A DEPOSITION OF NOVEL GRAPHENE OXIDE-HYDROXYAPATITE
COMPOSITE
ONTO
TITANIUM
DIOXIDE
NANOTUBES
AS
AN
ANTIBACTERIAL IMPLANT MATERIAL
Yardnapar Parcharoen1,*, Preecha Termsuksawad2, Sirinrath Sirivisoot1,#
Biological Engineering, Faculty of Engineering, King Mongkut’s University of Technology
Thonburi, Bangkok, Thailand
2
Division of Materials Technology, School of Energy, Environment and Materials, King
Mongkut’s University of Technology Thonburi, Bangkok, Thailand
1
*yardnapar.p@gmail.com, #Sirinrath.Sir@kmutt.ac.th
Abstract
Nanotubes modification for orthopedic implants has shown interesting biological
performances (such as improving cell adhesion, cell differentiation, and enhancing
osseointegration). The purpose of this study is to investigate effect of titanium dioxide (TiO2)
nanotube feature on performance of titanium (Ti) bone implants. TiO2 nanotubes were
prepared by anodization using ammonium fluoride electrolyte (NH4F) with glycerol
modifiers at various durations of anodization. In order to increase its bioactivity and enhance
osteointegration, titanium surfaces were coated with hydroxyapatite (HA) by
electrodeposition. Graphene oxide (GO) nano-flakes were used in the present study to inherit
many interesting properties, specifically antibacterial. In this study, an electrochemical
deposition of HA and GO was investigated. HA were electrodeposited first with electrolytic
deposition (ELD), then followed with GO by anodic electrophoretic deposition (EPD) on
titanium dioxide nanotubular sample or anodized titanium (ATi) (layer-by-layer method).
Prior to ELD, ATi surface was subjected to an alkaline treatment (P-ATi), which was
necessary to provide a template for nucleation of hydroxyapatite inside titanium dioxide
nanotubes (calls HA-P-ATi). The chemical composition and structure of nanocomposite
materials were characterized by field emission scanning electron microscope (FE-SEM),
energy dispersive x-ray spectrometer (EDS) and attenuated total reflectance-fourier transform
infrared spectroscopy (ATR-FTIR). The mechanical properties of the coatings were probed
by nanoindentation. The layer-by-layer deposited samples showed an advantage by
maintaining calcium phosphate ratio of HA closely mimicking to natural hydroxyapatite in
bone. Electrodeposited GO were tested antibacterial property with Staphylococcus aureus
and Escherichia coli. The result showed that the GO coating was antibacterial materials and
possessed biocompatibility to MC3T3-E1 osteoblast cells. In the present study, the results
suggest that electrophoretic deposition of GO on HA-P-ATi is a convenient method to
fabricate bioactive and antibacterial nanocomposite materials for orthopedic applications.
Keywords: TiO2 nanotubes, graphene oxide, hydroxyapatite, implant material, antibacterial
property.
Introduction
Bone is a bioceramic composite that has long held the attention of material engineers
who seek to duplicate its mechanical properties of both high strength and fracture toughness
(1). Among many materials, modulus of titanium and its alloys is close to that of bone. In
addition, their wear and corrosion resistance are higher than those of 316L stainless steels and
cobalt-chromium alloys. Good wear and corrosion resistance of titanium and its alloys is due
to TiO2 formation on the surface. This is reason why titanium and it alloys are broadly used
for bone implant. However, natural TiO2 has low osseointegration (2), causing growth of
fibrous soft tissue (should be hard bone) around the implant and in long term the contact
between the implant and the tissue will be loose (3). Therefore, researchers focus on surface
treatment of biomaterials in order to increase osseointegration of implants.
One method to increase osseointegration is to grow TiO2 nanotube by anodization. In
a non-toxic and environmental friendly system, a neutral fluoride solution (NH4F) was used
as an electrolyte in the present study. The TiO2 nanotubes (ATi) from this possess has high
adhesion to substrate due to intrinsic chemical bond between the nanotube and titanium. The
other approach to enhance osseointegration is to coat titanium with hydroxyapatite (HA) or
calcium phosphates (a main mineral in bones with Ca/P ratio of 1.67) (4). Many methods
have been developed to coat HA on titanium substrate such as soaking in simulated body
fluid (SBF) with ion concentrations nearly equal to those of the human blood plasma, plasma
spraying, high-velocity oxygen-fuel spraying, sol-gel deposition, hot isostatic pressing,
pulsed laser deposition, and electrochemical deposition (5-12). Electrodeposition of HA has
been investigated by many research groups in the late five years because this method can be
controlled easily. Morever, the HA-coated titanium from this process was claimed to have
good corrosion resistance, high strength and good biocompatibility (13).
