Preparation and Characterization of Antibacterial Phosphate Glasses


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Preparation and Characterization of Antibacterial Phosphate Glasses

  1. 1. Preparation and characterization of antibacterialP2O5–CaO–Na2O–Ag2O glassesA. A. Ahmed,1 A. A. Ali,1 Doaa A. R. Mahmoud,2 A. M. El-Fiqi11 Glass Research Department, National Research Centre, Dokki, Cairo 12622, Egypt2 Natural and Microbial Products Laboratory, National Research Centre, Dokki, Cairo 12622, EgyptReceived 16 October 2010; revised 5 February 2011; accepted 22 February 2011Published online 4 May 2011 in Wiley Online Library ( DOI: 10.1002/jbm.a.33101Abstract: 60P2O5–20CaO–(20 – x) Na2O–xAg2O and 60P2O5– An increase in the concentration of silver ions released from30CaO–(10 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5,1, and 2 mol silver-doped glasses into water was observed with increasing% were prepared using normal glass melting technique. The time of glass dissolution and with increasing Ag2O content.antibacterial activity of pressed disks of powdered glass The tested silver-free and silver-doped glasses demonstrated(undoped and silver-doped glass) was investigated against different antibacterial activity against the tested micro-organ-S.aureus, P.aeruginosa, and E.coli micro-organisms using isms. For silver-free glasses, an increase in IZD was observedagar disk-diffusion assays at 37 C for 24 h. The antibacterial with the increase in the glass dissolution rate and with theactivity was deduced from the inhibition zone diameter (IZD), decrease in pH of water. Also, the IZD showed an increasezone of no bacterial growth, measured under the stated ex- with increasing Ag2O content of silver-doped glasses. V 2011 Cperimental conditions. The antibacterial activity increases with Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 132–142, 2011.the increase in IZD and vice versa. Dissolution of glass inwater at 37 C, pH changes of water during glass dissolution, Key Words: antibacterial glasses, silver-doped phosphate-and concentrations of silver ions released from silver-doped based glasses, glass dissolution, controlled release silverglasses into water during their dissolution were determined. ions and antibacterial effectINTRODUCTION more, the degradation rate of such glasses can be tailoredAn antibacterial agent is defined as a substance that either to suit the end application.4 The main advantage of PBGskills bacteria (bactericidal agent) or inhibits their growth over other oxide glasses (e.g., silicate glasses) is its ability(bacteriostatic agent). The ideal antibacterial agent should to accommodate high concentrations of metal ions andhave a broad spectrum of antibacterial activity, that is, effec- remain amorphous, where metal ions have good solubilitytive against a broad range of Gþ and GÀ bacteria, long last- in phosphate glass melts rather than silicate glass antibacterial action, low bacterial resistance, safety, Thus, the metal ions are incorporated into the phosphateminimum side effects and it should not affect the physical glass structure and are not in a separate phase. Therefore,and chemical properties of the carrier.1 the ions are released in a controlled way as the glass In recent years, the use of inorganic antibacterial materi- degrades, and their rate of release is defined by the overallals has attracted interest for many applications.2 Most inor- rate of degradation of the glass. Accordingly, PBGs can actganic antibacterial materials are mainly metal ion (e.g., Agþ, as a delivery system for antibacterial metal ions, for exam-Cu2þ, and Zn2þ ions)-based inorganic materials, for example, ple, Agþ, Cu2þ, or Zn2þ and the higher reactivity of PBGsceramics, zirconium and calcium phosphates, and glasses. than silicate glasses (silica-based glasses are highly resistantThe inorganic antibacterial materials are used in many fields, to degradation) provide a better antibacterial activity. Fur-such as fiber, ceramic, plastic, composites, building materials, thermore, when such glasses, which melt at relatively lowsurface coating, and so forth. temperatures, are mixed with high-melting polymers, they A more recent field of research is the synthesis of anti- can be ensued a partial or complete fusion of the glasses sobacterial glasses. Generally, glass is a material with high that the glasses form a more intimate connection to thechemical durability due to its strong network structure. polymer, which can lead all the way to an extremely homo-However, it is possible to lower this chemical durability by geneous distribution in the polymer. A fusion of the glassesaltering its chemical composition. Phosphate-based glasses can be achieved during the processing of polymer-glass(PBGs) are degradable materials as they can dissolve gradu- composite materials, for example, plastic products with bio-ally or completely in water depending on their chemical cidal properties.composition, for example, binary sodium phosphate glasses A combination of the ability to create glass with lowcan dissolve within a few hours in distilled H2O.3 Further- chemical durability and the property that glass can retainCorrespondence to: A. A. Ali; e-mail: ali_nrc@hotmail.com132 V 2011 WILEY PERIODICALS, INC. C
