Cite this: DOI: 10.1039c6bm00270f [625531]

Biomaterials
Science
PAPER
Cite this: DOI: 10.1039/c6bm00270f
Received 18th April 2016,
Accepted 22nd June 2016
DOI: 10.1039/c6bm00270f
www.rsc.org/biomaterialsscienceBioactive and biocompatible copper containing
glass-ceramics with remarkable antibacterial
properties and high cell viability designed for
future in vivo trials
R. A. Popescu,a,b,dK. Magyari,*bA. Vulpoi,bD. L. Tranda fir,cE. Licarete,bM. Todea,b
R.Ștefan,dC. Voica,eD. C. Vodnar,fS. Simon,b,cI. Papucdand L. Baia*a,b
In the present study our interest is focused on finding the e fficiency of 60SiO 2·(32−x)CaO·8P 2O5·xCuO
(mol%) glass-ceramics, with 0 ≤x≤4 mol%, in terms of bioactivity, biocompatibility, antibacterial
properties and cell viability in order to determine the most appropriate composition for their further use
inin vivo trials. The sol –gel synthesized samples show a preponderantly amorphous structure with a few
crystallization centers associated with the formation of an apatite and calcium carbonate crystallinephases. The Fourier Transform Infrared (FT-IR) spectra revealed slightly modi fied absorption bands due to
the addition of copper oxide, while the information derived from the measurements performed by trans-
mission electron microscopy, UV-vis and electron paramagnetic resonance spectroscopy showed thepresence of ions and metallic copper species. X-Ray photoelectron spectroscopic analysis indicated the
presence of copper metallic species, in a reduced amount, only on the sample surface with the highest
Cu content. Regarding in vitro assessment of bioactivity, the results obtained by X-ray di ffraction, FT-IR
spectroscopy and scanning electron microscopy, demon s t r a t e dt h ef o r m a t i o no fac a l c i u mp h o s p h a t el a y e ro n
all investigated sample surfaces. The inhibitory e ffect of the investigated samples was more signi ficant on the
Pseudomonas aeruginosa than the Staphylococcus aureus strain, the sample with the lowest concen-
tration of copper oxide (0.5 mol%) being also the most e fficient in both bacterial cultures. This sample
also exhibits a very good bactericidal activity, for the other samples it was necessary to use a higher
quantity to inhibit and kill the bacterial species. The secondary structure of adsorbed albumin presentsfew minor changes, indicating the biocompatibility of the glass-ceramics. The cell viability assay shows a
good proliferation rate on samples with 0.5 and 1.5 mol% CuO, although all glass-ceramic samples
exhibited a good in vivo tolerance.
1. Introduction
Tissue engineering has developed greatly in recent years due
to significant clinical requirements. The increase of life expect-
ancy in the human population is the main cause of the contin-ued progress in this area.1,2A practical approach to this
multidisciplinary research aims to promote the regenerationability of host tissue over a suitable sca ffold. Teams of
scientists are working on combining properties of metallic
nanoparticles, functional cells and biodegradable sca ffolds
to obtain good healing solutions to diseased or damaged
tissues.
3–5
Bioactive glasses represent a class of biomaterials that have
a very good potential for hard and soft tissue regeneration
applications.6–8However, their principal deficiency is the total
or partial absence of angiogenesis and antibacterial pro-perties.
9Biomaterial-associated infections are frequent surgery
complications in tissue reconstruction.10These are caused
especially by Staphylococcus aureus and Staphylococcus epider-
midis , bacterial species capable to form an adherent biofilm to
implant surfaces.10Pseudomonas aeruginosa can be found
widely in the hospital environment, being resistant to manyaFaculty of Physics, Babes-Bolyai University, 400084 Cluj-Napoca, Romania
bInterdisciplinary Research Institute on Bio-Nano-Sciences, Babes-Bolyai University,
400271 Cluj-Napoca, Romania. E-mail: klara.magyari@ubbcluj.ro,
lucian.baia@phys.ubbcluj.ro
cNational Centre of Magnetic Resonance, Babes-Bolyai University, 400084 Cluj-
Napoca, Romania
dFaculty of Veterinary Medicine, University of Agricultural Science and Veterinary
Medicine, 400372 Cluj-Napoca, Romania
eNational Institute for Research and Development of Isotopic and Molecular
Technologies, 400293 Cluj-Napoca, Romania
fFaculty of Food Science and Technology, University of Agricultural Sciences and
Veterinary Medicine, 400372 Cluj-Napoca, Romania
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antibiotics. Frequently this Gram negative bacterium produces
multidrug resistant strains that are di fficult to remove.11,12
The majority of bioactive glasses and ceramics are prepared
by melt quenching or sol –gel derived methods. The sol –gel
method is very popular because the obtained materials present
a higher porosity associated with a high specific surface area.During the sol –gel synthesis hydrolysis and polycondensation
reaction of the molecules (sol formation), gelation (sol –gel
transformation), aging and drying occur.
13,14
It has been found that by adding some metal oxides one
can create a multifunctional sca ffold that will solve those two
drawbacks.15Silver, copper and zinc nanoparticles are very
active antibacterial agents, with low toxicity and good
stability,16–18which can be used to enhance a biomaterial ’s
qualities and create a high quality sca ffold.16
Copper ions have been reported to be an essential com-
ponent of the angiogenic response.19They act by stabilizing
the expression of hypoxia-inducible factor (HIF-1a) throughsimulating an oxygen deficit, which plays a very important role
in cell di fferentiation and in blood vessel formation.
