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Bioelectrochemistry
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Electrokinetic and bioactive properties of CuO∙SiO2 oxide composites
Magdalena Nowacka a, Anna Modrzejewska-Sikorska a, Łukasz Chrzanowski a, Damian Ambrożewicz a,Tomasz Rozmanowski b, Kamila Myszka c, Katarzyna Czaczyk c, Karol Bula d, Teofil Jesionowski a,⁎a Poznan University of Technology, Institute of Chemical Technology and Engineering, M. Sklodowskiej-Curie 2, PL-60965 Poznan, Polandb Poznan University of Technology, Institute of Chemistry and Technical Electrochemistry, Piotrowo 3, PL-60965 Poznan, Polandc Poznan University of Life Sciences, Department of Biotechnology and Food Microbiology, Wojska Polskiego 48, PL-60627 Poznan, Polandd Poznan University of Technology, Institute of Materials Technology, Piotrowo 3, PL-60965 Poznan, Poland
Article history:Received 7 September 2011Received in revised form 12 March 2012Accepted 18 March 2012Available online 28 March 2012
Keywords:CuO∙SiO2 oxide compositesElectrokinetic potentialBioactive and structural properties
CuO∙SiO2 hybrid oxide precipitated on a semi-technical scale was thoroughly characterised in terms ofphysicochemical properties. Its particle size distribution and SEM analysis were performed to establishdispersion and surface morphology. Chemical analysis provided information on the content of CuO andSiO2 oxides in the hybrid systems. The oxide systems were also subjected to elemental analysis. Zeta po-tential determinations were evaluated to obtain information regarding the interactions between colloidalparticles. The stability of copper silicates' water dispersions was estimated on the basis of zeta potentialmeasurements. The obtained oxide systems were used as components of polymer composites with poly-ester resins, which were subjected to mechanical tests and bactericidal tests against Pseudomonas aerugi-nosa, a well known biofilm-forming microorganism. The anti-adhesive activity of the CuO·SiO2 enrichedpolymers was assessed using a 9-degree scale of adhesion. A significant reduction in the P. aeruginosabiofilm development rate was achieved for Palatal A 400-01 resins enriched with both 2 and 8 phr ofthe filler. In the case of Aropol M 105 TB resins the introduction of CuO∙SiO2 caused inhibition of bacterialcolonisation but to a smaller extent. These results strongly indicate that the biological activity of Cu wasmaintained. The release of copper ions into the local environment was examined by atomic absorptionspectrometry (AAS). Maximum values of 1.621 and 5.934 mg/dm3 of released copper were detected.The surface composition of both resins studied by energy dispersive X-ray spectroscopy (EDS) contribut-ed to the data suggesting homogenous distribution of Si; however copper seemed to form local aggre-gates. The presented results may be of great significance for those dealing with materials tailored forspecific needs.
The development of new polymer composites (two-phase ma-terials with a polymer matrix) has recently been focused on thechoice of fillers, which essentially affect the polymer materialproperties. The properties of polymer composites depend on thetype, amount and form of the filler and the character of interac-tions between the filler and the polymer. Most often the fillersare inorganic substances, such as silicates; however the non-renewable resources of these minerals are susceptible to exhaus-tion. Therefore the search for new synthetic silicate materials iscontinuing, and the oxide system MO∙SiO2 is one of the proposedmaterials [1–7].
Composition of a small amount of metal oxide with a matrix ofhighly developed specific surface area made it possible to obtain
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new types of surfaces which exhibit high activity and reactivity. Theoxide composites MO·SiO2 combine the benefits of metal oxideswith the functional and application properties of silica. Combinationof two or more types of metal oxides with a silica matrix gives a com-plex system exhibiting many new properties, which were not ob-served for separate oxides. In the oxide system CuO·SiO2 theinteresting properties of copper oxide, such as antibacterial, antifun-gal and virucidal activity, were combined with the biocompatibility,non-toxicity and versatility of the silica surface. The composite isused in medicine and biology, e.g. in controlled drug administration,bioseparation and thermal therapy of cancer. An additional benefitof CuO·SiO2 oxide composite is the possibility of modifying its surface(and hence its properties) by simple chemical processes with the useof hydrophobic reagents and a large number of organofunctionalcompounds [8–17].