  2. 2. ORIGINAL ARTICLEmetal ions enable the production of antibacterial glasses. to get ride of gas bubbles. The melt was then cast on a pre-The incorporation of well-known silver, copper, or zinc anti- heated stainless steel plate in the form of rectangular slabsbacterial metal ions in several glass systems has a proven that were subsequently annealed in a muffle furnace main-negative influence on the growth of bacteria and fungi.5–8 tained at a temperature in the range 200–450 C for 20 min.Whereas in the presence of an aqueous medium or mois- The muffle furnace was then switched off and the glassture, the glass will gradually dissolve and at the same time, samples were left overnight to cool slowly to roomsilver, copper, or zinc ions are released during its dissolu- temperature.tion to provide an antibacterial effect. Generally, antibacte- The density (q) values of the prepared glasses wererial glasses can be manufactured either by addition of an determined on bulk glass samples at room temperatureantibacterial agent to the glass batch prior to their manufac- using the simple Archimedes’s method [standard testture or by post-treatment processes, for example, ion- method for density of glass by buoyancy (ASTM C693 Reap-exchange or surface coating. Antibacterial glasses are proved 1998)] with o-xylene as the buoyant liquid.becoming increasingly important in recent years because oftheir wide range of applications, for example, cosmetics, Glass characterizationelectrical appliances, fabrics and biomaterials as well as X-ray diffraction (XRD). The amorphous nature of thewastewater treatment.9 Water-soluble glasses with antibac- materials obtained was verified by using the XRD. The sam-terial effect have been developed for synthetic resins, filter- ples were finely ground in an agate mortar and the X-raying materials, inhibitors of aquatic microbes, cosmetics, or diffraction spectra were obtained using a Bruker D8medical applications. Antibacterial glass powders can be Advance X-ray diffractometer at room temperature witheasily blended into plastics and coatings much like the zeo- Ni-filtered Cu Ka radiation (k ¼ 0.15418 nm), generated atlite powders using conventional methods. 40 kV and 40 mA. Scans were performed with a step size of The general objective of the present study was the 0.02 and a step time of 0.4 s over an angular range 2yproduction of some antibacterial glasses that do not need from 4 to 70 .special precautions or preparation conditions, and whichcan have dissolution rates that can be controlled. For thispurpose, two base sodium calcium phosphate glasses, The glass dissolution test. The dissolution of the prepared60P2O5–20CaO–20Na2O and 60P2O5–30CaO–10Na2O were glasses was carried out in distilled water at 37 C for timeselected. The effect of doping such glasses with silver ions periods up to 6 h using the grain test method. This method(x ¼ 0, 0.5, 1, and 2 mol %) on their antibacterial activity has been recommended by ASTM and ISO No. 719 (1985)was also studied. specifications and adopted by International Commission on Glass as a routine test to evaluate the dissolution behaviorEXPERIMENTAL PROCEDURES of glasses.Glass preparationGlasses having the following compositions 60P2O5–20CaO–(20 – x) Na2O–xAg2O and 60P2O5–30CaO–(10 – x) Na2O– pH measurements. The pH changes of the distilled waterxAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol % were prepared. during the dissolution of some undoped and silver-dopedAll the batches used were prepared from chemically pure glasses were measured at 1-h intervals and up to 6 h usinggrade chemicals in the powder form. P2O5 was introduced IQ 140 pH-meter (IQ, USA). The pH electrode was calibratedas (NH4H2PO4) (99.0% Merck), calcium oxide (CaO) as cal- using pH calibration standards (Colourkey Buffer Solutionscium carbonate (CaCO3) (99.5% SRL), sodium oxide (Na2O) BDH, UK).as sodium carbonate (Na2CO3) (99.5% s.d.fine-chem), andsilver oxide (Ag2O) as silver nitrate (AgNO3) (99.9% FAAS determinations of silver ions released from glassSRL).The appropriate amounts of the starting materials of into water. The amount of silver ions released from glasseach batch equivalent to 50 g glass were accurately into distilled water was determined by using GBC Flameweighed, thoroughly mixed and then transferred into porce- Atomic Absorption Spectrometer (GBC Avanta R, GBC Scien-lain crucibles. Before melting, the batches were calcined tific Equipment Pyt., Australia).slowly in an electric muffle furnace at a temperature in therange of 350–550 C in order to get rid of the gaseousdecomposition products of the batch materials, for example, Antibacterial activity test. The antibacterial activity ofH2O, NH3, NO2, and CO2 and to minimize the evaporation undoped and silver-doped P2O5–CaO–Na2O glasses was eval-tendency of P2O5. Calcination was continued until the uated against bacterial species of American Type Culturedecomposition of the batch materials and evolution of gase- Collection (ATCC); S. aureus (ATCC, 25923), E. coli (ATCC,ous products came to an end. All the batches were melted 25922), and P. aeruginosa (ATCC, 27853) using the agar-in disposable porcelain crucibles inside an electrically disk diffusion assays. The antibacterial activity was deducedheated furnace in the range 800–1200 C. The melting time from the inhibition zone diameter (IZD), zone of no bacte-was continued for 1 up to 2 h depending upon the chemical rial growth, measured under the stated experimental condi-composition. During melting the melt was stirred manually tions. The antibacterial activity increases with the increaseby swirling about several times to ensure homogeneity and in IZD and vice versa.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUL 2011 VOL 98A, ISSUE 1 133
  3. 3. TABLE I. Glass Compositions, Density, Molar Volume, and Dissolution Rate of Studied Glasses Glass Composition (mol %) Density Molar Volume D.R (g cmÀ2 hÀ1)Glass No. P2O5 CaO Na2O Ag2O (gm cmÀ3) (cm3 molÀ1) Â 10À4I7 60 30 10 0 2.5064 43.1654 0.47I7Ag0.5 60 30 9.5 0.5 2.5352 43.0104 0.42I7Ag1 60 30 9 1 2.5605 42.9173 0.37I7Ag2 60 30 8 2 2.6064 42.8100 0.29I5 60 20 20 0 2.4820 43.8275 0.61I5Ag0.5 60 20 19.5 0.5 2.5096 43.6842 0.54I5Ag1 60 20 19 1 2.5401 43.4943 0.48I5Ag2 60 20 18 2 2.5869 43.3607 0.39RESULTS and dissolution rate decreases gradually with increasingDensity and molar volume Ag2O content. The pH changes measured during the dissolu-Table I shows the glass compositions studied and their den- tion of some studied glasses in distilled water at 37 C forsities, molar volumes, and dissolution rates. As shown in Ta- different time intervals up to 6 h are listed in Table II andble I, the replacement of Na2O by Ag2O leads to increase in are represented graphically in Figures 3 and 4. As can bethe density and decrease in the molar volume of studied observed from Figures 3 and 4, a fast drop in pH of distilledglasses. Whereas replacement of CaO by Na2O leads to water ($5.5) was seen through the first hour of glass disso-decrease in the density and increase in the molar volume. lution and then the pH decreased slowly with increasing