Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites

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Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites? Marija Vukomanovi#, Urška Repnik, Tina Zavašnik-Bergant, Rok Kostanjšek, Sreco D. Skapin, and Danilo Suvorov ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00170 • Publication Date (Web): 27 Aug 2015 Downloaded from http://pubs.acs.org on September 2, 2015

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ACS Biomaterials Science & Engineering

Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites? Marija Vukomanović,*† Urška Repnik,‡ Tina Zavašnik-Bergant,‡ Rok Kostanjšek,§ Srečo D. Škapin,† Danilo Suvorov† † Advanced Materials Department, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. ‡ Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. § Biotechnical Faculty University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia KEYWORDS. hydroxyapatite nanocomposites, silver nanoparticles, antibacterial activity, cytotoxicity, selectivity.

ABSTRACT. Because of the abounded presence of the silver-containing products in the market and intensively studied silver-containing biomaterials in the literature, we investigated a hypothesis that stabilizing silver within bioactive hydroxyapatite composite is capable to provide safe and effective antibacterial action. For that purpose nanocomposite made of bioactive, mineral hydroxyapatite (HAp) and antibacterial silver (Ag) is studied for interactions with both, bacterial and human cells. The nanocomposite provides controlled release of Ag-ions; induces severe damages in bacterial cells and is capable for strong bacteriostatic and bactericidal action.

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On the other hand, investigations of material’s interactions with human cells confirm that lower concentrations are highly compatible with osteosarcoma and fetal lung fibroblasts, but at higher concentrations (that provide bacteriostatic and bactericidal influences in bacteria) the material is toxic and capable to induce morphological changes similar to those observed in bacterial cells.

Introduction Intensive investigation of antibacterial materials in the last few decades mainly came with the need to improve modern protocols in medicine (especially surgery and dentistry) to avoid development of implantation-related infections.1 Since silver (Ag) is a natural antibacterial agent, highly effective against wide spectrum of pathogens, including Staphyloccocus aureus and its antibiotic-resistant species, it was frequently selected as antibacterial component.2 The idea that the manipulation of the physicochemical properties of Ag nanoparticles (surface charge and chemistry, stability, binding and agglomeration potential) can affect their interactions with cells (by controlling the direct contact, transport and interactions of these nanoparticles with cells) has led to intensive investigations.3,4 Starting from this idea it has been hypothesized that formation of nanocomposites with Ag antibacterial component incorporated within bioactive stabilizing component has capacity to provide safe and effective antibacterial biomaterial. This approach has been applied to the development of antibacterial hydroxyapatite/silver (HAp/Ag) materials. In these materials hydroxyapatite was added as a bioactive and biocompatible carrier of antibacterial silver in the form of: (i) surface adsorbed Ag-ions,5,6 (ii) doped Ag-ions,7-15 (ii) surface-attached Ag nanoparticles16-23 and combination of Ag nanoparticles and doped Ag ions.24 Both types of HAp/Ag materials, with Ag-ions and Ag nanoparticles, are efficient in action against bacteria. While the materials with Ag-ions have a bacteriostatic effect rather than the


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induction of bacterial death,9 the materials containing Ag nanoparticles have strong bactericidal effect22 and are intensively active against MRSA.19 After the incubation of Ag-doped HAp in the medium for up to 4 h, an Ag-release study confirmed the stability of the material that did not release or provided a low concentration of released Ag-ions (0.08-0.13 µg·ml-1) depending on the quantity of the incorporated ions.14 Prolonged incubation in the medium for up to 7 d confirmed the release of 0.8 µg·ml-1 and 0.35 µg·ml-1 during the first day of incubation (depending on the type of release medium), and the release was continued to the end of investigated time interval, up to the 1.2 µg·ml-1 and 2.3 µg·ml-1.11 For Ag nanoparticles attached to the surface of the HAp lower concentrations of Ag-ions was released that was attributed to the low solubility of metallic silver and the prevention of the solubility of the CaP coating by surface functionalization.18-23 The most relevant cytotoxicity investigations of these materials include their interactions with human cells at concentrations which are active against bacteria. In that context, investigation of the osteoblast propagation onto HAp/Ag material showed a decrease with an increase in the content of doped Ag.10 According to the hypothesis for stabilized Ag-containing biomaterials, these materials were made for antibacterial action provided by Ag-ions or Ag nanoparticles as well as for compatibility with mammalian cells due to mineral, apatite component. However, the way of providing compatible influence to human cells and bactericidal influence to bacterial cell has not been explained. In other words differences in the mechanisms of materials’ interactions with mammalian cells and bacteria have not been clarified. Starting from this point and having in mind the intensive promotion of Ag-containing materials for health care as well as its incredibly fast transfer to the market, our intention was to investigate hypothesis about formation of a safe and effective Ag-containing antibacterial biomaterial in


