescent staining technique were allowed to grow overnight in an incubator and [19, 29]. CLSM employed an Argon laser at 488 nm shaken at 180 rpm for 24–48 h at 37 °C. The OD value wavelength to monitor the emission at a magnification measurements of these 96-well plates were carried out of 100 × , and fluorescent staining utilized LIVE/ every 2 h for up to 24–48 h for microorganism growth DEAD BacLight bacterial viability kits (Kit L 13152, assessment utilizing a Microplate Reader (iMark, Bio- Molecular Probes, Invitrogen, USA) including two Rad, Hercules, CA, USA) at a wavelength of 595 nm [24, fluorescent indicators: SYTO-9 and propidium 25]. After incubation, the bacterial growth inhibition iodide (PI). With an appropriate mixture of SYTO-9 rate was calculated from OD values using the following and PI, microorganisms with intact cell membranes formula: growth inhibition rate (%) = [(OD value of stain fluorescent green, whereas those with damaged positive control well − OD value of experimental well) membranes stain fluorescent red. Four tested / (OD value of positive control well − OD value of microorganisms were prepared into suspensions (0.2 negative control well)] × 100% [26–28]. This study was OD) prior to the research. Sterile 24-well plates (Nest accomplished using three parallel assays. Biotechnology Co., Ltd, Wuxi, Jiangsu, China) used in 3
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 1. SEM images of the surface of the whiskers (a) and the composite powder (b)–(d).
this test had 50 μl suspensions of each microorganism and 1 ml of corresponding medium added to each well, then 250 μl of HAPw/n-ZnO composite suspension were added at a concentration of 1 mg ml−1 cultured for 12 h at 37 °C under shaking (150 rpm), while S. mutans was anaerobic cultured. The same samples containing only bacterial suspensions and medium were prepared as blank controls. After rinsing thrice with sterile distilled water, the samples were transferred to another 24-well plate and mixed thoroughly with 1 ml of live/dead bacteria stain in each well at room temperature in the dark for 15 min. Finally, 200 μl of each sample was shifted into a glass bottom microwell disher (P35G-1.5-14-C, MatTek, Ashland, MA, USA) to evaluate the live/dead bacteria proportion using CLSM. The graphs of each sample were randomly selected from three independent locations.
3. Results 3.1. Characterization of the material A morphological examination of the specimens was performed using SEM. In figure 1(a), the whiskers 4
were found to have a smooth surface, an average size of 0.5–2 µm in diameter and 4–40 µm in length, as well as a little bit of agglomeration. Figures 1(b) and (c) show representative micrographs of the composite powder that a mass of nanoparticles were dispersed on the surface of slender whiskers. While magnified to 80 000 × , it could be observed in figure 1(d) that nanoparticles with an average granular size of 20– 80 nm in diameter were uniformly directionally fused on the surface of smooth whiskers. The contents of relevant elements in the composite powder were spot analyzed by EDS. The energy spectrum in figure 2 indicates that the powder was primarily constituted of elements (O, P, Ca and Zn) with almost stoichiometric contents (O : P : Ca : Zn = 37.03 : 19.14 : 32.42 : 11.41). XRD analysis was used to study the structural properties of the specimen. Figure 3 shows XRD patterns that unambiguously demonstrate that the diffraction peaks of the powder are consistent with the standard diffraction peaks of hydroxylapatite and zincite, respectively. It also reveals that no typical peaks of possible impurities can be observed and that the asprepared sample crystallized well, corresponding to the hexagonal zincite structure of the ZnO phase.
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 2. Spot analysis of EDS for the powder in figure 1(c).
Figure 3. XRD patterns for the composite powder after sintering at 800 °C (HAPw, hydroxyapatite whisker; ZnO, zinc oxide).
