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Applied Surface Science 298 (2014) 225–235

Contents lists available at ScienceDirect

Applied Surface Science

jou rn al h om ep age: www.elsev ier .com/ locate /apsusc

ynthesis; characterization and antimicrobial effects of compositesased on multi-substituted hydroxyapatite and silver nanoparticles

urora Mocanua, Gabriel Furtosa, Sorin Rapunteanc, Ossi Horovitza, Chirila Florec,orina Garboa, Ancuta Danisteanua, Gheorghe Rapunteanc,ristina Prejmereanb, Maria Tomoaia-Cotisel a,∗

Department of Chemical Engineering, Babes-Bolyai University of Cluj-Napoca, 11 Arany J. Street, Cluj-Napoca 400028, RomaniaRaluca Ripan Institute of Research in Chemistry, Babes-Bolyai University, 30 Fantanele Street, Cluj-Napoca 400294, RomaniaUniversity of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca, 3-5, Manastur Street, Cluj-Napoca, 400372, Romania

r t i c l e i n f o

rticle history:eceived 8 December 2013eceived in revised form 26 January 2014ccepted 26 January 2014vailable online 3 February 2014

eywords:ulti-substituted nano hydroxyapatite

ilver nanoparticlesano compositesntibacterial effectsathogenic species

a b s t r a c t

Nano hydroxyapatite doped with zinc (0.2 wt%), silver (0.25 wt%) and gold (0.025 wt%), (HAP), has beenobtained by an innovative wet chemical approach, coupled with a reduction process for silver and gold.The synthesized multi-substituted nano HAP was freeze-dried and calcined at 650 ◦C. Nano HAP has beencharacterized by XRD, FTIR spectroscopy and imaging techniques: TEM, SEM and AFM. Then, nano HAPwas mixed with previously synthesized silver nanoparticles (AgNPs), in the amount of 9 wt%, to give anovel material (HAP-Ag). The AgNPs were prepared by the reduction of silver nitrate with glucose inalkaline medium. TEM and UV–Vis confirmed the formation of AgNPs with an average size of 12 nm.Further, organic matrix composites were obtained from a filler made of HAP and/or HAP-Ag and a mix-ture of monomers (such as bis-GMA and TEG-DMA), which were polymerized at various compositionsin AgNPs content up to 5.4 wt%. Antibacterial activities of these composites were investigated againstseveral different pathogenic species: Escherichia coli, Staphylococcus aureus, Staphylococcus spp., Bacillus

cereus, and Candida albicans, using the Kirby–Bauer disk-diffusion method. Antibacterial activities areenhanced with increasing of silver content within composites. These effects clearly reveal that AgNPscan be effectively utilized in combination with multi-substituted HAP and polymeric matrix, both usedas carriers, in order to improve their efficiency against various pathogenic species. These composites canbe considered a promising antimicrobial material for coating of orthopedic and dental implants or usedas bone cements in surgical applications.

. Introduction

The synthesis of silver-doped hydroxyapatite with antimicro-ial activity (i.e. antibacterial, antiviral, or antifungal activity) is ofreat interest, in view of its potential biomedical applications [1–4].he biological effect of silver nanoparticles (AgNPs) has been inten-ively studied, and different methods for their fabrication were

ested [5–14].

A multitude of ceramic materials with biomedical applicationsere synthesized. Among them, hydroxyapatite (HAP) is largely

Abbreviations: HAP, multi-substituted nano hydroxyapatite; AgNPs, sil-er nanoparticles; HAP-Ag, multi-substituted hydroxyapatite with adsorbedilver nanoparticles; bis-GMA, (2,2-bis[4-(2-hydroxy-3-methacryloyloxypropoxy)-henyl]propane; TEGDMA, triethyleneglycol dimethacrylate; TEOS, tetraethylrthosilicate; OMC, organic matrix composite; ICP–AES, inductively coupled plasmatomic emission spectrometry; SBF, simulated body fluid.∗ Corresponding author. Tel.: +40 264593833; fax: +40 264590818.

E-mail address: mtcotisel.ubbcluj@yahoo.ro (M. Tomoaia-Cotisel).

169-4332/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsusc.2014.01.166

© 2014 Elsevier B.V. All rights reserved.

used in orthopedics and dentistry, owing to its exceptional biocom-patibility and bioactivity [15–18]. Crystallographic and chemicalinvestigations showed synthetic HAP to be similar to the naturalone [19].

Since synthetic HAP is thermodynamically stable at physio-logical pH [20] and is osteoconductive, it was used for bone ordental reconstruction. Proteins, amino acids, and other organic sub-stances are easily adsorbed on HAP, which in turn might favor theadsorption and replication of bacteria on HAP [21]. As the resis-tance of microorganisms toward antibiotics is increasing, there isa continuous preoccupation of scientists in finding new bioactivecompounds, with high biocompatibility and antibacterial proper-ties, such as nanometrical silver [22].