20Another
important benefit of Cu2+ions is the low cost and the high
stability when are compared with growth factors.21Further-
more, these metallic ions are vital in human metabolism due
to the significant amount found in human endothelial cells inthe course of physiological angiogenesis.
22–24On the other
hand, a high concentration of copper ions promotes the for-
mation of free radicals and creates an important cytotoxicenvironment.
25
In some recent studies, various concentrations, up to
10 mol%,26,27of copper oxide were used, in order to demon-
strate the properties of this metal oxide in various glass
systems. It was found that copper has a positive influence on
the angiogenic mechanisms of blood vessel formation,21,22
enhances bone metabolic activity,24,25has a valuable anti-
bacterial action27,28and could play the role of an enzymatic
cofactor in metabolic signals during tissue formation.25
Our present work proposes to bring new insights regarding
the role played by the copper addition in the bioactive silicate
glasses, their antimicrobial properties as well as to the per-formance related to the sample ’s viability from the perspective
of their further use for in vivo trials. Accordingly, the present
work includes the structural and morphological characteri-zation of copper oxide containing SiO
2–CaO –P2O5glass-
ceramics samples using X-ray di ffraction analysis (XRD),
transmission electron microscopy (TEM), Fourier transforminfrared (FT-IR), UV-vis absorption, electron paramagnetic reso-
nance (EPR) and X-ray photoelectron spectroscopy (XPS). Fur-
thermore, in vitro bioactivity was tested following the growth of
the calcium apatite layer on the material ’s surface after immer-
sion in simulated body fluids (SBF). The antibacterial pro-
perties of the glasses were tested by using Staphylococcus
aureus and Pseudomonas aeruginosa ; copper ion release was
also studied. Finally, the in vitro biocompatibility of the
materials was analyzed using protein adsorption and humankeratinocyte cell viability. As serum albumin is an abundant
protein in blood plasma, it ’s adsorption on the biomaterialsurface and it ’s conformational changes after adsorption are
often studied in vitro in order to predict a material ’sin vivo
biocompatibility.
29
2. Materials and methods
2.1. Samples synthesis
The samples belonging to the 60SiO 2·(32−x)CaO·8P 2O5·xCuO
(mol%) compositional formula with x= 0; 0.5; 1.5; 2.5 and 4
were prepared by the sol –gel method. The precursors used
were tetraethylorthosilicate (TEOS), triethylphosphate (TEP),
calcium nitrate tetrahydrate (Ca(NO 3)2·4H 2O) and copper
nitrate trihydrate (Cu(NO 3)·3H 2O) hydrolyzed in the presence
of nitric acid; the (HNO 3+H 2O)/(TEOS + TEP) molar ratio was
equal to 8. Reactants were added consecutively at 1 hour inter-
vals, under continuous stirring. The solutions (sols) werepoured into closed containers that were kept at 37 °C until
gelation (gels) occurred ( ∼2 days). The resultant gels were aged
at 37 °C for 3 days and afterwards were dried at 120 °C for24 h. Material stabilization was carried out at 600 °C for 3 h.
This temperature was determined from the di fferential
thermal analysis results of the dried gels. Prior to characteri-zing the obtained samples they were milled by hand for 5 min
in a ceramic mortar.
2.2. Material characterization
2.2.1. X-ray di ffraction. The X-ray di ffraction analysis (XRD)
was carried out on a Shimadzu XRD 6000 di ffractometer using
Cu K αradiation ( λ= 1.54 Å), with an Ni-filter. The di ffracto-
grams were recorded in the 2 θrange from 10° to 80° with a
speed of 1° min
−1.
2.2.2. FT-IR spectroscopy. The FT-IR absorption spectra
were recorded with a JASCO 6000 (Jasco, Tokyo, Japan) spectro-
meter by using the well-known KBr pellet technique, at room
temperature, in the 400 –6000 cm−1spectral range with a spec-
tral resolution of 4 cm−1. The FT-IR spectra after protein
adsorption were recorded using a Jasco IRT-5000 FT-IR micro-
scope coupled to a Jasco FT-IR-5000 spectrometer in reflectionconfiguration in the 4000 –650 cm
−1spectral domain with a
4c m−1spectral resolution and by using a ×32 Cassegranian
objective imaging a sample area of ∼50 × 50 µm2. The recorded
spectra were smoothed by a 5-point Savitzky –Golay smoothing
function for background correction. Second-derivative spectral
analysis was performed by using the JASCO Spectra Managerin order to locate the position of the overlapped components
of amide I, which were further assigned to di fferent secondary
structures. The bands were deconvoluted with a Gaussianband profile, a linear baseline between 1600 cm
−1and
1710 cm−1being previously applied. The secondary structure
composition was determined from the areas of the individuallyassigned components and their fraction of the total area.
2.2.3. UV-Vis spectroscopy. Absorption measurements of
the glass samples were performed with an Analytic JenaSpecord 250 plus UV-Vis spectrometer. The spectral resolution
was 2 nm.Paper Biomaterials Science
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2.2.4. Transmission electron microscopy. TEM images
were recorded using a Tecnai F20 XTWIN field emission,
high resolution transmission electron microscope operating atan accelerating voltage of 200 kV and equipped with an Eagle
4k CCD camera. The samples were suspended in distilled
water and then added dropwise on carbon film coatedCu grids.