This study reports results concerning a thorough characterisationof the precipitated CuO∙SiO2 oxide systems together with analysis ofthe mechanical and bactericidal properties of composites based onthe above systems and polyester resins.
Table 1Chemical composition of the unmodified oxide composite CuO·SiO2.
Sample Content (%)
CuO SiO2 Na2O K2O H2O
CuO·SiO2 35.23 62.16 0.02 0.01 18.52
Fig. 2. Particle size distribution according to volume contribution in CuO∙SiO2 samplesunmodified (curve 1) andmodifiedwith 3 (curve 2), 5 (curve 3) and 10 (curve 4) wt./wt.of 3-glycidoxypropyltrimethoxysilane.
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2. Experimental
2.1. The synthesis of CuO∙SiO2
The unmodified CuO·SiO2 oxide system was obtained on a semi-technical scale. The substrates were a 5% solution of sodium silicate(filtered — technical grade 8.50% of Na2O, 27.18% of SiO2, density1.39 g/cm3, and silicate modulus, SiO2:Na2O molar ratio, equal to3.3) made by VITROSILICON SA, Poland and a 5% solution of copper ni-trate (analytical grade)made by Chempur, Poland. The process of pre-cipitation was performed at room temperature in a reactor of 10 dm3
capacity (QVF Mini Plant PilotTec, UK) equipped with a high-speedpropeller stirrer made by Eurostar Power Control-Visc Ika Werke,Germany (720 rpm). The precipitate was separated from the post-reaction mixture by filtration in vacuum and washed with distilledwater to remove the salt residue. The precipitate was dried in a sta-tionary drier at 105 °C.
2.2. Surface modification of CuO ∙SiO2
The CuO∙SiO2 oxide system was modified with 3, 5 or 10 weightparts by mass of 3-glycidoxypropyltrimethoxysilane. The silane washydrolysed in methanol for 10 min and then deposited on the surfaceof CuO∙SiO2 by atomisation (using a spray atomiser). The modifiedsystem was brought to a vacuum evaporator where it was stirredfor 1 h, after which the solvent was distilled off. The modified oxidesystem was subjected to extraction of unadsorbed silane with
Fig. 1. SEM images of CuO∙SiO2 unmodified (a) and modified with 3-glycidoxypropylt
methanol for three cycles and with deionised water for five cycles,and then subjected to convection drying.
2.3. Physicochemical analysis of CuO∙SiO2
The surface chemical composition was determined using energydispersive X-ray spectroscopy (EDS), Princeton Gamma-Tech, USA.The contents of Cu, Si, Na and K were measured and expressed interms of amounts of the relevant oxides. The morphology of theCuO·SiO2 oxide system was analysed on the basis of SEM images(Zeiss EVO40, Germany) to obtain information about the shape ofgrains, particle structure, character of agglomerations and degree ofdispersion. Particle size distributions were measured by a Mastersi-zer 2000 (Malvern Instruments Ltd., UK), employing the laser diffrac-tion technique and permitting measurements in the range0.2–2000 μm. Elemental analysis of the synthetic CuO·SiO2 oxide sys-tems was performed with the use of a Vario EL Cube analyser
rimethoxysilane in the amount of 3 wt./wt. (b), 5 wt./wt. (c), and 10 wt./wt. (d).
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(Elementar, Germany). The analyser is fully automatic and gives si-multaneous information on the content of C and H, working onmacro-concentrations as well as trace concentrations of these ele-ments while maintaining high accuracy. A weighed portion of thesample was subjected to combustion and then reduction, the volatileproducts were passed through appropriate catalysts into a chromato-graphic column in which they were separated and recorded by akatharometer. Electrophoretic mobility was measured by a ZetasizerNano ZS (Malvern Instruments Ltd., UK) equipped with an autotitra-tor. The apparatus combines the methods of electrophoresis and lasermeasurements of particle mobility based on the Doppler phenome-non. The velocity of particles in a 0.001 M NaCl solution measuredin an electric field is the electrophoretic mobility; knowing thisvalue the zeta potential was calculated from the Henry equation.