Orthopedic implants can cause post-operative infections dueto the adhesion of bacteria on their surface. In vitro investiga-

tions indicated that silver, zinc and copper ions in the coatingsof implants can prevent the adhesion of bacteria [1,23,24]. Goldnanoparticles also show antibacterial activity [25]. So implantswith deposited nanocomposites containing silver nanoparticles

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26 A. Mocanu et al. / Applied Su

an reduce post-operative inflammations and prevent infections26,27].

Nano HAP-silver composites may contain silver ions embed-ed in the HAP structure or silver nanoparticles adsorbed on theAP surface. A better antibacterial activity was reported for surfacedsorbed AgNPs, due to their interaction with the bacteria cells [28].he anti microbial effect of both elemental silver and of its salts isnown for some time; its inhibitory and bactericidal effect is mani-est against many microorganisms (bacteria, fungi, viruses) [29,30]nd was used in the treatment of wounds [31–33].

The comparative role of AgNPs and Ag+ ions in the antimicro-ial effect and in what regards toxicity against animal cells is still

matter for discussion [34,35]. While the toxicity of Ag+ ions isigh against microorganisms, it is relatively safe for animal cells29,36]. The toxicity could depend on the nanoparticles’ size andhape, whereupon particles smaller than 10 nm, and particularlyheir accumulation in the intracellular space could provoke cellysfunctions [37,38].

The mechanism of the antimicrobial effect of AgNPs is not yetlucidated [22]. A possibility is the gradual release of Ag+ ions fromgNPs, followed by their disruption on ATP production and DNAeplication [39–42]. Other considers that the AgNPs act by anchor-ng onto the bacterial cell wall, producing pits and penetratingnto the cytoplasm [27,34,37,40,41]. They accumulate in the cell

embrane, increasing its permeability, which eventually resultsn the death of the cell [43,44]. Another mechanism would be theeneration, by AgNPs and Ag+ ions, of reactive oxygen containingpecies (free radicals), such as superoxide radicals [45–48]. Modi-cations in the structure of peptidoglycan chains, with release ofuramic acid, as well as changes in the enzymatic equipment are

lso mentioned [37,44,49]. The association of metal nanoparticlesith various antibiotics, based on the synergism of their effects,

eems to be another promising area for research [50].As a continuation of our efforts to obtain silver nanoparticles

51] and new biological active materials based on nano hydroxya-atite [52–54], in the present investigation we have synthesized forhe first time a multi-substituted hydroxyapatite, containing zinc,old and silver in its inner HAP structure. In addition, this triple-ubstituted HAP was mixed with AgNPs and a new HAP-Ag materialas obtained, in which AgNPs are homogeneously adsorbed on

he surface of nano HAP particles. Moreover, new nano compos-tes have been developed by incorporating different amounts ofAP and/or HAP-Ag into an organic matrix, made by polymer-

zation of monomers, such as bis-GMA (60%) and TEGDMA (40%).hese composites are thoroughly investigated for their structurend antimicrobial properties.

. Material and methods

.1. Materials

The starting compounds include calcium nitrate,a(NO3)2·4H2O, diammonium hydrogen phosphate, (NH4)2HPO4,odium hydroxide (NaOH), potassium hydroxide (KOH), 4-2,4-dimethylheptan-3-yl)phenol (nonylphenol). They were pronalysi (p.a.) products obtained from Merck. Reagent gradeilver nitrate (AgNO3), tetrachloroauric(III) acid, HAuCl4·3H2O99.5%), sodium borohydride (NaBH4) and tetraethyl orthosil-cate, Si(OC2H5)4 (TEOS, 98 wt%) were also purchased from

erck. The Zn(NO3)2·6H2O p.a. (purum 99%) were purchasedrom Sigma-Aldrich., 5% glucose solution from Hemofarm SRL

Romania). All were used as received without further purification.eionized ultrapure water was used in all experiments. For thereparation of organic matrix composites bis-GMA (2,2-bis[4-2-hydroxy-3-methacryloyloxypropoxy)-phenyl]propane, Aldrich

cience 298 (2014) 225–235

Chemical, Milwaukee, WI, USA) and TEGDMA (triethyleneglycoldimethacrylate, Sigma Chemical, St. Louis, MO, USA) were used asmonomers; benzoyl peroxide (BPO) and N,N-dihydroxyethyl-para-toluidine (DHEPT) were used as the initiator and the activator,respectively; both were purchased from Merck–Schuchardt. TheMueller–Hinton agar was purchased from BioMerieux Inc., France.