2.2.5. EPR spectroscopy. The EPR measurements were per-
formed on powder samples using an ADANI X-band EPRspectrometer, in the magnetic field range 700 –4700 G, at room
temperature, on powdered samples.
2.2.6. Scanning electron microscopy. (SEM) imagines
were recorded using an FEI Quanta 3D FEG dual beam
electron microscope operating at an accelerating voltage of
30 kV.
2.2.7. X-Ray photoelectron spectroscopy. XPS spectra were
recorded with a SPECS PHOIBOS 150 MCD system employing
a monochromatic Al K αsource (1486.6 eV), a hemispherical
analyser and charge neutralization device. Samples were
fixed on double-sided carbon tape and care was taken to
ensure that the sample particles covered the tape. Experi-ments were performed by operating the X-ray source with a
power of 200 W, while the pressure in the analyse chamber
was in the range of 10
−9–10−10mbar. The binding energy
scale was charge referenced to the C 1s at 284.6 eV. Elemental
composition was determined from survey spectra acquired
at a pass energy of 60 eV. High resolution spectra wereobtained using an analyzer pass energy of 20 eV. Analysis of
the data was carried out with Casa XPS software. A Shirley
background was used for all curve-fitting along with theGaussian/Lorentzian product form (70% Gaussian and 30%
Lorentzian).
2.2.8. Specific surface area and pore volume. The results
were obtained from N
2-adsorption –desorption isotherms,
using a Sorptomatic 1990 apparatus. The specific surface area
was obtained by the Brunauer –Emmett –Teller (BET) method,
while the pore volumes were determined by the Barret –Joyner –
Halenda (BJH) method.
2.2.9. Inductively coupled plasma quadrupole mass spec-
trometry (ICP-Q-MS). The determinations were carried out by
inductively coupled plasma quadrupole mass spectrometry
(ICP-Q-MS). A Perkin Elmer ELAN DRC (e) was used with aMeinhart nebulizer and silica cyclonic spray chamber and con-
tinuous nebulization. The following instrumental parameters
of the spectrometer were used: nebulizer gas flow: 1.04Lm i n
−1, auxiliary gas flow: 1.20 L min−1, plasma gas flow: 15.00
L min−1,lens voltage: 14.50 V, ICP RF power: 1250 W, CeO/Ce =
0.029; Ba++/Ba+= 0.031. Ultra-pure de-ionized water (18.2
MΩcm−1) from a Milli-Q analytical reagent-grade water purifi-
cation system (Millipore) was used. All the plastic labware used
for sampling was either new or cleaned by soaking 24 h first in10% HNO
3then in ultra-pure water. A solution of 10 μgL−1
Mg, Cu, Cd, In, Ba, Ce, Pb, U in 1% HNO 3(Perkin Elmer
Atomic Spectroscopy Standard –Setup/Stab/Masscal Solution)
was applied as an external standard. The explored mass
domains were 6 < m/z< 14; 22 < m/z< 37; 40 < m/z< 238. Thebackground signal (blank) was determined with an ultrapure
water sample.
2.3. Assessment of bioactivity
In order to check the bioactivity, the obtained powders were
immersed for 7 days in simulated body fluid (SBF) in closableconical polypropylene tubes, and placed in an incubator at a
constant temperature of 37 °C under static conditions. The
SBF was prepared according to Kokubo ’s protocol.
30The solu-
tion was bu ffered at a pH of 7.4 at 37 °C. The weight of glass-
ceramic per volume of SBF used was 10 mg mL−1for each
sample. After 7 days, the powders were filtered, rinsed severaltimes with distilled water, and dried. The methods used to
evaluate the apatite presence were XRD, FT-IR spectroscopy
and SEM.
2.4. Ionic release
A quantity of 200 mg of powder samples were suspended in
1 mL deionized water and filtered after 24 h according to ISO
10993 part 5 and 12.
31,32The total content of copper was
obtained by dissolving 200 mg of sample in a 2 mL acidmixture (nitric acid/hydrofluoric acid 50/50). For these deter-
minations the ICP-Q-MS method was used. Errors are esti-
mated to be ±12.5% due to correlation between the arithmeticmean of the measured values and the accepted reference
value.
2.5. Antibacterial activity
2.5.1. Microorganisms and culture conditions. For the
bioassay two bacterial strains were used, one Gram positive:S. aureus (ATCC 49444) and one Gram negative: P. aeruginosa
(ATCC 27853). Tested microorganisms were obtained from the
Food Biotechnology Laboratory, Life Sciences Institute, Univer-sity of Agricultural Sciences and Veterinary Medicine, Cluj-
Napoca, Romania. The bacteria were cultured on Muller –
Hinton Agar and cultures were stored at 4 °C and subculturedonce a month.
