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Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb

evelopment of novel implants with self-antibacterial performancehrough in-situ growth of 1D ZnO nanowire

enhao Wang a,b, Tak Lung Li a,1, Hoi Man Wong a, Paul K. Chu c, Richard Y.T. Kao d,huilin Wu e,∗, Frankie K.L. Leung a,b, Tak Man Wong a,b, Michael K.T. To a,b,enneth M.C. Cheung a, Kelvin W.K. Yeung a,b,∗∗

Department of Orthopaedics and Traumatology, The University of Hong Kong, Pokfulam, Hong Kong, ChinaShenzhen Key Laboratory for Innovative Technology in Orthopaedic Trauma, The University of Hong Kong Shenzhen Hospital, 1Haiyuan 1st Road, Futianistrict, Shenzhen, ChinaDepartment of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong Kong, ChinaDepartment of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, ChinaHubei Collaborative Innovation Center for Advanced Organic Materials, Ministry-of-Education Key Laboratory for Green Preparation and Application ofunctional Materials, Hubei University, Wuhan, China

r t i c l e i n f o

rticle history:eceived 27 September 2015eceived in revised form 1 February 2016ccepted 16 February 2016vailable online 18 February 2016

eywords:nO nanowire

a b s t r a c t

To prevent the attachment of bacteria to implant surfaces, the 1D zinc oxide nanowire-coating hasbeen successfully developed on material surfaces by using a custom-made hydrothermal approach. Thechemical nature, surface topography and wettability of spike-like 1D ZnO nanowire-coating are compre-hensively investigated. The anti-adhesive and antimicrobial properties of 1D nanowire-coating are testedagainst Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by using in vitro live/deadstaining and scanning electron microscopy. We find that the adhesion of bacteria can be reduced via thespecial spike-like topography and that the release of Zn2+ ions can help suppress the growth of attached

elf-antibacterial effecturface modificationioluminescent bacteria

bacteria. Furthermore, the antimicrobial effect is also evaluated under in vivo conditions by using a ratmodel infected with bioluminescent S. aureus. The amount of live bacteria in the rat implanted with ananowire-coated sample is less than that of the control at various time points. Hence, it is believed that thenanowire-coated material is promising for application in orthopaedic implantation after the long-termanimal studies have been completed.

© 2016 Published by Elsevier B.V.

. Introduction

Medical-grade stainless steel and titanium alloys have beenpplied in various orthopaedic implants, such as bone fracturexation and artificial internal prostheses, due to their superiororrosion resistance and mechanical support as well as biocom-atibility [1,2]. However, post-operative implant-related bacterial

nfection has sometimes occurred when patients are surgically

mplanted with instruments made from those metallic materi-ls. For instance, periprosthetic infection (PPI) is one of the mostommon post-operative complications associated with surgical

∗ Corresponding author.∗∗ Corresponding author at: Department of Orthopaedics and Traumatology, theniversity of Hong Kong, Pokfulam, 999077, Hong Kong, China.

E-mail addresses: shuilin.wu@gmail.com (S. Wu), wkkyeung@hku.hkK.W.K. Yeung).

1 This author contributed equally.

ttp://dx.doi.org/10.1016/j.colsurfb.2016.02.036927-7765/© 2016 Published by Elsevier B.V.

implants [3]. Indeed, medical-grade stainless steel has a relativelyhigh infection rate as compare to the other common implantablebio-metals [4]. Despite of the best clinical practices and optimaloperation theater, the average post-operative implanted-relatedinfection rate in the United States was reported to be about 5%,which is equivalent to 100,000 cases per year. Additionally, theaverage spent per incidence was nearly 15,000 US dollars, whichresulted in a tremendous burden on the healthcare system [5].

In fact, the initial attachment of bacteria to the implant surfacewill lead to bacterial colonization, biofilm formation and eventu-ally implant-related bacterial infection in humans [3]. After theinstrument has been surgically implanted, a biological layer com-posed of host-derived adhesions will be established on the surface,and this conditioned layer can promote the adhesion of plank-

tonic bacterial cells [6]. When a biofilm is formed, a surface layercontaining macromolecules is able to resist the immune response,and the antibiotic dosage used will thus be 500–5000 times higherthan that needed in treating free-floating bacteria [7,8]. To prevent

624 W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633

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ig. 1. (A) TEM images of the ZnO nanowire and the corresponding selected area eleo Stainless Steel), ZnO nanowire coating with 5-h (abbr. to ZnO 5 h) and 17-h (abisplays the detailed morphology of the spike-like ZnO (the scale bar is 500 nm).

acterial adhesion and subsequent biofilm formation on themplant’s surface, a coating or surface modification is highly pre-erred in orthopaedic implants.