2.2. Preparation of samples

2.2.1. Synthesis of multi-substituted nano hydroxyapatite (HAP)Two solutions, A and B, were prepared. Solution A was a 0.2 M

calcium nitrate solution (2500 ml) with 1 ml nonylphenol. Zincnitrate, silver nitrate and tetrachloroauric(III) acid were added toreach the chosen Zn (0.2%), Ag (0.25%) and Au (0.025%) contentin the final inorganic powder (HAP). The pH of the solution was6.3. The other solution (B) was a 0.12 M di-ammonium hydrogenphosphate solution (2500 ml) with 1 ml nonylphenol The pH ofthis solution was adjusted in the range 11.5–12 by adding NaOHsolution. Sodium borohydride was added to assure the completereduction of silver and gold.

Solution A was quickly added to solution B under vigorousstirring (800 rpm). The temperature of both solutions (A and B)before mixing was 70 ◦C. The reaction mixture was maintainedstirred for 24 h at 70 ◦C for maturation, allowing the calcium phos-phates, precursors to HAP, to nucleate and grow. By the presenceof nonylphenol as surfactant, the nucleation and the growth ofHAP nuclei was controlled. Precipitate was separated by filtration,washed with distilled water until no NO3

− ions were detected, andfreeze-dried at −80 ◦C, under a pressure of 5 × 10−3 torr (0.67 Pa)to avoid particles agglomeration. Then, the obtained material wascalcined at 650 ◦C for 8 h. The obtained product (HAP) was grindedin a planetary ball mill Pulverisette 6.

2.2.2. Preparation of nano silver dispersionThe colloidal silver was synthesized by chemical reduction in

alkaline environment with glucose as reduction agent. The 10−3 Maqueous AgNO3 solution (2000 ml) was mixed with an equal vol-ume of an aqueous solution containing 0.104 g glucose and 80 �lTEOS dissolved in 20 ml ethanol. Both solutions had a temperatureof 50 ◦C, and after mixing, the pH of the solution was adjusted at pH11 using 10% potassium hydroxide solution. A dispersion of silvernanoparticles (AgNPs) resulted of pale yellowish brown color. Theprogression of the synthesis was controlled by recording UV–Visabsorption at different given time intervals and evaluated by TEMand AFM imaging.

2.2.3. Preparation of nano-HAP powder with 9 wt% content ofAgNPs (HAP-Ag)

The multi-substituted nano HAP powder prepared as above wasmoistened with the aqueous nano silver dispersion, obtained asdescribed above. The silver colloidal solution was added graduallyto nano HAP under continuous stirring, until a content of 9 wt% sil-ver nanoparticles was reached. The sample was further agitated onthe magnetic stirrer for 4–5 h at room temperature to obtain a com-plete homogenized system. A pale brown mixture was obtained.After filtration and freeze–drying process, the physical, chemicaland antimicrobial properties of the HAP-Ag product were deter-mined.

2.2.4. Preparation of organic matrix composites (OMC)An organic matrix was obtained from bis-GMA (60% by weight)

and TEGDMA (40% by weight). Two liquids (A with bis-GMA and

B with TEGDMA) were prepared by introducing the componentsrequested for the self-curing system. This system was based onBPO (initiator) and DHEPT (activator). BPO was dissolved in liquidA (1.5% by weight). DHEPT (1.5% by weight) was introduced in

A. Mocanu et al. / Applied Surface S

Table 1Composition of organic matrix composites (OMCs) and control samples.

Composition of OMCs (wt%) and control samples AgNPs (wt%) P/La

Monomers (100%): control 0 0/1Fillers (HAP 50%), monomers (50%): control 0 1/1Fillers (HAP 27.78%, HAP-Ag 22.22%), monomers (50%) 2 1/1Fillers (HAP 22.22%, HAP-Ag 27.78%), monomers (50%) 2.5 1/1Fillers (HAP 16.66%, HAP-Ag 33.34%), monomers (50%) 3 1/1Fillers (HAP 10.00%, (HAP-Ag 40%), monomers (50%) 3.6 1/1Fillers (HAP-Ag 50%), monomers (50%) 4.5 1/1Fillers (HAP-Ag 60%), monomers (40%) 5.4 1.5/1

a P represents nano powder, i.e. fillers (HAP and/or HAP-Ag); L stands for them0

lnaowwmoarA11sc(

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2

a

isc(aaa

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gnot

Hinton agar. It was liquefied by heating on water bath and poured

ixture of monomers (liquid); HAP is the multi-substituted nano HAP (i.e. HAP with.20% Zn, 0.25% Ag and 0.025% Au).

iquid B. The self-curing OMC has been obtained from two pastes,amely paste A and B, which were prepared by mixing liquid And liquid B with the same ratio of inorganic fillers. The filler wasbtained by mixing multi-substituted nano HAP (HAP) and HAPith 9 wt% AgNPs (HAP-Ag); the ratio of the two componentsas varied in order to realize 4, 5, 6, and 7.2 wt% AgNPs in theixed inorganic filler (Table 1). The final composition of OMCs, was

btained by varying the ratio between monomers and the filler. Thebove mentioned fillers were mixed with the monomers in the 1/1atio, to obtain OMCs with 2, 2.5, 3 and 3.6 wt% Ag. HAP-Ag (9 wt%g) powder was also used as filler, mixed with the monomers in the/1 ratio to give an OMC with 4.5 wt% AgNPs, respectively, in the.5/1 ratio for the OMC with 5.4 wt% Ag, as given in Table 1. A controlample was made of 50% HAP as filler and 50% monomers (silverontent 0.12 wt%). Since it did not show any antibacterial effect§3.3), OMCs only the content in AgNPs is taken into consideration.