2.5.2. Microdilution method. The modified microdilution
technique was used to evaluate antimicrobial activity. Bacterialspecies were cultured overnight at 37 °C in Tryptic Soy Broth
(TSB) medium. The bacterial cell suspensions were adjusted
with sterile saline to a concentration of approximately 3 × 10
5
CFU mL−1in a final volume of 100 µL per well. The inoculum
was stored at 4 °C for further use. Dilutions of the inoculum
were cultured on solid Muller –Hinton (MH) for bacteria to
verify the absence of contamination and to check the validity
of the inoculum. Determinations of minimum inhibitory con-
centrations (MICs) were performed by a serial dilution tech-nique using 96-well microtitre plates. Di fferent dilutions of
sample extract were carried out over the wells containing
100μL of Tryptic Soy Broth (TSB) and afterwards, 10 μLo f
inoculum was added to all the wells. The microplates were
incubated for 24 h at 37 °C. The MIC of the samples was
detected following the addition of 20 μL (0.2 mg mL
−1) of resa-
zurin in solution to each well, and the plates were incubated
for 2 h at 37 °C. A change from blue to pink indicatesBiomaterials Science Paper
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reduction of resazurin and therefore bacterial growth. The MIC
was defined as the lowest extract concentration that prevented
this color change. The minimum bactericidal concentrations(MBCs) were determined by serial subcultivation of 2 μL into
microtitre plates containing 100 μL of broth per well and
further incubation for 48 h at 37 °C. The lowest concentrationwith no visible growth was defined as the MBC, indicating
99.5% killing of the original inoculum. Gentamycin
(0.06 –125 µg mL
−1) was used as a positive control for bacterial
growth.
2.6. Protein adsorption
Bovine serum albumin (BSA) (Sigma-Aldrich, molecular mass
66 kDa) solution with 2 mg mL−1concentration was prepared
in phosphate bu ffer with pH 7.4. The powder samples (10
mg mL−1) were immersed in BSA solution for 3 h at 37 °C. The
powders were then removed from the protein solution, rinsed
three times with distilled water, and dried at room tempera-ture. The method used to assess protein adsorption was FT-IR
spectroscopy.
2.7. Cell viability
Human keratinocytes cells (HaCaT, Cell Line Service,
Germany) were cultured in Dulbecco ’s modified Eagle ’s
medium (Lonza) supplemented with 2 mM
L-glutamine, Pen/
Strep 100 U mL−1and 10% FCS and incubated in a humidified
incubator with 5% CO 2atmosphere at 37 °C.
The cytotoxic e ffect of glass-ceramics was assayed using
WST-1 dye (water soluble tetrazolim salt, Millipore), with a
method based on the enzymatic cleavage of the tetrazoliumsalt WST-I to formazan by mitochondrial dehydrogenases
active in the living cells. Therefore, HaCaT cells were seeded
in a 96-well plate, at a density of 10
4cells per well. The follow-
ing day di fferent amounts of samples were added to the test
wells and cells were placed in the incubator for additional24 h. Each sample concentration was tested in triplicate. Cells
without a powder sample were used as positive control. At the
end of the incubation period, the medium was removed fromall wells and 100 µl of fresh medium containing 10% WST-1
solution were added to each well, cells being further
incubated for another 60 min at 37 °C. Empty wells withmedium containing WST-1 reagent were used as blank. After
60 minutes of incubation, the optical absorbance was
measured at 440 nm, using a microplate reader (FlostarO-mega, BMG, Germany). A reference wavelength was used at
650 nm.
3. Results and discussion
3.1. Structural characterization
The XRD patterns of the obtained samples revealed a pre-
ponderantly amorphous structure (Fig. 1a). The irregular shape
of the recorded XRD patterns could be due to the presence ofthe second line centred at 2 θ∼32° that indicates the existence
of a few crystallization centers associated with the formation of
the apatite phase.
33–37This line has the highest intensity for
the samples with an amount of copper oxide equal to or less
than 1.5 mol%. For concentrations higher than 2.5 mol% CuO
this line is no longer clearly distinguishable, but instead onecan see the clear signature of a calcium carbonate crystalline
phase (strongest line at 2 θ= 29.8°). This change may seem
undesirable, but as it was previously reported,
38this phase
may be formed in appropriate conditions instead of a calcium
phosphate phase, but has no negative e ffect on the sample ’s
bioactivity due to the fact that is a precursor for carbonateapatite layer formation in SBF.
34,38In fact, by carefully analyz-
ing the patterns one can see that the calcium carbonate crystal-
line phase gives small reflections for all investigated samples.The diffractograms of the copper containing samples show no
obvious peaks that can be associated with the presence of
Fig. 1 XRD patterns (a) and FT-IR spectra (b) of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%) samples.Paper Biomaterials Science
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copper based phases, as previously reported.15Possible
reasons for this peak absence can be either the detection limit
(the amount of copper is under 1.5 atom percent), or the homo-genous distribution of copper in the glassy matrix.
