Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants Kaveh Memarzadeh,1,2 Amir S. Sharili,1 Jie Huang,2 Simon C. F. Rawlinson,1 Robert P. Allaker1 1
Queen Mary, University of London, Barts and The London School of Medicine and Dentistry, Institute of Dentistry 4 Newark Street, London, E1 2AT, United Kingdom 2 Department of Mechanical Engineering, University College London, Torrington Place, London, WC1E 7JE, United Kingdom Received 2 April 2014; revised 18 May 2014; accepted 23 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35241 Abstract: Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA compositecoated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-a and IL-6 cyto-
kines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial C 2014 Wiley Periodicals, Inc. J Biomed Mater and biocompatible. V Res Part A: 00A:000–000, 2014.
Key Words: nanotechnology, ZnO, coatings, antimicrobial, biocompatibility, bone
How to cite this article: Memarzadeh K, Sharili AS, Huang J, Rawlinson SCF, Allaker RP. 2014. Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Orthopedic and dental implants are susceptible to failure due to a variety of causes with bacterial infection being of particular concern.1,2 This occurs despite extensive measures to reduce such infections. Some medical devices incorporate antibiotics to help resist infection; however, due to certain limitations, for example, decreased release at the later stages of implantation, microbial resistance continues to rise.3 Development of innovative methods which deliver an alternative approach to antimicrobial function is therefore much in demand. In particular, the potential of antimicrobial nanoparticles (NPs) as future agents has received much attention.4 These agents possess physiochemical properties that make them unique among all antimicrobial agents. Their shape, increased surface area and size, combined with their toxic nature towards prokaryotic organisms make them extremely attractive as multifunctional agents.5 As coatings for medical bone implants evolve, a niche for new products that outperform the previous generation of biomaterials is created. nHA is a direct result of such evolution.6 This material is known to increase osteoblast prolifer-
ation, collagen production as well as maintaining normal osteoblast morphology.7 In this study, using a novel coating method, we investigate the potential of composites of nHA and nZnO as innovative coating materials for future orthopedic and dental implants. Conventional coating methods such as plasma spraying utilize extreme heat that can change properties of NPs, often resulting in the production of a rough, thick (40–50 mm) and uneven surface.8 Therefore, by applying a method that allows for formation of a homogenized, micro-fabricated coating, it has been possible to produce thin layers of nanocoatings that are uniform in nature. This unique quality allows for the exposure of NPs to both bacteria and osteoblasts which may result in unique outcomes not achievable by conventional coatings. The combination of this novel approach with the advantage of zinc being an important trace element in the body can be distinctively useful for bone-related implants. More recently, it has been demonstrated that the addition of ZnO to bonecomposites can enhance the proliferation, adhesion, and differentiation of osteoblasts.9 Furthermore, zinc embedded calcium phosphate (CaP) ceramics have also been shown to
Additional Supporting Information may be found in the online version of this article. Correspondence to: Kaveh Memarzadeh; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
1
FIGURE 1. Transmission electron micrograph of both ZnO (A) and TiO2 (B) nanoparticles. A: Red lines indicate rod-shaped structures of the NPs whereas the black triangles showcase the sharp-edged particles. B: TiO2 NPs are similar in size but lack sharp edged confirmations. Red triangles indicate round shaped TiO2 particulates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
promote the growth of bone cells when compared with nonembedded Zn CaP ceramics.10 The antibacterial effects of nZnO are currently under investigation and recent studies suggest that this material is strongly bactericidal, making it suitable as a coating for bone medical implants.11,12 Preliminary observations suggest that this agent has a degree of selectivity towards Staphylococcus aureus, a species that is predominantly responsible for surgical site infections.13 Possible advantages of NPs as compared with conventional antibiotics are their ability to promote bactericidal effects via different routes, namely: ion release, direct contact, and reactive oxygen species generation.14,15 This investigation was designed to aid our understanding of the relationship between coated nZnO and conventional nHA in relation to pathogenic bacteria and osteoblasts and also propose that nZnO, on its own, can be a suitable candidate for future medical bone implants. MATERIAL AND METHODS
Nanoparticles Metal oxide nanoparticles (NPs) were synthesized using the flame pyrolysis method as used by Johnson Matthey plc (JMTC). The average surface area was determined by Brunauer Emmett Teller (BET) analysis at JMTC. nHA particles were made by the process of wet precipitation between calcium hydroxide (Ca(OH)2) and orthophosphoric (H3PO4) acid.16 Handling and storage of these NPs was performed in a manner to avoid biological and environmental harm. The NPs tested were: Zinc Oxide (nZnO) (Fig. 1) Titanium dioxide (nTiO2, Fig. 1) and nHA. It is essential to state that nTiO2 was employed for comparative purposes in the cytotoxicity assay and was not utilized in all studies.
2
MEMARZADEH ET AL.
Nanoparticulate suspensions and composites Nanoparticulate suspensions. Different concentrations of nZnO suspension (Table I) were made in 100% ethanol and sonicated thoroughly for EHDA spraying. Nanoparticulate composites. For preparation of the composite mixtures (Table I), two different suspensions of nZnO and nHA were made with equal concentrations of 10,000 mg/mL. From these suspensions a known volume was added to produce the required percentile of NPs within each composite.
TABLE I. A Summary of all Coated Samples, Utilized Both for Biocompatibility and Antimicrobial Studies nZnO Suspensions
nHA Suspensions
mg/mL
100%
10,000
75% 50% 25% -
10,000 10,000 10,000 10,000
100%
-
10,000
100% 100%
-
5000 2500
25% 50% 75% 100%
1 1 1
Composite Coatings
nZnO Coatings
Composite coatings, except for 100% nHA were employed for their antimicrobial activity. All composites were used for ALP, proliferation, and focal adhesion studies. The nZnO coatings at different concentrations were used to observe the morphological state of the cells over the course of time. 1 5 indicates addition of one concentration to another.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
Glass substrates Glass slides and 13 mm coverslips were used in this study (VWR international). A diamond cutter was used to separate 1 cm2 samples from each glass slide. Each sample was dipped in 100% ethanol for a period of 1 min and air dried for 5 min before EHDA spraying. Additionally, uncoated glass samples were considered as controls. EHDA deposition of nZnO coating EHDA was used to coat all samples. Briefly, EHDA consists of an epoxy resin-embedded nozzle connected to both a power supply (Glassman High Voltage) and a 10cc syringe pump (Harvard Apparatus, PHD 4400 Programmable) containing suspended NPs. The applied voltage (3.2–4.2 kV) creates an electrical field between the nozzle and the substrate, which allows for the formation of a uniquely shaped stable cone jet. Such a jet, together with a steady flow rate of 5 mL/min is required to create a uniform spray which forms nanosized droplets that encapsulate the NPs. The spraying is carried out for 2 min with a 30 mm distance between the tip of the nozzle and the substrate. Subsequently, the coated samples were heat treated at 500 C for 1 h and allowed to cool gradually to help ensure the mechanical integrity of the coating. Antimicrobial studies Bacterial species. Staphylococcus aureus (Oxford strain; NCTC. 6571) was used in this study. This bacterium was stored at 280 C on beads in cryopreservative fluid. S.aureus is the predominant species involved in orthopedic-related infections.17 Nano-coated bactericidal samples. The coated samples were dipped in 100% ethanol for 1 min and flamed to ensure sterility. Test species of bacteria were grown overnight in Tryptone Soya Broth (TSB). The OD (optical density) was adjusted to 0.1 at 540 nm in Phosphate Buffered Saline (PBS) or Adult Bovine Serum (ABS, SIGMA-A). 30 mL of the OD adjusted bacterial suspension (Population of 1 3 106/mL) was transferred onto the surface of the coated and uncoated samples. Samples were placed in a humidified chamber then incubated at 37 C with 10% CO2 for a period of 24 h. Subsequently each sample was carefully removed and placed in 3 mL of PBS. Approximately 5 glass beads were added to each tube to aid the detachment of the bacteria while spinning the tubes for a period of 30 s at 1500 RPM. Control experiments demonstrated such treatment did not adversely affect viability of bacteria. Totally, 50 mL of the suspension was then removed and a 1 : 10 serial dilution performed. Neat and dilutions were plated onto Tryptone Soya Agar (TSA) plates and incubated for 24 h, after which colonies were counted. Biocompatibility and cell proliferation studies Cell culture. UMR-106 (Rattus norvegicus, osteosarcoma, ATCC), MG-63 (Homo sapiens, osteosarcoma, ATCC) and human mesenchymal stem cells (hMSC, University College London, Eastman Dental Institute) were used in this study.