2.4. Polymer composite analysis
Prior to the experiments the dumbbell surface was prepared by pol-ishing with water sand papers with increasing gradation. Copper releaseinto water was evaluated by atomic absorption spectrometry (Unicam989QZ, UK). The examined resin samples (surface area 16 cm2, unfilledand filled with CuO∙SiO2) were shaken for 24 and 48 h in 100 cm3 deio-nisedwater. The resulting aqueous phasewas then examined for Cu con-tent. Surface content of Cu, Si and Owas analysed with energy dispersiveX-ray spectroscopy (EDS). The mechanical properties of the polymercomposites were tested in terms of tensile strength, flexural modulusand elongation to break according to standard procedure — tensile testaccording to ISO 527-2, Flexural test according to ISO 178 (Instronmodel 4481, United States).
2.5. Bacterial strain and growth conditions
Pseudomonas aeruginosa strain ATCC 10145 was obtained from theAmerican Type Culture Collection (Rockville, MD, USA). Microorgan-isms were grown for 48 h at 37 °C with shaking conditions(100 rpm) on an LB medium according to Bertani [18]. The LB medi-um contained: peptone 10 g/dm3; yeast extract 5 g/dm3; NaCl 5 g/dm3.The pH of the medium was set at 7.0.
2.6. Bacterial adhesion analysis
The standard dumbbells were produced from CuO·SiO2 enrichedpolymers. In the next step 1 cm×6 cm dumbbell pieces were treatedwith 70% ethanol in water for 10 min at room temperature. After
Table 3Results of elemental analysis of CuO·SiO2.
Sample Type of modifier
Amount(wt./ wt.
CuO·SiO2 – –
3-glicydoxypropyltrimethoxysilane(A-187)
3510
rinsing with distilled water the plates were inserted into appropriatebacterial cultures for 48 h. After 24 or 48 h the plates were removedand washed with PBS solution (pH 7.2) in order to remove unat-tached cells from the dumbbell surfaces. The plates were stainedwith 0.01% solution of acridine orange (2 min at room temperature).For observation of bacterial adhesion to CuO·SiO2 enriched polymers,a fluorescence microscope was used (Carl-Zeiss, Axiovert 200,Germany). To determine the level of bacterial adhesion to the surfaceof the dumbbells, the method described by Le Thi et al. was used [19](Table 8). This technique is based on the estimation of 50 visual fieldsaccording to a 9-degree scale:
1st degree: from 0 to 5 bacterial cells in the visual field,2st degree: from 5 to 50 bacterial cells in the visual field,3st degree: only single bacterial cells (above 50 bacteria cells inthe visual field), no microcolonies,4th degree: single bacteria cells+macrocolonies,5th degree: large but not confluent microcolonies+single bacte-ria cells,6th degree: confluent microcolonies+single bacteria cells,7th degree: 1/4 visual fields covered by the biofilm,8th degree: 1/2 visual fields covered by the biofilm,9th degree: visual fields totally covered by the biofilm.
Each experimental variant was repeated three times.
3. Results and discussion
3.1. Dispersive and morphological properties
The results of chemical composition of the CuO·SiO2 filler(obtained on a semi-technical scale) measured using the energy-dispersive X-ray spectroscopy (EDS) method, and expressed interms of the appropriate oxides are given in Table 1. The contents ofCuO and SiO2 were 35.23 and 62.16% respectively. Sodium and potas-sium oxides coming from the water glass solution are residues of saltsnot washed after precipitation of the pigment. Their total content didnot exceed 0.05%.
The surface of the CuO·SiO2 filler was modified by 3-glycidoxypropyltrimethoxysilane (A-187) at different concentra-tions, and SEM images of the samples were taken to analysetheir morphological and dispersive properties. Particle size distri-butions of the samples were measured, and the results are
Fig. 3. Zeta potential versus pH for CuO∙SiO2 unmodified (curve 1) and modified with 3(curve 2), 5 (curve 3) or 10 (curve 4) wt./wt. of 3-glycidoxypropyltrimethoxysilane.
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presented in terms of the percentage contributions of particleswith given diameters.
Fig. 1 shows SEM images of CuO·SiO2 samples unmodified andmodified with the adhesive silane in different concentrations.