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more detail and to perform a systematic investigation of the properties of the HAp/Ag composite to clarify the way of material’s interaction with human and bacterial cells. Materials and Methods Synthesis of HAp/Ag composite. The HAp/Ag composite was synthesized using a homogeneous sonochemical precipitation method followed by a thermal treatment.24 A water solution of AgNO3 (Sigma Aldrich, Germany) (50 ml of 0.5 mg·ml-1, 2.5 mg·ml-1 or 5 mg·ml-1 solution) was added to 100 ml of a mixture of HAp precursors – water solutions of Ca(NO3)2·5H2O (Sigma, Aldrich, Germany) and NH4H2PO4 (Sigma Aldrich, Germany) to form the HAp/Ag composite with 1 wt%, 5 wt% and 10 wt% of metallic component. After heating the mixture of precursors to Tmax.=90°C, 10 ml of a 12 wt% solution of urea (Alfa, Aesar, Germany) was added and the sonication was started. The following ultrasonic parameters were applied: ultrasonic field power, P=600W; frequency of the field, f=20 kHz; effective time of pulsed sonication, t=3h; and pulsation-to-relaxation time, on : off = 02 : 01 sec. When the sonication was finished, the samples were aged for 12 h. The precipitates were separated from the supernatant by centrifugation (1 h at 4000 rpm) and then air dried. All of the powders were calcined for 4h at 300°C in an Ar/H2 (96:4%) atmosphere. Physico-chemical properties of HAp/Ag composites. For the phase composition identification the samples were recorded in the 2θ range from 2° to 70° with a step size of 0.02° and a time of 2sec/step using the Bruker AXS D4 Endeavor diffractometer. The optical properties were analyzed based on the absorption spectra measured on a UV-vis-NIR spectrophotometer (Shimadzu UV-3600) in the range between 200 and 900 nm with a spectral resolution of 0.1 nm. The morphological and structural properties of the HAp/Ag composite were investigated using


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scanning (SUPRA 35 VP Carl Zeiss) and transmission (JEOL JEM-2100) electron microscopes. The surface potential was measured by z-potential using a Zeta-Plus Analyzer (Brookhaven Instruments, USA). The measurements were performed using electrophoretic mobility approach and calculated using Smoluchowski’s theory. The samples were measured in triplicate using suspensions in a diluted sodium chloride solution (1 mM) at pH=7.0. The specific surface area was measured using the BET and for this purpose five-point repetitive measurements were applied. In vitro investigation of the release of metal-ions. Investigation of the release of Ag-ions from the HAp/Ag composite was performed under pH = 7.4, 5.4 or 3.4 using PBS buffer (Sigma Aldrich, Germany) with acidity maintained by HCl (Sigma Aldrich, Germany)). For that purpose 10 ml of 0.6 mg/ml HAp/Ag composite (with 10 wt% of Ag) dispersed in release medium was used. The dynamic conditions (60 rpm) and constant temperature (37°C) were ensured by the use of a shaking water bath (Memert, WNB 7–45, Germany). The total release of the ions was investigated during a period of 10d and performed in two parallel measurements. The quantities of Ag-ions released under the described conditions were determined from solutions using the inductively coupled plasma atomic emission spectrometric (ICP AES) (Thermo Jarrell Ash, model Atomscan 25) method, while the powders were used to investigate phase composition. Disc-diffusion test (Kirby-Bauer method). E. coli (K-12 MG1655) and S. aureus were collected during the log phase and 100 µl of their suspension in the media broth (1 OD) was plated on agar (30 ml per plate) with a wet cotton swab. HAp/Ag composites with three concentrations of the Ag (10 wt%, 5 wt% and 1 wt%) were compacted into 50-mg discs. The discs were manufactured by direct compressing of the powders using 8-mm stainless steel die by hand-pressing. They were initially tested for checking susceptibility of composites with different


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content of Ag. The 8-mm disks of HAp/Ag composites (10 wt%, 5 wt% and 1 wt% of Ag) and HAp (used as negative control) were put on the surface of the agar plates with bacteria, turned upside down and incubated at 37 °C for the next 24 h. The zones of inhibition of bacteria growth were photographed as well as observed with the optical microscope (Olympus BX series) using a phase-contrast technique and evaluated according to the Schmalz criteria.25 The test was performed in duplicate. Live/Dead BacLight bacterial viability test. Detection of dead and live bacteria and their distribution on the surface of the disks and within the zone of inhibition were investigated using fluorescence microscopy. Fluorescent Live/Dead BacLight Bacterial Viability Kit (Molecular Probes, Inc.) consisting of two fluorescent dyes, i.e. SYTO 9 and propidium iodide (PI), was used for the staining. Agar beds were prepared on glass slides and 50 µl of E. coli and S. aureus suspension in medium broth with OD~1 were plated on top with a cotton swab. Disks made of bacteria susceptible composites were put either on freshly inoculated agar or on confluent bacteria grown overnight. After 24h incubation at 37°C the disks were removed and the bacteria were stained with SYTO-9 and PI dyes for 30 min in dark. The analysis of the fluorescently stained bacteria was performed using an Olympus IX81 inverted research microscope with a motorized fluorescence attachment. The emission signal was filtered using U-N49002 (green fluorescence) and U-M41002 (red fluorescence) filter cubes. Micrographs were taken with a Hamamatsu CCD camera ORCA-R2 and Olympus Cell F software was used to evaluate the specific fluorescence signal. The examinations were performed in two parallel series of investigated composites. Minimum inhibitory and minimum bactericidal concentration (MIC and MBC) tests. After initial testing of the susceptibility, the composite with the highest content of Ag (10 wt% ) was