Table 1. MIC and MBC of the three samples (mg ml−1). Samples MIC (MBC)
S. aureus
Escherichia coli
Candida albicans
Streptococcus mutans
HAPw/n-ZnO
0.625 (1.250)
1.250 (2.500)
0.312 (0.625)
0.078 (0.156)
Commercial n-ZnO
0.312 (1.250)
1.250 (2.500)
0.625 (0.625)
0.156 (0.312
HAPw
– (–)
– (–)
– (–)
– (–)
‘–’ indicates no antimicrobial activity.
3.2. Determination of MIC and MBC The MIC and MBC values of the tested pathogenic microorganisms for the three samples are depicted in table 1. The MIC of the HAPw/n-ZnO composite and commercial n-ZnO is 0.078–1.250 mg ml−1 and 0.156–1.250 mg ml−1, respectively. The MBC of the HAPw/n-ZnO composite and commercial n-ZnO is 0.156–2.500 mg ml −1 and 0.312–2.500 mg ml −1, respectively. Additionally, the two samples had the same MIC of E. coli (1.250 mg ml−1) as well as the same MBC of S. aureus (1.250 mg ml−1), E. coli (2.500 mg ml−1) and C albicans (0.625 mg ml−1). However, HAPw exhibited no antimicrobial activity. 5
3.3. Influence of different illuminants on the kinetic growth of the microorganisms The inhibition of the growth of microorganisms after incubation with the presence of the samples was evaluated using kinetic measurements. The variation curve of OD values at 595 nm shown in figure 4 shows the inhibiting action on the bacteria and lower OD values mean a higher antibiotic influence. In the figure, the curves of the positive control reflected normal microbial growth, which, free of the samples, apparently, the lag phase, the logarithmic phase and the stationary phase (the decline phase was not given) of the microorganisms were observed under both darkness
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 4. The growth curves of the tested microorganisms in the presence of HAPw, commercial n-ZnO and HAPw/n-ZnO at an initial concentration of 1 mg ml−1; cultures were set up at an initial OD value equivalent to 0.2 and incubated for 24–48 h. (a) and (b) were under darkness and simulated solar irradiation, respectively. The data are shown with mean ± SD (black ●, positive control; blue ■, HAPw; green ▲, commercial n-ZnO; red ▼, HAPw/n-ZnO; violet ◆, negative control).
and simulated solar excitation. We noted in figure 4(a) that within 24–48 h of cultivation under darkness, the OD values of HAPw groups were extremely analogous to the positive controls during the kinetic measurement, and HAPw/n-ZnO and commercial n-ZnO had significant microbial inhibitory capabilities on the growth of the four microorganisms, being more effective on S. mutans, C albicans and S aureus than E. coli. By contrast, when exposed to simulated solar excitation in figure 4(b), OD value alterations in the HAPw group resembled the positive control, whereas the antibiotic efficacy of HAPw/n-ZnO and commercial n-ZnO manifested a certain degree of enhancement against the tested microorganisms compared to 6
darkness. In addition, the bacterial growth inhibition rates represented in figure 5 illustrated the maximum antimicrobial effectiveness of the samples after 24– 48 h incubation. When analyzed by ANOVA and the LSD test, the growth inhibition rate among HAPw/nZnO, commercial n-ZnO and HAPw had a significant difference (P commercial n-ZnO and against S aureus commercial n-ZnO > HAPw/nZnO (P
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My IOPscience
Synthesis, characterization, antimicrobial activity and mechanism of a novel hydroxyapatite whisker/nano zinc oxide biomaterial
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015001 (http://iopscience.iop.org/1748-605X/10/1/015001) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 132.203.227.61 This content was downloaded on 29/12/2014 at 07:28
Please note that terms and conditions apply.