As follows from the analysis of particle size distributions in Fig. 2and the data from Table 2, the unmodified CuO∙SiO2 system (curve1) contains 10% of particles with diameters smaller than 2.8 μm, 50%of particles with diameters smaller than 9.5 μm, and 90% of particleswith diameters smaller than 31.0 μm. The mean diameter of particlesin this sample is 13.7 μm — D[4.3]. For the modified samples a smallshift in the particle diameters towards higher values was noted.The mean particle diameters (D[4.3]) in the samples modified with3 (curve 2), 5 (curve 3) and 10 (curve 4) wt./wt. of 3-glycidoxypropyltrimethoxysilane were 18.5, 16.3 and 14.6 μm respec-tively. The largest diameters of CuO∙SiO2 were obtained after modifi-cation with 3 wt./wt. of A-187 silane. In this sample 10% ofagglomerates had diameters smaller than 2.7 μm, 50% had diameterssmaller than 10.7 μm, and 90% of particles had diameters smallerthan 46.5 μm.
Elemental analysis results of unmodified and modified CuO∙SiO2
oxide systems provided information on the effectiveness of the mod-ification process and enabled calculation of the degree of coverage(see Table 3).
The results confirmed an increase in the content of carbon and hy-drogen with increasing quantity of the silane used for modification.The degree of coverage increased from 0.157 μmol/m2 for the sample
Table 4Chemical composition and Cu elution results for polymer composites based on Palatal A 40
Sample Amount of CuO∙SiO2 filler(phr)
Modifier
Type Amount (wt./wt.)
Palatal A 400-01 – – –
2 A-187 38
Aropol M 105 TB – – –
2 A-187 38
modified with 3 wt./wt. of silane to 0.483 μmol/m2 for the samplemodified with 10 wt./wt. of A-187 silane.
3.2. Electrokinetic characteristics
The electrokinetic properties of silicates significantly dependon the type of substance adsorbed on their surface. Even asmall amount of a compound adsorbed on the surface ofCuO∙SiO2 significantly affects the surface charge density andhence the zeta potential and stability of dispersion. High valuesof zeta potential (positive or negative) are typical for stable sys-tems. To meet the demands of different branches of industry,the surface of synthetic silicates must be modified in a target-specific way. The modification is usually performed with organo-functional silanes, of which the type and amount determine thephysicochemical properties of the final products. Fig. 3 presentsa comparison of the dependencies of zeta potential on pH forunmodified CuO∙SiO2 and for the samples modified with 3, 5 or10 wt./wt. of A-187 silane.
The electrokinetic curve obtained for the unmodified sample(the reference curve) runs in the range of negative zeta valuesover the whole range of pH changes. The unmodified CuO∙SiO2
(curve 1) is characterised by high stability for pH values rangingfrom 5 to 11. The modification with 3 wt./wt. of A-187 silane(curve 2) resulted in a shift of the electrokinetic curve towardshigher potential values, so that the isoelectric point was reachedat pH of 4. The zeta potential of this sample varied from 5 to(−15) mV for pH from 1.7 to 11. For the sample with the contentof A-187 silane increased to 5 wt./wt. (curve 3), the electrokineticcurve was shifted towards lower pH relative to the curve deter-mined for the sample modified with 3 wt./wt. of A-187 silane. Theisoelectric point was reached at pH=3. For the sample modifiedwith 5 wt./wt. the zeta potential values range from 5 to (−20)mV, for pH varying from 1.7 to 11. For the sample modified with10 wt./wt. of A-187 silane (curve 4) the isoelectric point was shiftedto a pH of 2.3. The zeta potential values changed from 4 to (−20)mV for pH varying from 1.7 to 11.
3.3. Polymer composite analysis
The bioactivity of the CuO∙SiO2 systemwas tested in polymer com-posites prepared with the two polyester resins Palatal A 400-01 andAropol M 105 TB, filled with CuO∙SiO2 in quantities of 2 and 8 phr.The composites were also characterised by chemical compositionand in terms of their mechanical properties by determination oftheir tensile strength, flexural strength and temperature of softeningpoint to Vicat (VST).
The chemical composition of the obtained modified polymers ispresented in Table 4. For pure Palatal A 400-01 0.006% (wt.) Si con-tent was determined. Introduction of CuO∙SiO2 resulted in an increase
54 M. Nowacka et al. / Bioelectrochemistry 87 (2012) 50–57
of Si content to 0.81 and 2.12% (wt.) for 2 and 8 phr CuO∙SiO2 filledpolymers respectively. Copper was detected at the level of 0.84 and2.25% (wt.) respectively (Fig. 4).
Fig. 4. Surface composition of pure Palatal A 400-01 (a) and filled with 2 phr (b) or8 phr (c) of CuO∙SiO2.