Most investigations were carried out using UMR-106 cells. hMSCs were utilized for qualitative adhesion observations. MG-63 cell-lines were utilized for both qualitative and quantitative adhesion observations as well as ELISA. Frozen cells were gently thawed and cultured in highglucose DMEM with 2% FCS, 1% penicillin, and 1% L-glutamine. The cells were incubated for 48 h at 37 C with 5% CO2 and then detached using Trypsin/EDTA. Trypsinized cells were resuspended with 5 mL of medium, placed in 10 mL tubes and centrifuged at 800 3 g for 5 min. Supernatant was removed from each tube; fresh media was then added to cell pellets. Osteoblasts were then dispersed, suspended, counted, and seeded onto coated samples with a population of 1 3 104 cells. Samples were incubated at 37 C, 5% CO2 for 2 h to allow cell-substrate adhesion. Proliferation assay. The proliferation of the UMR-106 cells R -AbD Serowas assessed using Alamar Blue (alamarBlueV tec). The reagent was diluted with normal growth medium (1 : 10 Dilution) and stored at 4 C prior to use. Approximately 1 3 104 osteoblasts were seeded onto coated glass samples in a 24-well plate (n 5 4 per condition). Seeded cells were left for a period of 15–20 min in the incubator (37 C, 5% CO2) to ensure adhesion and 1 mL of medium was added to each well for overnight incubation. At 1, 5, and 10 days (media was replaced every 2 days); 10% volume of the medium in each well was replaced with ab medium. The plate was then incubated at 37 C, 5% CO2 for a period of 4 h and 100 mL of the supernatant was removed from relevant wells and added to a 96-well (Falcon) plate. Fluorescence was measured using a fluorescent plate reader (FLUstar OPTIMA) at wavelengths of 570 nm (excitation) and 600 nm (emission). Cytotoxicity assay. Lactate Dehydrogenase (LDH) release from UMR-106 cell lines was measured using a commercially available kit (LDH-Cytotoxicity Assay Kit II, abcam, UK) and carried out according to the manufacturer’s instructions. LDH release: Osteoblasts exposed to NP suspension supernatants. To examine the possible effects of released ions on osteoblast viability, nZnO particulates were suspended in 2% FCS DMEM (10,000 mg/mL) and left in a shaking incubator for 1, 5, and 10 days (37 C, 200 RPM). nTiO2 particulates were also tested in a similar manner. At each time point, samples were removed and transferred into a 50-mL corning centrifuge tube and spun at a speed of 1000 RPM (160 3 g) for 5 min. The supernatant from each sample was removed and filtered using a syringe filter unit (0.22 mm). Osteoblasts were seeded at a density of 1 3 104 per well in a 96-well plate. 100 mL of DMEM (with 2% FCS) was added to each well and incubated at 37 C, 5% CO2 for 24 h to allow a cell monolayer to develop. The medium in each well was replaced with 100 mL of the filtered supernatant or Lysis Buffer (Positive control) and incubated for 40 min. The plate was centrifuged at 600 3 g for 10 min and 10 mL of the supernatant was transferred to a new 96-well plate. 100 mL of the LDH reaction mix was
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
3
added to each well and incubated for a further 30 min. Absorbance for all samples and controls was measured at a wavelength of 450 nm using a plate reader (FLUstar OPTIMA, BMG Labtech, Germany). LDH release: Osteoblasts exposed to nano-coated sample supernatants. The coated samples were placed in a 24well plate containing 2 mL of 2% FCS DMEM to each well. The samples were then left in a shaking incubator (37 C, 200 RPM) for periods of 1, 5, and 10 days. In parallel, 1 3 104 cells per well were seeded into a separate 24-well plate and incubated at 37 C, 5% CO2. 1 mL of DMEM from each time point was subsequently added to cells. Osteoblasts were then incubated for a further 24 h and 100 mL of each well was removed and dispensed into a 96-well plate and the LDH release was measured. Alkaline phosphatase activity. The differentiation of the UMR-106 cells on the nZnO-coated samples was determined by measuring alkaline phosphatase release. A population of 1 3 104 cells were seeded onto coated glass samples in a 24-well plate and incubated at 37 C, 5% CO2 for 1, 5, and 10 days respectively. For the ALP assay, one tablet of 4nitrophenyl phosphate disodium (SIGMA-A, Dorset, UK) was mixed with 8 mL 0.1M Trizma Hydrochloride (pH 5 9.5) and 15 mL of 2M MgCl2. Before addition of the reaction solution to the cells, each well was washed with PBS three times. 1 mL of the reaction solution was added to each well containing the cell-seeded-coated samples. The 24-well plate was left at room temperature for 10 min for the reaction to take place. NaOH (0.5M) was then used to stop the reaction. Subsequently, 100 mL aliquots were removed from each well and transferred into a 96-well plate and absorbance read at 405 nm using a plate reader (FLUstar OPTIMA). Cytokine release measurements. ELISA was performed in order to quantify cytokine release. MG-63 human osteosarcoma cells were seeded onto glass samples (as described previously) and cytokine release (TNF-a and IL-6) was subsequently measured according to the manufacturer’s protocol (Thermo Scientific). Microscopy Characterization. Transmission electron microscopy. ZnO along with TiO2 NPs were placed in 5 mL of distilled water and sonicated to provide a homogenous suspension. Carbon film grids (400 Mesh Cu, Agar scientific) were briefly dipped into the nanoparticulate suspension and left at room temperature to air dry. The grids were transferred into the TEM (Philips CM 12) and subsequently analyzed. Cell morphology. Optical microscopy. Standard inverted light microscopy (Nikon Eclipse TE2000-5) was used to observe cellular growth at every stage. Approximately 1 3 105 population of cells were seeded onto coated samples and their morphology and distribution was monitored over time. Scanning electron microscopy (SEM). Both coated and uncoated substrates that were seeded with cells were placed into 3% Glutaraldehyde (SIGMA-Aldrich, Dorset, UK)
4
MEMARZADEH ET AL.