time of dissolution. Figures 5 and 6 display the variation of pH of distilled water after 6 h with glass dissolution rateGlass dissolution and pH measurement (D.R). From Figures 5 and 6 it can be seen that the pH isThe dissolution of 60P2O5–30CaO–(10 À x) Na2O–xAg2O dependant on the glass dissolution rate. As shown in theseand 60P2O5–20CaO–(20 À x) Na2O–xAg2O glasses, x ¼ 0, figures, the pH decreases with the increase of glass dissolu-0.5, 1, and 2 mol %, in distilled water were investigated at tion rate. Figure 7 illustrates the relationship between pH of37 C for different time intervals up to 6 h and the pH distilled water after 6 h and silver oxide content in thechanges were measured during dissolution. The results of glass. From Figure 7, it can be seen that, the pH slightlyweight loss measurements for studied glasses are displayed increases with the gradual increase in Ag2O content.graphically in Figures 1 and 2. From these figures it can beseen that, the weight loss increases linearly with time.According to Figures 1 and 2, the weight loss is almost pro- Silver ions release profilesportional to the time of dissolution, thus the dissolution The concentrations of silver ions released into distilledrate could be calculated by fitting a straight line through water during the dissolution of some P2O5–CaO–Na2O–Ag2Othe data and at the same time passing through the origin. glasses at 37 C for different time intervals up to 6 h wereThe slope of this line gives a glass solubility value in terms determined using the flame atomic absorption spectrometryof the dissolution rate (g cmÀ2 hÀ1).The calculated dissolu- (FAAS). The results obtained are listed in Table II and dis-tion rates of the studied glasses are given in Table I. As played graphically in Figures 8 and 9. It can be seen fromseen in these Figures 1 and 2 and Table I, the weight loss Table III and Figures 8 and 9 that the concentration of silverFIGURE 1. Variation of weight loss with time during the dissolution of FIGURE 2. Variation of weight loss with time during the dissolution of60P2O5–30CaO–(10 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol 60P2O5–20CaO–(20 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol%, in distilled water at 37 C. %, in distilled water at 37 C.134 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
  4. 4. ORIGINAL ARTICLETABLE II. pH Values of Studied Glasses in Different Times pHGlass Code No. 1h 2h 3h 4h 5h 6hI7 3.67 3.62 3.58 3.54 3.49 3.44I7Ag0.5 3.72 3.67 3.63 3.58 3.53 3.48I7Ag1 3.83 3.76 3.72 3.67 3.61 3.56I7Ag2 4.04 3.96 3.90 3.83 3.78 3.72I5 3.53 3.49 3.45 3.40 3.36 3.33I5Ag0.5 3.60 3.54 3.49 3.45 3.41 3.37I5Ag1 3.72 3.63 3.58 3.52 3.46 3.42I5Ag2 3.88 3.79 3.73 3.66 3.60 3.55ions released during the glass dissolution increases with timeof dissolution and with increasing concentration of Ag2O con-tent in the glasses. From Tables I and III it can be seen that FIGURE 4. pH variation with time during the dissolution of 60P2O5– 20CaO–(20 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %.the rate of release of silver ions (it was calculated by fitting astraight line through the data, in Figures 8 and 9, and at thesame time passing through the origin. The slope of this line, depends on the glass composition, Ag2O content and type ofwhich gives the silver ion concentrations released into water the tested micro-organism. S. aureus was found to be thein terms of its release rate ppm hÀ1), still increases in spite most susceptible micro-organism to the tested antibacterialof that the glass dissolution rate slightly decreases with grad- glasses. The degree of susceptibility of the tested micro-ual increase of Ag2O content in glass. organisms to the tested antibacterial glasses was in this order S. aureus P. aeruginosa E. coli. Figures 11 and 12 also show the variation of the IZD with the Ag2O content inAntibacterial activity the glass. As displayed in Figures 11 and 12, a gradualThe antibacterial effects of undoped and Ag2O-doped P2O5– increase in the IZD (the antibacterial activity is proportionalCaO–Na2O glasses were tested in vitro against S.aureus as to the size of inhibition zone) was seen with increasingGþ, P.aeruginosa and E.coli as GÀ micro-organisms using Ag2O content in the glass. The biggest zone of inhibitionagar disk-diffusion assays. The results of agar disk-diffusion among silver-doped glasses was observed for the highest sil-assays conducted for 24 h at 37 C are shown in Figure 10. ver releasing glass against S. aureus micro-organism.The antibacterial activity of the glass was confirmed by thepresence of an inhibitory zone (i.e., zone of no bacterialgrowth) around each tested glass disk. The measured IZDs DISCUSSION(minus the diameter of the glass disk, 12 mm) as a function Density and molar volumeof Ag2O content are given in Figures 11 and 12, which show The density of phosphate glasses is affected by the packingthat all tested glasses (even silver free glasses) demonstrate degree of structural units, which depends on the phosphatedifferent antibacterial effects against the tested micro-organ- chain length and whether the branching group (PO5/2; Q3)isms as indicated by the clear zone around each glass disk. is present in the glass structure. The presence of branchingFigure 10 (a–f) also show that the glass antibacterial effect groups and a longer phosphate chain length will lead to aFIGURE 3. pH variation with time during the dissolution of 60P2O5– FIGURE 5. pH variation with D.R for 60P2O5–20CaO–(20 – x) Na2O–30CaO–(10 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %. xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %, in distilled water at 37 C.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUL 2011 VOL 98A, ISSUE 1 135