In the past, antibiotic loaded implant surfaces have been exten-ively investigated [9–11]. However, their clinical effectivenessas doubtful due to the diversity of bacterial strains involved in

eriprosthetic infection and concerns about the development ofntibiotic-resistant bacteria [12,13]. Alternatively, the use of zincxide (ZnO) as a broad-spectrum bactericidal agent against variousacterial strains such as Escherichia coli [14], Staphylococcus aureus15] and S. epidermidis [16], has attracted a great deal of attention17,18]. The bactericidal effect could be partially attribute to theelease of Zn2+ ions because these metallic ions are able to inhibithe active transportation and metabolism of sugars and also dis-upt the enzymatic activity of bacteria by displacing magnesiumons [19]. Furthermore, zinc ions have been considered an essentiallement in helping promote bone development through the stim-lation of osteoblastic proliferation and mineralization, as well ashe up-regulation of osteoblastic genes [20–22].

In this study, we have successfully fabricated a coating of one-imensional (1D) ZnO nanowires on a stainless steel surface; thisurface is able to resist bacterial attachment and growth. By adjust-ng the fabrication parameters, such as time and temperature, 1DnO nanowires of various sizes were then formed on the metal sur-ace. The surface topography, wettability and dissolution propertyf the coated layer have been comprehensively characterized. Also,he coating was tested against S. aureus, Pseudomonas aeruginosand E. coli, and its antibacterial property was evaluated via live/deadacterial staining. Additionally, a minimum inhibitory concentra-ion (MIC) test of Zn2+ ions against these bacteria was performed asell. In addition to the characterization of its antimicrobial ability,

ts biocompatibility has also been verified by culturing osteoblastsith conditioned media using a cell adhesion assay and an MTT

ssay. Lastly, the antibacterial property of this 1D ZnO nanowire-oated material was examined by using rat models infected by S.

ureus.

diffraction (SAED) pattern. (B) SEM images of polished stainless steel surface (abbr. ZnO 17 h) reaction times, respectively (the scale bar is 1.00 �m). The inset in (B)

2. Materials and methods

2.1. Sample preparation

Medical-grade 316L stainless steel rods (Carpenter, USA) wereprepared in a disc shape: 5 mm in diameter and 1 mm in thickness.Various grades of abrasive silicon carbide papers (Pace Technolo-gies) were used to grind the rough samples before polishing with1um diamond paste (Pace Technologies). Subsequently, the pol-ished samples were then cleaned with acetone and distilled watervia sonication.

The 1D ZnO nanowires on the stainless steel surface were fab-ricated by using a custom-made hydrothermal method, a modifiedversion of the protocols used by Rakhshani and Thomas [23] andYang and Cui [24]. In brief, the polished disc samples were heatedto 500 ◦C for 60 min in order to enhance the formation of an oxidelayer on the surfaces. Afterwards, they were immersed in a zincacetate dehydrate solution (Sigma, USA, 10 mM) in ethanol beforea second heat treatment at 350 ◦C for 20 min. After the secondheat treatment, a ZnO seed layer was formed on the top of theoxide layer. Subsequently, the ZnO nanowires were then grownbased on the seed layer when the samples were immersed in achemical bath containing zinc nitrate hydrate (25 mM) and hex-amethylenetramine (25 mM) diluted with deionized water at 90 ◦Cfor 5 h (ZnO 5 h) or 17 h (ZnO 17 h). The same procedure was alsoapplied to the stainless steel rod samples in order to build the 1Dnanowire-coating on the material surface for in vivo assessment.

2.2. Characterizations of 1D ZnO nanowires

The crystal structure and chemical composition of ZnOnanowires on the stainless steel surface were investigated usinga scanning transmission electron microscope (FEI Tecnai G2 20

Scanning TEM) and the selected area electron diffraction (SAED)technique. A scanning electron microscope (Hitachi S4800 FEGSEM) was employed so as to visualize the surface morphology ofthe treated and untreated stainless steel.

W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633 625

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ig. 2. (A) SEM images of different 1D ZnO nanowire morphologies (upper: ZnO

umber of Zn2+ ions released, (C) SEM images of 1D ZnO nanowire morphology (uorresponding amount of Zn2+ ions released. It is obvious that the ZnO nanowire ar

The surface free energy of ZnO nanowire-coating was calculatedased on the Owens-Wendt geometric equations, using the contactngle with two liquids [25] as shown below.

1 + cos �LV)�LV = 2

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here � is the contact angle and �LVis the surface free energy,hich can be divided into a dispersive component �d

LVand a polaromponent �p

LV.In this experiment, deionized water and ethylene glycol were

pplied. The surface free energy, dispersive component and polaromponent of deionized water are 72.8, 21.8 and 51 nJ cm−2,hereas those values for ethylene glycol are 48.3, 29.3 and

9.0 nJ cm−2, respectively [26]. The contact angle between the liq-id and ZnO nanowire-coating was measured by using a contactngle goniometer (Rame-Hart Inc., USA). The experiment was donet an ambient temperature, and the contact angles were measured

ased on the captured images of droplets and averaged.