Orthopedic implants have greatly improved the quality of life of many people
suffering from aging related diseases, such as osteoarthritis and osteoporosis. The overall
infection rate associated with primary surgeries is estimated to be in the range of 0.5 to 5%.
However, after revision surgery, the re-infection rate can significantly increase up to 14%.
The cost for treating such infections is estimated to be at least $50,000 per patient (14). The
great morbidity and economic burden, suspensions to prevent infections have been very
limited. The most common pathogens causing infections are Gram-positive Staphylococcus
aureus (S. aureus) and coagulase-negative staphylococci (20–30%, e.g. Staphylococcus
epidermis) (15). To prevent or treat implant from infections, antibiotics are used. In these
aspects, the use of commercial antimicrobial agents for the modification of implant materials
has been limited due to their toxicity, need of complex post treatment processes,
environmental pollution, and multidrug-resistance (MDR) property of bacteria (16, 17). Such
as, both S. aureus and Staphylococcus epidermis are known to have a high potential for
developing resistance to traditional antibiotics, especially after the formation of biofilms on
implant surfaces (18).
To overcome the MDR effect of bacteria, several researches have been attempted in
the recent decades. The advantage of nanoparticles for antibacterial activity is that it
overcomes the MDR of bacteria. Inorganic nanoparticles such as Ag, Au, TiO2, ZnO, and
carbon nanotubes (CNTs) exhibited antibacterial activity towards several multidrug resistant
bacteria (19). CNTs are one type of carbon nanomaterials and can be considered as rolled
graphene (20). Another carbon nanomaterials, graphene was found to be biocompatible for
growth, adhesion, and proliferation of many cells such as L-929 mouse fibroblasts cells (21),
neuron cells, and osteoblasts (22), and graphene oxide was used as an effective nanocargo to
deliver water-insoluble drugs into cells (23, 24). Recently, the antibacterial activity of
graphene oxide nanosheets was reported (25, 26). Moreover, the production of graphene
oxide is inexpensive, the massive production is possible when its applications come to market
(27). Therefore, the objective of the present study is to explore the cytotoxicity and
antibacterial property of graphene oxide nanocomposite.
In the present study, to determine the optimal condition for fluoride anodization, the
growing of TiO2 nanotube layers in different times was studied. After that, nanoindentation
was conducted to probe the mechanical properties, such as Young’s modulus and hardness.
Then HA was deposited on NaOH pretreated-titanium dioxide nanotubes (P-ATi) using ELD
method. The graphene oxide nanowalls (GO) were deposited on HA-P-ATi using EPD in an
isopropyl alcohol suspension containing graphene oxide nanowalls. Electrophoretic
deposition (EPD) is a well-accepted, relatively simple and low-cost technique for preparing
ceramic and composite coatings and self-supported layers (28-33). Then, the antibacterial
property of the GO coated HA-P-ATi was studied. To understand that how GO nanowalls
could cause bacteria damage, the oxidative stress of both Gram-negative Escherichia coli (E.
coli) and Gram-positive S. aureus bacteria was examined by using cyclic voltammetry (CV)
method.
Methodology
Fabrication of Anodized titanium
1) Preparation of titanium samples: Conventional titanium of 99.9% purity was used as a
substrate to grow oxide nanotube arrays. The surface was firstly polished to mirror finish
using silicon carbide paper (TOA, Thailand) of successively finer roughness (400, 600, 800,
1000, 1500 and 2000 grits), and following by polishing with 0.05-µm alumina powder
(Allied, USA) to a mirror quality smooth. After polishing, the titanium substrate was washed
with deionized (DI) water and sonicated for five minutes in acetone (Sigma, Thailand) and
another five minutes in 95% ethanol (Sigma, Thailand), and then anodized immediately.