  5. 5. FIGURE 6. pH variation with D.R for 60P2O5–20CaO–(20 – x) Na2O– FIGURE 8. Concentrations of Agþ ions released during dissolutionxAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %. of 60P2O5–30CaO–(10 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %.lower density and loose structure of the glasses.10 The indicates that Agþ ions find rooms in the empty spaces ofreplacement of CaO by Na2O at constant P2O5 content the phosphate glass network and may cause further contrac-resulted in decrease in density. This trend can be explained tion of these rooms.rather simply as being due to the replacement of a heavierone (Caþ) by a lighter cation (Naþ). Also the phosphatechains will be bound tighter since the field strength, defined Glass dissolutionas the ratio of the ion valence (z) to the square of the bond It is well known that the reaction between a glass and anlength (a) between the ion and oxygen, z/a2, of the divalent aqueous solution is affected by several factors such as theCa2þ is higher than that of the monovalent Naþ, leading to composition of the glass, the pH of the solution, glass sur-an increase in density of the glasses. The density of the face area to solution volume ratio, temperature and time ofP2O5–CaO–Na2O glasses increases as Ag2O replaces Na2O, the reaction. When a glass comes into contact with water orwhich can be attributed to the replacement of lighter so- an aqueous solution, the release of cations from glass to thedium ions (molecular mass of Na2O ¼ 62) with the heavier aqueous solution usually proceeds by two main types ofsilver ions (molecular mass of Ag2O ¼ 231.77) in the glass chemical reactions depending on whether the cation occu-network. The replacement of CaO by Na2O at constant P2O5 pies a network forming or modifying site.11–13content resulted in increase in molar volume. The decreaseof molar volume with increasing CaO content is expectedsince the incorporation of such divalent Caþ2 cations that Leaching (selective dissolution). Leaching represents theare smaller than the monovalent Naþ cations will change type of chemical reaction in which network modifiers arethe glass structure (shorten the phosphate chain length) selectively extracted from glass by attacking solutions,and make it more compact. The decrease in the molar vol- where Hþ or H3Oþ ions from the aqueous solution replacesume with the gradual increase in the Ag2O concentration network modifiers through an ion exchange reaction. This FIGURE 9. Concentrations of Agþ ions released during dissolutionFIGURE 7. pH variation with Ag2O content for dissolution of some of 60P2O5–20CaO–(20 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, andP2O5–CaO–Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %. 2 mol %.136 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
  6. 6. ORIGINAL ARTICLETABLE III. Concentration of Released Silver and Release Rate of Silver Ions into Distilled Water During the Dissolution Concentrations of Released Silver Ions (ppm)Glass Code No. 1h 2h 4h 6h Silver Ions Release Rate (ppm hÀ1)I7Ag0.5 0.308 0.389 0.532 0.647 0.1216I7Ag1 0.477 0.874 1.481 2.019 0.3470I7Ag2 1.490 1.792 2.230 2.654 0.6629I5Ag0.5 0.673 0.956 1.669 2.489 0.4244I5Ag1 1.356 1.913 2.596 3.274 0.5775I5Ag2 1.978 2.338 3.112 3.796 0.6901type is addressed by a parabolic relation between the Hydration reaction. The glass exchanges its sodiumweight loss and time. ions with the hydrogen ions in water to carry out Na-H ion exchange reaction, resulting in the formation of a hydratedEtching (network dissolution). Etching represents the type layer on the glass surface at the glass–water interface.of chemical reaction by which network forming cations passinto the aqueous solution as a result of the breakdown of Network breakage. Under the attack of hydrogen ionsthe glass network structure at the leached layer solution and water molecules, the PAOAP bonds in hydrated layerinterface. This type is addressed by a linear relation break up and result in the destruction of the glass networkbetween the weight loss and time. and the release of chains of phosphates with different Van Wazer and Holst14 proposed a number of mecha- degrees of polymerization into the solution.nisms for the dissolution of phosphate glasses in relation to Bunker et al.15 proposed that the dominating reaction intheir structure. According to their polymeric structural the dissolution process of phosphate glass is the Na-H ionmodel, the basic unit in the network is the tetrahedral PO4 exchange reaction and divided the dissolution process intogroup that can be bonded to a maximum of three neighbor- two kinetic periods according to the profiles of dissolveding groups through the bridging oxygens. The addition of amount (q) versus time (t):modifier oxides disrupts the bridging PAOAP bonds and 1. A decelerating dissolution period where q is a linearlowers the number of branching PO4 tetrahedra. At a P2O5 function of t1/2.content of 50 mol %, the glass structure consists of long lin- 2. A uniform dissolution period where q is a linear functionear PO4 chains without branching tetrahedra. Dissolution of of t.phosphate glass, unlike silica glass, does not involve the ini-tial leaching of alkali ions from the surface. It is dominated Throughout the whole process, the phosphate glass dis-by congruent, or matrix leaching15 rather than the selective solves congruently which means that the dissolution prod-leaching that addresses the dissolution of silicate glasses.16 ucts in the solution have identical composition with that ofMoreover, the dissolution of phosphate glasses is minimized the bulk glass. On the other hand, Liu et al.19 proposed thatbetween pH 5 and 9, whereas that of silicate increases dras- the network breakage is the dominating reaction in the dis-tically at pH 9 and accelerated as the pH is elevated to 12. solution process after a careful study of both the pH changeThe reaction is highly complex and involves many processes. of solution and FTIR analysis of phosphate glass with theFor instance, water penetration and subsequent decomposi- composition of 50P2O5–25CaO–25Na2O and its solutiontion of a complex mixture leading to the formation of sub- before and after the dissolution process. They concludedstances completely different from the original glasses, and that the Na-H ion exchange reaction plays a role no moremoreover, these substances affect the course of the reaction. than moistening the glass surface and initiating the network According to the generally accepted theory of glass dis- breakage process. Gao et al.20 outlined that phosphate-con-solution,17,18 phosphate glasses dissolve in aqueous media trolled release glasses dissolve congruently and uniformly inin the following two interdependent steps that are similar aqueous media and their dissolution rates are dependent onto those of silicate glasses: the solution pH, temperature, and concentrations of phos- phate ions and calcium ions in the medium, but independ- ent on the stirring speed of the solution. The formation and development of hydrated layer depends on the diffusion and penetration of water molecules inside the bulk glass. (1) The nature of the hydration reaction is the dissociation of sodium ions from the [PO4] units, and the dissolution pro- cess is realized through the breakage of PAOAP bonds in the hydrated layer. Also the chelation effect of polyphos- phates with divalent ions on the glass-media interface has significant influence on the dissolution rate of phosphate (2) glasses.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUL 2011 VOL 98A, ISSUE 1 137