The dissolution of ZnO nanowires was studied by monitoring themount of zinc ions released from 1D ZnO nanowire-coatings. ThenO 5 h and ZnO 17 h samples, as well as untreated stainless steel,

wer: ZnO 17 h) after incubation in TSB for 30 min at 37 ◦C, (B) the corresponding ZnO 5 h, lower: ZnO 17 h) after incubation in DMEM at 37 ◦C for 72 h and (D) theas started to degrade in both media.

were immersed in an appropriate volume of tryptic soy broth (TSB)and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively, forpredetermined time. Inductively-coupled plasma mass spectrom-etry (ICP-MS) was applied to determine the ion concentrations ofthe resultants. The morphology of the samples after immersion wasalso examined via scanning electron microscopy (SEM).

2.3. Bacterial culture

S. aureus (S. aureus, ATCC 29213), P. aeruginosa (P. aeruginosa,ATCC 27853) and E. coli (E. coli, ATCC 25922) were maintained on atryptic soy agar (TSA, Difco) plate. For each bacterial assay, a singlecolony of each bacterium was picked and spread on a fresh TSA platebefore incubation at 37 ◦C for 24 h. A single colony was then picked

from the fresh TSA plate and inoculated in 5 ml of TSB and placed ina shaking platform at 250 rpm and 37 ◦C for 18 h. After incubationovernight, the desired bacterial concentration was determined andadjusted via the optical density measurement.

626 W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633

Fig. 3. The results of bacterial adhesion test for S. aureus (1 × 107 CFU/ml, 20 �l) on different nanowire coatings after incubation at 37 ◦C for 30 min. (A) SEM images of theattachment of S. aureus cells to an untreated stainless steel surface, ZnO 5 h coating and ZnO 17 h coating (the scale bar is 1.00 �m). The appendages released from bacterialc ructu( red bya in thi

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ells were highlighted by red circles. This revealed that the loose and disorganized stB) The plot of the number of live S. aureus cells on various sample surfaces measuttachment than the ZnO 17 h sample. (For interpretation of the references to color

.4. Anti-adhesion test of 1D ZnO nanowire-coating

To verify the anti-adhesion ability, 20 �l of medium contain-ng 107 CFU/ml bacterial cells was applied on the surfaces andnoculated at 37 ◦C for 30 min. The samples were then rinsed fourimes using 0.85% sodium chloride solution before the live/deaduorescent staining kit (Molecular Probe, L7012) was applied. Thettached bacterial cells were stained with the mixture of dyes in theark at room temperature for 20 min, and the number of live bac-eria was then investigated by analyzing the attachment of cells inifferent areas via fluorescent microscopy (Nikon, Eclipse 80i) and

CCD camera with a 40X objective lens. Finally, the results werealculated via the following equation:

Attached bacteria in percentage = No. of live bacteria attachedo ZnO nanowire-coated stainless steel surface/No. of live bacteriattached to untreated stainless steel × 100%

The killing efficiency was also calculated:Killing efficiency in percentage = 100% − Attached bacteria in

ercentage

.5. Morphological examination of the attached bacterial cells onD ZnO nanowire-coating

The concentration of bacterial cells used and the inocula-ion process in this experiment were exactly the same as those

escribed in the aforementioned test. The attached bacterial cellsere fixed via formalin immersion for an hour and then dehydrated

hrough a graded series of ethanol solutions (30%, 50%, 70%, 90% and00%) for two periods of 15 min at each concentration. After being

re of the ZnO 17 h sample provided more anchoring points for bacterial attachment. live/dead staining. The ZnO 5 h sample is more effective in suppressing bacterial

s figure legend, the reader is referred to the web version of this article.)

washed, the samples were treated via critical point drying processand sputter coating for further examination. The morphologies ofthe attached bacterial cells were observed under scanning electronmicroscopy.

2.6. Antimicrobial property of Zn2+ ions alone

To assess the minimal inhibitory concentration (MIC) of Zn2+

ions against different bacterial cells, a water-based Zn2+ ion solu-tion was diluted to concentrations ranging from 0 to 200 ppm andstored in TSB solution. 50 �l of diluted TSB medium containing dif-ferent Zn2+ ion concentrations, together with 50 �l of 106 CFU/mlbacterial medium, were mixed in triplicate wells in a 96-well plate,and the pure TSB or bacteria-containing medium served as the neg-ative and positive controls, respectively. The 96-well plate was thenincubated at 37 ◦C overnight, and the absorbance of each well wasrecorded via a microplate reader (Beckman, DTX880) at a wave-length of 595 nm. The MIC results for the Zn2+ ions against differentbacilli were determined systematically and compared with thosefor the negative control and positive controls.

2.7. Evaluation of coating cytocompatibility

In order to assess the cytotoxicity of the released Zn2+ ions,the cell adhesion test was then performed by using the selected

concentrations of Zn2+ ion. According to the results obtained in aprevious dissolution test, the water-based Zn2+ ion solution wasdiluted to 0.4 ppm and 0.5 ppm (i.e., the amounts of Zn2+ ionsreleased from ZnO 5 h and ZnO 17 h samples, respectively) via

W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633 627

Fig. 4. The results of bacterial adhesion test for P. aeruginosa (1 × 107 CFU/ml, 20 �l) on different nanowire coatings after incubation at 37 ◦C for 30 min. (A) SEM images oft atingr on dii

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he attachment of P. aeruginosa cells to untreated stainless steel surface, ZnO 5 h coeleased from the bacterial cells. (B) The plot of the number of live P. aeruginosa cellsn suppressing the bacterial attachment of P. aeruginosa cells.