2) Anodic oxidation to form TiO2 nanotube arrays: The titanium dioxide nanotube layers
were prepared by anodization. Platinum and the titanium plate were used as negative and
positive electrodes, respectively. All oxide films were grown at 5 °C and in 0.36 M
ammonium fluoride (NH4F) electrolytes with medium modifier, consisting of 90% of
glycerol. Anodization was conducted with a voltage of 35/-4V which was adapted following
the work from Chanmanee et al (34). The samples were anodized for 30, 90 and 150 minutes.
After anodization, the samples were carefully cleaned with DI water and then were dried by
nitrogen gas (N2). The titanium dioxide-grown porous layers were annealed at 450 °C for 30
minutes to achieve its anatase phase.
A measurement of Young’s modulus
A NanoTest System, manufactured by Micro Materials (Courtesy Micro Materials
Ltd.), was used for nanoindentation testing. The NanoTest System is a pendulum-besed
depth-sensing system, with the sample mounted vertically and the load applied
electromagnetically. For this machine, depth resolution is less than 0.1 nm and force
resolution is better than 100 nN. All TiO2 nanotube layers were tested with the Berkovich
indenter is calibrated by performing the indentation testing on standard fused silica with
different load range of 0.5-200 mN. The applied force (P) was increase data constant loading
rate up to the maximum force (Pmax) and then unloaded to zero at the same loading rate
(Figure 3b). The indenter impressions after experiments were observed using FESEM to
clarify the fracture characteristic of the nanotubes.
Graphene oxide (GO)-calcium phosphate (CaP) depositing with layer by layer method on
ATi
The electrolyte to deposite of hydroxyapatite was prepared by dissolving 1.67 mM
phosphate containing salt in the form of NH4H2PO4 (Sigma, Thailand) and 2.5 mM calcium
containing salt in the form of Ca(NO3)2 (Sigma, Thailand) in distilled water. Without a preanodization surface treatment of anodized Ti (ATi), the subsequent HA coating was not
homogeneous (35). The pre-treated ATi was used as a cathode and platinum as an anode
electrode. The electrodeposition of HA was carried out at a constant potential of 2.5 V at 65
°C for 10 minutes. Then GO (Graphene square, Korea) was deposited by anodic-EPD on HA
electrodeposited P-ATi layers. Concentrations of GO and applied direct current (DC) voltage
were 200 μg/ml of total EPD in isopropanol electrolyte at 10 V, respectively. The deposition
time was in 10 minutes.
Physical and chemical characterizations of coating
The surface morphology of the samples was observed by a scanning electron
microscopy (SEM, Nova NanoSEM 450FEG, USA). In addition, the elemental compositions
of the sample surfaces were detected by an energy dispersive X-ray spectrometer (EDS,
Bruker, USA) equipped on the SEM system. Attenuated Total Reflectance-Fourier transform
infrared (ATR-FTIR, Bruker, USA) spectra (4000−400 cm−1) were determined with diamond
crystal on a Thermo Scientific to analyze the surface chemical functionality of the GO.
Responses of bacteria
The HA-P-ATi samples which coated with GO 200 μg/ml (GO200) by anodic-EPD
method and the HA-P-ATi substrate were compared each other. All samples were
investigated against E. coli and S. aureus bacteria as Gram-negative and Gram-positive
models, respectively. Before each microbiological experiment, all the samples and glassware
were sterilized by autoclaving at 120 °C for 15 minutes. The bacteria were cultured in a
Mueller-Hinton broth at 37 °C for 24 h with 200 rpm rotating. Then, the cultured bacteria
were adjusted into concentrations as ~106 no./cc by using Mueller-Hinton broth to study on
samples with and without GO coating. Each sample was placed into a sterilized 24 wells
plate. Then, 1 ml of the diluted bacterial suspension was spread on surface of the sample. The
bacteria were cultured in a Mueller-Hinton broth at 37 °C for 18 h with 200 rpm rotating. To
measure bacteria absorbance by use a spectrophotometer and calculating percent relative of
cell survival of bacteria (1). The reported data were the average value of three separate
similar runs.
% Relative cell survival = Absorbance of sample X 100%
Absorbance of control
(1)
Determination of oxidative stress using the electrochemical determination
Three-electrode system was used in this study. The HA-P-ATi and GO200 substrates
were used as the working electrodes. A silver/silver chloride (Ag/AgCl; 6.0726.107;
Methrome) and a platinum wire were used as reference and counter electrode, respectively.