The OMCs were prepared by hand-mixing of pastes A with theorresponding pastes B, at room temperature, as published else-here [55]. Due to the radical polymerization of the monomerichase the organic matrix composites became solid in 7–10 min.fter curing, the specimens were removed from the mould and theamples with visible voids were excluded from the further inves-igation. For antimicrobial effects two controls were used, as givenn Table 1.

.3. Methods for the characterization of samples

The investigated samples were made of HAP with or withoutdsorbed silver nanoparticles.

SEM samples have been prepared by deposition of each powdern thin films on carbon coated SEM grids or on an adhesive metallicupport. Before SEM imaging, samples were gold coated for a betteronductivity in the AGAR Auto Sputter Coater. The thin gold coatingthickness 10 nm) was sputtered in three sputtering cycles takingbout 10 s each. These metallized samples were examined by SEMt different magnifications, using a Microscope Quanta 3D FEG (FEI),pplying the secondary electron imaging technique (SEI)

For the TEM imaging, samples were prepared by re-dispersinghe materials at a wanted concentration in deionized water, thusorming aqueous colloidal dispersions. From these dispersions, thearticles were adsorbed on the specimen grids, while the excessolution was removed with filter paper and the samples were airried. The samples were observed with a transmission electronicroscope (JEOL-JEM 1010). TEM images have been recorded with

EOL standard software.AFM samples were prepared by vertical adsorption on optical

lass of the powders, re-dispersed in deionized water, for 5 min and

atural drying. AFM images were recorded using a AFM-JEOL 4210perated in tapping mode, thus allowing for the simultaneouslyopography, phase and amplitude images for each sample.

cience 298 (2014) 225–235 227

The X-ray powder diffraction was performed by using a D8Advance type diffractometer from Bruker, equipped with Cu source.A Ge (1 1 1) monochromator placed in the incident allows to obtainonly Cu K�1 radiation (� = 1.54056 A). An ultrarapid LynxEye detec-tor was used to record the diffracted radiations. A 40 kV voltage wasapplied to the X ray tube, while the current intensity was of 40 mA.

UV–Vis spectra for the colloidal aqueous solution of AgNPs weremeasured with a Jasco UV/Vis V-650 spectrophotometer, with10 mm quartz cuvettes, in the 250–800 nm wavelengths range, atroom temperature, about 22 ± 2 ◦C.

FTIR spectra were recorded on powder samples pelleted in aKBr matrix. Measurements were performed using a JASCO 6100spectrometer, in the spectral domain of 4000–400 cm−1, with aresolution of 4 cm−1, and 700 accumulations.

For evaluating of silver content from the HAP-Ag sample, a smallquantity of HAP-Ag powder was completely dissolved in concen-trated (67%) nitric acid. The silver concentration from the obtainedsolution was measured by inductively coupled plasma atomic emis-sion spectrometry (ICP–AES) (Spectra AA 220FS, Varian, USA).

2.3.1. In vitro silver (Ag+) release testDisks (diameter 8 mm and thickness of 1.00 mm) made from

the OMCs with compositions given in Table 1 were soaked into10 ml of simulated body fluid (SBF) in a polystyrene sterile flasksfor 1, 3, 7, 14, 21 and 40 days at 37 ◦C. SBF solutions were preparedaccording to Kokubo’s SBF solution [56], containing the followingions (mmol/dm3): Na+ (142.0); K+ (5.0); Mg2+ (1.5); Ca2+ (2.5); Cl−

(147.8); HCO3− (4.2); HPO4

2− (1.0); SO42− (0.5), and buffered at

the physiologic pH 7.40 at 37 ◦C, with tris(hydroxymethyl)aminomethane and hydrochloric acid. After every chosen period, SBFsolution used for each sample storage was collected and silverions released were determined with an inductively coupled plasmaoptical emission spectrometer (ICP–OES) OPTIMA 3500 DV (Perkin-Elmer, USA).