BS-supplemented DMEM. An aliquot contained 5 × 104 murineC3T3-E1 pre-osteoblasts was seeded on a 6-well plate and cul-

uried with the Zn2+ ion-containing medium for 6 h. Afterwards,he cells were rinsed with PBS several times before being fixedith 2.5% glutaraldehyde. To realize the cell morphology, the

ytoskeleton protein F-actin was stained with phalloidin fluores-ein isothiocyanate (Sigma, USA) and the nuclei were stained withoechst33342 (Sigma, USA). The cell morphology was observed bysing fluorescent microscopy.

The cell viability against various concentrations of Zn2+ ionsas examined with the use of an MTT assay. An aliquot of 200 �l

f medium suspension containing 4 × 105 cells/ml was triplicateeeded in the 96-well plate and cultured with FBS supplementedMEM. The normal and conditioned mediums were changed everyay during the cell culture. On Day 1 and Day 3, the MTT solutionas added and the plate was incubated at 37 ◦C in the incubator

or 4 h. Afterwards, 10% sodium dodecyl sulphate (SDS, Sigma, USA)as added to each well and incubated overnight in order to dissolve

he crystal. Lastly, the cell viability was determined via absorbanceeading at a wavelength of 570 nm with a reference of 640 nm underhe plate reader (Beckman, DTX880).

.8. In vivo antibacterial test in rat model

For the animal testing, six 3-week old female Sprague-Dawley

ats (SD rats) weighting an average of 50 g and supplied by the Lab-ratory Animal Unit of the University of Hong Kong were divided

nto two: a treatment group and a control group (n = 3). Treatednd untreated stainless steel rod samples (1 mm in diameter and

and ZnO 17 h coating (the scale bar is 1.00 �m). No appendages were found to befferent sample surfaces, measured by live/dead staining. Both samples are effective

4 mm in length) were prepared and examined via SEM before beingimplanted into the left femora of the rats through their trochleargrooves. Bioluminescent S. aureus bacterial cells were injected intothe femur, together with the implants. The anaesthetic, surgicaland post-operative care protocols used in this study were provedby the University Ethics Committee of the University of Hong Kongand the Licensing Office of the Department of Health of the HongKong Government.

To monitor the severity of infection, a special bioluminescentS. aureus was used, and an optical bioluminescence imaging sys-tem (Xenogen IVIS 100, Caliper Life Sciences, USA) was applied forreal-time monitoring. The bioluminescent signal emitted was cap-tured at different time points, and the signal intensity was directlyproportional to the number of live bacterial cells. Hence, the real-time relative amount of live bacteria in the implanted site couldbe determined by comparing the intensity of the bioluminescentsignals of different groups.

2.9. Statistical analysis

The experiments were performed in triplicate, and all datawere analyzed via the one-way ANOVA and expressed asmeans ± standard deviations. A p value < 0.05 was considered to bestatistically significant.

628 W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633

Fig. 5. The results of bacterial adhesion test for E. coli (1 × 107 CFU/ml, 20 �l) on different nanowire coatings after incubation at 37 ◦C for 30 min. (A) SEM images of thea nO 17c ) The

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ttachment of E. coli cells to untreated stainless steel surface, ZnO 5 h coating and Zells were collapsed after being exposed to the ZnO 5 h and ZnO 17 h coatings. (Bive/dead staining. Both samples are highly effective in suppressing E. coli cell attac

. Results

.1. Morphology examination of 1D ZnO nanowire-coating

Fig. 1A revealed a rod-shaped wire on the surface of theedical-grade stainless steel after hydrothermal treatment for 5 h,

s well as the selected area electron diffraction (SAED) patternf the chemical composition and crystal structure. The rod wasbout 30 nm in diameter and 350 nm in length. The clear lat-ice fringes revealed that the rod was a single crystal and thathe measured lattice parameters of the rod were a = 3.2539 Å and

= 5.2098 Å. These measurements matched the calculated latticearameters (a = 3.247 Å and c = 5.201 Å) of pure hexagonal-phasenO (wurtzite-type, space group: P63m), as stated by the Jointommittee on Power Diffraction Standards (JCPDS No. 36–1451),ell. This proves that the synthesized ZnO nanowire was a wurtzite

rystal structure in a hexagonal shape.The surface topography of stainless steel before and after the

oating was displayed in Fig. 1B. The stainless steel surface wasmooth, whereas the surface became rough after hydrothermalreatment. The 1D ZnO nanowires were grown perpendicular tohe sample surface and were evenly distributed. The inset in Fig. 1Blearly suggested that the nanowires were in a hexagonal rod shapend that the length and diameter of the nanowires matched thosebtained from the TEM examination (Fig. 1A). Furthermore, the ZnOanowires on the surface of the ZnO 5 h sample were found to be

round 50 nm in diameter and organized. However, the nanowiresrown on the surface of the ZnO 17 h sample were around 150 nmn diameter and disorganized.

h coating (the scale bar is 1.00 �m). No appendages were observed, and the E. coliplot of the number of live E. coli cells on different sample surfaces, measured by

t.