The changes of oxidative signal was recorded with potentiostat PGSTAT 302N (Methrohm,
Switzerland) with its user interface NOVA. Before the measurements, all electrodes were
cleaned with deionized water. All electrodes were connected to the electrochemical
workstation and immersed in an electrolyte solution. The electrolyte was 1x PBS solution.
The experimental conditions were as it follows: scan rate=100 mV/s; potential step=0.00244
V; start potential=-1 V; upper vertex potential=1 V, lover vertex potential=-1 V and stop
potential=-1 V.
MTT assay
The 80% confluent cells were sub-cultured through trypsinization (0.25% trypsin and
0.53M EDTA; Invitrogen Corporation). Cells were seeded and cultured on plastic
polystyrene (PS) (control), HA-ATi, and HA-P-ATi samples which coated with GO 200
μg/ml (GO200). The samples were placed in 24-well culture plates at the density of plates
5x104 cells/cm2. Cell viability was tested using a commercially available 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma, Thailand). The
solution of MTT was dissolved in salt solutions without phenol red to form a yellowish
solution before adding on cell-seeded samples at day 3 of cultures. Mitochondrial
dehydrogenases of viable cells cleave the tetrazolium ring, yielding purple formazan crystals
on cell-seeded samples after incubating for 2h. The resulting purple solution after incubating
with cells was measured at 540 nm using a microplate reader (Tecan, Infinite200PRO,
Switzerland).
Results
Anodized titanium
The insets in Figure 1 show top- and side-view SEM images of nanotube arrays,
which were anodized at 5˚C, +35/ -4V for 30, 90, and 150 minutes. The wall thickness,
diameter and length of nanotubes were measured using Image J and were shown in Figure 2.
The figure suggested that an increase of time significantly increases tube length. The tube
lengths of anodized TiO2 formed for 0.5, 1.5, 2.0 hrs are about 260, 550 and 900 nm,
respectively. Comparing the morphology of each condition, the structure of titanium oxide
prepared at 30 minute is irregular.
Figure 1. SEM images of nanotube arrays anodized at various time: a) 0.5, b) 1.5 and c) 2.5 hours, which
lengths are approximately 260, 550 and 900 nm, respectively. The inner images shows side views of TiO 2
nanotube arrays. All scale bars are 100 nm.
Figure 2. Wall thickness, diameter and length of nanotubes formed with different anodization times analyzed by
Image J.
Nanoindentation
In this section we describe nanomechanical characterization of the TiO2 nanotube
structures on Ti. Figure 3c shows the extracted reduce Young’s modulus obtained from
instrument continuously monitors the load and the corresponding depth with time in load
partial unload mode (Figure 3a) by varying anodization time. Total nanoindentation depth is
much larger than the thickness of the coating (>1,500 nm) (Figure 3d). The term reduce is
used because the measured modulus and hardness are not only a function of the coating, but
are also influenced by the substrate and indenter tip.
Figure 3. (a) Continuously monitors the load and the corresponding depth with time in load partial unload
mode. (b) A typical load–displacement curve (36). (c) Reduced Young’s modulus vs. nanoindentation
displacement (depth) for Ti samples anodized different times (0.5, 1.5, 2.0 hrs). (d) FESEM micrographs of
typical nanoindentations on the surface of ATi.
Physical and chemical characteristics of graphene oxide (GO)-calcium phosphate
(CaP)coating
The Figures 4a-b show the SEM images of the bioinspired nano-HA formation on
ATi and GO coating on HA-P-ATi, respectively. Figures 4c show SEM image with high
magnification of Figure 4b. The presence of GO on HA-P-ATi was confirmed as shown in
FTIR spectra in Figure 4d. The FTIR spectrum of ATi does not exhibit any significantly
peaks. The spectra of GO coated HA-P-ATi exhibits typical peaks for both GO and HA. The
peak at 3,350 cm-1 attributes to O–H stretching vibration, the peak at 1,620 cm-1 attributes to
C=O stretching vibration, the peak at 1,408 cm-1 attributes to COO–, the peak at 1,226 cm-1
attributes to vibration of C–O (epoxy), and the peak at 1,030 cm-1 attributes to PO43- that is
HA peak position. The FTIR spectrum of HA-P-ATi coating shows no peaks at 1,620 cm-1,
1,408 cm-1 and 1,226 cm-1. The results strongly indicate that GO was coated on HA-P-ATi by
anodic-EPD method in the present study.