IOP Publishing
10.1088/1748-6041/10/1/015001
Biomed. Mater. 10 (2015) 015001
Paper
received
14 September 2014
Synthesis, characterization, antimicrobial activity and mechanism of a novel hydroxyapatite whisker/nano zinc oxide biomaterial
accep ted for publication
22 October 2014 published
22 December 2014
Jian Yu1,2, Wenyun Zhang1,5, Yang Li1,2, Gang Wang1, Lidou Yang1, Jianfeng Jin3, Qinghua Chen4 and Minghua Huang4 1
Department of Stomatology, Kunming General Hospital of Chengdu Military Region, #212 Daguan Road, Xishan District, Kunming, Yunnan Province, People’s Republic of China 2 Clinical College of Kunming Medical University, Kunming, People’s Republic of China 3 Kunming Municipal Stomatological Hospital, Kunming, People’s Republic of China 4 Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Kunming, People’s Republic of China 5 Author to whom correspondence should be addressed. E-mail: [email protected] Keywords: hydroxyapatite whisker, zinc oxide, antibacterial mechanism, biomaterial
Abstract Postoperative infections remain a risk factor that leads to failures in oral and maxillofacial artificial bone transplantation. This study aimed to synthesize and evaluate a novel hydroxyapatite whisker (HAPw) / nano zinc oxide (n-ZnO) antimicrobial bone restorative biomaterial. A scanning electron microscope (SEM), energy dispersive spectroscopy (EDS) and x-ray diffraction (XRD) were employed to characterize and analyze the material. Antibacterial capabilities against Staphylococcus aureus, Escherichia coli, Candida albicans and Streptococcus mutans were determined by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), and kinetic growth inhibition assays were performed under darkness and simulated solar irradiation. The mode of antibiotic action was observed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). The MIC and MBC were 0.078–1.250 mg ml−1 and 0.156–2.500 mg ml−1, respectively. The inhibitory function on the growth of the microorganisms was achieved even under darkness, with gram-positive bacteria found to be more sensitive than gram-negative, and enhanced antimicrobial activity was exhibited under simulated solar excitation compared to darkness. TEM and CLSM images revealed a certain level of bacterial cell membrane destruction after treatment with 1 mg ml−1 of the material for 12 h, causing the leakage of intracellular contents and bacteria death. These results suggest favorable antibiotic properties and a probable mechanism of the biomaterial for the first time, and further studies are needed to determine its potential application as a postoperative anti-inflammation method in bone transplantation.
1. Introduction Postoperative infections remain a severe risk of artificial bone transplantation in oral and maxillofacial regions, which leads to failures and psychological trauma for patients [1–3]. An increasing number of experiments involving hard tissue substitutes loaded with antibiotics were investigated to provide antimicrobial efficacy against relevant pathogens [4–6]. Nevertheless, the presence of certain disadvantages associated with the composite constrains its application. It was reported that bone cement as a drug carrier had limited drug release and imperfect biodegradation, and might bring about necrosis between the surface of the cement and the surrounding tissue [7, 8] as well as dose-dependent © 2015 IOP Publishing Ltd
cytotoxicity from the additives [9]. Therefore, employing bone repair materials possessing excellent biological and mechanical performance incorporated with appropriate antibacterial agents was imperative for clinical work. Hydroxyapatite (HAP), a pervasively used bioceramic, constitutes the major component of skeleton and teeth in human body and is supposed to have enormous potential on account of its favorable biocompatibility and osteoinductive activity [10, 11]. HAP whisker (HAPw) is a type of monocrystal fiber with high purity and modulus, principally utilized for reinforcing the biomechanical properties of HAP [12, 13]. Despite being a promising artificial bone substitute, the independent antibiotic capability of HAPw is scarcely
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
investigated. Acknowledged as a representative of inorganic antimicrobial biomaterials, zinc oxide (ZnO) has been given great significance in recent years because of the superiority of its broad antibacterial spectrum, biosecurity, durability and stability when compared to organic antimicrobial biomaterials [14–16]. Furthermore, continuous studies with respect to ZnO nanostructures were carried out to indicate the probable antibiotic mechanism and photocatalysis of ZnO [17–19]. However, the exact antimicrobial mechanism of ZnO nanoparticles against a wide range of microorganisms is still an extensive field of research that is generally unexplored. In this paper, our objective is to prepare a novel biomaterial that couples the ideal mechanical and biological functions of HAPw with the superior antibacterial ability of ZnO. A sol–gel technology was applied to synthesize a hydroxyapatite whisker/nano zinc oxide (HAPw/n-ZnO) biocomposite in which nanometer-sized ZnO were fused on the surface of HAPw. The properties of the composite were characterized and analyzed using SEM, EDS and XRD. Both gram-positive and gram-negative bacteria were considered for antibacterial assays, including determination of MIC, MBC and kinetic growth inhibition under darkness and simulated solar irradiation. The mode of antibiotic action on the tested microorganisms was evaluated using TEM and CLSM. These results were employed to discuss the antimicrobial activity and possible mechanism of HAPw/n-ZnO biomaterial for the first time.