The morphologies of the untreated and treated medical-gradestainless steel rods for in vivo animal model were demonstratedin Fig. S1. By comparing the surface topographies of the untreatedand treated samples, the 1D ZnO nanowire-coating was also suc-cessfully built onto the surface of the stainless steel rod.

3.2. Contact angle analysis

To study surface hydrophilicity, contact angle measurements forthe treated (i.e., ZnO 5 h and ZnO 17 h) and untreated stainless steelsurfaces were obtained. The surface free energy of each sample wascalculated and subsequently summarized in Table 1. The contactangle dropped significantly after the surface was coated with 1DZnO nanowires. In contrast, the surface free energy of the ZnO 5 hsample was higher than that of the ZnO 17 h sample. Because thedispersive components of all samples decreased, it is believed thatthe surface free energy increased due to the change in its polarcomponent.

3.3. Dissolution of 1D ZnO nanowire-coating

In order to study the dissolution of 1D ZnO nanowire-coatingin different solutions, the surface morphology and release of zincions for the treated samples after TSB and DMEM immersion wereobserved under scanning electron microscopy (SEM) and induc-tively coupled plasma mass spectrometry (ICP-MS), respectively.

After being incubated in TSB at 37 ◦C for 30 min, the surfacebecame rough, and the hexagonal structure gradually degraded,as revealed in Fig. 2A. Subsequently, the media were extracted, andthe release of zinc ions was measured by using ICP-MC, while the

W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633 629

Fig. 6. (A) Fluorescent microscopic images of pre-osteoblastic cells (5 × 104 cells) cultured with normal medium and conditioned media containing 0.4 ppm and 0.5 ppm Zn2+

ions at 37 ◦C for 6 h (the scale bar is 100 �m). The cell morphology is normal. (B) The cell viability (4 × 105 cells/ml) of pre-osteoblasts cultured in normal and conditionedmedia at 37 ◦C for different time points. No cytoxicity was observed.

Table 1The measurement of contact angle, surface free energy, and dispersive component and polar component for untreated stainless steel surface, ZnO 5 h coating and ZnO 17 hcoating. The treated samples demonstrated more hydrophilic surfaces than the untreated control.

Samples Water contact angle (◦) Surface energy(nJ cm−2) Dispersive component(nJ cm−2) Polar component(nJ cm−2)

uras

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sb

Control 82.7 24.71

ZnO 5 h 24.7 76.27ZnO 17 h 35.6 65.44

ntreated sample served as a control. The amounts of Zn2+ ionseleased from the Zn 5 h and ZnO 17 h samples were 18.2nmolnd 29.9nmol, respectively. The background value of the untreatedample was 0.7nmol.

Another immersion test was performed in DMEM solution at7 ◦C for 72 h. The morphological change in the ZnO nanowires wasimilar to that of the samples immersed in TSB. After immersion for

days, the nanowires were further degraded, as shown in Fig. 2C.he hexagonal structure of the ZnO nanowires in the ZnO 17 hample even disappeared completely. The amounts of Zn2+ ionseleased from the ZnO 5 h and ZnO 17 h samples were 31.9 nmolnd 37.8 nmol as exhibited in Fig. 2D. The results suggested that theD ZnO nanowires could gradually degrade in various solutions tollow zinc ions release.

.4. Antimicrobial-adhesion tests

.4.1. S. aureusS. aureus is a kind of Gram-positive bacilli and also the major

ource of implant-associated infection in orthopaedics [27]. Aftereing inoculated on untreated and treated surfaces for 30 min, the

13.59 11.133.27 73.005.07 60.37

adhered morphology of S. aureus on various sample surfaces wasanalyzed via SEM and shown in Fig. 3A. Due to the tightly packedsurface structure of the ZnO 5 h sample, the bacterial cells could notpenetrate into the surface layer formed by the nanowire, whereasthe ZnO 17 h sample layer trapped relatively more bacterial cellsdue to the specific orientation of the ZnO nanowire-coating. Mean-while, the hair-like appendages extended out from the bacteriamembrane on the untreated and ZnO 17 h coated surfaces. Fur-thermore, quantitative analysis showed that nearly 95% of S. aureuscould not attach to the surface of the ZnO 5 h sample, whereas 55%of bacteria could not adhere to the surface of the ZnO 17 h sample,as compared with the untreated control (Fig. 3B).