The FT-IR spectra exhibited absorption bands characteristic
for bioactive silicate based glasses and glass-ceramics (Fig. 1b).Thereby, one can see features that can be assigned to the Si –
O–Si stretching (1040 and 860 cm
−1) and Si –O–Si bending
vibrational modes (465 cm−1).34The maximum intensity of the
Si–O–Si stretching vibrational signal showed a slight increment
with the addition of copper oxide. The band that arises at
1440 cm−1can be associated with the existence of the carbo-
nate groups,34while the 560 and 580 cm−1twin signals show
the presence of phosphate groups.34For 2.5 and 4 mol% CuO
samples some changes can be observed in the shape of thedoublet located around 600 cm
−1that becomes broader and
gives rise to a single distinguishable absorption signal. A poss-
ible reason might be the signals from phosphate groups thatoverlap with those from CuO vibrational modes. Copper oxide
is known to give bands at 603 and 497 cm−1due to the Cu –O
stretching modes along the [101] direction.39Moreover, other
IR active modes for Cu –O were reported in the spectral range
between 605 and 660 cm−1, which may be associated with the
existence of another phase, i.e.,C u 2O.39It is also worth
emphasizing the absorption bands given by carbonate
vibrations between 1400 and 1500 cm−1.
In Fig. 2a and d there can be seen TEM images taken from
the sample with 0.5 mol% CuO and Fig. 2g taken from the
sample with 4 mol% CuO, revealing a mostly amorphous
porous structure. The dark spots represent crystalline struc-tures, and in order to determine the nature of these crystalline
particles HRTEM images were also recorded (Fig. 2b, e and h,
respectively). The Fourier transformed image (Fig. 2c) made onFig. 2b, displays measured interplanar distances of 0.311 and
0.202 nm that are in good agreement with the (104) and (018)
lattice planes of calcium carbonate,
40while the one made on a
Fig. 2 TEM (a, g-scale bar 200 nm, d-scale bar 10 nm), HRTEM (b, e, h-scale bar 5 nm) and FFT on HRTEM images (c, f, i) of the 60SiO 2·(32−x)
CaO·8P 2O5·xCuO (mol%) samples, with x= 0.5 (a –f) and x=4( g –i).Biomaterials Science Paper
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nanoparticle with a diameter of 8 –10 nm from the second
HRTEM image (Fig. 2e) reveals an interplanar distance of d=
0.226 nm (Fig. 2f), which can be attributed to the (111) latticeplane of metallic copper.
41In the 4 mol% CuO containing
sample we could also find crystals that could be attributed to
HAP with a characteristic d-spacing of 0.385 nm of the (111)
lattice plane,42as calculated from the FFT (Fig. 2i) image from
the corresponding high resolution image (Fig. 2i).
In order to obtain further insight, UV-vis absorption
measurements were performed. By looking at the UV-vis
spectra displayed in Fig. 3 one can see that the samples con-
taining CuO exhibit a dominant broad band centered at800 nm characteristic of d –d transitions of Cu
2+in octahedral
coordination.43As expected, this signal increases in intensity
as the copper content becomes higher. The broadness of theabsorption band may be due to the overlap of the signals given
by metallic Cu with those originating from the copper ions,
which are expected to be formed; the latter signal usuallygiving rise to broad electronic absorption features at wave-
lengths higher than 700 nm.
44,45
For clarifying the distribution of the copper ions in the
glass matrix, EPR spectra were recorded and are presented in
Fig. 4. The obtained asymmetric EPR spectra for low copper
concentration are characteristic for Cu2+(3d9) ions in axially
distorted octahedral symmetric sites.46In most environments
for Cu( II), the ground state magnetism is essentially spin-only
and the orbital motion is said to be “quenched ”. Since Cu( II)
has one unpaired electron in its 3d9configuration, the
“effective ”spin is equal to the actual spin of the free ion S=
1/2.47The B ∥band can be hyperfine split to four signals due to
the Cu( II) unpaired electron interaction with Cu( II) nucleusspin ( I= 3/2). The obtained Cu( II) EPR spectra are described
by a spin Hamiltonian with axial symmetry. The spectralparameters obtained for all investigated compounds show that
g
e<g⊥<g∥,47,48where gedenotes the Lande factor for a free
electron.
The hyperfine structure is quite well resolved only in the
parallel band for the sample with 0.5 mol% CuO and illus-
trates that at this concentration copper ions are far enoughapart to avoid magnetic interaction and have a randomized
distribution inside the glass. The two hyperfine lines at the
parallel orientation and g-factor position, indicated by arrows,are presented in the inset of Fig. 4; the values of corresponding
parameters being g
∥= 2.33 and A∥= 124 G. The perpendicular
band with g⊥= 2.03 has its hyperfine structure unresolved. The
EPR spectra revealed a partially resolved hyperfine structure
with two (1.5 –4 mol% samples) and three (0.5 mol% samples)
B∥lines below 3000 G, and the fourth hyperfine line over-
lapped with the second component of the experimental
spectra; without a hyperfine structure due to the dipolar inter-
action between Cu2+ions,48it is very broad, with a peak-to-
peak linewidth around 300 G.
The changes in line-shape of the 1.5 –4 mol% EPR are due
the clustering tendency of copper ions with increasing con-centration in the samples, unlike the 0.5 mol% CuO sample
where the EPR spectrum shows the presence of high
amounts of isolated Cu
2+ions.48Once the CuO content
increases, the EPR line becomes more symmetric, and the
parallel and perpendicular signals convolute in one signal as
a result of the prevalence of dipole –dipole interactions.
Taking into account this result and that derived from the
UV-vis spectra analysis, i.e.the broad band associated with
the presence of Cu2+ions, one can infer that Cu2+ions are
present inside of all copper containing samples, being pre-
ponderantly isolated in the sample with the smallest copper
content.
Fig. 3 UV-vis spectra of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%)
samples.