  7. 7. FIGURE 10. Photos of Petri dishes after conducting agar disk-diffusion assays at 37 C for 24 h with S. aureus, P. aeruginosa, and E.coli as testmicro-organisms. [Color figure can be viewed in the online issue, which is available at]138 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
  8. 8. ORIGINAL ARTICLE expected for the higher phosphate containing compositions, due to the breakdown of more PO4 groups thus creating more acidic species in the solution. Thus, the pH drop can be explained in terms of the glass compositions. The P2O5– CaO–Na2O glass compositions containing 60 mol % P2O5 belong to ultraphosphate glasses (acidic glass compositions). Accordingly, due to their high P2O5 contents the acid forma- tion reaction is much more dominant over the base forma- tion one and these glasses have an acidic reaction to water, and this account for the drop in the pH of the attacking water during glass dissolution. The above mentioned shift toward the acidic range was observed to be sharp at the be- ginning of the experiment, whereas it tends to level-off with time. According to Bunker et al.,15 this was attributed to the increase in the leachant from the glass to solution thatFIGURE 11. Variation of IZD with Ag2O content 60P2O5–30CaO–(10 – x) became concentrated enough to form a buffer solution. TheNa2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %. capacity of the buffer is sufficient to neutralize any change in the solution pH caused by further glass dissolution. As shown in Figures 1 and 2, the dissolution rates of Glasses having high dissolution rates showed a highersome P2O5–CaO–Na2O glasses in water decreases gradually decrease in pH than that showed by the glasses having lowas Ag2O replaces Na2O. In general, an explanation of the role dissolution rates. This may be due to that the high solubleof Ag2O in minimizing the dissolution rate in water is indi- glasses showed the breakdown of more PO4 groups, andcated by the solubility of Ag2O and Na2O in water. Ag2O has thus creating more acidic species in the solution than thatvery low solubility, even in hot water (0.0054 g per 100 cm3 showed by the low soluble glasses. Glasses containingof water), while Na2O react violently with water to form xAg2O showed similar trend to that of P2O5–CaO–Na2ONaOH. NaOH has solubility in hot water of 347 g per 100 glasses, but the pH drop shifted to slightly higher valuescm3 of water.21 Also Agþ ions retard the chemical attack by than that of P2O5–CaO–Na2O glasses. This observation canwater due to its large size and greater polarizability. Agþ is be explained by the gradual improvement in the chemicalsimilar to Pb2þ, has a large polarizability, hence the PAOAAg durability of P2O5–CaO–Na2O glasses due to the replacementgroup like the PAOAPb group has some covalent character, of Na2O by small amounts of Ag2O. As shown in Figures 6which may also explain the improvement in the chemical du- and 7, the pH shifted to slightly higher values as the disso-rability of the glass when Ag2O replaces the Na2O. lution rate decreases. The shift was obvious for glasses con- taining 2 mol % Ag2O due to their lowest dissolution rates.pH changesWhen glass reacts with an aqueous solution, both chemical Silver ions releaseand structural changes occur. In addition, as the dissolution An increase in concentrations of silver ions released into so-proceeds, accumulation of dissolution products causes both lution was observed with increasing time of dissolution,the chemical composition and pH of the solution to glass dissolution rate and with increasing Ag2O contents. Aschange.22 In general, during the dissolution of P2O5–CaO– expected, the highest levels of silver ions release wereNa2O glasses in distilled water, the pH of the solution is observed for the glass compositions having high dissolutionaffected by the relative dominance of one of the followingreactions over the other23: P2 O5 þ 3H2 O ¼ 2H3 PO4 Na2 O þ H2 O ¼ 2NaOH CaO þ H2 O ¼ CaðOHÞ2 Thus, the change of the solution pH toward acidity oralkalinity can be attributed to the relative dominance of oneof these reactions over the other. In the present work,P2O5–CaO–Na2O glasses I5, and I7 showed a sharp drop inpH of water after the first hour of glass dissolution (the so-lution became acidic quickly), and then a gradual slowdecrease in pH of water with time of dissolution wasobserved (Figs. 3 and 4). The contents of basic and acidicoxides in the glass composition play a significant role incontrolling the dominance of either base formation or acid FIGURE 12. Variation of IZD with Ag2O content 60P2O5–20CaO–(20 –formation reaction over each other. The pH drop was x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol %.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUL 2011 VOL 98A, ISSUE 1 139