2.3.2. Antimicrobial testingCylindrical samples (disks) with smooth surfaces were prepared

from the OMCs with various Ag content, as indicated in Table 1. Thecontrol disks were made from the polymer without filler and sepa-rately from polymer and multi-substituted HAP as filler (in the ratio1:1), given in Table 1. The antimicrobial effectiveness was testedby the disk diffusion assay in nutritive agar with glucose, accord-ing to the model of antibiogram execution. The tested pathogenicbacteria strains were: Escherichia coli (E. coli, ATCC 10536), Staphy-lococcus aureus (Staphylococcus a., ATCC 6538 P), Staphylococcusspecies (Staphylococcus spp., strain isolated from a gangrenous mas-titis lesion of a goat), Bacillus cereus (ATCC 14579) and Candidaalbicans (ATCC 90028). The strains were inoculated in glucose brothmedia (nutritive broth) and agar. The incubation period was con-ducted in a thermostat, at 37 ◦C temperature, in aerobic conditions,for 18–24 h. The culture characteristics of bacteria were the usualfor the growth of bacteria, and their morphological characters werealso the typical ones. In this respect bacterial smears were treatedby Gram’s method of staining to distinguish bacteria. For each disksample with a particular silver nanoparticles concentration, theinoculation density of bacteria was kept constant in each of thethree Petri dishes used, in order to assure conditions for experi-mental repeatability.

For each tested microbial species, an inoculum was prepared inliquid medium, corresponding to a turbidity of 0.5 (≈106 UFC/ml)on the McFarland standard, for estimating the number of bacteriain suspensions. The culture medium used for testing was Mueller

in amounts of 25 ml in Petri dishes (diameter 90 mm), giving an uni-form layer (4 mm thick). The plates inseminated with the inoculumwere introduced in the thermostat, with half-open lid for 15 min in

228 A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235

TEM im

oattaodbd

3

3

witaamt

n

pii

sdg6t

Fig. 1. Characterization of silver nanoparticles (AgNPs): (a) UV–Vis spectrum; (b)

rder to eliminate the excess moisture. In the following, on thegar plate surface the disk samples were radially distributed, withhe control sample at the center. The incubation was made in thehermostat at 37 ◦C temperature for 18–24 h. The plates were readfter 24 and 48 h, for the identification of the presence or absencef zones of inhibition. When zones of inhibition were present, theiriameter was measured and also the outskirts of the zones of inhi-ition were assessed. The plates were further observed for other 7ays, to notice any modifications in time.

. Results and discussion

.1. Physical and chemical characterization of the powders

The AgNPs used for adsorption on the phosphate particlesere characterized by UV–Vis spectroscopy (Fig. 1a), and TEM

magery. The characteristic surface plasmon resonance absorp-ion band is present in the visible spectrum, with the maximumt 411 nm (Fig. 1a). One of the TEM images for these particles,long with the histogram of their size distribution, obtained fromeasurements on TEM images are given in Fig. 1b and c, respec-

ively.The average size of the AgNPs is 12.0 ± 5.0 nm, the diameters of

anoparticles ranging mostly between 4 and 24 nm.TEM images for the multi-substituted HAP particles and for the

articles with AgNPs adsorbed at their surface (HAP-Ag) are givenn Fig. 2. The particles constitute random aggregates, the sizes of thendividual particles going from some dozens of nm to over 100 nm.

The sizes of a great number (hundreds) of particles were mea-ured on the TEM images, and the histograms providing the size

istribution of HAP particles without and with adsorbed AgNPs areiven in Fig. 3. The average size (diameter) of the HAP particles is0 ± 21 nm (Fig. 3a), while for the HAP particles with AgNPs (Fig. 3b)he average size was found to be 65 ± 17 nm. The ANOVA analysis

age; the bar in the image is 100 nm; (c) histogram of size distribution for AgNPs.

showed that at the 0.05 level, these means are NOT significantlydifferent.

SEM images are given in Fig. 4 for HAP particles, and forHAP particles with adsorbed AgNPs. The particles with apparentlymicrometrical size in Fig. 4a and b, are as a matter of fact conglom-erates of nanoparticles, as seen in Fig. 4c–g.

Here again the sizes of a great number of particles were mea-sured on the SEM images, and the corresponding histograms givenin Fig. 5. The average diameter of the HAP particles is 73 ± 19 nm.(Fig. 5a), and for the HAP particles with AgNPs (Fig. 5b) it is79 ± 26 nm. In this case also the ANOVA analysis showed that at the0.05 level, these means are NOT significantly different. The some-what lower size of particles found from the TEM images is the resultof re-dispersing the sample in water.

AFM images for the HAP particles without or with silvernanoparticles, on samples prepared by vertical adsorption on glassfor 5 min are given in Figs. 6 and 7, respectively.

The sizes of the particles, as visualized in the AFM images, andfrom the cross section profiles, are consistent with those given bySEM and TEM investigations.

The FTIR spectra of HAP without and with adsorbed AgNPs arecompared in Fig. 8. For clarity reasons, the spectral domain wassplit into two sections: from 4000 to 2500 cm−1, and from 2000 to400 cm−1 (with different scales in the two parts).

The spectra are almost identical, showing that the adsorption ofAgNPs did not modify the internal structure of the HAP.