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tested in a concentration range 0.1-6.0 mg/ml for quantification of antimicrobial activity (MIC and MBC determination). HAp was used as a negative control. The powders were dispersed in the growth medium to form stock solutions (4 mg·ml-1). These solutions were subsequently diluted by the addition of distinct volumes of growth medium to form series of solutions with different concentrations of each material. The series contained solutions in the range 0.0-6.0 mg·ml-1. A 1000 µl of each solution was mixed with the same volume of both bacterial cultures (E. coli and S. aureus). Dispersions containing different concentrations of materials and a constant concentration of bacteria (estimated 1·105 cells·ml-1 for E. coli and 1·104 cells·ml-1 for S. aureus) were incubated overnight in 15-ml tubes with shaking at 37°C. To determine MIC, after overnight incubation the cultures were analyzed for the absence of turbidity due to the inhibition of the bacterial growth. To determine MBC, 100 µl of overnight cultures were plated on agar in Petri dishes (Ø=10 mm) using a wet cotton swab and another 100 µl were added to 1.9 µl of fresh medium. Both sets of samples were incubated at 37°C until the next day when they were inspected for the growth of bacteria. All the analyzed concentrations were tested in duplicates for each material. The statistical analysis of the results was made with a Student’s ttest. Sigma plot software version 11.0 was applied for the processing of the data and the differences were considered as significant at a p-value below 0.05. SEM investigation of bacteria. The morphological and structural properties of E. coli and S. aureus were analyzed after exposure of the bacteria to HAp/Ag. Fifteen mg of material were compacted into disks (Ø=8 mm) and kept overnight on bacteria plated in agar at 37°C. The sampling of bacteria on the agar surface exposed to the tested materials was performed from the inhibition zone near the surface of the disks, while bacteria growing in the healthy colony far from the disk were used as negative controls. Parts of the agar in the inhibition and control zones


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were carefully cut from the plate and fixed in 1% formaldehyde and 0.5% glutaraldehyde in a 0.1-M phosphate buffer solution (pH = 7.4) at 4°C overnight. After fixation the samples were rinsed with the PBS solution (three times for 10 min), post-fixed using a 1% aqueous solution of osmium-tetroxide for 1 h and rinsed with distilled water. For dehydration the samples were immersed in 50%, 70%, 90% and 100% ethanol solutions and finally in 100% acetone during intervals of 20 min for each solution. In the next step they were dried using the critical point drying method in a CPD030 dryer (Balzers) using liquid carbon-dioxide as the transition medium. Before the SEM examination the samples were coated with platinum in a sputter coater SCD 050 (BAL-TEC). Cytocompatibility tests. In vitro cytotoxicity tests were performed in human foetal lung fibroblasts (IMR-90) (ECCAC no. 85020204) and human osteosarcoma (U-2 OS) (ECCAC no. 92022711) cell lines. Non-transformed IMR-90 fibroblasts were maintained in Eagle’s minimal essential medium (EMEM, PAA) supplemented with 2 mM stable glutamine (PAA), 1% nonessential amino acids (NEAA, PAA), 10% foetal bovine serum (FBS, Sigma), 100 U·ml-1 penicillin and 0.1 mg·ml-1 streptomycin (PAA). U-2 OS were maintained in McCoy's 5a medium (Sigma) supplemented with 1.5 mM stable glutamine, 10% FBS, 100U·ml-1 penicillin and 0.1 mg·ml-1 streptomycin. All the cells were maintained under standard conditions at 37°C and 5% CO2. To analyze cell viability after exposure to HAp/Ag composite cells were plated in 24-well plates at least one day before the treatment. HAp with and without Ag content were dispersed in complete culture media and added to the cell cultures at different concentrations. After 24 h of exposure to the materials the cells were trypsinized and stained with annexin V (BD Biosciences, Pharmingen, San Diego/CA, USA) and propidium iodide (Sigma-Aldrich; St. Louis/MO, USA) according to the manufacturer’s instructions. Samples were than analyzed using a FASCalibur