3.4.2. P. aeruginosaAmong various kinds of Gram-negative bacilli, P. aeruginosa is

another of the most frequently clinically isolated bacteria foundin orthopaedics [28]. The results indicate that there were only

5.1% and 6% of live P. aeruginosa attached to the surface of theZnO 5 h sample and ZnO 17 h sample, respectively, as comparedwith the control (Fig. 4B). Obviously, the 1D ZnO nanowire-coatingreduced the adhesion of P.aeruginosa by about 90%. It is believed

630 W. Wang et al. / Colloids and Surfaces B: Biointerfaces 141 (2016) 623–633

Fig. 7. (A) X-ray and bioluminescent signal images of the rat model implanted with bacteria-contaminated Ti implant for in vivo antibacterial test. The red circle highlightswhere the Ti implant is placed in the rat. The intensity of the bioluminescent signal emitted from the operated rats was observed. The red colour represents high levels ofa ensityd or intet

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ctivity on the part of live bacteria under in vivo conditions. (B) The plot of signal intemonstrated less bacteial activity from Day 0–7 as compared with the control. (Fhe web version of this article.)

hat the P. aeruginosa cells cannot easily penetrate into the matrixf nanowires because of their size. Hence, the treated sample canffectively resist the attachment of large numbers of bacteria. Also,o hair-like appendage was found on adhered bacterial cells.

.4.3. E. coliThe anti-adhesion ability of 1D nanowire-coating had been

ested against another Gram-negative bacterium—E. coli. Becausehe dimensions of E. coli cells and P. aeruginosa cells are similar tone another, the anti-adhesion coating can therefore significantlyesist the attachment of these bacterial cells. It was found that thereated surfaces of the ZnO 5 h and ZnO 17 h samples could alsoffectively decrease bacterial adhesion by 95and 97%, respectivelyFig. 5B). Furthermore, it was found that the all the E. coli cells hadollapsed after becoming attached to the surfaces of the treatedamples.

The killing efficiencies of each coating for different bacteria haveeen summarized in Table S1. Additionally, the fluorescent images

f different coatings acting against various bacterial cultures arerovided in Fig. S3. In general, the coatings were found to be able toill 90% of bacteria, except for the ZnO 17 h sample in the S. aureusulture.

of live bacteia recorded in the rat models, along the timepoints. The coated samplerpretation of the references to color in this figure legend, the reader is referred to

3.5. Antimicrobial properties of Zn2+ ions

To study the antimicrobial properties of Zn2+ ions, minimalinhibitory concentration (MIC) tests against S. aureus, P. aeruginosaand E. coli were performed. For the S. aureus cells, it was noticed thatthe OD values of the well containing 87.5–200 ppm of Zn2+ ionswere close to those of the negative control and that the p valuebetween these groups and the negative control was greater than0.05, indicating that the bacteria concentrations of these groupswere similar to that of the negative control group. Meanwhile, withanalyzing the absorbance of wells containing 0–75 ppm as com-pared to that of negative control group, the p values were smallerthan 0.05, suggesting that the bacteria concentrations in these wellswere significant different from that in the negative control group.These results suggest that the MIC value of Zn2+ ions that was ableto inhibit the growth of S. aureus was 87.5 ppm. By applying thesame analytical methods, the MIC values of Zn2+ ions against P.aeruginosa and E. coli were 137.5 ppm and 125 ppm, respectively.

3.6. Evaluation of coating cytocompatibility

The cytotoxicity of the Zn2+ ions released from the 1D nanowire-coating was investigated. In accordance with the previous study,the zinc ion concentration released from the nanowire matrix was

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.4 and 0.5 ppm respect to the ZnO 5 h and ZnO 17 h samples,espectively. Hence, the conditioned media containing Zn2+ ions at.4 and 0.5 ppm were prepared for mammalian cell culture, respec-ively. The cell morphology after culturing with these conditionednd normal media for 6 h was shown in Fig. 6A, and the osteoblastiability after culturing on day 1 and day 3 was also shown in Fig. 6B.

The cell morphology after incubation with the control mediumid not differ from that of the cells cultured with conditionededia contained 0.4 ppm Zn2+ or 0.5 ppm Zn2+ ions, indicating that

ell attachment and spreading were not affected by the Zn2+ ionseleased from the nanowire matrix. Furthermore, the cell viabil-ty of pre-osteoblasts cultured with Zn2+ containing medium waslightly higher than that of the medium without Zn2+ ions. How-ver, the difference was not statistically significance except for theells cultured with ZnO 5 h samples at Day 3.

.7. In vivo antibacterial test in rat model

Fig. 7A demonstrated the post-operative X-ray image of the ratmplanted with a stainless steel rod in the femur and the biolu-

inescent signals emitted at the operation site. The signals wereecorded and plotted at predetermined time points from Day 0o Day 9, as shown in Fig. 7B. The signal from the nanowire-oated rod was significantly reduced as compared with that fromhe untreated control sample after 2 h of operation. Subsequently,he intensity of the bioluminescent signal found in the nanowire-oated rod was lower on Day 3 as compared with that of thentreated control, suggesting that fewer live bacteria were found

n the nanowire-coated group. Furthermore, the signal found in theanowire-coated group became undetectable, whereas the signal

ntensity of untreated group remained at a significant level. Hope-ully, the signal intensity of the untreated group also flattened after

days of implantation.