In the spectra of all the samples, the absorbance bands corre-sponding to the vibrations of PO4 and HO groups could be identified.The symmetric P–O stretching mode �1 should be inactive in IR foran ideal tetrahedral symmetry (Td) of the PO4 group, but it appears

with a low intensity in our spectra, at 982 cm−1, as a result of thelowering of symmetry due to the deformation of the tetrahedronin these phosphates as against the free PO4

3− ion. The strongestIR absorption band corresponds to the asymmetric P–O stretching

A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235 229

s with

mama1

aTynd

gc

simsts

stoHco

(8

Fig. 2. TEM images of HAP particles (a), and HAP particle

ode �3. Since in the free phosphate ion it is triply degener-te, at lower symmetry the band can be split into three separateodes. In the spectra of our samples the main maximum appears

t 1045–1049 cm−1, while other two maxima appear at 1091 and122 cm−1.

The absorptions at 567 and 604 cm−1 can be assigned to thesymmetric P–O bending �4; it is again a triply degenerate mode ford symmetry, but is split into at least two components in hydrox-apatites. The bands characteristic for hydroxyapatite are ratherarrow, suggesting a relatively good spatial orderliness, i.e. a highegree of crystallinity.

The same fundamental vibration modes are found in the SiO4roup, with frequencies near to those of the PO4 vibrations, so weannot discriminate between them.

The broad band (maximum at 3435 cm−1) is due to the O–H. . .Otretching vibrations of absorbed water with hydrogen bondingn the samples. Another band also due to absorbed water has the

aximum at 1630 cm−1. The first band overlaps with the band oftructural OH (from hydroxyapatite) at 3571 nm−1. A band due alsoo structural OH (libration vibration) is observed at 631 cm−1. Theyuggest the presence of a number of–OH group in hydroxyapatite.

X Ray diffraction: Fig. 9 presents the XRD pattern of the nanoHAPample and of the nanoHAP sample with 9 wt% of AgNPs. They showhe crystallinity degree of these powders While in the HAP samplenly the diffraction pattern characteristic to pure non substitutedAP is evidenced, in the HAP-Ag sample the diffraction patternharacteristic for Ag is also revealed. Therefore, the crystallinity

f silver in the nanoparticles is also pronounced.

The silver content in the HAP samples with adsorbed AgNPsHAP-Ag), measured by ICP–OES, was calculated to be about.34 wt %.

20 40 60 80 100 12 0 14 0

0

6

12

19

25

a b

%

Diameter, nm

Fig. 3. Histograms of size distributions for HAP particles (a), and HAP

adsorbed AgNPs (b). The bars in the images are 200 nm.

3.2. In vitro silver release test

The evolution of silver ion release from the OMCs in simulatedbody fluid (SBF), in time is shown in Fig. 10. The rate of silver ionsrelease is highest in the first day, when Ag+ ions in the solutioncome from the outmost layer of AgNPs or even from adsorbed Ag+

on the surface of the AgNPs. Then, the release rate decreases andthe plots in Fig. 10 become virtually straight lines after 7 daysof incubation, i.e. the release rate is constant after this period.This result is supported by a study of the kinetics of silver ionsrelease from AgNPs in aqueous medium [57]. The rates of silverions release, calculated from the parameters of linear regression(the slopes of the linear plots for the days 7 to 40) are given inTable 2, along with the correlation coefficient,r. The release rate inthe first day of contact with the simulated body fluid is also givenin Table 2. The total silver content in the samples was of several mg,so the silver within each sample was far from depletion even after40 days.

3.3. Antimicrobial effectiveness tests

The reading and interpretation of the agar plates were made at24 and 48 h. The inhibitory effect was manifested by the formationof circular, clear zones of inhibition around the disks, their sizedepending on the AgNPs content. No effect was observed for thecontrol samples (pure polymer, or polymer with multi substitutedHAP with Zn, Ag and Au as filler, in the ratio 1:1, but without AgNPs).

The diameters of the inhibition zones (mm) were measured. Sincethe differences between the diameters of a zone did not exceed1 mm, we considered the largest size observed in one of the threeplates (Table 3).

20 40 60 80 10 0 1200,0

2,5

5,0

7,5

10,0

12,5

%

Diameter, nm

particles with adsorbed AgNPs, HAP-Ag (b), from TEM images.

230 A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235

F s (b, df

swTaa

ig. 4. SEM images of HAP particles without (a, c, e and g) and with adsorbed AgNP), respectively, 400 nm (g and h).

The inhibitory effect was observed for all the five microbialpecies tested. Inhibition zone diameter increases simultaneously

ith the increase in silver nanoparticles content of the samples.

he best efficacy was attained for the highest AgNPs contents, 4.5%nd 5.4%. There were not significant differences between readingst 24 and 48 incubation hours.