in 0.1M Cocadylate buffer (SIGMA-A) for a period of 24 h. Each sample was then dehydrated using ascending concentrations of ethanol: 20, 50, 70, and 90% for 10 min and 100% ethanol for 30 min. Finally the samples were dried using hexamethyldisilazane (HMDS) (SIGMA-A). Samples that lacked the presence of cells were cleaned with 100% ethanol and processed without the dehydration process. Furthermore, glass samples were mounted onto stubs and made conductive by the application of conducting carbon cement (Agar Scientific) between the sample and the stub. All samples were gold coated and directly transferred into the scanning electron microscope (FEI Quanta 3D FEG). Immunofluorescence. Cells were fixed with 4% paraformaldehyde (PFA), incubated for 10 min in permeabilization buffer (phosphate-buffered saline (PBS), 0.2% Triton X-100) and subsequently blocked with 2% bovine serum albumin (BSA). Samples were incubated with primary antibody (4 mg/mL mouse anti-paxillin; Santa Cruz, USA) for 24 h at 4 C. Subsequently NP coated coverslips were washed with R 568 conjuPBS and incubated for 45 min with Alexa FluorV gated rabbit anti-mouse secondary antibody (Invitrogen R 488-phalloidin staining Molecular Probes, UK). Alexa FluorV was applied to visualize F-actin (Invitrogen Molecular Probes, UK). Imaging was performed with a Leica DM4000 epi-fluorescence microscope (Leica, Solms, Germany). Statistical analysis All quantitative results were presented as standard error of mean (mean 6 SEM). Comparisons of two sample means were carried out using Student’s t-test in GraphPad Prism software. Statistical significance was established when p < 0.05. RESULTS
Nanoparticulate characterization Conventional TEM was used to characterize the size and shape of the nanoparticles. Micrographs revealed a size range between 10 and 100 nm for nZnO particulates. These nanoparticulates possess elongated rod shaped structures with sharp edges at their distal extremes (Fig. 1), whereas the nTiO2 particulates are between 20 and 110 nm and lack elongated structures and sharp edged features. The surface area for both ZnO and TiO2 NPs were measured by BET method and found to be 47 m2/g and 87 m2/g respectively. Antimicrobial effects of coated nZnO NP coatings were analyzed using SEM and light microscopy. Micrographs revealed differences between a coated and an uncoated glass substrate (Supporting Information Fig. 1). Antibacterial properties of coated nZnO and nHA mixtures exposed to ABS-bacterial suspension demonstrated a reduction in populations of bacteria as the concentration of nZnO increased. Furthermore there was a significant (p < 0.05) dose dependent reduction when the control samples were compared with both the lowest and highest concentrations of coated nZnO [Fig. 2(A)]. A similar property was observed when PBS-suspended bacteria were
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
nZnO in culture was further investigated by immunofluorescent imaging of focal adhesion protein paxillin. Higher concentrations of nZnO did not have a detrimental effect on osteoblast spreading, punctate paxillin staining, and stress fibers (Fig. 5). Thus nZnO coatings allows osteoblast attachment and focal adhesion formation.
FIGURE 2. Bactericidal effects of nZnO-coated substrates A: Antimicrobial effect of coated nZnO 1 nHA composites on S.aureus in adult bovine serum (ABS). The coated glass samples are coated with different concentrations of nHA and nZnO. B: Antimicrobial effect of coated nZnO 1 nHA composites on S.aureus in PBS. Coated mixtures have bactericidal effects as the concentration of nZnO increases. Significance was established as compared with the control (uncoated substrate), **p < 0.001, *p < 0.05, mean 6 SEM, n 5 4.
placed onto the surface of the coated material [Fig. 2(B)]. Furthermore, the antimicrobial activity was more significant between the control and the 100% coated nZnO samples (Fig. 2). We further characterized the morphology of bacteria on the surface of the coated material using scanning electron microscopy (SEM). Images revealed bacterial colonization on the surface of the uncoated samples [Fig. 3(A)], whereas coated samples [Fig. 3(C)] displayed restricted bacterial colonization [Fig. 3(B)]. The increased amount of debris on the coated surfaces is due to the presence of dead bacteria [Fig. 3(B)]. Effect of nZnO on osteoblast adhesion and morphology Osteoblasts (UMR-106) were cultured on different concentrations of nZnO coatings (2,500 mg/mL, 5,000 mg/mL, 10,000 mg/mL—Table I). Bright field microscopy revealed a qualitative insight into the greater number of cells at 2, 24, and 48 h and minimal cell death (Fig. 4). Cell interaction with nZnO coated samples was further characterized by SEM. UMR-106 and MG-63 cells revealed spindle shaped morphology, characteristic of a normal osteoblast [Supporting Information Fig. 2(a)]. Cell morphology was largely normal in hMSCs, with no signs of cellular stress [Supporting Information Fig. 2(b)]. Osteoblasts displayed filopodia projections that were in close contact with the nZnO surface or in proximity to neighboring cells (Supporting Information Fig. 3). Adhesion to coated
Osteoblast cytotoxicity, viability, and proliferation LDH production and release from supernatants derived from suspended NPs (TiO2 and ZnO) [Fig. 6(A)] and coated nZnO (in cell culture medium) were measured using UMR106 cells [Fig. 6(B)]. No significant LDH release was observed under both conditions. Moreover, osteoblasts seeded on NP surface coatings demonstrated an increase in proliferation [Fig. 6(C)] and alkaline phosphatase release [Fig. 6(D)], with the higher nZnO percentages showing an increased activity in both. In addition, measurement of cytokine release (TNF-a and IL-6) revealed that no inflammatory response was induced when cells were allowed to adhere on coated surfaces. Coated and uncoated sample release were below the level of detection, i.e., absorbance (450 nm) was below 0.15 in all cases at 24 h and 5 days. IL-6 and TNF-a were detectable with absorbance readings of above 0.18. DISCUSSION
An ideal bone implant or prosthesis would allow bone growth on the coated surface of the implant, together with minimizing the likelihood of S.aureus attachment and proliferation. This study provides evidence to support these functions. Using different experimental approaches in vitro, we show that ZnO NPs together with nHA can function under different simulated environments to promote antimicrobial activity. We further employed a coating technique of EHDA with glass samples. This method provides a uniform coating of NPs that is both stable and more sustainable than other conventional coating methods. Our antimicrobial investigations with S.aureus demonstrate that the majority of coatings tested have significant antimicrobial activity. This effect is more apparent when the concentration of nZnO is increased which is in agreement with our previous comparative findings where coated nZnO displayed an increased antimicrobial activity when submerged in a suspension of S.aureus for a prolonged period of time.18 These results along with earlier findings19 indicate that coated nZnO can be particularly effective against given species of bacteria. The antimicrobial effect of ZnO is a recently described phenomenon and no previous studies have addressed the potential of uniformly coated nZnO as an antimicrobial orthopedic coating. Observations with osteoblast like cells (UMR-106 and MG-63) and hMSCs revealed that cell morphology is not compromised when cells are exposed to different concentrations of nZnO-coated onto glass. Moreover, nZnO immobilized on a surface can provide a suitable substrate for osteoblasts to grow, adhere and be metabolically active which is in agreement with previous studies reporting that
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
5
FIGURE 3. A qualitative assessment on the antimicrobial effect of nZnO-coated onto glass samples. A: Bacteria present on a surface without the coating. B: The white arrow indicates a single bacterium present on the coated surface. C: High resolution image of coating showing uniform characteristics.
osteoblasts could maintain structural integrity and adhesive properties when introduced onto NP coated coverslips.20,21
In general, when eukaryotic cells are introduced to a toxic environment/material, the phospholipid bilayer can be ruptured and intracellular compartments of the cell escape
FIGURE 4. Osteoblast growth on nZnO-coated surfaces. Seeded UMR-106 (osteoblast-like cells) on the surface of nZnO-coated glass samples. The samples were coated with increasing concentrations of nZnO to observe any morphological changes. Phase images show increased numbers of cells at 2, 24, and 48 h. Red arrows indicate Initial attachment of osteoblasts to the substrates after 2 h of incubation and black arrows show the similarity of osteoblast confluency between a coated and uncoated substrate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
6
MEMARZADEH ET AL.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
FIGURE 5. Effect of nZnO and nHA on actin cytoskeleton and focal adhesions. Fluorescent images of actin filaments (arrow heads in A, D, G, J) and focal adhesions (arrows in B, E, H, K). Human osteoblast-like cells (UMR-106) cultured for 24 h on 100% nZnO, nHA, and 50% nZnO coatings show more marked focal adhesions (E, H, K) compared with control (B). Scale bars 5 50lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to the extracellular medium.22 Release of the enzyme LDH is a helpful indicator of cytotoxicity. In the present study, when exposed to supernatants of both nTiO2 and nZnO, although eliciting a greater response than nTiO2, the supernatant derived from nZnO did not elicit significant toxicity when compared to osteoblasts placed in normal media. There was also no significant increase in toxicity between days 5 and 10. Similarly, low LDH release was detected when cells were cultured onto nZnO coated surfaces. For early bone formation to occur, the conditions surrounding the implant should provide a suitable environment for osteoblast attachment and proliferation. Therefore, it is essential that the implanted prosthesis has the capability to be both osteoconductive and biocompatible. Our study showed an increase of proliferative activity after 5 and 10 days for 100% nZnO-coated samples. This phenomena was not observed when compared with other coated samples. Alkaline phosphatase (ALP) activity was also assessed in osteoblastic cells UMR-106. A time dependent increase in ALP was observed with all samples and was associated with samples having a higher percentage of nZnO. This effect could be due to the role that Zn21 plays in regulating the metabolism of bone as well as the charge present on the surface of each component (nHA
and nZnO), that will have an effect on the biological properties of these particles.23–25 The combination of these benefits to bone cells, together with antimicrobial properties against S.aureus and other pathogens makes nZnO an outstanding candidate for promoting bone growth and inhibiting infection at an implant site. Therefore, the use of a nZnO coating for future orthopedic and dental implants (even without the support of additional composite materials), could lead to improved bone growth and a more biocompatible, osteoconductive, and antimicrobial implant. This in turn may help prevent failure of joint replacements resulting from aseptic loosening and infection, and have a significant impact on patient quality of life and costs arising from revision procedures. It is likely that, such novel multifunctional implant coating system will shortly be entering a clinical trial phase that should be of major benefit.