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flow cytometer (BD; Franklin Lakes/NJ, USA) and the CellQuestTM software, version 3.3 (FACSComp Software, BD; Franklin Lakes/NJ, USA). The acquisition was stopped when at least 5000 events were acquired in the cell region and the percentages of viable cells (annexin V and PI double negative) were determined. Morphological changes in cells exposed to different concentrations of HAp/Ag composites for 1 h were observed in a light microscope. Untreated cells and cells incubated with HAp without metallic component was used as controls. Two independent experiments were performed in duplicates. Results The major starting point of the research was to investigate interactions of HAp/Ag nanocomposite designed to: (i) control the release of Ag-ions, (ii) prevent nanosize-induced toxicity and (iii) be bioactive due to the apatite component, with both bacterial and human cells. We tested the hypothesis that formation of nanocomposites that contain silver along with bioactive component has potential to mitigate toxic influence of this metal to human cells and to provide safe antibacterial protection. During these tests special emphasize has been put at MIC and MBC concentrations, able to stop bacterial growth and provide their death, because these concentrations are relevant for effective, practical usage of the material as potential antibacterial agent. Physicochemical properties of HAp/Ag nanocomposite The major physicochemical properties of investigated HAp/Ag material are summarized in Figure 1. The material contains hexagonal HAp (ICPDS no. 09-0432) together with cubic and hexagonal Ag(0)- phases (ICPDS no. 04-0783 and ICPDS no. 41-1002) (Figure 1a). It shows absorption of the metallic part of the composite superimposed on the absorption of HAp in the


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UV/vis region (Figure 1b), similar to the HAp/Pt.26 The maximum around 456 nm corresponds to surface plasmon oscillations (SPR) of Ag nanoparticles within the composites. Compared to the single, spherical Ag nanoparticles with a SPR absorption around 410 nm,27 for the HAp/Ag it is a broad, low-intensity maximum that is red-shifted due to the influence of the dielectric constant of the contacting HAp phase. HAp influences and inhibits the free oscillations of the Ag nanoparticles and a change in their optical properties is a consequence of their attachment and embedment within the carrier.

Figure 1. Phase composition (XRD) (a), optical (UV/Vis) (b) morphological (SEM) (c) and structural (TEM) (d-f) properties of the HAp/Ag nanocomposite. Morphologically, HAp carriers are thin sub-micrometre-sized plates uniformly covered by Ag nanoparticles (Figure 1c-f). A detailed investigation of the formation, growth and structure


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performed during the development of the composite revealed that smaller Ag nanoparticles are embedded within the HAp plates and they are cubic, larger ones are attached on the surface and they have a hexagonal structure, while a part of the Ag is present in the form of ions doped within the HAp structure.24 According to the ICP, FTIR and detailed SEM/TEM investigations, our previous investigation confirmed presence of the metallic Ag at HAp that corresponds to Ag nanoparticles attached onto HAp plates. In depth analysis confirmed a presence of metallic and ionic Ag in apatite plates indicating presence of Ag nanoparticles embedded into HAp as well as Ag-ions incorporated into apatite structure.24 Incorporation of the Ag within apatite resulted in decrease of the Ca/P ratio from 1.62 for the pure, Ca-deficient HAp formed by homogeneous sonochemical precipitation method to 1.52 for Ag-doped apatite.24,28 At physiological pH the surface charge of the pure HAp obtained by the homogeneous sonochemical precipitation method is ξ (HAp) =-7 mV.29 Formation of the composite with Ag results in an increase of the magnitude of the negative charge of the composite (ξ (HAp/Ag) =-15 mV) contributing to a change in the surface properties of the material. Similarly, the surface of the pure HAp obtained by homogeneous sonochemical precipitation is 30 m2·g-1,29 while formation of the composite with Ag nanoparticles attached at the surface increases the specific surface area of the material to 69 m2·g-1. The presence of the Ag nanoparticles attached onto HAp results in formation of the nanostructured surface and is the main reason for increase of the specific surface area of the composite. It also contributes to the change of the surface chemistry inducing the change of the surface potential. The increase of the magnitude of the negative charge in HAp/Ag composite in comparison to the pure HAp could be assigned to the change of the structural and morphological properties of the material obtained by formation of the composite that results in the change of the density of the negative charge at the surface.


