This document describes a study on the preparation and characterization of antibacterial phosphate-based glasses doped with silver ions. Glasses with compositions of 60P2O5-20CaO-(20-x)Na2O-xAg2O and 60P2O5-30CaO-(10-x)Na2O-xAg2O with x=0, 0.5, 1, 2 mol% Ag2O were prepared. The glasses were characterized based on their density, dissolution rate in water, and pH changes during dissolution. The antibacterial activity against various bacteria was also evaluated based on inhibition zone diameters. The dissolution rate and antibacterial activity increased with higher Ag2O content and lower
Preparation and Characterization of Antibacterial Phosphate Glasses
1. Preparation and characterization of antibacterial
P2O5–CaO–Na2O–Ag2O glasses
A. A. Ahmed,1 A. A. Ali,1 Doaa A. R. Mahmoud,2 A. M. El-Fiqi1
1
Glass Research Department, National Research Centre, Dokki, Cairo 12622, Egypt
2
Natural and Microbial Products Laboratory, National Research Centre, Dokki, Cairo 12622, Egypt
Received 16 October 2010; revised 5 February 2011; accepted 22 February 2011
Published online 4 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33101
Abstract: 60P2O5–20CaO–(20 – x) Na2O–xAg2O and 60P2O5– An increase in the concentration of silver ions released from
30CaO–(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 observed
agar disk-diffusion assays at 37 C for 24 h. The antibacterial with the increase in the glass dissolution rate and with the
activity was deduced from the inhibition zone diameter (IZD), decrease in pH of water. Also, the IZD showed an increase
zone of no bacterial growth, measured under the stated ex- with increasing Ag2O content of silver-doped glasses. V 2011
C
perimental 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 in
water 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 silver
glasses into water during their dissolution were determined. ions and antibacterial effect
INTRODUCTION more, the degradation rate of such glasses can be tailored
An antibacterial agent is defined as a substance that either to suit the end application.4 The main advantage of PBGs
kills 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 and
have a broad spectrum of antibacterial activity, that is, effec- remain amorphous, where metal ions have good solubility
tive against a broad range of Gþ and GÀ bacteria, long last- in phosphate glass melts rather than silicate glass melts.
ing antibacterial action, low bacterial resistance, safety, Thus, the metal ions are incorporated into the phosphate
minimum 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 overall
als has attracted interest for many applications.2 Most inor- rate of degradation of the glass. Accordingly, PBGs can act
ganic 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 PBGs
ceramics, zirconium and calcium phosphates, and glasses. than silicate glasses (silica-based glasses are highly resistant
The 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 low
surface 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 so
bacterial glasses. Generally, glass is a material with high that the glasses form a more intimate connection to the
chemical 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 glasses
altering 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 low
can dissolve within a few hours in distilled H2O.3 Further- chemical durability and the property that glass can retain
Correspondence to: A. A. Ali; e-mail: ali_nrc@hotmail.com
132 V 2011 WILEY PERIODICALS, INC.
C
2. ORIGINAL ARTICLE
metal 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 slabs
bacterial 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 glass
ture, the glass will gradually dissolve and at the same time, samples were left overnight to cool slowly to room
silver, 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 were
rial glasses can be manufactured either by addition of an determined on bulk glass samples at room temperature
antibacterial agent to the glass batch prior to their manufac- using the simple Archimedes’s method [standard test
ture 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 of
their wide range of applications, for example, cosmetics,
Glass characterization
electrical appliances, fabrics and biomaterials as well as
X-ray diffraction (XRD). The amorphous nature of the
wastewater 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-ray
ing materials, inhibitors of aquatic microbes, cosmetics, or
diffraction spectra were obtained using a Bruker D8
medical applications. Antibacterial glass powders can be
Advance X-ray diffractometer at room temperature with
easily blended into plastics and coatings much like the zeo-
Ni-filtered Cu Ka radiation (k ¼ 0.15418 nm), generated at
lite 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 2y
production of some antibacterial glasses that do not need
from 4 to 70 .
special precautions or preparation conditions, and which
can have dissolution rates that can be controlled. For this
purpose, two base sodium calcium phosphate glasses, The glass dissolution test. The dissolution of the prepared
60P2O5–20CaO–20Na2O and 60P2O5–30CaO–10Na2O were glasses was carried out in distilled water at 37 C for time
selected. 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 behavior
EXPERIMENTAL PROCEDURES of glasses.