Figure 4. SEM images of HA coating on P-ATi (a) and GO coated on HA-P-ATi by anodic EPD method with
200 μg/ml of GO in electrolyte and high magnification of GO coating, respectively (b and c). (d) FTIR peak of
anodized titanium ATi , HA-P-ATi, and HA-P-ATi coated with GO 200 μg/ml in isopropanol electrolyte.
A percentage of relative cell survival and toxicity of GO through oxidative stress of the
bacteria
This experiment was focused on the studying oxidative stress in bacterial culture of E.
coli and S. aureus induced by GO on HA-P-ATi. E. coli and S. aureus were used as a model
bacterium to evaluate antibacterial activities of the four concentrations of GO materials. E.
coli and S. aureus (106 to 107 no./cc) were incubated with the samples in Mueller-Hinton
broth at 37 °C for 18 h with 200 rpm rotating. The % survival of bacterial cells was
determined by the direct microscropic method. The Mueller-Hinton broth without materials
was used as a control. As shown in Figure 5b and d, the GO200 dispersion exhibits a
moderate cytotoxicity with the cell inactivation percentage at 25% of S. aureus cells. The GO
coated shows a slight stronger antibacterial activity with S. aureus when compared to E. coli,
which the cell inactivation percentage was about 20%.
Cyclic voltammetry (CV) is an elementary electrochemical method implemented in
all of electrochemical analyzers. For this reason it is very accessible and easy-to-use for
various users. These techniques are reliable enough to assay a broad spectrum of antioxidants
without any pretreatment of samples or use of specific reagents (37). Using this method we
were able to demonstrate oxidative signal in both E. coli and S. aureus after treated with
different concentrations of GO (Figures 5a and c). For a better understanding of induced
oxidative stress, voltammograms were re-plotted with peak current to compare oxidative
power of materials on each bacterial species (Figure 5b and d). The higher oxidative of S.
aureus over E. coli was also found in this investigation.
Figure 5. Cyclic voltammograms of detected E. coli (a) and S. aureus (c) oxidative stress. And cytotoxicity of
GO coating on HA-P-ATi to E. coli, and S. aureus after exposed to the GO coating on HA-P-ATi samples, for
18 h.
Responses of pre-osteoblast cells on different coatings
To evaluate the biological response to the materials under investigation, the cell
density at 3,500 cells of osteoblast were seeded per each wells (1 ml), all the experiments
were carried out at least in triplicate. A MTT test was carried out to evaluate cell vitality in
different samples. The highest values obtained were observed in PS. In comparing, cell
vitality was lower in both GO200 and HA-P-ATi samples but these two samples were
exhibited biocompatibility property to osteoblast cell (Figure 6).
Figure 6. MTT test on osteoblast cultures after one and five days from cell seeding. PS=polystyrene (control).
Discussion and Conclusions
The reason of non-uniform TiO2 layers is that when the time is too short, growth rate
is higher than dissolution rate because activity of titanium to form oxide layer is still high
compared with activity of oxide to dissolve in the electrolyte. At the initial state, the surface
is locally activated and pores start to grow randomly (35). With high growth rate leads to
irregular oxide structure. When anodization time is longer, the individual pores start
interfering with each other, and start to self-order. At this stage, the growth rate is equal to the
dissolution rate; then, the steady state is established. Therefore, when anodization time is 90
or 150 minutes, the TiO2 layers were highly order and the structure changes to nanotube
form.
Increasing depth, the measured modulus came up to that to the Ti substrate (100 GPa,
(38)). Average coating thickness for the samples are chosen by a dotted horizontal line in the
plots of reduces modulus vs. displacement (Figure 3c). The elastic modulus of the indented
sample can be inferred from the initial unloading contact stiffness, S=dP/dh, the slope of the
initial portion of the unloading curve (Figure 3b) (36). The compression tests were repeated
for five points to ensure the reproducibility of the data. From the reduced Young’s modulus
vs. nanoindentation displacement in Figure 3c, at displacement lower than 200 nm, the result
is not suitable to be analysis because there are high error values. In different time at 0.5, 1.5,
and 2.5 hours that obtained different thickness of nanotube layers as 260, 550, and 900 nm in
order and estimated Young’s modulus is around 60-80 GPa. Reduced Young’s modulus
shown different value, in case of lowest tube thickness found highest modulus and become
lower in order of increasing in tube thickness. The reason for higher Young’s modulus is the
effect of substrate. The result can be concluding that the compressive stresses and estimate
the elastic modulus are significant contribution from the substrate. Following the morphology
and modulus results of nanotubes, they indicates that anodization time of 1.5 hours, which
results in tube length of 550 nm, is appropriate to be used as substrate for further studies.