  9. 9. rates. Also, glass compositions having high Ag2O content of pH or an increase of the phosphate ion concentration ofreleased more Agþ ions into solution than glasses having the media, and this resulted in promoting antibacterial ac-low Ag2O content. The silver ion release data correlated tivity. Therefore, the results of silver-free glass dissolution,well with that of the glass solubility data obtained. their pH changes during dissolution, and their antibacterial effects correlate well with each other. The antibacterialAntibacterial activity effect of the silver free-glasses may be attributed to otherIt was thought that these silver-free glasses would not show factors,27,28 for example, the ionic strength of the mediumany antibacterial effects against the tested micro-organisms. where high concentrations of calcium, sodium, and phos-Interestingly, these silver free glasses namely, I5 and I7 pro- phates ions likely to be released from the glass during dis-duced inhibitory zones of different sizes depending on the solution could cause perturbations of the membrane poten-chemical composition of the glass, the glass dissolution rate, tial of bacteria and such as osmotic effects caused by theand the type of the tested micro-organism. This antibacterial nonphysiological concentration of ions such as sodium, cal-effect of these silver-free glasses can be explained in terms cium, and phosphate dissolved from the glass and lead toof the glass composition, the glass dissolution rate, and the change in the osmotic pressure in the vicinity of the glass.pH changes of the medium. Valappil et al.24 observed a zone The exact mechanism of the antibacterial action of these sil-of inhibition for S. aureus, MRSA, and C. difficile in testing ver-free glasses is unknown. Therefore, we conclude thatthe antibacterial activity of gallium free 45P2O5–16CaO– the antibacterial effect observed with these glasses can be39Na2O glass. They attributed the antibacterial effect of this explained by the dramatic changes in the physicochemicalgallium free glass to the change in pH during the glass deg- characteristics of the culture medium (pH, ionic strength,radation. Pickup et al.25 investigated the antibacterial activ- and osmotic pressure), which occur as a consequence of theities of a Ga-doped sol-gel PBGs of composition (CaO)0.30 glasses dissolution. Thus, the antibacterial action of glass is(Na2O)0.20-x (Ga2O3)x (P2O5)0.50 where x ¼ 0 and 0.03 mol influenced by its chemical composition and the dissolution%. They observed a small zone of inhibition (7 mm) for the conditions in its surroundings.gallium-free glass and they attributed it to either a change The addition of Ag2O to P2O5–CaO–Na2O glass has beenin pH as the glass dissolves or by reduced water activity as found to potentate its antibacterial activity. Silver-dopedions leach out. The glass compositions investigated contain glasses showed increased antibacterial activities (depending60 mol % of P2O5 and due to their high P2O5 contents, upon the Ag2O content) more than silver-free glasses asthese glasses have acidic composition and this means that shown in Figure 10. This increase is attributed to the releasethe dissolution of such glasses change the pH and produce of the Agþ ions that are well-known as antibacterial metalacidic media (since more acidic phosphate ions are ions.29 An increase in the antibacterial activity as representedreleased). The pH value of the medium (in which the glass by the increase in the IZD was seen with increasing the Ag2Odissolves) was found to be related to the glass dissolution content in the glass as displayed in Figures 11 and 12. Thisrate. The dissolution rate of P2O5–CaO–Na2O glasses was in good agreement with the results of Agþ ions releasedepends on the contents of P2O5, CaO, and Na2O in the seen in water, whereas an increase in concentrations of silverglass. From the results of dissolution of such glasses, it was ions released from glass into water was seen with thefound that the glass dissolution rate is directly proportional increase in Ag2O content. Generally, in an aqueous mediumto either P2O5 or Na2O contents and inversely proportional or in presence of moisture, the silver-doped glass graduallyto CaO content. The glass dissolution study showed that the dissolves depending on its dissolution rate and during its dis-dissolution rate of I5 greater than I7. It is well known that26 solution, the silver ions (the antibacterial active agents)the acidity or alkalinity of the medium affects the growth of incorporated into its structure are released into the mediumbacteria. The pH affects the rate of enzyme action and plays and inhibit the growth of bacteria. Thus, the mechanism fora role in determining the ability of bacteria to grow or sur- antibacterial action of silver-doped glasses is bacterial growthvive in particular environments. Most bacteria survive near inhibition by the silver ions released from the glass.neutral conditions and grow optimally within a narrow Bellantone et al.30 investigated the antibacterial effects ofrange of pH between 6.7 and 7.5. Thus, the decrease in pH Ag2O-doped bioactive glasses on S. aureus, E. coli, andduring the dissolution of P2O5–CaO–Na2O glasses could P. aeruginosa. The antibacterial action of the silver-doped bio-explain the bacterial growth inhibition produced by such active glass was attributed to the leaching out of Agþ ionsglasses. The more the phosphate ions released, the lower is from the glass matrix. Kim et al.31 investigated the antimicro-the pH and greater the antibacterial effect. Thus, the low pH bial effects of various ceramics against E. coli using viableproduced by glass dissolution was certainly a critical factor count and growth rate studies. They concluded that Agþ ionsfor glass antibacterial effect. Overall, the antibacterial effect interfered with the metabolism of the micro-organism, thusof silver-free glasses was influenced by glass composition, inhibiting its growth. The exact mechanism of antibacterialglass dissolution rate, and the dissolution conditions in the action of silver ions is still unknown. Antibacterial mechanismsglass surroundings. This might explain at least some of the of silver ions might differ according to the species of bacteria.differences in the antibacterial action of glass with varying Generally, most antibacterial agents exert their antibacterialchemical compositions. The antibacterial activity of silver- action by four principal modes of action. These modes include:free glasses was closely related to the dissolution rate of inhibition of bacterial cell wall synthesis, inhibition of proteinthe glasses because high dissolution rates cause a decrease synthesis, inhibition of synthesis of bacterial RNA and DNA, or140 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