CONCLUSIONS
The present study offers strong evidence in support of coated and stabilized nZnO as a potential antimicrobial agent for future medical device applications, particularly as a bone implant coating material. Further analysis of ZnO
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
7
FIGURE 6. Cytotoxicity, viability, and proliferation assays. A: Lactate dehydrogenase (LDH) release from cells exposed to supernatants of suspended nZnO and nTiO2. Each suspension was incubated for 1, 5, and 10 days, and its supernatant tested on UMR-106 cells. B: Lactate dehydrogenase (LDH) release from the seeded UMR-106 cells (1 3 104). High-control 5 Lysis buffer 1 osteoblasts, Low Control 5 cell culture media 1 osteoblasts, Background control 5 cell culture media. C: Percentage cytotoxicity instigated upon UMR-106 cells by supernatants of suspended nZnO and nTiO2. D: Percentage cytotoxicity instigated by coated nZnO glass samples. E: Alkaline phosphatase (ALP) activity in UMR-106 cells. ALP activity was measured from the seeded cells on coated mixtures at 1, 5, and 10 days. F: Proliferation assay (AlamarBlue % reduction) of UMR-106 cells. Cultured cells were monitored on coated samples for periods of 1, 5, and 10 days. (*) asterisks indicate statistical significance within the given time point. Results show: **p < 0.001,*p < 0.05, mean 6 SEM, n 5 4.
nanoparticulates would allow us to confirm its biocompatibility and provide mechanistic insight. ACKNOWLEDGMENTS
The authors would like to thank Orthopaedic Research UK for funding this project and also Johnson Matthey plc for providing the nanoparticles. KM wishes to acknowledge the following individuals for kindly providing their advice and expertise as regards to the content of this manuscript: Steve Gschmeissner, Sadia Sajid, Sepideh Bahadorian, and Mojgan Hamedi. REFERENCES 1. Mack D, Rohde H, Harris L, Davies A, Horstkotte M, Knobloch J. Biofilm formation in medical device-related infection. Int J Artificial Organs 2006;29:343–402. 2. Otto M. Staphylococcal infections: Mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annual Rev Med 2013;64:175–188.
8
MEMARZADEH ET AL.
3. Aviv M, Berdicevsky I, Zilberman M. Gentamicin-loaded bioresorbable films for prevention of bacterial infections associated with orthopedic implants. J Biomed Mater Res A 2007;83:10–19. 4. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv 2009;27:76–159. 5. Salata OV. Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2004;2:3. 6. Bryington M, Hayashi M, Kozai Y, Vandeweghe S, Andersson M, Wennerberg A, et al. The influence of nano hydroxyapatite coating on osseointegration after extended healing periods. Dental Mater 2013;29:514–520. 7. Shi Z, Huang X, Cai Y, Tang R, Yang D. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomaterialia 2009;5:338–345. 8. Sun L, Berndt C, Gross K, Kucuk A. Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: A review. J Biomed Mater Res 2001;58:570–592. 9. Saha N, Dubey A, Basu B. Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites. J Biomed Mater Res Part B Appl Biomater 2012:100:256–264.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
10. Atsuo I, Haruo K, Makoto O, Masako I, Hajime O, Kunio I, et al. Zinc-releasing calcium phosphate for stimulating bone formation. Mater Sci Eng C 2002;22. 11. Seil J, Webster T. Reduced Staphylococcus aureus proliferation and biofilm formation on zinc oxide nanoparticle PVC composite surfaces. Acta Biomaterialia 2011;7:2579–2584. 12. Vargas-Reus M, Memarzadeh K, Huang J, Ren G, Allaker R. Antimicrobial activity of nanoparticulate metal oxides against periimplantitis pathogens. Int J Antimicrobial Agents 2012;40:135– 139. 13. Darouiche R. Treatment of infections associated with surgical implants. New Engl J Med 2004;350:1422–1429. 14. Padmavathy N, Vijayaraghavan R. Enhanced bioactivity of ZnO nanoparticles—An antimicrobial study. Sci Technol Adv Mater 2008:9;035004. ndez-Sierra J, Ruiz F, Pena DC, Martınez-Gutie rrez F, 15. Herna n A, et al. The antimicrobial sensitivity of StrepMartınez A, Guille tococcus mutans to nanoparticles of silver, zinc oxide, and gold. Nanomedicine 2008;4:237–240. 16. Thian ES, Li X, Huang J, Edirisinghe MJ, Bonfield W, Best SM. Electrospray deposition of nanohydroxyapatite coatings: A strategy to mimic bone apatite mineral. Thin Solid Films 2011;519: 2328–2331. 17. Rao N, Ziran BH, Lipsky BA. Treating osteomyelitis: antibiotics and surgery. Plast Reconstr Surg 2011;127 (Suppl 1):177S–187S.
18. Memarzadeh K, Vargas M, Huang J, Fan J, Allaker RP. Nano metallic-oxides as antimicrobials for implant coatings. Key Eng Mater 2012;489:493–494. 19. Xie Y, He Y, Irwin P, Jin T, Shi X. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microbiol 2011;77:2325–2331. 20. Park J, Kim Y-J, Yeom J, Jeon J, Yi G-C, Je J, et al. The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration. Adv Mater (Deerfield Beach, Fla) 2010;22: 4857–4918. 21. Colon G, Ward B, Webster T. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2. J Biomed Mater Res A 2006;78:595–604. 22. Lin W, Huang Y, Zhou D, Ma Y. In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicol Appl Pharmacol 2006; 217:252–259. 23. Kawamura H, Ito A, Miyakawa S, Layrolle P, Ojima K, Ichinose N, et al. Stimulatory effect of zinc-releasing calcium phosphate implant on bone formation in rabbit femora. J Biomed Mater Res 2000;50:184–190. 24. Prentice A, Bates C. Adequacy of dietary mineral supply for human bone growth and mineralisation. Eur J Clin Nutr 1994;48 (Suppl 1): S161–S176. 25. Xu Y, Schwartz F, Traina S. Sorption of zn21 and cd21 on hydroxyapatite surfaces. Environ Sci Technol 1994;28:1472–1480.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
9
Queen Mary, University of London, Barts and The London School of Medicine and Dentistry, Institute of Dentistry 4 Newark Street, London, E1 2AT, United Kingdom 2 Department of Mechanical Engineering, University College London, Torrington Place, London, WC1E 7JE, United Kingdom Received 2 April 2014; revised 18 May 2014; accepted 23 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35241 Abstract: Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA compositecoated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-a and IL-6 cyto-
kines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial C 2014 Wiley Periodicals, Inc. J Biomed Mater and biocompatible. V Res Part A: 00A:000–000, 2014.