Since controllable calcium phosphate ratio was easily formed under electrodeposition
method (35), this study was coating P-ATi with HA by ELD, and following by second layer
of GO with anodic electrophoretic deposition (anodic-EPD). Then these were tested
antibacterial property against two models of bacteria (E. coli and S. aureus) and
biocompatibility with osteoblasts. It is believed that bacterial adhesion is dependent on
various intermolecular and surface interactions such as electrostatic, van der waals,
hydrophobic forces, hydrogen bonding, and covalent bonding (39). These forces are
influenced by the physicochemical properties of the substratum and the bacterial surface.
Bacterial cells normally compose of negatively charge on cell wall. These negative net
charges on the bacterial cell surface affected the resistance to cationic antimicrobial peptides
such as defences from human phagocytes (40). E. coli exhibited more negatively charged
(from O-antigenic polysaccharide) and less soft surface than that of S. aureus (41, 42). The
presence of long O-antigen chains reduces bacterial adhesion to various substrates because of
the presence of an energetic barrier during the adsorption process, which is caused by the
affinity of hydrophilic neutral O-antigen chains to water and the steric entropic barrier of LPS
chains on the cell membrane surface (43). It is suggested that S. aureus were more contacted
on GO than E. coli lead to higher percent survival of E. coli than S. aureus. Although, the
flagella apparently are play an essential role during the initial reversible stages of attachment
by overcoming the repulsive forces. However, the nonmotile bacterium S. aureus adhered to
the tested substrates at pH=6 in greater number than E. coli (44). The short-range chemical
interactions (ionic, hydrogen, and covalent bonding) occur between the biomaterial and the
bacteria (45). Therefore, from these results was confirmed that the adhesion of bacteria of S.
aureus (Figure 5) over E. coli is the one reason of higher peak currents.
Nanostructure of biomaterials is an important factor to cell functions (such as
synthesis of extracellular matrix proteins and formation of calcium mineral depositions) (46).
Moreover, the adhesion of osteoblast cells is a crucial prerequisite to subsequent such of that
cell functions. According to Webster et al., the use of nanostructured ceramics (such as
alumina, titania, and HA) significantly improves osteoblast adhesion (47, 48). Therefore, the
effect of nanostructured HA-P-ATi on pre-osteoblast proliferation was expected to be
highest. However, in Figure 6, MTT activity on HA-P-ATi was high but when compared with
that on GO coated sample it was lower than this novel material. These results clearly showed
that the GO coated, beyond not exerting any cytotoxic effects on the cells, actually promotes
osteoblast cell attachment and proliferation. Several literature reports focusing primarily on
biocompatibility of GO films were published (21, 49, 50), from which the results are
generally consistent with what is shown here on the GO film enhancing mammalian cell
attachment and proliferation. Taken together these results indicate that GO is a great support
for mammalian cell attachment, growth, and proliferation.
The length of the nanotubes could be significantly controlled from about 250 nm to 1
µm. The 550 nm (1.5 hrs of anodization) was the best tube orientation and high modulus than
the other. This best condition was used to prepare the ATi as a substrate of HA coating. Then
GO were successfully electrodeposited onto the HA-P-ATi using an EPD method. The results
from the FTIR analysis showed the relative presence of carbon on the surface of the HA-PATi after EPD of GO nanowalls. The direct contact of bacteria with material causes oxidative
stress leading to irreversible damages. Several studies have demonstrated that the use of
negatively charged material surface is of particular benefit in reducing bacteria colonization.
Furthermore GO acts as an enhancer of osteoblast cell growth. The results in the present
study for the first time suggest that GO and HA nanocomposites can decrease bacteria
susceptibility of S. aureus and E. coli, and the present study significantly shows the results of
biological properties of graphene oxide for its uses in biomedical applications.
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