  10. 10. ORIGINAL ARTICLEinhibition of a metabolic pathway. In bacteria, silver ions are the time of glass dissolution. This behavior of glass disso-known to react with bacterial nucleophilic amino acid residues lution indicated that the mechanism of total dissolutionin proteins, and attach to sulphydryl, amino, imidazole, phos- of the glass network with no selective leaching of cationsphate, and carboxyl groups of membranes or enzymes, result- from glass is the predominating mechanism ofing in protein denaturation.32,33 Silver is also known to inhibit dissolution.a number of oxidative enzymes such as yeast alcohol dehydro- 3. The dissolution rate of P2O5–CaO–Na2O–Ag2O glassesgenase,34 the uptake of succinate by membrane vesicles,35 and was found to slightly decrease with the gradual replace-the respiratory chain of E. coli, as well as causing metabolite ment of Na2O by Ag2O.efflux36 and interfering with DNA replication.32 Holt and 4. Measurements of pH changes during dissolution ofBard37 examined the interaction of silver ions with the silver-free and silver-doped glasses in water revealed arespiratory chain of E. coli. They found that an addition of decrease of water pH with increasing time of glass disso- 10 lM AgNO3 to suspended or immobilized E. coli resulted lution. It was found that the magnitude of the pH dropin stimulated respiration before death, signifying uncoupling increases with the increase in glass dissolution rate. Sil-of respiratory control from ATP synthesis. This was a symp- ver-doped glasses showed less pH drop than silver-freetom of the interaction of Agþ with enzymes of the respiratory glasses and this was attributed to the Ag2O addition thatchain. Feng et al.38 studied the antibacterial effect of silver slightly decreased the glass dissolution rate.ions on E. coli and S. aureus and suggested that the antibacte- 5. In agar disk-diffusion assays, all the tested silver-free andrial mechanism was due to DNA not being able to replicate, silver-doped glasses demonstrated different antibacterialand proteins becoming inactivated after contact with effects (depending on the glass composition and the typeAgþ ions. of the tested micro-organism) against S. aureus, P. aerugi- One of the primary targets of Agþ ions, specifically at nosa, and E. coli micro-organisms as indicated by thelow concentrations, appears to be the Naþ-translocating clear zone (zone of no bacterial growth) around eachNADH: ubiquinone oxidoreductase system.39,40 Silver has tested glass disk.also been shown to be associated with the cell wall,41 cyto- 6. For silver-free glasses, an increase in bacterial growthplasm and the cell envelope.42 Chappell and Greville43 IZD was observed with the increase in the glass dissolu-acknowledged that low levels of Agþ ions collapsed the pro- tion rate and with the decrease in pH, whereas for silver-ton motive force on the membrane of bacteria, and this was doped glasses an increase in bacterial growth IZD wasreinforced by Mitchell’s work.44,45 Dibrov et al.46 showed observed with increasing Ag2O content.that low concentrations of Agþ ions induced a massive pro- 7. S. aureus as a Gþ bacterium was found to be the mostton leakage through the bacterial membrane, resulting in susceptible micro-organism to the tested antibacterialcomplete de-energization and, ultimately, cell death. Overall, glasses. The degree of susceptibility of the tested micro-there is consensus that surface binding and damage to organisms to the tested antibacterial glasses was foundmembrane function are the most important mechanisms for in this order S. aureus P. aeruginosa E. coli.the killing of bacteria by Agþ ions. 8. Finally, the results of this work suggested that the pre- The results of antibacterial activity showed that the sil- pared silver-free and silver-doped glasses hold promisever-free and silver-doped glasses exhibited different antibac- as antibacterial glasses and could offer many advantagesterial effects against the tested bacteria and the sensitivity over conventional organic antibacterial agents.of GÀ and Gþ bacteria to the antibacterial glasses was dif-ferent. A remarkable difference was seen between GÀ bacte-rium (E. coli) and Gþ bacterium (S. aureus), while a small REFERENCESdifference was found between the two GÀ bacteria. It is 1. Uzarski JR. M.Sc. Thesis, Investigations of Bacteria Viability on Surfaces Using x-functionalized Alkanethiol Self-Assembled Mono-known that the cell wall of GÀ bacteria is composed of high layers. The Virginia Polytechnic Institute, State University; 2006.proportion of phospholipids, lipopolysaccharides, and pro- 2. Jeon HJ, Yi SC, Oh SG. Preparation and antibacterial effects ofteins (the cell wall of GÀ bacterium is chemically more Ag-SiO2 thin films by sol-gel method. Biomaterials 2003;24:complex than that of Gþ bacteria), whereas peptidoglycan is 4921–4928. 3. Ahmed I, Abou Neel EA, Valappil SP, Nazhat SN, Pickup DM,the major component of the cell wall of Gþ bacteria. This Carta D, Carroll DL, Newport RJ, Smith ME, Knowles JC. Thefact would possibly contribute to the difference of antibacte- structure and properties of silver-doped phosphate- basedrial effects between GÀ and Gþ bacteria. glasses. J Mater Sci 2007;42:9827–9835. 4. Ahmed I, Lewis M, Olsen I, Knowles JC. Phosphate glasses for tis- sue engineering: Part 1. Processing and characterisation of a ter- nary-based P2O5-CaO-Na2O glass system. Biomaterials 2004;25:CONCLUSIONS 491–499.1. For P2O5–CaO–Na2O–Ag2O glasses, it was found that the 5. Mulligan AM, Wilson M, Knowles JC. Effect of increasing silver content in phosphate-based glasses on biofilms of Streptococcus density increases with replacement of Na2O by Ag2O. The sanguis. J Biomed Mater Res 2003;67A:401–412. molar volumes of glasses in the quaternary P2O5–CaO– 6. Ahmed I, Ready D, Wilson M, Knowles JC. Antimicrobial effect of Na2O–Ag2O systems calculated from their measured den- silver-doped phosphate-based glasses. J Biomed Mater Res 2006; 79A:618–626. sities showed the reverse trend to that of density. 7. Mulligan AM, Wilson M, Knowles JC. The effect of increasing2. The weight loss during the dissolution of P2O5–CaO– copper content in phosphate-based glasses on biofilms of Strep- Na2O–Ag2O glasses in water was almost proportional to tococcus sanguis. Biomaterials 2003;24:1797–1807.JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUL 2011 VOL 98A, ISSUE 1 141