Key Words: nanotechnology, ZnO, coatings, antimicrobial, biocompatibility, bone
How to cite this article: Memarzadeh K, Sharili AS, Huang J, Rawlinson SCF, Allaker RP. 2014. Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants. J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Orthopedic and dental implants are susceptible to failure due to a variety of causes with bacterial infection being of particular concern.1,2 This occurs despite extensive measures to reduce such infections. Some medical devices incorporate antibiotics to help resist infection; however, due to certain limitations, for example, decreased release at the later stages of implantation, microbial resistance continues to rise.3 Development of innovative methods which deliver an alternative approach to antimicrobial function is therefore much in demand. In particular, the potential of antimicrobial nanoparticles (NPs) as future agents has received much attention.4 These agents possess physiochemical properties that make them unique among all antimicrobial agents. Their shape, increased surface area and size, combined with their toxic nature towards prokaryotic organisms make them extremely attractive as multifunctional agents.5 As coatings for medical bone implants evolve, a niche for new products that outperform the previous generation of biomaterials is created. nHA is a direct result of such evolution.6 This material is known to increase osteoblast prolifer-
ation, collagen production as well as maintaining normal osteoblast morphology.7 In this study, using a novel coating method, we investigate the potential of composites of nHA and nZnO as innovative coating materials for future orthopedic and dental implants. Conventional coating methods such as plasma spraying utilize extreme heat that can change properties of NPs, often resulting in the production of a rough, thick (40–50 mm) and uneven surface.8 Therefore, by applying a method that allows for formation of a homogenized, micro-fabricated coating, it has been possible to produce thin layers of nanocoatings that are uniform in nature. This unique quality allows for the exposure of NPs to both bacteria and osteoblasts which may result in unique outcomes not achievable by conventional coatings. The combination of this novel approach with the advantage of zinc being an important trace element in the body can be distinctively useful for bone-related implants. More recently, it has been demonstrated that the addition of ZnO to bonecomposites can enhance the proliferation, adhesion, and differentiation of osteoblasts.9 Furthermore, zinc embedded calcium phosphate (CaP) ceramics have also been shown to
Additional Supporting Information may be found in the online version of this article. Correspondence to: Kaveh Memarzadeh; e-mail: [email protected]
C 2014 WILEY PERIODICALS, INC. V
1
FIGURE 1. Transmission electron micrograph of both ZnO (A) and TiO2 (B) nanoparticles. A: Red lines indicate rod-shaped structures of the NPs whereas the black triangles showcase the sharp-edged particles. B: TiO2 NPs are similar in size but lack sharp edged confirmations. Red triangles indicate round shaped TiO2 particulates. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
promote the growth of bone cells when compared with nonembedded Zn CaP ceramics.10 The antibacterial effects of nZnO are currently under investigation and recent studies suggest that this material is strongly bactericidal, making it suitable as a coating for bone medical implants.11,12 Preliminary observations suggest that this agent has a degree of selectivity towards Staphylococcus aureus, a species that is predominantly responsible for surgical site infections.13 Possible advantages of NPs as compared with conventional antibiotics are their ability to promote bactericidal effects via different routes, namely: ion release, direct contact, and reactive oxygen species generation.14,15 This investigation was designed to aid our understanding of the relationship between coated nZnO and conventional nHA in relation to pathogenic bacteria and osteoblasts and also propose that nZnO, on its own, can be a suitable candidate for future medical bone implants. MATERIAL AND METHODS
Nanoparticles Metal oxide nanoparticles (NPs) were synthesized using the flame pyrolysis method as used by Johnson Matthey plc (JMTC). The average surface area was determined by Brunauer Emmett Teller (BET) analysis at JMTC. nHA particles were made by the process of wet precipitation between calcium hydroxide (Ca(OH)2) and orthophosphoric (H3PO4) acid.16 Handling and storage of these NPs was performed in a manner to avoid biological and environmental harm. The NPs tested were: Zinc Oxide (nZnO) (Fig. 1) Titanium dioxide (nTiO2, Fig. 1) and nHA. It is essential to state that nTiO2 was employed for comparative purposes in the cytotoxicity assay and was not utilized in all studies.
2
MEMARZADEH ET AL.
Nanoparticulate suspensions and composites Nanoparticulate suspensions. Different concentrations of nZnO suspension (Table I) were made in 100% ethanol and sonicated thoroughly for EHDA spraying. Nanoparticulate composites. For preparation of the composite mixtures (Table I), two different suspensions of nZnO and nHA were made with equal concentrations of 10,000 mg/mL. From these suspensions a known volume was added to produce the required percentile of NPs within each composite.
TABLE I. A Summary of all Coated Samples, Utilized Both for Biocompatibility and Antimicrobial Studies nZnO Suspensions
nHA Suspensions
mg/mL
100%
10,000
75% 50% 25% -
10,000 10,000 10,000 10,000
100%
-
10,000
100% 100%
-
5000 2500
25% 50% 75% 100%
1 1 1
Composite Coatings
nZnO Coatings
Composite coatings, except for 100% nHA were employed for their antimicrobial activity. All composites were used for ALP, proliferation, and focal adhesion studies. The nZnO coatings at different concentrations were used to observe the morphological state of the cells over the course of time. 1 5 indicates addition of one concentration to another.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
Glass substrates Glass slides and 13 mm coverslips were used in this study (VWR international). A diamond cutter was used to separate 1 cm2 samples from each glass slide. Each sample was dipped in 100% ethanol for a period of 1 min and air dried for 5 min before EHDA spraying. Additionally, uncoated glass samples were considered as controls. EHDA deposition of nZnO coating EHDA was used to coat all samples. Briefly, EHDA consists of an epoxy resin-embedded nozzle connected to both a power supply (Glassman High Voltage) and a 10cc syringe pump (Harvard Apparatus, PHD 4400 Programmable) containing suspended NPs. The applied voltage (3.2–4.2 kV) creates an electrical field between the nozzle and the substrate, which allows for the formation of a uniquely shaped stable cone jet. Such a jet, together with a steady flow rate of 5 mL/min is required to create a uniform spray which forms nanosized droplets that encapsulate the NPs. The spraying is carried out for 2 min with a 30 mm distance between the tip of the nozzle and the substrate. Subsequently, the coated samples were heat treated at 500 C for 1 h and allowed to cool gradually to help ensure the mechanical integrity of the coating. Antimicrobial studies Bacterial species. Staphylococcus aureus (Oxford strain; NCTC. 6571) was used in this study. This bacterium was stored at 280 C on beads in cryopreservative fluid. S.aureus is the predominant species involved in orthopedic-related infections.17 Nano-coated bactericidal samples. The coated samples were dipped in 100% ethanol for 1 min and flamed to ensure sterility. Test species of bacteria were grown overnight in Tryptone Soya Broth (TSB). The OD (optical density) was adjusted to 0.1 at 540 nm in Phosphate Buffered Saline (PBS) or Adult Bovine Serum (ABS, SIGMA-A). 30 mL of the OD adjusted bacterial suspension (Population of 1 3 106/mL) was transferred onto the surface of the coated and uncoated samples. Samples were placed in a humidified chamber then incubated at 37 C with 10% CO2 for a period of 24 h. Subsequently each sample was carefully removed and placed in 3 mL of PBS. Approximately 5 glass beads were added to each tube to aid the detachment of the bacteria while spinning the tubes for a period of 30 s at 1500 RPM. Control experiments demonstrated such treatment did not adversely affect viability of bacteria. Totally, 50 mL of the suspension was then removed and a 1 : 10 serial dilution performed. Neat and dilutions were plated onto Tryptone Soya Agar (TSA) plates and incubated for 24 h, after which colonies were counted. Biocompatibility and cell proliferation studies Cell culture. UMR-106 (Rattus norvegicus, osteosarcoma, ATCC), MG-63 (Homo sapiens, osteosarcoma, ATCC) and human mesenchymal stem cells (hMSC, University College London, Eastman Dental Institute) were used in this study.