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In-vitro properties of the HAp/Ag nanocomposite The properties of the composite obtained after incubation under simulated physiological conditions were investigated over a period of ten days (Figure 2) and compared with the properties of the material obtained after ageing in media with higher acidity. The material has the ability to release Ag-ions and the release kinetics starts with a burst release during the first day, followed by a slow release during the next few days and an increased release by the end of the investigation period, similarly to the release of antibiotic from HAp used as a drug-carrier.30

Figure 2. Release of the Ag-ions within media with different acidity (pH = 3.4, pH = 5.4 and pH = 7.4) during aging for different time intervals; 0.6 mg/ml of HAp/Ag (10 wt% of Ag). The initially released concentration of Ag-ions was the highest for the medium at pH=7.4 and it was decreased with an increase in the acidity. However, after the initial release, the kinetics of the ion release was increased with an increase in the acidity. The overall concentrations of Agions released during the investigations were in the range between 15 and 25 µg·ml-1 depending on the acidity of the release media. The total quantity of released Ag-ions was 150–250 µg, which is 25-42 wt% of the total Ag-content within the composite. The solubility of the HAp


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increases with an increase in the acidity 18 and, since the total amount of released Ag-content exceeds the quantity of Ag incorporated within the HAp,24 the released ions is attributed to the simultaneous dissolution of both HAp-embedded and HAp-attached Ag nanoparticles. Apatite layer containing Ag is instable and prone to dissolve, resulting in release of Ag-ions. Increased dissolution of apatite layer is certainly a consequence of doping/embedding silver that decreased its crystallinity.24 Along with the investigation of the supernatants that contained released Agions, the composite remained after the release of the ions was also analyzed. Investigations confirmed a change of the phase composition in comparison to the initially applied material (Figure 3).

Figure 3. Phase compositions of HAp/Ag (10 wt % of Ag) after aging in media under different acidity ((a) pH = 3.4, (b) pH = 5.4 and (c) pH = 7.4) for different time intervals (2, 4, 6, 8 and 10 d).


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In all three cases when the media had different pH values, a new phases corresponding to the structure of silver-chloride (AgCl) (ICPDS no. 31-1238) and sodium-chloride (NaCl) (ICPDS no. 05-0628) were identified together with the phases corresponding to the structures of the HAp (ICPDS no. 09-0432), cubic and hexagonal Ag phases (ICPDS no. 04-0783 and ICPDS no. 411002), which were initially present. Formation of the additional phase started at the beginning of the aging period and it was continued to the end along with the release of Ag-ions that confirmed instability of the material under tested in vitro conditions. Antibacterial properties of HAp/Ag nanocomposite A susceptibility test (Figure 4) was performed in E. coli and S. aureus using composites containing 1 wt% (Figure 4a2,a6), 5 wt% (Figure 4a3,a7) and 10 wt% (Figure 4a4,a8) of Ag together with the pure HAp (Figure 4a1,a5) as a negative control. Inhibition of bacterial growth was not detected around the disks with HAp, while in the case when Ag was present formation of the rings around the disks indicated the suppression of bacterial growth in their surroundings. The higher content of Ag in the material resulted in a higher defending concentration and a greater width of the rings around the tested disks. The inhibition zones were mostly formed due to the diffusion of dissolved antibacterial Ag-ions through the agar medium since the Ag nanoparticles were attached and incorporated within the HAp carrier (Figure 1). The zones of inhibition of bacterial growth were subsequently investigated for dead and intact bacteria. A distinction between viable and death cells was performed by in situ double fluorescent staining of cells using SYTO 9 and PI, which both bind to DNA. SYTO 9, a cell membrane-permeable green fluorescent dye, binds to nucleic acids of both dead and viable cells and stains all bacteria. PI, red fluorescence dye, which is impermeable to intact membranes


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readily penetrates mechanically destroyed or damaged membranes and labeled only dead bacteria. The fluorescence signal was detected around disc made of pure HAp (c01,c02 and c09, c10), used as a reference, and followed around the disks made of HAp/Ag (1 wt Ag) (c03,c04 and c11, c12), HAp/Ag (5 wt Ag) (c05,c06 and c13, c14) and HAp/Ag (10 wt Ag) (c07,c08 and c15, c16) for both E coli and S. aureus, respectively.


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Figure 4. The growth of E. coli and S.aureus near the disks made of HAp (a1, b1, c01, c02 and a5, b5, c09, c10) as well as near discs made of HAp/Ag composites with 1 wt% (a2, b2, c03, c04 and a6, b6, c11, c12), 5 wt% (a3, b3, c05, c06 and a7, b7, c13, c14) and 10 wt% (a4, b4, c07, c08 and a8, b8, c15,