Glass preparation
Glasses having the following compositions 60P2O5–20CaO–
(20 – x) Na2O–xAg2O and 60P2O5–30CaO–(10 – x) Na2O– pH measurements. The pH changes of the distilled water
xAg2O glasses, x ¼ 0, 0.5, 1, and 2 mol % were prepared. during the dissolution of some undoped and silver-doped
All the batches used were prepared from chemically pure glasses were measured at 1-h intervals and up to 6 h using
grade chemicals in the powder form. P2O5 was introduced IQ 140 pH-meter (IQ, USA). The pH electrode was calibrated
as (NH4H2PO4) (99.0% Merck), calcium oxide (CaO) as cal- using pH calibration standards (Colourkey Buffer Solutions
cium carbonate (CaCO3) (99.5% SRL), sodium oxide (Na2O) BDH, UK).
as sodium carbonate (Na2CO3) (99.5% s.d.fine-chem), and
silver oxide (Ag2O) as silver nitrate (AgNO3) (99.9% FAAS determinations of silver ions released from glass
SRL).The appropriate amounts of the starting materials of into water. The amount of silver ions released from glass
each batch equivalent to 50 g glass were accurately into distilled water was determined by using GBC Flame
weighed, 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 the
range of 350–550 C in order to get rid of the gaseous
decomposition products of the batch materials, for example, Antibacterial activity test. The antibacterial activity of
H2O, 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 Culture
decomposition 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 deduced
heated 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 increase
by 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. 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 10À4
I7 60 30 10 0 2.5064 43.1654 0.47
I7Ag0.5 60 30 9.5 0.5 2.5352 43.0104 0.42
I7Ag1 60 30 9 1 2.5605 42.9173 0.37
I7Ag2 60 30 8 2 2.6064 42.8100 0.29
I5 60 20 20 0 2.4820 43.8275 0.61
I5Ag0.5 60 20 19.5 0.5 2.5096 43.6842 0.54
I5Ag1 60 20 19 1 2.5401 43.4943 0.48
I5Ag2 60 20 18 2 2.5869 43.3607 0.39
RESULTS and dissolution rate decreases gradually with increasing
Density 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 for
sities, molar volumes, and dissolution rates. As shown in Ta- different time intervals up to 6 h are listed in Table II and
ble I, the replacement of Na2O by Ag2O leads to increase in are represented graphically in Figures 3 and 4. As can be
the density and decrease in the molar volume of studied observed from Figures 3 and 4, a fast drop in pH of distilled
glasses. 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 rate
Glass dissolution and pH measurement
(D.R). From Figures 5 and 6 it can be seen that the pH is
The dissolution of 60P2O5–30CaO–(10 À x) Na2O–xAg2O
dependant on the glass dissolution rate. As shown in these
and 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 of
37 C for different time intervals up to 6 h and the pH
distilled water after 6 h and silver oxide content in the
changes were measured during dissolution. The results of
glass. From Figure 7, it can be seen that, the pH slightly
weight 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 be
seen that, the weight loss increases linearly with time.