2. Materials and methods 2.1. Preparation of the materials HAPw was provided by Faculty of Materials Science and Engineering, Kunming University of Science and Technology, Yunnan, China. Zinc acetate [(CH 3COO) 2Zn·2H 2O], polyethylene glycol 6000 (PEG-6000), anhydrous ethanol, aqueous ammonia and glacial acetic acid were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd, Tianjin, China. The HAPw/n-ZnO biomaterial was synthesized using a sol–gel technique. First, zinc acetate and PEG-6000 powders were dissolved into anhydrous ethanol solution then bathed in water for 2 h at 70 °C in clarified liquid obtained with a rotary evaporator, which was the precursor product [Zn5(OH)8(Ac)2·2H2O] of nanometer zinc oxide. Subsequently, HAPw (after drying for 24 h at 80 °C), with a weight ratio of 2:1 of zinc acetate and PEG-6000, was dissolved into anhydrous ethanol solution to obtain a dispersion liquid by means of ultrasonic oscillating and magnetic stirring. Third, with magnetic stirring at room temperature, the clarified liquid was titrated slowly into the dispersion liquid with a constant flow pump, while simultaneously applying aqueous ammonia or glacial acetic acid to maintain PH = 6.4. After titration, the mixed solution was transferred to the rotary evaporator and bathed in 2
water for 6 h at 70 °C, followed by ultrasonic dispersion for 20 s and heating to 78–80 °C to be dehydrated, then proceeding with thorough desiccation in a thermostatic drying oven. Finally, the obtained mixture was heated to 800 °C at 2 °C per min and sintered for 1 h at 800 °C. After being cooled naturally to an ambient temperature, the composite powders were obtained. 2.2. Characterization of the composite The surface microstructure of HAPw and the composite powder were observed using a scanning electron microscope (SEM, FEI Quanta 200, USA), and the contents of the relevant elements in the composite were spot analyzed with energy dispersive spectroscopy (EDS). To determine the components and structure of the composite powder, x-ray diffraction (XRD) analysis was performed with an x-ray diffractometer (D/max2200, Rigaku Corporation, Japan), which measured Cu-Kα (λ = 0.15406 Å) radiation in the 2θ range from 0° to 90° with a scanning speed of 4° per min at 36 kV and 30 mA. 2.3. Microorganisms and culture conditions The following microorganisms were used in this experiment. Gram-negative: Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231) and Streptococcus mutans (ATCC 25175). Gram-positive: Escherichia coli (ATCC 8739). These bacterial strains were obtained from the Department of Microbiology and Immunology, Kunming Medical University, Yunnan, China. S. aureus and E. coli were grown in nutrient agar medium and C. albicans and S. mutans were grown in sabouraud dextrose agar (SDA) medium and tryptone peptone yeast (TPY) agar medium, respectively. S. aureus, E. coli and C. albicans were aerobic cultured at 37 °C for 24–48 h, while S. mutans was under anaerobic condition (80% N2, 10% H2 and 10% CO2). All microorganisms were maintained by subculture at 4 °C in a refrigerator. The media used in the research were all purchased from Qingdao Hope Bio-Technology Co., Ltd, Shandong, China. 2.4. Antimicrobial assay of the HAPw/n-ZnO biocomposite The antimicrobial activity of the HAPw/n-ZnO biocomposite was investigated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To evaluate the MIC, HAPw and commercial n-ZnO (20–50 nm in diame ter, Kunming Nuob ei Biotechnology Co., Ltd, Yunnan, China) were used for comparison. The three samples were suspended in sterile distilled water and prepared for suspension at the required concentrations of 20–0.039 mg ml−1 prior to the experiment, using the method of two-fold serial dilutions [20]. Bacterial suspensions with a turbidity equivalent to a 0.5 McFarland standard were prepared and then further diluted to 1:10 twice to obtain a final concentration of 1.5 × 106 colony-forming units
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