Most investigations were carried out using UMR-106 cells. hMSCs were utilized for qualitative adhesion observations. MG-63 cell-lines were utilized for both qualitative and quantitative adhesion observations as well as ELISA. Frozen cells were gently thawed and cultured in highglucose DMEM with 2% FCS, 1% penicillin, and 1% L-glutamine. The cells were incubated for 48 h at 37 C with 5% CO2 and then detached using Trypsin/EDTA. Trypsinized cells were resuspended with 5 mL of medium, placed in 10 mL tubes and centrifuged at 800 3 g for 5 min. Supernatant was removed from each tube; fresh media was then added to cell pellets. Osteoblasts were then dispersed, suspended, counted, and seeded onto coated samples with a population of 1 3 104 cells. Samples were incubated at 37 C, 5% CO2 for 2 h to allow cell-substrate adhesion. Proliferation assay. The proliferation of the UMR-106 cells R -AbD Serowas assessed using Alamar Blue (alamarBlueV tec). The reagent was diluted with normal growth medium (1 : 10 Dilution) and stored at 4 C prior to use. Approximately 1 3 104 osteoblasts were seeded onto coated glass samples in a 24-well plate (n 5 4 per condition). Seeded cells were left for a period of 15–20 min in the incubator (37 C, 5% CO2) to ensure adhesion and 1 mL of medium was added to each well for overnight incubation. At 1, 5, and 10 days (media was replaced every 2 days); 10% volume of the medium in each well was replaced with ab medium. The plate was then incubated at 37 C, 5% CO2 for a period of 4 h and 100 mL of the supernatant was removed from relevant wells and added to a 96-well (Falcon) plate. Fluorescence was measured using a fluorescent plate reader (FLUstar OPTIMA) at wavelengths of 570 nm (excitation) and 600 nm (emission). Cytotoxicity assay. Lactate Dehydrogenase (LDH) release from UMR-106 cell lines was measured using a commercially available kit (LDH-Cytotoxicity Assay Kit II, abcam, UK) and carried out according to the manufacturer’s instructions. LDH release: Osteoblasts exposed to NP suspension supernatants. To examine the possible effects of released ions on osteoblast viability, nZnO particulates were suspended in 2% FCS DMEM (10,000 mg/mL) and left in a shaking incubator for 1, 5, and 10 days (37 C, 200 RPM). nTiO2 particulates were also tested in a similar manner. At each time point, samples were removed and transferred into a 50-mL corning centrifuge tube and spun at a speed of 1000 RPM (160 3 g) for 5 min. The supernatant from each sample was removed and filtered using a syringe filter unit (0.22 mm). Osteoblasts were seeded at a density of 1 3 104 per well in a 96-well plate. 100 mL of DMEM (with 2% FCS) was added to each well and incubated at 37 C, 5% CO2 for 24 h to allow a cell monolayer to develop. The medium in each well was replaced with 100 mL of the filtered supernatant or Lysis Buffer (Positive control) and incubated for 40 min. The plate was centrifuged at 600 3 g for 10 min and 10 mL of the supernatant was transferred to a new 96-well plate. 100 mL of the LDH reaction mix was
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
3
added to each well and incubated for a further 30 min. Absorbance for all samples and controls was measured at a wavelength of 450 nm using a plate reader (FLUstar OPTIMA, BMG Labtech, Germany). LDH release: Osteoblasts exposed to nano-coated sample supernatants. The coated samples were placed in a 24well plate containing 2 mL of 2% FCS DMEM to each well. The samples were then left in a shaking incubator (37 C, 200 RPM) for periods of 1, 5, and 10 days. In parallel, 1 3 104 cells per well were seeded into a separate 24-well plate and incubated at 37 C, 5% CO2. 1 mL of DMEM from each time point was subsequently added to cells. Osteoblasts were then incubated for a further 24 h and 100 mL of each well was removed and dispensed into a 96-well plate and the LDH release was measured. Alkaline phosphatase activity. The differentiation of the UMR-106 cells on the nZnO-coated samples was determined by measuring alkaline phosphatase release. A population of 1 3 104 cells were seeded onto coated glass samples in a 24-well plate and incubated at 37 C, 5% CO2 for 1, 5, and 10 days respectively. For the ALP assay, one tablet of 4nitrophenyl phosphate disodium (SIGMA-A, Dorset, UK) was mixed with 8 mL 0.1M Trizma Hydrochloride (pH 5 9.5) and 15 mL of 2M MgCl2. Before addition of the reaction solution to the cells, each well was washed with PBS three times. 1 mL of the reaction solution was added to each well containing the cell-seeded-coated samples. The 24-well plate was left at room temperature for 10 min for the reaction to take place. NaOH (0.5M) was then used to stop the reaction. Subsequently, 100 mL aliquots were removed from each well and transferred into a 96-well plate and absorbance read at 405 nm using a plate reader (FLUstar OPTIMA). Cytokine release measurements. ELISA was performed in order to quantify cytokine release. MG-63 human osteosarcoma cells were seeded onto glass samples (as described previously) and cytokine release (TNF-a and IL-6) was subsequently measured according to the manufacturer’s protocol (Thermo Scientific). Microscopy Characterization. Transmission electron microscopy. ZnO along with TiO2 NPs were placed in 5 mL of distilled water and sonicated to provide a homogenous suspension. Carbon film grids (400 Mesh Cu, Agar scientific) were briefly dipped into the nanoparticulate suspension and left at room temperature to air dry. The grids were transferred into the TEM (Philips CM 12) and subsequently analyzed. Cell morphology. Optical microscopy. Standard inverted light microscopy (Nikon Eclipse TE2000-5) was used to observe cellular growth at every stage. Approximately 1 3 105 population of cells were seeded onto coated samples and their morphology and distribution was monitored over time. Scanning electron microscopy (SEM). Both coated and uncoated substrates that were seeded with cells were placed into 3% Glutaraldehyde (SIGMA-Aldrich, Dorset, UK)
4
MEMARZADEH ET AL.
in 0.1M Cocadylate buffer (SIGMA-A) for a period of 24 h. Each sample was then dehydrated using ascending concentrations of ethanol: 20, 50, 70, and 90% for 10 min and 100% ethanol for 30 min. Finally the samples were dried using hexamethyldisilazane (HMDS) (SIGMA-A). Samples that lacked the presence of cells were cleaned with 100% ethanol and processed without the dehydration process. Furthermore, glass samples were mounted onto stubs and made conductive by the application of conducting carbon cement (Agar Scientific) between the sample and the stub. All samples were gold coated and directly transferred into the scanning electron microscope (FEI Quanta 3D FEG). Immunofluorescence. Cells were fixed with 4% paraformaldehyde (PFA), incubated for 10 min in permeabilization buffer (phosphate-buffered saline (PBS), 0.2% Triton X-100) and subsequently blocked with 2% bovine serum albumin (BSA). Samples were incubated with primary antibody (4 mg/mL mouse anti-paxillin; Santa Cruz, USA) for 24 h at 4 C. Subsequently NP coated coverslips were washed with R 568 conjuPBS and incubated for 45 min with Alexa FluorV gated rabbit anti-mouse secondary antibody (Invitrogen R 488-phalloidin staining Molecular Probes, UK). Alexa FluorV was applied to visualize F-actin (Invitrogen Molecular Probes, UK). Imaging was performed with a Leica DM4000 epi-fluorescence microscope (Leica, Solms, Germany). Statistical analysis All quantitative results were presented as standard error of mean (mean 6 SEM). Comparisons of two sample means were carried out using Student’s t-test in GraphPad Prism software. Statistical significance was established when p < 0.05. RESULTS
Nanoparticulate characterization Conventional TEM was used to characterize the size and shape of the nanoparticles. Micrographs revealed a size range between 10 and 100 nm for nZnO particulates. These nanoparticulates possess elongated rod shaped structures with sharp edges at their distal extremes (Fig. 1), whereas the nTiO2 particulates are between 20 and 110 nm and lack elongated structures and sharp edged features. The surface area for both ZnO and TiO2 NPs were measured by BET method and found to be 47 m2/g and 87 m2/g respectively. Antimicrobial effects of coated nZnO NP coatings were analyzed using SEM and light microscopy. Micrographs revealed differences between a coated and an uncoated glass substrate (Supporting Information Fig. 1). Antibacterial properties of coated nZnO and nHA mixtures exposed to ABS-bacterial suspension demonstrated a reduction in populations of bacteria as the concentration of nZnO increased. Furthermore there was a significant (p < 0.05) dose dependent reduction when the control samples were compared with both the lowest and highest concentrations of coated nZnO [Fig. 2(A)]. A similar property was observed when PBS-suspended bacteria were
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
nZnO in culture was further investigated by immunofluorescent imaging of focal adhesion protein paxillin. Higher concentrations of nZnO did not have a detrimental effect on osteoblast spreading, punctate paxillin staining, and stress fibers (Fig. 5). Thus nZnO coatings allows osteoblast attachment and focal adhesion formation.