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c16) of Ag, respectively. Inserted images are details of merged images of red and green fluorescence. In the case of E. coli, the region around the disk made of HAp showed dominant green fluorescence (Figure 4c01,c02), indicating that the majority of the bacteria were alive and that the material was non-toxic for bacteria. For the HAp/Ag only a part of the bacteria exhibited green fluorescence (Figure 4c03,c05,c07), while a large number of them showed an intensive red fluorescence (Figure 4c04,c06,c08) for all three nanocomposites with 1, 5 and 10 wt% of Ag, revealing the large population of dead bacteria. The change of the density of bacteria around disks made of nanocomposites with different concentration of Ag was also observed. Results were comparable for investigations of the growth of S. aureus. Testing the HAp for interactions with S. aureus showed that all the bacteria were labeled with SYTO9, exhibiting green fluorescence (Figure 4c09,c10), indicating the growth of a healthy colony that was not affected by the material. Testing the materials with 1, 5 and 10 wt% of Ag showed that, besides green fluorescence (Figure 4c11,c13,c15), there was a significant number of bacteria labeled with PI and exhibiting red fluorescence (Figure 4c12,c14,c16) that confirmed the existence of dead bacteria. The merged images of green and red fluorescence are also shown, indicating the ratio of intact and damaged bacterial cells (inserts in Figure 4). For the both types of bacteria it shows increase of the content of damaged bacteria with increase of the concentration of Ag in nanocomposite. The material was able to suppress the adherence of E. coli and S. aureus on the surface of the disk made of the HAp/Ag composite as well as affecting the growth and causing the death of the bacteria in its close surroundings. Quantification of antibacterial activity of HAp/Ag nanocomposites


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During the quantification of the antibacterial action of the HAp/Ag composite composite powder was dispersed finely within the medium containing the bacteria, which provided better contact between the bacteria and the material and did not depend only on the diffusion of Ag through the medium, as was the case with the disk-diffusion method (Figure 5A). Both types of bacteria were plated with different concentrations of HAp/Ag (10 wt% of Ag) nanocomposite and corresponding calculations of colony forming units (CFU) along with log CFU reduction was performed. In comparison to the reference corresponding to the pure HAp, increasing the concentration of the nanocomposite provided decrease of the CFU and increase of the log CFU reduction. Using minimal inhibitory concentration (MIC) method it was determined that the minimal concentration of the material able to inhibit the growth of E. coli was in the range 0.6 0.7 mg·ml-1 (Figure 5) while the minimal concentration of this material able to inhibit the growth of S. aureus was in the range 0.7 - 0.8 mg·ml-1 (Figure 5). In the following step, minimal bactericidal concentration (MBC) method was applied and material was also tested for the concentration that had a bactericidal effect on these two bacteria. In the case of E. coli the bactericidal concentration of the composite was 0.8 mg·ml-1, while this concentration for S. aureus was 1.25 mg·ml-1 (Figure 5).


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Figure 5. Growth Inhibition of bacteria plated with different concentrations of HAp/Ag (10% wt of Ag), quantification of grown bacteria (CFU/ml) and log CFU reduction for E. coli (a1-10)


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and S. aureus (b1-10) (A); MIC and MBC concentrations of HAp/Ag for both types of bacteria (B). Morphological changes in bacteria induced by HAp/Ag nanocomposites Interactions between the HAp/Ag and bacteria were also investigated from the standpoint of the morphological changes induced in the bacterial cells after exposure to the material (Figure 6). In contrast to some previous studies,31,32 we compared the bacterial morphology of the uninfluenced bacteria from the zone of confluent growth and bacteria from the inhibition zone around the tested substances on the same agar plate to eliminate possible artificial influences (Figure 4). Uninfluenced E. coli cells were 1-2-µm-long rods with a rough appearance of the surface, distinctive for the Gram negative bacteria (Figure 6a,6b). Compared with these, the morphology of the bacteria exposed to HAp/Ag was significantly changed, with a heavily damaged, even ruptured cell surface and a collapsed structure of the cell (Figure 6c,6d). In the case of S. aureus, uninfluenced bacterial cells were spheres, 200-500 nm in diameter, with relatively smooth surface characteristic for the peptidoglycanic cell wall of Gram positive bacteria (Figure 6e,6f). In the inhibition zone, only a small group of bacterial cells of S. aureus, embedded in the agar surface, were observed (Figure 6g,6h). The bacterial cells were often covered by a filamentous network (insert and area within the dotted circle in Figure 6g), which may be associated with a protective role in the Gram positive bacteria.33 Besides these surface anomalies and in spite of the morphological integrity of most of the observed S. aureus cells in the inhibition zone, cells with wrinkled and punctured cell walls were occasionally observed (indicated by arrows in Figure 6h).


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Figure 6. SEM images of: E. coli before (a,b) and after (c,d) treatment by HAp/Ag composite as well as S. aureus before (e,f) and after (g,h) treatment by HAp/Ag. Cytotoxic properties of HAp/Ag nanocomposite Interactions with human cells were investigated for HAp/Ag composites with 1 wt%, 5 wt% and 10 wt% of Ag component and compared to the pure HAp used as a negative probe (Figure 7). The interactions between the cells and composites were quantitatively presented as a percentage of viable cells obtained after an overnight exposure to the gradually increased


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concentrations of composites. The material with the lowest content of silver (HAp/Ag 1 wt%), tested in different concentrations in the range 0.4-1.6 mg·ml-1, had a relatively low toxicity and showed only a slight decrease in the compatibility with cells in comparison to the pure HAp (Figure 7a). After a 24h exposure to the material, around 80% of the tested cells were viable. Further investigations performed with a composite with 5 wt% of Ag showed a high percentage of survival cells for lower concentrations and a significant drop in their number for higher concentrations of the investigated material (Figure 7b). For concentrations up to 1 mg·ml-1 the viability was around 80% and it was decreased below 50% for higher concentrations. In the last step the material with 10 wt% of silver was tested (HAp/Ag 10 wt%) (Figure 7c).