Fig. 4 EPR spectra of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%)
samples.Paper Biomaterials Science
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Roughness, porosity and pore size distribution play a criti-
cal role in protein adsorption, cell adhesion and bone for-
mation.29,49Data obtained by BET analyses showed a
progressive increase of the surface area and average pore
volume distribution, starting from the initial undoped matrix
until 1.5 and 2.5 mol% CuO, respectively. Fig. 5a reveals thehighest surface area in the 1.5 mol% CuO containing sample
and Fig. 5b shows the highest average pore volume at 2.5 mol
% CuO. Increasing the surface area and pore volume of amaterial may greatly accelerate the kinetic deposition process
of hydroxyl-carbonate apatite and therefore enhance the bone
formation bioactivity of the material.
36,50The pore volume dis-
tribution follows the same trend as the specific surface area of
the samples; in this case the 2.5 mol% CuO containing
sample presents the highest measured pore volume value(Fig. 5b). The decrease of the surface area starting with
2.5 mol% CuO can be due to the increase of the size of the
nanostructures that build up the sample structure, while thedecrease of the average pore volume for the sample with
4 mol% CuO may occur because of the partial blocking of
several pores (as a consequence of the nanostructure sizeincrease).
To validate the derived hypothesis related to the existence
of isolated copper ions for the small Cu content and theirgradual clusterization as the Cu content increases, XPS spectra
were recorded. In order to examine the chemical state of
copper in the glass-ceramics, the high resolution spectra areusually deconvoluted and the assessment of the metallic
copper (Cu
0) and Cu( II) oxide contributions is analyzed.51In
the case of the presently investigated samples, one shouldnote that due to the low content of copper the sample with the
smallest Cu amount exhibited no signal in the high resolution
spectrum and consequently it is not presented in Fig. 6. Actu-ally, one can see that all the recorded high resolution spectra
show a bad signal to noise ratio as a result of the reduced
amount of copper. Therefore, we decided to analyze thecopper band located around 934 eV without deconvoluting the
signal into its components, but performing a smoothing of the
signal. One can see that the maximum value of this band is
shifted to higher binding energies as the copper content
Fig. 5 The BET surface area (a) and pore volume distribution with median pore radius values inserted (b) of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO
(mol%) samples.
Fig. 6 The XPS high resolution spectra recorded on 60SiO 2·(32−x)
CaO·8P 2O5·xCuO (mol%) samples.Biomaterials Science Paper
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decreases, i.e. 934.09 eV for x= 4 mol%, 934.4 eV for
x= 2.5 mol% and 934.95 eV for x= 1.5 mol%, proving once
again that the copper species inside the silicate network aremore strongly bonded as the Cu amount becomes higher.
Indirectly, this is a confirmation of the hypothesis that copper
ions are less isolated with increasing copper content; ions clus-ters and particles are formed, and consequently copper can be
released more easily from the samples as the copper content
increases.
3.2. Assessment of bioactivity
The immersion of the investigated samples in SBF was per-
formed to evaluate the bioactivity of these materials. It isknown that SBF mimics the blood plasma, simulating the
apatite-like layer growth for in vitro studies.
52After 7 days of
immersion in SBF, the hydroxyapatite (HA) phase is appears atthe recorded signals at 2 θ∼26° and 32° in the XRD patterns,
but is overlap with apatite phase existing in the samples
(Fig. 7a). To easily assign this apatite phase, XRD pattern ofHA is also included as reference.
53
In the FT-IR spectra of the samples immersed in SBF
(Fig. 7b), the bands at 604 and 564 cm−1that are specific for
crystalline HA33,53,54increased in intensity compared with
those obtained for the non-immersed ones. Moreover, the
sample with 4 mol% CuO (Fig. 7b) reveals a well-defineddoublet (604 and 564 cm
−1) specific for crystalline HA, bands
that could not be distinguished as a two absorption signal in
the FTIR spectra of this sample ’s non-immersed counterpart.
Considering that this doublet is also present in the spectrum
of bioactive glass, in order to clarify the existence of HA crystal-
lite after immersion in SBF, the ratio between the intensities ofthe typical FT-IR band for crystalline HA at 604 cm
−1and the
Si–O–Si bending vibration at 465 cm−1was calculated, before
and after immersion in SBF (Fig. 7c).34The ratio increases
after immersion in SBF, a result that indicates the growth of
an HA layer on the surface. One should emphasize that
this increase is in accordance with the results derived fromXRD data.Fig. 8a –e show the SEM micrographs of the 60SiO
2·(32−x)
CaO·8P 2O5·xCuO (mol%) samples before soaking in SBF, and
confirm the existence of a crystalline surface morphology thatstrengthens the findings derived from XRD and FT-IR analysis.
The formation of HA deposition on the glass-ceramics sur-
faces after immersion in SBF was also observed by SEM.Fig. 8f –j show the surface morphology of glass specimens incu-
bated in SBF for 7 days. The surface of almost all samples
(Fig. 8f –h, j) show clusters with a cauliflower like morphology
covering the entire surface, the exception being the sample
with 2.5 mol% CuO, which shows the formation of submicron
apatite grains.