For Aropol M 105 TB the initial Si content was 1.06% (wt.), andit increased to 1.59 and 4.38% (wt.) for 2 and 8 phr CuO∙SiO2 filledpolymers respectively. EDS analysis revealed copper at 1.02 and3.30% (wt.) for 2 and 8 phr respectively. It should be noted that Siatoms are dispersed homogeneously on the samples' surfaces(Figs. 5–7).
Tables 5 and 6 present parameters characterising the mechanicalproperties of the composites based on polyester resins. Addition ofCuO∙SiO2 to the resins Palatal A 400-01 and Aropol M 105 TB resultedin a decrease in tensile strength. Significant deterioration was notedin the maximum breaking strain and extension on breaking, whichfor Palatal A 400-01 with CuO∙SiO2 decreased by 14.8 and 50.0%,while for Aropol M 105 TB it decreased by 37.3 and 48.7%. A positiveeffect, that is an increase in the maximum flexural stress (stress atyield) by 8.9%, was observed only for Palatal A 400-01.
The softening temperatures of the composites measured accord-ing to Vicat are given in Table 7. The results were different depend-ing on the type of resin. The addition of CuO∙SiO2 as a filler ofPalatal A 400-01, in a quantity of 2 or 8 phr, resulted in an increasein the softening temperature from 100.5 °C (for pure resin) to141.5 °C for resin with 2 phr of CuO∙SiO2 and 154 °C for resin with8 phr of CuO∙SiO2. For the composites with Aropol M 105 TB, theaddition of 2 or 8 phr of CuO∙SiO2 led to a significant decrease inthe softening temperature. This negative effect could be related tothe curing process of the resin.
3.4. Bacterial adhesion analysis
P. aeruginosa is considered to be an opportunistic human patho-gen, commonly found in terrestrial environments. It is responsiblefor frequent clinical infections, especially among patients sufferingfrom cystic fibrosis [20]. Its virulence is also commonly linkedwith the secretion of polymeric substances responsible for bacterialadhesion prior to surface colonisation and specific features of cell
Fig. 5. SEM image and surface composition area of pure Palatal A400-01resin.
Fig. 6. SEM image and surface composition areas of Palatal A400-01resin filled with2 phr of CuO∙SiO2.
Fig. 7. SEM image and surface composition areas of Palatal A400-01resin filled with8 phr of CuO∙SiO2.
55M. Nowacka et al. / Bioelectrochemistry 87 (2012) 50–57
structure such as pili [21,22]. Bacterial attachment to the surface iscrucial for the survival and development of bacterial biofilms,which contribute to enhanced biological, chemical and mechanicalresistance. In order to assess the anti-adhesive activity of theCuO·SiO2 enriched polymers, the 9-degree scale of adhesion previ-ously proposed by Le Thi et al. [19] was employed. A certain degreebecame dominant when the adhesion occurred with a minimum of20%. The appearance of higher adhesion degrees on the tested poly-mers was also examined.
In the case of Aropol M 105 TB polymer there was a slight de-crease in the dominant adhesion degree from an average 6th forpure polymer to 4th for CuO∙SiO2 modified polymer. Instead ofconfluent microcolonies, which were visible on pure Aropol M105 TB polymer, introduction of CuO∙SiO2 into the polymer matrixreduced bacterial colonisation and only single bacterial cells in thepresence of macrocolonies were observed. This effect was visibleafter both 24 and 48 h. A significant reduction in the P. aeruginosa
biofilm development rate was achieved for Palatal A 400-01 resinsenriched with 2 and 8 phr of the filler. The 1st and 2nd degreeswere the most frequent stages of adhesion (Table 8). This corre-sponds to an average of 5 to 50 bacterial cells in the visual field,which represents a considerable reduction in microbial adhesion.Pure Palatal A400-01 resins were colonised at the 6th degree of ad-hesion. Such results strongly indicate that the biological activity ofCu was maintained, especially for Aropol M 105 TB polymer. This isof great significance for those dealing with materials tailored forspecific needs. Copper oxide is generally considered to have anti-bacterial, antiviral and antifungal properties [23]. For example, per-manent biocidal properties of fabrics containing 3–10% copperwere reported by Gabbay and Borkow [24]. In our experiments asimilar range of copper oxide concentrations were used. The subse-quent increase in copper concentration is likely to decrease the me-chanical properties of CuO-silicate blended polymers. Neverthelessthere is a recent tendency to explore properties of nanoparticles,
Table 5Mechanical properties of the CuO∙SiO2 composites with polyester resins Palatal A 400-01 and Aropol M 105 TB (σ — standard deviation) obtained in tensile tests.