, f and h). The bars in the images are 20 �m (a and b), 5 �m (c and d), 1 �m (e and

For E. coli the size of the inhibition area was between 10 and15 mm, as there is no clear demarcation between the edge of the

area and the culture. Therefore the contour of the zones of inhi-bition appears a little irregular (Fig. 11a). For S. aureus the zonesof inhibition were in the 11–16 mm range. It can be assessed thatthis species of staphylococcus presented the highest sensitivity

A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235 231

20 40 60 80 100 12 0 14 0 1600

10

20

30

40

50ab

%

Diameter, nm

20 40 60 80 100 120 140 160 1800

8

16

24

32

40

%

Diameter, nm

Fig. 5. Histograms of size distributions for HAP particles (a), and HAP particles with adsorbed AgNPs (b), from SEM images.

F glass;p

aTtb

Fs

ig. 6. 2-D topographic (a) and phase (b) AFM images of HAP particles adsorbed onrofile along the arrow in panel (a).

gainst AgNPs, showing the largest zones of inhibition (Fig. 11b).he Staphylococcus spp, species was less sensitive as compared withhe reference strain. It was not inhibited at 2 wt% AgNPs content,ut the inhibitory effect was observed in the other levels, the largest

ig. 7. 2-D topographic (a) and phase (b) AFM images of HAP-Ag particles adsorbed on gection profile along the arrow in panel (a).

scanned area 500 × 500 nm; 3D-view (c) of the topography in (a); (d) cross section

area (14 mm) appears at 4.5 wt% AgNPs (Fig. 11c). For B. cereus(Fig. 11d) there was also no inhibitory effect at 2 wt% AgNPs, butinhibition was present at higher concentration (14 mm at 5.4 wt%AgNPs). For C. albicans, readings were made at 48–72 h, since yeast

lass; scanned area 1000 × 1000 nm; 3D-view (c) of the topography in (a); (d) cross

232 A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235

4000 3800 3600 3400 3200 3000 2800 26000.0

0.1

0.2

0.3

0.4

a b

HAP+A gNPs

HAP

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an

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.

Wavenumber, cm-1

2000 1800 1600 1400 1200 1000 800 600 4000.0

0.5

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1.5

2.0

2.5

3.0

3.5

HAP+AgNPs

HAP

Absorb

ance, a.u

.

Wavenu mber, cm-1

Fig. 8. FTIR spectra of HAP and HAP with adsorbed AgNPs for the spectral domains with wave numbers from 4000 to 2500 cm−1 (a) and from 2000 to 400 cm−1 (b).

Table 2Silver ions release from OMCs samples (disks) in simulated body fluid at 37 ◦C.

AgNPs content in sample (%) Ag+ release rate (days 7 to 40), mg/(dm3 day) Correlation coefficient, r Ag+ release rate, first day, mg/(dm3 day)

2 7.07 × 10−3 ± 0.24 0.9989 90 × 10−3

2.5 8.36 × 10−3 ± 0.44 0.9972 90 × 10−3

3 8.26 × 10−3 ± 0.17 0.9996 150 × 10−3

3.6 9.88 × 10−3 ± 0.47 0.9978 220 × 10−3

4.5 17.12 × 10−3 ± 2.35 0.9816 270 × 10−3

5.4 26.75 × 10−3 ± 1.79 0.9955 360 × 10−3

FH

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ig. 9. RXD patterns for HAP, and HAP with 9 wt% AgNPs (characteristic peaks forAP are marked with *).

trains develop slower as compared to bacteria (Fig. 11e). There

as an inhibitory effect for all concentrations (zones of inhibition

rom 10 to 15 mm).Excepting E. coli, where the zones of inhibition were not well

elimited, for all the other tested microorganisms, these zones were

able 3ilver content of the disk samples and the diameter of the zones of inhibition at 48 h.

Testedstrains

AgNPs content and diameter of the zones of inhi

2% 2.5% 3%

Escherichia coli 10 12 12

Staphylococcus aureus 11 12 13

Staphylococcus spp. 0 8 10

Bacillus cereus 0 10 11

Candida albicans 10 11 12

Fig. 10. Silver release in time from organic matrix composites (OMCs).

circular and clear-cut, meaning the inhibition was total. After keep-ing the plates at +4 ◦C, the zones of inhibition retained the same size.Removing the disks, the medium beneath them was transparent,without culture.

bition (mm)

3.6% 4.5% 5.4% 0%

12 14 15 013 15 16 010 12 14 011 12 14 012 15 15 0

A. Mocanu et al. / Applied Surface Science 298 (2014) 225–235 233

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ig. 11. Inhibition zones for Escherichia coli ATCC 10536 (a), Staphylococcus aureus Ad), Candida albicans ATCC 90028 (e), and after removing the disks for Staphylococc%; disk 2: 2.5%; disk 3: 3%; disk 4: 3.6%; disk 5: 4.5%; disk 6: 5.4%; W stands for the

.