(CFU) ml−1. We added 1 ml of suspensions of three samples with each concentration into separate sterile test tubes containing 0.9 ml of the corresponding broth medium inoculated with 0.1 ml of each bacterial suspension, then allowed it to culture overnight at 37 °C. The tubes were checked for visible turbidity after 24–48 h of culturing for the tested microorganisms and were referenced by positive and negative growth control tubes. The positive control tube was free of the three samples and the negative control tube did not contain bacterial inoculum. The MIC was the lowest concentration in each sample, and no visible growth of microorganism could be detected after incubation with regard to the controls [21]. To avoid the possibility of misinterpretations owing to any turbidity of insoluble samples, the MBC was determined by application of 0.1 ml from each test tube with no turbidity and incubation onto respective agar plates at 37 °C for 48 h. The MBC was regarded as the lowest concentration of each sample that produced no colony growth of microorganisms on the plate [21]. All experiments were performed in triplicate and protected from light.
2.6. Statistical analysis The kinetic growth curves of the tested microorganisms were obtained using the OD values at each time point, and all the data recorded were the means of three parallel assays and were shown as mean ± SD. One way analysis of variance (ANOVA) and the least significant difference (LSD) test via SPSS 22.0 software were used to compare the bacterial growth inhibition rate of the three samples under different light conditions (α = 0.05).
2.7. Morphological observations of the microbial cells Transmission electron microscopy (TEM, JEM-1011, JEOL, Japan) was applied to examine morphological changes of four tested microbial cell treatments with the HAPw/n-ZnO composite. First, the microorganism suspensions (0.2 OD) treated with 1 mg ml−1 of HAPw/ n-ZnO composite were incubated in EP tubes overnight at 37 °C for 12 h, while S. mutans was anaerobic cultured, using the same samples untreated with the composite as blank controls. After incubation, each EP tube was centrifuged at 8000 × g for 15 min then discarded the 2.5. Kinetics of microbial growth study under supernatant. The samples were first fixed with 2.5% different light irradiation glutaraldehyde solution for 2 h then rinsed three times To examine the inhibition on the kinetics of (5 min each) with 0.1 m phosphate buffer solution microbial growth in the presence of the HAPw/ (PBS, pH = 7.2), followed by secondary fixation with n-ZnO composite under different illuminants, the 1% osmic acid for 2 h, and then rinsed three times four cultured microorganisms were suspended (5 min each) with 0.1 m PBS at room temperature. and the optical density (OD) value was adjusted to Subsequently, the specimens were dehydrated using OD595nm = 0.2 using a visible spectrophotometer (WFJ ethanol solutions (20, 50, 70, 80, 90 and 100%) and 7200, UNICO, Shanghai, China). Three samples, the acetone solution (100%), followed by infiltration and HAPw/n-ZnO composite, commercial n-ZnO and embedment of epoxy resin 618, which polymerized at HAPw, were suspended in sterile distilled water then 60 °C for 48 h. Finally, the samples were processed into concentrations of 1 mg ml−1 were obtained. Sterile 96- ultrathin sections using Leica ultramicrotome (Leica well plates (Nest Biotechnology Co., Ltd, Wuxi, Jiangsu, Ultracut R, Germany), then stained with lead citrate China) were employed in this test [22, 23]. To every 8 for 10 min and uranyl acetate for 20 min to observe the wells of the plate we added 50 μl of each sample and micrographs obtained from TEM at 100 kV. 170 μl of corresponding liquid medium containing 10 μl of each microorganism, while preparing appropriate 2.8. Confocal laser scanning microscopic analysis wells only with inoculum containing bacterium as Inspection and analysis of the overall live/dead bacteria the positive control and with liquid medium and each fluorescent proportion of the microorganisms sample as the negative control. The inoculated plates that were treated with (and without) the HAPw/ were exposed to