3.3. Ions release
Copper ions are very important components for angiogenic
processes,
15as well as having a beneficial e ffect in killing
microorganisms.15ICP-Q-MS analysis was performed to obtain
the amount of copper ion released in deionized water in 24 h
(Fig. 9a). In order to quantify the total copper ion content in
the glass-ceramic samples, before performing the ICP-Q-MSmeasurements, the samples were dissolved in an acidic
mixture (Fig. 9b). The obtained results show that the copper
content values are comparable with those desired ones. Thepercentage release of Cu in 24 h was shown to be initially con-
centration-dependent; the higher the initial content the higher
the amount released in 24 hours.
3.4. Antibacterial activity
The antibacterial activity of the glass-ceramic samples was
studied on Pseudomonas aeruginosa and Staphylococcus aureus ,
two of the most resistant bacteria to many antibiotics, whichare present ubiquitously in hospitals. Minimum inhibitory
and bactericidal concentration (MIC, MBC) are used as stan-
dard qualitative methods for determining the antimicrobialeffect of a material. Copper ions were reported to be the most
active form concerning antibacterial action.
55,56The high
response in contact with bacteria or cells is caused by themigration of Cu
2+to the surrounding environment.57MIC
Fig. 7 XRD patterns (a), FT-IR spectra (b) and the 604/467 cm−1intensity ratio of FT-IR spectra (c) of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%)
samples after immersion in SBF for 7 days. The XRD pattern and FT-IR spectrum of HA were inserted only for comparison purpose.Paper Biomaterials Science
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represents the lowest concentration point of antimicrobial
material that will stop the visible growth of a microorganism
after 24 h incubation,58while MBC represents the lowest con-
centration point of an antibacterial agent required to kill a par-ticular bacterium.
58The inhibitory e ffect of the investigated
samples was more significant on Pseudomonas aeruginosa thantheStaphylococcus aureus strain; the lowest concentration of
copper oxide was also the most e fficient in both bacterial cul-
tures. The sample with 0.5 mol% CuO also shows a very goodbactericidal activity, for the other samples it is necessary to
use a higher quantity to inhibit and kill the bacterial species
(Fig. 10a). The obtained MIC and MBC revealed the greaterefficiency of the 0.5 mol% CuO containing sample for both
bacteria and of all investigated samples with copper for
Pseudomonas aeruginosa in comparison with gentamycin (see
Fig. 10).
It has been found that copper ions released by the CuO
nanoparticles adhere to the negatively charged bacterial cellwall and destroy it, causing protein denaturation and cell
death.
59,60Copper ions inside the bacterial cells may link to
DNA acid molecules and cross-link the nucleic acid strands,this results in a disorderly helical structure.
60Moreover,
copper ions absorbed by the bacterial cells have also been
found to damage important biochemical processes, therebyleading to an overall diminishing in the bacteria growth
rate.
60–62The presence of isolated species in the sample with
0.5 mol% CuO can lead to an increase of the antimicrobialefficiency related to the copper amount inside the samples. In
fact, for the samples with higher CuO content, the copper
species are more easily released, but their action from an anti-bacterial perspective is not as e ffective as in the case of the
sample with 0.5 mol% CuO, most probably due to their larger
dimensions.
3.5. Assessment of biocompatibility
Furthermore, the samples were subjected to protein adsorp-
tion biocompatibility studies by means of FTIR spectroscopy.
The obtained results after BSA adsorption on the glass surfaces
show characteristic bands of the proteins: amide I (C –O
stretch) at 1650 cm
−1and amide II (N –H in-plane bending) at
1550 cm−1(Fig. 11) denoting the presence of proteins on the
samples surface.63
It is known that the conformation of proteins changes
upon adsorption onto a material ’s surface due to the electro-
static and hydrogen bonding interaction between proteins andmaterials.
64,65After BSA adsorption onto all glass-ceramic sur-
faces the question was the following: how is the secondary
structure a ffected by the copper oxide concentration of the
glass-ceramics? One of the methods for analysis of the second-
ary structure is the deconvolution of the amide I absorption
band from the FT-IR spectrum.63The secondary structure of
lyophilized BSA is dominated by α-helices (1649 –1657 cm−1),
with a small amount of β-sheet (1618 –1641 and
1674 –1695 cm−1) and β-turn (1662 –1686 cm−1).66The second-
ary structure of BSA, after its adsorption on the glass-ceramic
surfaces, evidenced only minor changes in helical structure
that are within the error limits (Fig. 12). Other changes appearin the β-sheet and β-turn structure, that is, the β-sheet/ β-turn
ratio decreases with increasing copper oxide content in the
glass structure. At the same time, in the case of samples with1.5, 2.5 and 4 mol% CuO, one can observe the band around
1608 cm
−1associated with the amino acid side chain residues.
Fig. 8 SEM images of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%)
samples before (a) x= 0, (b) x= 0.5, (c) x= 1.5, (d) x= 2.5, (e) x=4( a –e)
and after immersion (f) x=0 ,( g ) x= 0.5, (h) x= 1.5, (i) x= 2.5, ( j) x=4
in SBF for 7 days, with a scale bar of 5 µm.Biomaterials Science Paper
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It is known that BSA has great a ffinity to bind copper( II) ions,67
which may cause a few changes in the conformation of the
proteins. However, these changes are minor and it can be con-cluded that all investigated samples are biocompatible.