Figure 7. Viability of U-2 OS cells in the presence of HAp/Ag composites with 1 wt% (a), 5 wt% (b) and 10 wt% (c) of metallic component. In this case a very low percentage of viable cells was obtained. It was lower than 50% for the whole range of investigated concentrations, confirming the significant level of toxicity of the material. The obtained results show the concentration-dependent toxicity induced by the HAp/Ag composite, where lower concentrations are more compatible with the investigated cells. However, the MIC and MBC concentrations that are able to prevent the bacterial growth and to have a bactericidal impact on them are of essential importance for the application of the material


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and have to be selected for the toxicity assessment. As was already mentioned, the MIC and MBC for E. coli are between 0.6 and 0.8 mg·ml-1 and for S. aureus they are between 0.8 and 1.25 mg·ml-1 (Figure 5). As marked on the viability graph (Figure 7c), for these concentrations the observed viability of the U-2 OS cells is lower than 20%, which confirms the very high toxicity of the investigated material under concentrations that are applicable in practice. Moreover, the results confirm the complete non-selectivity of the material that uses general toxicity to affect both mammalian and bacterial cells. The same tests were repeated for IMR-90 human lung fetal fibroblast cells. The results were very similar, showing the intensive toxicity of the MIC concentrations. Morphological changes in human cells induced by HAp/Ag nanocomposite The interactions of the HAp/Ag composite with two types of human cells, U-2 OS osteosarcoma and IMR-90 fetal lung fibroblasts, was also investigated from the standpoint of in situ observed morphological changes obtained after exposure over a period of 1 hour (Figure 8). Figure 8a summarizes the morphological properties of the U-2 OS cells: incubated without material (Figure 8a1), incubated with pure HAp without any metallic content (Figure 8a2) as well as the morphology of the cells exposed to different concentrations of HAp/Ag composite (Figure 8 a3-a6). U-2 OS osteosarcoma with characteristic polygonal / epithelial adherent morphology were observed in non-treated culture and in culture with HAp alone (Figure 8a1,a2). Cells incubated in the presence of HAp/Ag (10 wt%) changed the morphology and turned into floating dead cells.34


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Figure 8. Morphology of U-2 OS (a1-6) and IMR-90 cells (b1-6) without materials as well as in the presence of HAp and different concentrations of HAp/Ag composite. For lower concentrations (0.4 mg·ml-1 and 0.6 mg·ml-1) (Figure 8a3,a4) a 1h exposure initiated the morphological changes observed in only a small portion of cells. It means that even low concentrations of HAp/Ag (10%) composite that contain only 40 µg·ml-1 of Ag-content (29-48% of which is initially dissolved and released in vitro) are prone to initiate apoptosis or the first step


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of necrosis. At 0.8 mg·ml-1 (which is about the MIC concentrations of the composite) and for all other higher concentrations (Figure 8a5,a6) a 1h exposure induced significant morphological changes in the majority of the observed cells. They turned into swollen, interconnected and fused structures that clearly indicated necrosis. Similar was obtained for IMR-90 human fibroblasts (Figure 8b). Normal IMR-90 cells (Figure 8b1), cells incubated with HAp (Figure 8b2) and cells incubated with low concentration (0.2 mg·ml-1) of HAp/Ag (Figure 8b3) are elongated and spindle-shaped. At higher concentrations (0.4 mg·ml-1, 0.5 mg·ml-1 and 0.6 mg·ml-1) (Figure 8b4-8b6) clear signs of cell damage indicated by cell retraction, rounding up and eventual detachment. A low concentration of material (0.4 mg·ml-1) induced the first signs of toxicity. This concentration is only half of the MIC concentration of HAp/Ag in action against S. aureus and about half of the MBC concentration of E. coli. Interactions with human cells are comparable to interactions with bacterial cells that led to the conclusion about their non-selective nature and the same mechanism of action of HAp/Ag for both types of cells. Discussion The possible interactions of the HAp/Ag nanocomposite with the environment - its potential for antibacterial action, as well as its potential to induce toxic influence on mammalian cells - are investigated in terms of its ability to release metallic ions. The material had a capacity to release 2·10-2 to 3·10-2 µg·cm-2 of Ag-ions during a 10d period in PBS. Recently, it has been pointed out that silver wound dressings currently used in clinical applications release up to approximately 14 µg·ml-1 (or up to 42 µg·cm-2) of silver in the culture medium during 24h 6 and highlighted that CaP coatings that release less than 15 µg·cm-2 retained a high percentage of viability of the