two types of light conditions, darkness n-ZnO composite were performed using confocal and simulated solar irradiation, and were removed laser scanning microscopy (CLSM, Leica TCS SP5, from the latter after the initial 2 h. Then the plates Germany) and the fluorescent staining technique were allowed to grow overnight in an incubator and [19, 29]. CLSM employed an Argon laser at 488 nm shaken at 180 rpm for 24–48 h at 37 °C. The OD value wavelength to monitor the emission at a magnification measurements of these 96-well plates were carried out of 100 × , and fluorescent staining utilized LIVE/ every 2 h for up to 24–48 h for microorganism growth DEAD BacLight bacterial viability kits (Kit L 13152, assessment utilizing a Microplate Reader (iMark, Bio- Molecular Probes, Invitrogen, USA) including two Rad, Hercules, CA, USA) at a wavelength of 595 nm [24, fluorescent indicators: SYTO-9 and propidium 25]. After incubation, the bacterial growth inhibition iodide (PI). With an appropriate mixture of SYTO-9 rate was calculated from OD values using the following and PI, microorganisms with intact cell membranes formula: growth inhibition rate (%) = [(OD value of stain fluorescent green, whereas those with damaged positive control well − OD value of experimental well) membranes stain fluorescent red. Four tested / (OD value of positive control well − OD value of microorganisms were prepared into suspensions (0.2 negative control well)] × 100% [26–28]. This study was OD) prior to the research. Sterile 24-well plates (Nest accomplished using three parallel assays. Biotechnology Co., Ltd, Wuxi, Jiangsu, China) used in 3
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Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 1. SEM images of the surface of the whiskers (a) and the composite powder (b)–(d).
this test had 50 μl suspensions of each microorganism and 1 ml of corresponding medium added to each well, then 250 μl of HAPw/n-ZnO composite suspension were added at a concentration of 1 mg ml−1 cultured for 12 h at 37 °C under shaking (150 rpm), while S. mutans was anaerobic cultured. The same samples containing only bacterial suspensions and medium were prepared as blank controls. After rinsing thrice with sterile distilled water, the samples were transferred to another 24-well plate and mixed thoroughly with 1 ml of live/dead bacteria stain in each well at room temperature in the dark for 15 min. Finally, 200 μl of each sample was shifted into a glass bottom microwell disher (P35G-1.5-14-C, MatTek, Ashland, MA, USA) to evaluate the live/dead bacteria proportion using CLSM. The graphs of each sample were randomly selected from three independent locations.
3. Results 3.1. Characterization of the material A morphological examination of the specimens was performed using SEM. In figure 1(a), the whiskers 4
were found to have a smooth surface, an average size of 0.5–2 µm in diameter and 4–40 µm in length, as well as a little bit of agglomeration. Figures 1(b) and (c) show representative micrographs of the composite powder that a mass of nanoparticles were dispersed on the surface of slender whiskers. While magnified to 80 000 × , it could be observed in figure 1(d) that nanoparticles with an average granular size of 20– 80 nm in diameter were uniformly directionally fused on the surface of smooth whiskers. The contents of relevant elements in the composite powder were spot analyzed by EDS. The energy spectrum in figure 2 indicates that the powder was primarily constituted of elements (O, P, Ca and Zn) with almost stoichiometric contents (O : P : Ca : Zn = 37.03 : 19.14 : 32.42 : 11.41). XRD analysis was used to study the structural properties of the specimen. Figure 3 shows XRD patterns that unambiguously demonstrate that the diffraction peaks of the powder are consistent with the standard diffraction peaks of hydroxylapatite and zincite, respectively. It also reveals that no typical peaks of possible impurities can be observed and that the asprepared sample crystallized well, corresponding to the hexagonal zincite structure of the ZnO phase.