3.6. Cell viability
Given the fact that all samples are biocompatible, the next step
to obtain the optimal concentration of CuO in the glass-ceramic for further in vivo trials of these materials was to
evaluate the e ffect of the material on cell viability. MTT tests
were performed to compare continuously the proliferation ofHaCaTs cultured on di fferent sample concentrations. The
obtained results show that the cytotoxic e ffect is almost absent
or minimal after 24 h exposure in both sample dilutions(Fig. 13).
The results concerning toxicity of copper on di fferent cell
cultures are contradictory in the reported studies. Some ofthem report that CuO is a cytotoxic agent, inducing apoptosis
and autophagy,
60,68others argue about a very good proliferation
Fig. 9 Copper ion release of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%) samples soaked for 24 h in deionized water (a) and the total copper ion
content of the samples dissolved in acid mixture (b).
Fig. 10 Minimum inhibitory concentration (MIC) (a) and minimum bactericidal concentration (MBC) (b). Note that the glass-ceramic without CuO
content did not exhibit relevant behavior for presenting the data in the above MIC and MBC diagrams.
Fig. 11 FT-IR spectra of the 60SiO 2·(32−x)CaO·8P 2O5·xCuO (mol%)
samples after BSA adsorption.Paper Biomaterials Science
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rate after several times of exposure.21However, these results
depend on several parameters, such as: time of measurement,
type of cells, concentration and pre-treatments of the material,
among others.26The pre-treatment of the glass by previous
soaking in culture medium to seed the cells can avoid high
early ion release and pH increase.69In the present study the proliferation of HaCaT cells
were significantly promoted by the samples with 0.5 and
1.5 mol% CuO when compared to the glass-ceramic without
CuO content. The viability is very close to 100% in theother two samples ( x= 2.5 and 4), suggesting a good
in vivo tolerance.
Fig. 12 Deconvolution of the amide I (1710 –1600 cm−1) adsorption band of the lyophilized BSA, before and after its adsorption on the 60SiO 2·(32−x)-
CaO·8P 2O5·xCuO (mol%) sample surface, and their distribution on secondary structure in lyophilized adsorbed BSA onto a glass surface.Biomaterials Science Paper
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By coming back to the information derived from EPR and
XPS analyses, a possible explanation regarding the excellentresults obtained for the cell viability can be drawn. Thus, the
isolated copper ions are well-bonded on the porous silicate
network and are the predominant copper species in thesamples with x= 0.5 and 1.5 mol% CuO. Once the copper
content increases, the number of copper ions and metallic
clusters increases, to the detriment of the isolated copperions, leading to a higher rate of Cu release. Thus, the struc-
tural environment formed by the copper ions that are “well-
connected ”to the bioactive silicate porous network would lead
to excellent performances regarding the viability of the
samples with the smallest CuO content, i.e. x = 0.5 and
1.5 mol%.
4. Conclusions
The sol –gel method was used to obtain SiO 2–CaO –8P2O5glass-
ceramics with four di fferent concentrations of CuO (0.5, 1.5,
2.5 and 4 mol%) in order to establish the required properties
that could o ffer a suitable material for bone tissue engineer-
ing. The UV-vis, EPR spectra and TEM analysis revealed that
all samples contain ions and metallic copper species. X-Ray
photoelectron spectroscopic analysis showed that copper met-allic species are present in a reduced amount only on the
sample surface with the highest Cu content. The BET analysis
shows the highest surface area for the 1.5 mol% CuO contain-ing sample and the highest average pore volume for 2.5 mol%
CuO.
The information derived from the XRD, FTIR and SEM
measurements performed on the immersed samples in SBF
for 7 days prove the existence of HA crystallites, a result that
confirms the in vitro bioactivity of all investigated samples.
It was found that the inhibitory e ffect of the investigated
samples was more significant on Pseudomonas aeruginosa thanStaphylococcus aureus strain, the sample with the lowest con-
centration of copper oxide (0.5 mol%) was also the most
efficient in both bacterial cultures. This sample also exhibited
a very good bactericidal activity, while for the other investi-
gated samples a higher amount was necessary to inhibit and
kill the bacterial species. The albumin absorption has detectedthat the binding of copper species causes a few minor changes
in the conformation of proteins, the final conclusion being
that all investigated samples are biocompatible. Regarding thecytotoxicity of the samples, the e ffect is almost absent or
minimal after 24 h exposure in both sample dilutions, as well
as in the samples with 0.5 and 1.5 mol% CuO content, thusrevealing a very high proliferation rate.
Based on the fact that the investigated glass-ceramics
exhibit good bioactivity and biocompatibility, the samples with0.5 and 1.5 mol% CuO content having excellent viability, and
glass-ceramics with minimum 0.5 mol% content showing
good antibacterial e ffects against Staphylococcus aureus ,i t
could be concluded that the 0.5 mol% CuO concentration is
the most appropriate for further in vivo trials.
Acknowledgements
This paper is a result of a doctoral research made possible by
the financial support of the Sectoral Operational Programme
for Human Resources Development 2007 –2013, co-financed
by the European Social Fund, under the project
POSDRU/187/1.5/S/155383- “Quality, excellence, transnational
mobility in doctoral research ”& by a grant of the Romanian
National Authority for Scientific Research and Innovation,
CNCS –UEFISCDI, project number PN-II-RU-TE-2014-4-1597.
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