The inoculation of bacteria in the glucose broth from the zonesf inhibition (after removing the disks) was tested. After incuba-ion at 37 ◦C for 48 h and a new verification after 7 days, it wastated that the inoculated medium remained sterile (no culture waseveloped). From these observations we conclude that the inducedntimicrobial effect is bactericidal (Fig. 11f).

This study gives an understanding on the silver ions release fromrganic matrix composites (OMCs) containing multi-substitutedAP (0.2 wt% Zn, 0.25% Ag and 0.025 wt% Au), AgNPs and polymer

obtained from bis-GMA and TEGDMA). The data show significant

ntimicrobial effects of the slow Ag+ ions release in vitro against theve pathogens. Our results are consistent with literature data onhe effect of Ag on E. coli [2,8,10,11,27,37,38,43,44,58,59], S. aureus2,11,27,36,44,49,58,59], and C. albicans [27,60].

583 P (b), Staphylococcus spp. (gangrenous mastitis) (c), Bacillus cereus ATCC 14579,eus (f). Disks are numbered according to their silver nanoparticles content: disk 1:l samples: two control disks were used (Table 1). Insets: magnified images for disk

The mechanisms about their action mostly agreed that they canbind to proteins on the bacterial wall and may enter in cytoplasm.Silver ions are considered highly reactive on bacteria. They canalso bind to bacterial DNA and RNAs and inhibit bacterial replica-tion. Thus, they will cause structural changes in bacteria leading tocell distortion and death. They preferably do attack the respiratorychain, cell division finally leading to cell death.

These results suggest that by using our composites, the silverions release can be controlled and in vivo might improve the safetyof using AgNPs being mixed with HAP and organic matrix in nano

composites. Therefore, silver ions are released from AgNPs, andthey determine the bactericidal activity when AgNPs are incor-porated in carriers, like HAP and organic matrix. The exposure ofHAP/AgNPs/organic matrix composites to the pathogens allowed

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34 A. Mocanu et al. / Applied Su

ilver ions to accumulate around the disks of composites and diffusen agar, assuring an inhibition zone.

. Conclusions

Nano hydroxyapatite substituted with zinc, silver and goldHAP) was obtained by the wet chemical route. The average diam-ter of HAP particles is about 70 nm, as given by TEM and SEMmages. The HAP nanoparticles are mixed with silver nanoparti-les (AgNPs) of 12 nm in average size to a content of 9 wt%. Theize and shape of HAP particles, both without and with AgNPs wasevealed by TEM, SEM and AFM images, while the silver content wasonfirmed by ICP–OES measurements. While FTIR spectra presentnly the characteristic absorption peaks of hydroxyapatite, the XRDpectra of the HAP-Ag samples reveal also the diffraction patternsharacteristic for Ag, showing the crystallinity of silver in nanopar-icles.

For antibacterial characterization of HAP and HAP-Ag, organicatrix composites were obtained from these powders (multi

ubstituted nano hydroxyapatite and its mixture with silveranoparticles) incorporated within a polymeric matrix obtained

rom bis-GMA (60 wt%) and TEGDMA (40 wt%). The AgNPs con-ent of the composites was varied from 0 to 5.4 wt%. The rate ofilver ions release from these composites in simulated body fluids high in the first day, decreases in the next days, and becomesractically constant after 7 days. The antimicrobial effectivenessests of the AgNPs containing composites, made against five differ-nt pathogenic species showed a clear inhibitory effect in all theicrobial species tested.This antimicrobial effect increases with the increase in AgNPs

ontent of the composites. Moreover, the medium remained sterileven after removing the silver containing composite disks, suggest-ng, that the antimicrobial effect of our compounds is of bactericidalature. Therefore, the bactericidal effect of these composites indi-ates that these composites are strongly active against the fiveacterial strains: E. coli (ATCC 10536), S. aureus (ATCC 6538 P),taphylococcus sp. (strain isolated from a gangrenous mastitis lesionf a goat), B. cereus (ATCC 14579) and C. albicans (ATCC 90028).owever, multi substituted HAP with Zn (0.200%), Ag (0.250%) andu (0.025%) without silver nanoparticles did not show any antibac-

erial effect at these concentrations. These results suggest that ouromposites should be considered antibacterial materials and mighte used in implants for dental and orthopedic surgical applications.he released Ag + ions reacted with the five pathogens inactivatingheir metabolism, inhibiting their growth and finally determininghe bactericidal activity of these composites containing HAP andgNPs.

Future experiments are planned for in vivo evaluationhat/HAP/AgNPs/organic matrix nano composites might representotential agents for coating of implants and thus avoiding or con-rolling bacterial infections—in orthopedic and dental surgery.

cknowledgments

We are grateful to the National Research Plan for funding of thisork through Projects nos.171/2012 and 257/2011. We thank Dr.

. Barbu-Tudoran for assistance with SEM and TEM imaging.

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