Table 6Mechanical properties of CuO∙SiO2 composites with Palatal A 400–01 and Aropol M 105 TB (σ — standard deviation) obtained in 3-point bending flexural tests.
Table 7Temperature of softening according to Vicat determined for composites based on Pala-tal A 400-01 and Aropol M 105 TB polyester resins.
Material Amount of CuO∙SiO2 filler(phr)
Modifier Softening temperature(°C)
Type Amount(wt./wt.)
Palatal A400-01
– – – 100.52 A-187 3 111.5
10 141.58 3 154.0
10 113.0Aropol M105 TB
– – – 70.02 A-187 3 64.5
10 65.58 3 64.0
10 60.5
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and among them the antimicrobial properties of copper, e.g. [25]. Asreported by Ren et al. [9] the minimum bactericidal concentration ofCuO in nanoparticle form for P. aeruginosa is within 5000 μg/mL. Al-though nanoparticles in the size range 1–10 nm have been shown topossess the greatest activity against bacteria [26,27] our resultsclearly indicate that even larger particles entrapped in the polymer
Table 8Degrees of Pseudomonas aeruginosa adhesion to the CuO∙SiO2 dumbbell surface.
Examined material Amount of CuO∙SiO2 filler(phr)
Modifier Incubat(h)
Type Amount(wt./wt.)
Palatal A 400-01 – – – 2448
2 A-187 3 2448
8 2448
Aropol M 105 TB – – – 2448
2 A-187 3 2448
8 2448
matrix are still active against surface colonising bacteria. The releaseof copper ions into the local environment is required for mainte-nance of microbial activity, which was previously suggested by Renet al. [9]. However according to results obtained from the AAS ex-periments there is no simple correlation with the amount of copperreleased from the polymer matrix and microbial growth inhibition.Most likely this effect was caused by additional factors such as sur-face smoothness, although we made a great deal of effort to providethe same smoothness for all samples. Another issue might be the re-lease of organic constituents of polymers. This issue is currentlybeing investigated by our team.
4. Conclusions
Analysis of the dispersion data has shown that modification ofCuO∙SiO2 systems with 3-glycidoxypropyltrimethoxysilane resultedin an increase in the CuO∙SiO2 particle diameters, irrespective ofthe amount of silane used. The CuO∙SiO2 particles with the greatestdiameters (a mean diameter (D[4.3]) of 18.5 μm) were obtainedafter modification with 3 wt./wt. of the silane. Elemental analysisdemonstrated an increase in the content of carbon and hydrogenwith increasing quantity of silane used for the modification,which is confirmed by the calculated degree of coverage increasing
ion time Dominating adhesion degree Presence of higher adhesion degree(6, 7, 8, 9)
6 6, 7, 86 6, 7, 82, 1 –
2 –
1 –
1 –
6 6, 7, 86 64, 5 64 74 –
4 –
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from 0.157 μmol/m2 for the filler modified with 3 weight parts bymass of silane to 0.483 μmol/m2 for that modified with 10 wt./wt.of A-187.
As suggested by the data on the mechanical properties of thecomposites, positive effects of CuO∙SiO2 addition were observedonly for Palatal A 400-01. For the composites with Aropol M 105 TB,irrespective of the amount of filler added, the mechanical strengthwas considerably decreased with respect to that of pure resin. More-over, the addition of CuO·SiO2 significantly increased the softeningtemperature of Palatal A 400-01, which substantially extends the pos-sibilities for application of these polymer composites. Surface compo-sition of both resins studied by EDS contributed to the data suggestinghomogeneous distribution of Si; however copper seemed to formlocal aggregates (Fig. 4).
As revealed by AAS, introduction of copper in the form of CuO∙SiO2
did not prevent copper from being released into solution. Thereforethe bacteriostatic action of copper could be observed in the form oflimitation of bacterial adhesion.
Acknowledgements
This work was supported by the Ministry of Science and HigherEducation research grant no. N N209 032738 (2010–2011).
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