. Discussion

Due to the increase in antibiotic-resistant bacilli, a great dealf attention has been focused on the development of zinc (Zn)elated coatings [14–16,18,29]. The hydrothermal method is annexpensive and feasible surface modification technique for fab-icating ZnO coatings on various substrates. In the present study,he growth of nanowires involved two steps: ZnO seed layer for-

ation and the growth of ZnO nanowires. Indeed, we believe thathe ZnO seed layer plays a very important role in the subsequentrowth of nanowires. When the duration of heat treatment in therst step was reduced, the ZnO nanowire-coating could only cover

he stainless steel surface partially and the coating was unevenlyistributed (Fig. S2). Heat treatment will also trigger the formationf a metal oxide layer on the stainless steel surface. When the ZnOeed is bonded to the metal oxide layer, the subsequent reactionime during heat treatment would be the sole parameter to alterhe diameter and orientation of nanowires in this study.

The degree of hydrophobicity of the biomaterial surface is gen-rally considered highly correlated to the bacterial attachment30]. According to the literature, the membranes of bacterial cellsre generally hydrophobic [31,32]. With respect to the theory ofhermodynamics, when a bacterial cell adheres to a hydrophilicurface, this will definitely help minimize the overall free energyf the entire system; thus, this approach is energetically favorable31]. In addition to hydrophobicity, surface free energy (SFE) isnother parameter determining the adhesion of cells and bacte-

ia [33]. However, according to the Baier curve [34], the minimaldhesion rate can be achieved when the SFE lies between 20 and0 nJ cm−2. When the SFE is out of the range, the adhesion ofells and bacteria will theoretically increase with increased SFE.

Biointerfaces 141 (2016) 623–633 631

However, the 1D ZnO nanowire-coating was found to exhibit alower contact angle and relatively higher SFE as compared withthose of the untreated stainless steel surface, indicating that it wasrelatively hydrophilic and favorable for bacterial attachment. Nev-ertheless, the ZnO nanowire-coating demonstrated the ability toresist the adhesion of various bacteria.

The literature reported that the lowest level of bacterial adhe-sion was observed on the surface, with smoothness down tonanometric levels, indicating that larger surface areas at the bacte-ria substrates interfaces would promote greater microbial adhesion[35]. Therefore, a specifically designed surface with nanoarchitec-ture that is able to reduce the bacterial anchoring surface maydemonstrate excellent anti-adhesion activity against various bac-terial strains [36]. According to a previous study [37] superiorantibacterial properties were demonstrated by randomly orientedZnO nanoarrays as compared with those of oriented and par-tially oriented nanoarrays. Jasson et al. [38] also fabricated a ZnOnanorod-coating on a titanium surface and evaluated the adhesionand viability of P. aeruginosa and S. epidermidis as compared withthose for titanium substrates sputtered with ZnO (flat control). Theresults suggested that only the ZnO nanorod-coating could effec-tively resist the adhesion of bacterial cells. In this research, thespike surface, especially the ZnO 5 h nanowire-coated surface, caneffectively reduce the contact area between the bacterial cell andthe material surface. As shown in Figs. 3 A-5 A, the tips of thenanowires are the only anchoring points for the bacteria. However,when a bacterial cell penetrated into the matrix of ZnO nanowires,particularly that of the ZnO 17 h sample, the appendage was thenreleased, as shown in Fig. 3A. The loose and disorganized structuremay provide more anchoring points for S. aureus, and therefore,some bacteria can survive in this ZnO nanowires matrix.

In addition to the specific surface topography formed by 1DZnO nanowire-coating, the antibacterial activity of the coating maybe partially attributed to the release of zinc ions from the ZnOnanowires. In a previous study, Zn2+ ions were found to inhibitthe active transport and metabolism of sugars, as well as disruptenzyme systems [19]. Meanwhile, through ion channels, Zn2+ ionswere transported to the bacteria cytosol and accumulated in thecytosol, which could be detrimental to bacteria [39]. Therefore, theantimicrobial property of zinc ions would be relied on the sus-tainable release of Zn2+ ions over a period of time [40]. In thedissolution test of ZnO nanowire-coating, various solutions wereused to incubate the coated samples. Zinc ions were detected in thesolutions, suggesting that Zn2+ ions were gradually released fromthe nanowire coating, so as to offer the ability to resist bacterialgrowth after surgical implantation.

Furthermore, the reactive oxygen species (ROS) that are gen-erated from the surface of ZnO, such as superoxide and hydroxylradicals [41,42], are suspected to be among the antimicrobial fac-tors [43]. In the past, Sawai et al. [41] and Yamatomo et al. [44]found that the release of peroxide hydrogen (H2O2) from ZnO helpkill bacteria. However, both noticed that the H2O2 was more effec-tive in killing E. coli as compared to S. aureus. It is believed that thisdiscrepancy is due to the different cell membrane structures of S.aureus and E. coli [45]. In our experiments, gram-negative bacterialcells, e.g., P. aeruginosa cells and E. coli cells were likely suppressedby the generated ROS, in addition to Zn2+ ions, whereas the ROSeffect was less effective for S. aureus cells were less sensitive to thenanowire matrix, no collapse was observed.