FIGURE 2. Bactericidal effects of nZnO-coated substrates A: Antimicrobial effect of coated nZnO 1 nHA composites on S.aureus in adult bovine serum (ABS). The coated glass samples are coated with different concentrations of nHA and nZnO. B: Antimicrobial effect of coated nZnO 1 nHA composites on S.aureus in PBS. Coated mixtures have bactericidal effects as the concentration of nZnO increases. Significance was established as compared with the control (uncoated substrate), **p < 0.001, *p < 0.05, mean 6 SEM, n 5 4.
placed onto the surface of the coated material [Fig. 2(B)]. Furthermore, the antimicrobial activity was more significant between the control and the 100% coated nZnO samples (Fig. 2). We further characterized the morphology of bacteria on the surface of the coated material using scanning electron microscopy (SEM). Images revealed bacterial colonization on the surface of the uncoated samples [Fig. 3(A)], whereas coated samples [Fig. 3(C)] displayed restricted bacterial colonization [Fig. 3(B)]. The increased amount of debris on the coated surfaces is due to the presence of dead bacteria [Fig. 3(B)]. Effect of nZnO on osteoblast adhesion and morphology Osteoblasts (UMR-106) were cultured on different concentrations of nZnO coatings (2,500 mg/mL, 5,000 mg/mL, 10,000 mg/mL—Table I). Bright field microscopy revealed a qualitative insight into the greater number of cells at 2, 24, and 48 h and minimal cell death (Fig. 4). Cell interaction with nZnO coated samples was further characterized by SEM. UMR-106 and MG-63 cells revealed spindle shaped morphology, characteristic of a normal osteoblast [Supporting Information Fig. 2(a)]. Cell morphology was largely normal in hMSCs, with no signs of cellular stress [Supporting Information Fig. 2(b)]. Osteoblasts displayed filopodia projections that were in close contact with the nZnO surface or in proximity to neighboring cells (Supporting Information Fig. 3). Adhesion to coated
Osteoblast cytotoxicity, viability, and proliferation LDH production and release from supernatants derived from suspended NPs (TiO2 and ZnO) [Fig. 6(A)] and coated nZnO (in cell culture medium) were measured using UMR106 cells [Fig. 6(B)]. No significant LDH release was observed under both conditions. Moreover, osteoblasts seeded on NP surface coatings demonstrated an increase in proliferation [Fig. 6(C)] and alkaline phosphatase release [Fig. 6(D)], with the higher nZnO percentages showing an increased activity in both. In addition, measurement of cytokine release (TNF-a and IL-6) revealed that no inflammatory response was induced when cells were allowed to adhere on coated surfaces. Coated and uncoated sample release were below the level of detection, i.e., absorbance (450 nm) was below 0.15 in all cases at 24 h and 5 days. IL-6 and TNF-a were detectable with absorbance readings of above 0.18. DISCUSSION
An ideal bone implant or prosthesis would allow bone growth on the coated surface of the implant, together with minimizing the likelihood of S.aureus attachment and proliferation. This study provides evidence to support these functions. Using different experimental approaches in vitro, we show that ZnO NPs together with nHA can function under different simulated environments to promote antimicrobial activity. We further employed a coating technique of EHDA with glass samples. This method provides a uniform coating of NPs that is both stable and more sustainable than other conventional coating methods. Our antimicrobial investigations with S.aureus demonstrate that the majority of coatings tested have significant antimicrobial activity. This effect is more apparent when the concentration of nZnO is increased which is in agreement with our previous comparative findings where coated nZnO displayed an increased antimicrobial activity when submerged in a suspension of S.aureus for a prolonged period of time.18 These results along with earlier findings19 indicate that coated nZnO can be particularly effective against given species of bacteria. The antimicrobial effect of ZnO is a recently described phenomenon and no previous studies have addressed the potential of uniformly coated nZnO as an antimicrobial orthopedic coating. Observations with osteoblast like cells (UMR-106 and MG-63) and hMSCs revealed that cell morphology is not compromised when cells are exposed to different concentrations of nZnO-coated onto glass. Moreover, nZnO immobilized on a surface can provide a suitable substrate for osteoblasts to grow, adhere and be metabolically active which is in agreement with previous studies reporting that
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
5
FIGURE 3. A qualitative assessment on the antimicrobial effect of nZnO-coated onto glass samples. A: Bacteria present on a surface without the coating. B: The white arrow indicates a single bacterium present on the coated surface. C: High resolution image of coating showing uniform characteristics.
osteoblasts could maintain structural integrity and adhesive properties when introduced onto NP coated coverslips.20,21
In general, when eukaryotic cells are introduced to a toxic environment/material, the phospholipid bilayer can be ruptured and intracellular compartments of the cell escape
FIGURE 4. Osteoblast growth on nZnO-coated surfaces. Seeded UMR-106 (osteoblast-like cells) on the surface of nZnO-coated glass samples. The samples were coated with increasing concentrations of nZnO to observe any morphological changes. Phase images show increased numbers of cells at 2, 24, and 48 h. Red arrows indicate Initial attachment of osteoblasts to the substrates after 2 h of incubation and black arrows show the similarity of osteoblast confluency between a coated and uncoated substrate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
6
MEMARZADEH ET AL.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
FIGURE 5. Effect of nZnO and nHA on actin cytoskeleton and focal adhesions. Fluorescent images of actin filaments (arrow heads in A, D, G, J) and focal adhesions (arrows in B, E, H, K). Human osteoblast-like cells (UMR-106) cultured for 24 h on 100% nZnO, nHA, and 50% nZnO coatings show more marked focal adhesions (E, H, K) compared with control (B). Scale bars 5 50lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to the extracellular medium.22 Release of the enzyme LDH is a helpful indicator of cytotoxicity. In the present study, when exposed to supernatants of both nTiO2 and nZnO, although eliciting a greater response than nTiO2, the supernatant derived from nZnO did not elicit significant toxicity when compared to osteoblasts placed in normal media. There was also no significant increase in toxicity between days 5 and 10. Similarly, low LDH release was detected when cells were cultured onto nZnO coated surfaces. For early bone formation to occur, the conditions surrounding the implant should provide a suitable environment for osteoblast attachment and proliferation. Therefore, it is essential that the implanted prosthesis has the capability to be both osteoconductive and biocompatible. Our study showed an increase of proliferative activity after 5 and 10 days for 100% nZnO-coated samples. This phenomena was not observed when compared with other coated samples. Alkaline phosphatase (ALP) activity was also assessed in osteoblastic cells UMR-106. A time dependent increase in ALP was observed with all samples and was associated with samples having a higher percentage of nZnO. This effect could be due to the role that Zn21 plays in regulating the metabolism of bone as well as the charge present on the surface of each component (nHA
and nZnO), that will have an effect on the biological properties of these particles.23–25 The combination of these benefits to bone cells, together with antimicrobial properties against S.aureus and other pathogens makes nZnO an outstanding candidate for promoting bone growth and inhibiting infection at an implant site. Therefore, the use of a nZnO coating for future orthopedic and dental implants (even without the support of additional composite materials), could lead to improved bone growth and a more biocompatible, osteoconductive, and antimicrobial implant. This in turn may help prevent failure of joint replacements resulting from aseptic loosening and infection, and have a significant impact on patient quality of life and costs arising from revision procedures. It is likely that, such novel multifunctional implant coating system will shortly be entering a clinical trial phase that should be of major benefit.