  11. 11. 8. Boyd D, Li H, Tanner DA, Towler MR, Wall JG. The antibacterial Bactericidal effects of bioactive glasses on clinically important effects of zinc ion migration from zinc-based glass polyalkenoate aerobic bacteria. J Mater Sci Mater Med 2008;19:27–32. cements. J Mater Sci Mater Med 2006;17:489–494. 29. Lansdown ABG. Silver. I: Its antibacterial properties and mecha- 9. Geng P, Zhang W, Tang H, Zhang X, Jin L, Feng Z, Wu Z. Com- nism of action. J Wound Care 2002;11:125–130. parison of antibacterial ability of copper and stainless steel. Front 30. Bellantone M, Williams HD, Hench LL. Broad-Spectrum Bacteri- Chem China 2007;2:209–212. cidal Activity of Ag2O-Doped Bioactive Glass. Antimicrob Agents10. Shih PY, Chin TS. Preparation of lead-free phosphate glasses with Chemother 2002;46:1940–1945. low Tg and excellent chemical durability. J Mater Sci Lett 2001;20: 31. Kim TN, Feng QL, Kim JO, Wu J, Wang H, Chen GC, Cui FZ. Anti- 1811–1813. microbial effects of metal ions (Agþ, Cu2þ, Zn2þ) in hydroxyapa-11. Paul A. Chemistry of Glasses, Chapter 6, 2nd ed. London: tite. J Mater Sci Mater Med 1998;9:129–134. Chapman and Hall; 1990. 32. Russell AD, Hugo WB. Antimicrobial activity and action of silver.12. Clark DE, Pantano CG, Hench LL. Corrosion of Glass. New York: Prog Med Chem 1994;31:351–370. Magazines for Industry; 1977. 33. Percival SL, Bowler PG, Russell D. Bacterial resistance to silver in13. Douglas RW, El Shamy TM. Reactions of Glasses with Aqueous wound care. J Hosp Infect 2005;60:1–7. Solutions. J Am Ceram Soc 1967;50:1–8. 34. Snodgrass PJ, Vallee BI, Hoch FL. Effects of silver and mercu-14. Van Wazer JR, Holst KA. Structure and Properties of the Con- rials on yeast alcohol dehydrogenase. J Biol Chem 1960;235: densed Phosphates. J Am Chem Soc 1950;72:639. 504–508.15. Bunker BC, Arnold GW, Wilder JA. Phosphate glass dissolution in 35. Rayman MK, Lo TCY, Sanwal BD. Transport of succinate in Esche- aqueous solutions. J Non-Cryst Solids 1984;64:291. richia coli: characteristics of uptake and energy coupling with16. Jedlicka AB, Clare G. Chemical Durability of Commercial Silicate transport in membrane preparations. J Biol Chem 1972;247: Glasses. 1. Materials Characterization. J Non-Cryst Solids 2001; 6332–6339. 281:6–24. 36. Schreurs WJA, Rosenberg H. Effect of silver ions on transport17. Fernandez E, Gil FJ, Ginebra MP, Driessens FCM, Planell JA, Best and retention of phosphate by Escherichia col. J Bacteriol 1982; SM. Calcium phosphate bone cements for clinical applications. 152:7–13. Part I: solution chemistry. J Mater Sci Mater Med 1999;10: 37. Holt KB, Bard AJ. Interaction of silver(I) ions with the respiratory 169–176. chain of Escherichia coli: An electrochemical and scanning elec-18. Isard JO, Allnatt AR, Melling PJ. An improved model of glass dis- trochemical microscopy study of the antimicrobial mechanism of solution. Phys Chem Glasses 1982;23:185–189. micromolar’’ Ag. Biochemistry 2005;44:13214–13223.19. Liu QX, Chen XM, Li X. The hydrolysis of Na2OÁCaOÁ2P2O5 bio- 38. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A mechanistic glass. J Wuhan Univ Technol 1996;18:26–29. study of the antibacterial effect of silver ions on Escherichia coli20. Gao H, Tan T, Wang D. Dissolution mechanism and release and Staphylococcus aureus. J Biomed Mater Res 2000;52:662–668. kinetics of phosphate controlled release glasses in aqueous me- 39. Semeykina AL, Skulachev VP. Submicromolar Agþ increases pas- dium. J Control Release 2004;96:29–36. sive Naþ permeability and inhibits the respiration supported for-21. Peng YB, Day DE. High Thermal Expansion Phosphate Glasses. mation of Naþ gradient in Bacillus FTU vesicles. FEBS Lett 1990; Part 2. Glass Technol 1991;32:200–205. 269:69–72.22. Clark DE, Pantano CG, Hench LL. Corrosion of Glass. New York: 40. Hayashi M, Miyoshi T, Sato M, Unemoto T. Properties of respira- Magazines for Industry; 1979. tory chain-linked Na (+)-independent NADH-quinone reductase in23. El-Shafi NA, Ibrahim S, Ali AA. Chemical durability of some a marine Vibrio alginolyticus. Biochim Biophys Acta 1992;1099: P2O5-PbO-Al2O3-R2O glasses. Eur J Glass Sci Technol B 2009;50: 145–151. 45–51. 41. Rosenkranz HS, Carr HS. Silver sulfadiazine: effect on the growth24. Valappil SP, Ready D, Abou Neel EA, Pickup DM, Chrzanowski W, and metabolism of bacteria, Antimicrob Chemother 1972;2: O’Dell LA, Newport RJ, Smith ME, Wilson M, Knowles JC. Antimi- 367–372. crobial Gallium-Doped Phosphate-Based Glasses. Adv Funct 42. Goddard PA, Bull AT. Accumulation of silver by growing and non Mater 2008;18:732–741. growing population of Citrobacter intermedius B5. Appl Microbiol25. Pickup DM, Valappil SP, Moss RM, Twyman HL, Guerry P, Smith Biotechnol 1989;31:314. ME, Wilson M, Knowles JC, Newport RJ. Preparation, structural 43. Chappell JB, Greville GD. Effect of silver ions on mitochondrial characterisation and antibacterial properties of Ga- doped solgel adenosine triphosphatase. Nature 1954;174:930–931. phosphate-based glass. J Mater Sci 2009;44:1858–1867. 44. Mitchell P. Coupling of phosphorylation to electron and hydrogen26. Singleton P. Introduction to Bacteria, 2nd ed. New York: Wiley; transfer by a chemiosmotic type of mechanism. Nature 1961;191: 1992. 144–148.27. Stoor P, Soderling E, Salonen JI. Antibacterial effects of bioactive ¨ 45. Mitchell P. Chemiosmotic coupling in oxidative and photosyn- glass paste on oral microorganisms. Acta Odontol Scand 1998;56: thetic phosphorylation. Biol Rev 1966;41:445–502. 161–165. 46. Dibrov P, Dzioba J, Gosink KK, Hase C. Chemiosmotic mechanism28. Munukka E, Lepparanta O, Korkeamaki M, Vaahto M, Peltola T, of antimicrobial activity of Agþ in Vibrio cholerae. Antimicrob Zhang D, Hupa L, Ylanen H, Salonen JI, Viljanen MK, Eerola E. ¨ Agents Chemother 2002;8:2668–2670.142 AHMED ET AL. 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