Indeed, various nanostructured forms of ZnO such as nanorods[37,38,40], nanotubes [46], nanobelts [47], and nanoflowers [48]have been investigated for antimicrobial properties. However, the

antimicrobial abilities of these nanostructured coatings were onlyseen in in vitro tests, verification by using an in vivo model hasnever been reported in the literature. In this study, the aim of theanimal experiment was to demonstrate the antibacterial effect of

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D ZnO nanowire-coating under in vivo conditions rather than toropose a treatment protocol for implant-related osteomyelitis. Inhe results of the animal test, the 1D ZnO nanowire-coated samplesemonstrated lower initial attachment and suppression of bacte-ial growth as compared with those of the untreated stainless steel.t was speculated that the surface with 1D ZnO nanowire-coatingiscouraged the attachment of bacterial cells at the time of implan-ation. Then, the generated ROS and released Zn2+ ions were able touickly kill and suppress the growth of bacteria as compared withhe untreated control.

Because the medical application of zinc-supplemented andinc-incorporated biomaterials has increased [17,36,49], concernsbout the cytotoxicity of Zn2+ ions have increased as well.amaswamy et al. [50] demonstrated that the Zn2+ ions extracted

rom zinc containing bioglass would not result in any toxicity foruman osteoblast-like cells. The highest concentration of extractedn2+ ions detected was 0.004 mM (approx. 0.3 ppm). Addition-lly, Brauer, et al. [49] studied the cytotoxicity of conditionedulture medium (i.e., DMEM) contained various concentrationsf ZnCl2 against MC3T3-E1 pre-osteoblasts. Cell viability signifi-antly dropped when the concentration exceeded 300 �M (approx.0 ppm). According to the result obtained in the dissolution testf 1D ZnO nanowire-coating in the DMEM solution, the concen-rations of the released Zn2+ ions from the ZnO 5 h and ZnO 17 hamples were 0.4 ppm and 0.5 ppm, respectively. Obviously, theoncentration of released Zn ions in our study was much lower than00 �M. Also, our in vitro results suggest that the ZnO nanowire-oating would not induce substantial toxicity for mammalian cells.evertheless, a long-term animal study of ZnO nanowire-coated

tainless steel must be initiated prior to human clinical trials.

. Conclusion

A layer of 1D ZnO nanowire-coating has been successfully estab-ished on a medical-grade stainless steel surface (plain or rod)y using a hydrothermal approach in which the dimensions andtructure of the nanowires depend on treatment parameters suchs time and temperature. The coating featured a specific spikedurface structure, and the ability to sustainably release Zn2+ ionsemonstrates reliable antimicrobial properties against the bacilliommonly seen in implant-associated infections in orthopaedics.lso, the concentration of released zinc ions is unlikely to lead to

cytotoxic effect to mammalian cells. More importantly, antibac-erial effect has been successfully demonstrated in animal model,uggesting that the 1D nanowire-coated stainless steel is promisingor clinical application in orthopaedics.

cknowledgements

This project was financially supported by The University ofong Kong Foundation Seeding Fund for Applied Research, Specialrophase for Key Basic Research of Ministry of Science and Tech-ology of China (973 Program, No. 2014CB660809), the Nationalatural Science Foundation of China (Project Nos. 51422102,1271715 and 31370957)

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02.36.

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1

Appendix A. Supplementary data

Fig. S1. (A) SEM images of untreated (‘U’) and 1D ZnO nanowire-coated (‘T’) medical-grade

stainless steel rods. (B-D) High-resolution SEM images of untreated and treated sample surface.

The surface topography of the untreated stainless steel rod was flat, while a nanowire matrix was

clearly seen after the treatment (the scale bar is 1.00µm)

2

Fig. S2. SEM images of sample surface treated via other parameters, where (A) shows the low

magnification and (B) shows the high one. The ZnO nanowire coating was unevenly distributed

on the stainless steel surface when the heat treatment duration was reduced

3

Fig. S3. The fluorescent images of live/dead staining of untreated, ZnO_5hrs and ZnO_17hrs

against various bacterial strains (1*107 CFU/ml, 20µl) at 37°C for 30 minutes (the scale bar is

20μm). Each green dot represents one live bacterium, while each red dot refers to one dead

bacterium. Obviously, the ZnO_5hrs sample was able to significantly suppress all three types of

bacteria. However, the ZnO_17hrs sample was more effective against P.aeruginosa and E.coli as

compared with S.aureus

4

Table S1. The killing efficiencies of the ZnO_5hrs and ZnO_17hrs samples against S.aureus,

P.aeruginosa and E.coli (1*107 CFU/ml, 20µl, 37°C for 30 minutes). The bacterial killing effects

of the ZnO_5hrs and ZnO_17hrs samples are generally more than 90%, except for the

ZnO_17hrs sample cultured with S.aureus

S.aureus P.aeruginosa E.coli

ZnO_5hrs 93.5% 94.9% 95.4%

ZnO_17hrs 55.3% 94.0% 97.5%