CONCLUSIONS
The present study offers strong evidence in support of coated and stabilized nZnO as a potential antimicrobial agent for future medical device applications, particularly as a bone implant coating material. Further analysis of ZnO
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
7
FIGURE 6. Cytotoxicity, viability, and proliferation assays. A: Lactate dehydrogenase (LDH) release from cells exposed to supernatants of suspended nZnO and nTiO2. Each suspension was incubated for 1, 5, and 10 days, and its supernatant tested on UMR-106 cells. B: Lactate dehydrogenase (LDH) release from the seeded UMR-106 cells (1 3 104). High-control 5 Lysis buffer 1 osteoblasts, Low Control 5 cell culture media 1 osteoblasts, Background control 5 cell culture media. C: Percentage cytotoxicity instigated upon UMR-106 cells by supernatants of suspended nZnO and nTiO2. D: Percentage cytotoxicity instigated by coated nZnO glass samples. E: Alkaline phosphatase (ALP) activity in UMR-106 cells. ALP activity was measured from the seeded cells on coated mixtures at 1, 5, and 10 days. F: Proliferation assay (AlamarBlue % reduction) of UMR-106 cells. Cultured cells were monitored on coated samples for periods of 1, 5, and 10 days. (*) asterisks indicate statistical significance within the given time point. Results show: **p < 0.001,*p < 0.05, mean 6 SEM, n 5 4.
nanoparticulates would allow us to confirm its biocompatibility and provide mechanistic insight. ACKNOWLEDGMENTS
The authors would like to thank Orthopaedic Research UK for funding this project and also Johnson Matthey plc for providing the nanoparticles. KM wishes to acknowledge the following individuals for kindly providing their advice and expertise as regards to the content of this manuscript: Steve Gschmeissner, Sadia Sajid, Sepideh Bahadorian, and Mojgan Hamedi. REFERENCES 1. Mack D, Rohde H, Harris L, Davies A, Horstkotte M, Knobloch J. Biofilm formation in medical device-related infection. Int J Artificial Organs 2006;29:343–402. 2. Otto M. Staphylococcal infections: Mechanisms of biofilm maturation and detachment as critical determinants of pathogenicity. Annual Rev Med 2013;64:175–188.
8
MEMARZADEH ET AL.
3. Aviv M, Berdicevsky I, Zilberman M. Gentamicin-loaded bioresorbable films for prevention of bacterial infections associated with orthopedic implants. J Biomed Mater Res A 2007;83:10–19. 4. Rai M, Yadav A, Gade A. Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv 2009;27:76–159. 5. Salata OV. Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2004;2:3. 6. Bryington M, Hayashi M, Kozai Y, Vandeweghe S, Andersson M, Wennerberg A, et al. The influence of nano hydroxyapatite coating on osseointegration after extended healing periods. Dental Mater 2013;29:514–520. 7. Shi Z, Huang X, Cai Y, Tang R, Yang D. Size effect of hydroxyapatite nanoparticles on proliferation and apoptosis of osteoblast-like cells. Acta Biomaterialia 2009;5:338–345. 8. Sun L, Berndt C, Gross K, Kucuk A. Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: A review. J Biomed Mater Res 2001;58:570–592. 9. Saha N, Dubey A, Basu B. Cellular proliferation, cellular viability, and biocompatibility of HA-ZnO composites. J Biomed Mater Res Part B Appl Biomater 2012:100:256–264.
NANOPARTICULATE ZnO AS A COATING MATERIAL
ORIGINAL ARTICLE
10. Atsuo I, Haruo K, Makoto O, Masako I, Hajime O, Kunio I, et al. Zinc-releasing calcium phosphate for stimulating bone formation. Mater Sci Eng C 2002;22. 11. Seil J, Webster T. Reduced Staphylococcus aureus proliferation and biofilm formation on zinc oxide nanoparticle PVC composite surfaces. Acta Biomaterialia 2011;7:2579–2584. 12. Vargas-Reus M, Memarzadeh K, Huang J, Ren G, Allaker R. Antimicrobial activity of nanoparticulate metal oxides against periimplantitis pathogens. Int J Antimicrobial Agents 2012;40:135– 139. 13. Darouiche R. Treatment of infections associated with surgical implants. New Engl J Med 2004;350:1422–1429. 14. Padmavathy N, Vijayaraghavan R. Enhanced bioactivity of ZnO nanoparticles—An antimicrobial study. Sci Technol Adv Mater 2008:9;035004. ndez-Sierra J, Ruiz F, Pena DC, Martınez-Gutie rrez F, 15. Herna n A, et al. The antimicrobial sensitivity of StrepMartınez A, Guille tococcus mutans to nanoparticles of silver, zinc oxide, and gold. Nanomedicine 2008;4:237–240. 16. Thian ES, Li X, Huang J, Edirisinghe MJ, Bonfield W, Best SM. Electrospray deposition of nanohydroxyapatite coatings: A strategy to mimic bone apatite mineral. Thin Solid Films 2011;519: 2328–2331. 17. Rao N, Ziran BH, Lipsky BA. Treating osteomyelitis: antibiotics and surgery. Plast Reconstr Surg 2011;127 (Suppl 1):177S–187S.
18. Memarzadeh K, Vargas M, Huang J, Fan J, Allaker RP. Nano metallic-oxides as antimicrobials for implant coatings. Key Eng Mater 2012;489:493–494. 19. Xie Y, He Y, Irwin P, Jin T, Shi X. Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl Environ Microbiol 2011;77:2325–2331. 20. Park J, Kim Y-J, Yeom J, Jeon J, Yi G-C, Je J, et al. The topographic effect of zinc oxide nanoflowers on osteoblast growth and osseointegration. Adv Mater (Deerfield Beach, Fla) 2010;22: 4857–4918. 21. Colon G, Ward B, Webster T. Increased osteoblast and decreased Staphylococcus epidermidis functions on nanophase ZnO and TiO2. J Biomed Mater Res A 2006;78:595–604. 22. Lin W, Huang Y, Zhou D, Ma Y. In vitro toxicity of silica nanoparticles in human lung cancer cells. Toxicol Appl Pharmacol 2006; 217:252–259. 23. Kawamura H, Ito A, Miyakawa S, Layrolle P, Ojima K, Ichinose N, et al. Stimulatory effect of zinc-releasing calcium phosphate implant on bone formation in rabbit femora. J Biomed Mater Res 2000;50:184–190. 24. Prentice A, Bates C. Adequacy of dietary mineral supply for human bone growth and mineralisation. Eur J Clin Nutr 1994;48 (Suppl 1): S161–S176. 25. Xu Y, Schwartz F, Traina S. Sorption of zn21 and cd21 on hydroxyapatite surfaces. Environ Sci Technol 1994;28:1472–1480.
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MONTH 2014 VOL 00A, ISSUE 00
9