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mouse fibroblasts.23 On the other hand, some reports show a significantly low concentration of released Ag-ions (up to 0.045 µg·ml-1) that had the ability to induce a considerable cytotoxic effect in macrophage cells, although in this case the mineral phase was not present.11 Therefore, the presence of the mineral phase and the ability to release Ag-ions in a content within a clinically acceptable level indicate the possibility of good cytocompatibility of the investigated HAp/Ag material. Earlier studies showed that the majority of the HAp/Ag materials containing Ag nanoparticles had an equal influence on the growth and death of both S. aureus and E. coli (or some other type of Gram negative bacteria) 17,18,23 while the Ag-doped material had a stronger activity against E. coli and mainly contributes to the suppression of bacterial growth.15 The mode of the antibacterial action of our investigated material is more similar to the mode of action of the HAp-doped material with an improvement that concerned both bacteriostatic and bactericidal action. This improvement is a consequence of the presence of both Ag ions and Ag nanoparticles within the composite. Oxidative stresses, induced by the release of Ag-ions, and electrostatic interactions, induced by the charge of the surface of Ag-nanoparticles, are concerned with one of the main mechanisms for the antibacterial activity of Ag-containing materials.35 Both mechanisms have a special role in the antibacterial activity of Ag, since they interact with membrane proteins and affect their conformation. Destroying the structure of transmembrane proteins affects the integrity of the membrane and disintegrates it by the formation of mechanical defects.36,37 Our material has the capacity to release Ag-ions and possesses Ag nanoparticles that are negatively charged and prone to having electrostatic interactions with positively charged domains in macromolecules of the bacterial cell wall. The coupling of these two mechanisms is capable of inducing damage in the bacterial wall, as was shown in Figure 6; however it is also prone for the similar influence on human cells, as was


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shown in Figure 8. This mechanism of antibacterial action is non-selective and has the potential to be applied in mammalian cells in the same manner as for bacteria, resulting in toxicity. Another aspect of the toxicity of this material concerns its long-term instability. As already mentioned material has affinity to precipitate in vitro and shows instability in the simulated physiological condition. The newly formed, AgCl phase with unknown properties is a highly insoluble salt of silver (Ksp=1.6·10-10). Its precipitation under physiological conditions indicates a potential for the accumulation of Ag in cells or organisms and is a potential source of material’s toxicity. This property raises a question of possibility for its bioaccumulation. On the other hand, nanocomposites formed of mineral carriers and Ag nanoparticles are prone for microorganismtriggered release of Ag.38 It is a consequence of mineral bioresorption and uptake by growing microorganisms. Direct result of this behavior is a release of Ag nanoparticles. Besides, incorporation of Ag into HAp decreases its crystallinity which contributes to its easier dissolution that releases embedded/incorporated Ag-content. In this way Ag decreases bioactivity of pure HAp. This fact leads to the beginning of the problem regarding toxicity of free Ag nanoparticles and about capacity of bioactive HAp to compensate toxicity of Ag. Possibility for long-term instability and release of Ag nanoparticles restarts the problem regarding toxicity of Ag that actually initiated basic idea of nanocomposite formation and hypothesis that stabilization of Ag by bioactive, mineral component is a potential solution. Conclusion The formation of nanocomposites made of Ag as antibacterial and HAp as bioactive component is not a safe way to form Ag-based antibacterial material. In spite of the design of the material developed to provide optimal responses during interactions with cells, it has been confirmed that


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there is no possibility to combine safety and antibacterial efficacy in a single Ag-containing material. The main reason is in non-selectivity of Ag component that uses the same mechanism to interact with both, mammalian and bacterial cells. Therefore, the presence of a mineral phase, able to stimulate and promote the cellular growth, was not able to minimize the toxic influence of the effective, antibacterial contents of Ag component. In the case when the safety was reached, the material was not any more efficient in antibacterial action. Directing a future research to the components able to provide bacteria-specific interactions will be an excellent solution and promising way for enhancement of the antibacterial potential and joining safety with antibacterial activity in this class of advanced bionanomaterials. AUTHOR INFORMATION Corresponding Author *Advanced Materials Department, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia, e-mail: [email protected] , phone: +386 1 477 35 47. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This research was supported by Slovenian Human Resources Development and Scholarship Fund under Grant Number 11011-20/2009 and Slovenian Research Agency (Program P1-0140). REFERENCES 1. Goodman, S. B.; Yao, Z.; Keeney, M.; Yang, F. The future of biologic coatings for orthopaedic implants. Biomaterials 2013, 34, 3174-3183.


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Table of Contents Graphic Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites? Marija Vukomanović, Urška Repnik, Tina Zavašnik-Bergant, Rok Kostanjšek, Srečo D. Škapin, Danilo Suvorov


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Is Nano-Silver Safe within Bioactive Hydroxyapatite Composites

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