IOP Publishing
Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 2. Spot analysis of EDS for the powder in figure 1(c).
Figure 3. XRD patterns for the composite powder after sintering at 800 °C (HAPw, hydroxyapatite whisker; ZnO, zinc oxide).
Table 1. MIC and MBC of the three samples (mg ml−1). Samples MIC (MBC)
S. aureus
Escherichia coli
Candida albicans
Streptococcus mutans
HAPw/n-ZnO
0.625 (1.250)
1.250 (2.500)
0.312 (0.625)
0.078 (0.156)
Commercial n-ZnO
0.312 (1.250)
1.250 (2.500)
0.625 (0.625)
0.156 (0.312
HAPw
– (–)
– (–)
– (–)
– (–)
‘–’ indicates no antimicrobial activity.
3.2. Determination of MIC and MBC The MIC and MBC values of the tested pathogenic microorganisms for the three samples are depicted in table 1. The MIC of the HAPw/n-ZnO composite and commercial n-ZnO is 0.078–1.250 mg ml−1 and 0.156–1.250 mg ml−1, respectively. The MBC of the HAPw/n-ZnO composite and commercial n-ZnO is 0.156–2.500 mg ml −1 and 0.312–2.500 mg ml −1, respectively. Additionally, the two samples had the same MIC of E. coli (1.250 mg ml−1) as well as the same MBC of S. aureus (1.250 mg ml−1), E. coli (2.500 mg ml−1) and C albicans (0.625 mg ml−1). However, HAPw exhibited no antimicrobial activity. 5
3.3. Influence of different illuminants on the kinetic growth of the microorganisms The inhibition of the growth of microorganisms after incubation with the presence of the samples was evaluated using kinetic measurements. The variation curve of OD values at 595 nm shown in figure 4 shows the inhibiting action on the bacteria and lower OD values mean a higher antibiotic influence. In the figure, the curves of the positive control reflected normal microbial growth, which, free of the samples, apparently, the lag phase, the logarithmic phase and the stationary phase (the decline phase was not given) of the microorganisms were observed under both darkness
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Biomed. Mater. 10 (2015) 015001
J Yu et al
Figure 4. The growth curves of the tested microorganisms in the presence of HAPw, commercial n-ZnO and HAPw/n-ZnO at an initial concentration of 1 mg ml−1; cultures were set up at an initial OD value equivalent to 0.2 and incubated for 24–48 h. (a) and (b) were under darkness and simulated solar irradiation, respectively. The data are shown with mean ± SD (black ●, positive control; blue ■, HAPw; green ▲, commercial n-ZnO; red ▼, HAPw/n-ZnO; violet ◆, negative control).
and simulated solar excitation. We noted in figure 4(a) that within 24–48 h of cultivation under darkness, the OD values of HAPw groups were extremely analogous to the positive controls during the kinetic measurement, and HAPw/n-ZnO and commercial n-ZnO had significant microbial inhibitory capabilities on the growth of the four microorganisms, being more effective on S. mutans, C albicans and S aureus than E. coli. By contrast, when exposed to simulated solar excitation in figure 4(b), OD value alterations in the HAPw group resembled the positive control, whereas the antibiotic efficacy of HAPw/n-ZnO and commercial n-ZnO manifested a certain degree of enhancement against the tested microorganisms compared to 6
darkness. In addition, the bacterial growth inhibition rates represented in figure 5 illustrated the maximum antimicrobial effectiveness of the samples after 24– 48 h incubation. When analyzed by ANOVA and the LSD test, the growth inhibition rate among HAPw/nZnO, commercial n-ZnO and HAPw had a significant difference (P commercial n-ZnO and against S aureus commercial n-ZnO > HAPw/nZnO (P