According to Figures 1 and 2, the weight loss is almost pro- Silver ions release profiles
portional to the time of dissolution, thus the dissolution The concentrations of silver ions released into distilled
rate could be calculated by fitting a straight line through water during the dissolution of some P2O5–CaO–Na2O–Ag2O
the data and at the same time passing through the origin. glasses at 37 C for different time intervals up to 6 h were
The slope of this line gives a glass solubility value in terms determined using the flame atomic absorption spectrometry
of 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 from
seen in these Figures 1 and 2 and Table I, the weight loss Table III and Figures 8 and 9 that the concentration of silver
FIGURE 1. Variation of weight loss with time during the dissolution of FIGURE 2. Variation of weight loss with time during the dissolution of
60P2O5–30CaO–(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. ORIGINAL ARTICLE
TABLE II. pH Values of Studied Glasses in Different Times
pH
Glass Code No. 1h 2h 3h 4h 5h 6h
I7 3.67 3.62 3.58 3.54 3.49 3.44
I7Ag0.5 3.72 3.67 3.63 3.58 3.53 3.48
I7Ag1 3.83 3.76 3.72 3.67 3.61 3.56
I7Ag2 4.04 3.96 3.90 3.83 3.78 3.72
I5 3.53 3.49 3.45 3.40 3.36 3.33
I5Ag0.5 3.60 3.54 3.49 3.45 3.41 3.37
I5Ag1 3.72 3.63 3.58 3.52 3.46 3.42
I5Ag2 3.88 3.79 3.73 3.66 3.60 3.55
ions released during the glass dissolution increases with time
of 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 a
straight line through the data, in Figures 8 and 9, and at the
same time passing through the origin. The slope of this line, depends on the glass composition, Ag2O content and type of
which gives the silver ion concentrations released into water the tested micro-organism. S. aureus was found to be the
in terms of its release rate ppm hÀ1), still increases in spite most susceptible micro-organism to the tested antibacterial
of 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 in
Antibacterial activity the glass. As displayed in Figures 11 and 12, a gradual
The antibacterial effects of undoped and Ag2O-doped P2O5– increase in the IZD (the antibacterial activity is proportional
CaO–Na2O glasses were tested in vitro against S.aureus as to the size of inhibition zone) was seen with increasing
Gþ, P.aeruginosa and E.coli as GÀ micro-organisms using Ag2O content in the glass. The biggest zone of inhibition
agar 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 the
presence of an inhibitory zone (i.e., zone of no bacterial
growth) around each tested glass disk. The measured IZDs DISCUSSION
(minus the diameter of the glass disk, 12 mm) as a function Density and molar volume
of Ag2O content are given in Figures 11 and 12, which show The density of phosphate glasses is affected by the packing
that all tested glasses (even silver free glasses) demonstrate degree of structural units, which depends on the phosphate
different 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 branching
Figure 10 (a–f) also show that the glass antibacterial effect groups and a longer phosphate chain length will lead to a
FIGURE 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. FIGURE 6. pH variation with D.R for 60P2O5–20CaO–(20 – x) Na2O– FIGURE 8. Concentrations of Agþ ions released during dissolution
xAg2O 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 of
replacement 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 heavier
one (Caþ) by a lighter cation (Naþ). Also the phosphate
chains will be bound tighter since the field strength, defined Glass dissolution
as the ratio of the ion valence (z) to the square of the bond It is well known that the reaction between a glass and an
length (a) between the ion and oxygen, z/a2, of the divalent aqueous solution is affected by several factors such as the
Ca2þ 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 of
P2O5–CaO–Na2O glasses increases as Ag2O replaces Na2O, the reaction. When a glass comes into contact with water or
which can be attributed to the replacement of lighter so- an aqueous solution, the release of cations from glass to the
dium ions (molecular mass of Na2O ¼ 62) with the heavier aqueous solution usually proceeds by two main types of
silver 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.11–13
content resulted in increase in molar volume. The decrease
of molar volume with increasing CaO content is expected
since the incorporation of such divalent Caþ2 cations that Leaching (selective dissolution). Leaching represents the
are smaller than the monovalent Naþ cations will change type of chemical reaction in which network modifiers are
the 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 replaces
ume with the gradual increase in the Ag2O concentration network modifiers through an ion exchange reaction. This
FIGURE 9. Concentrations of Agþ ions released during dissolution
FIGURE 7. pH variation with Ag2O content for dissolution of some of 60P2O5–20CaO–(20 – x) Na2O–xAg2O glasses, x ¼ 0, 0.5, 1, and
P2O5–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. ORIGINAL ARTICLE
TABLE 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 0.1216
I7Ag1 0.477 0.874 1.481 2.019 0.3470
I7Ag2 1.490 1.792 2.230 2.654 0.6629
I5Ag0.5 0.673 0.956 1.669 2.489 0.4244
I5Ag1 1.356 1.913 2.596 3.274 0.5775
I5Ag2 1.978 2.338 3.112 3.796 0.6901
type is addressed by a parabolic relation between the Hydration reaction. The glass exchanges its sodium
weight loss and time. ions with the hydrogen ions in water to carry out Na-H ion
exchange reaction, resulting in the formation of a hydrated
Etching (network dissolution). Etching represents the type layer on the glass surface at the glass–water interface.
of chemical reaction by which network forming cations pass
into the aqueous solution as a result of the breakdown of Network breakage. Under the attack of hydrogen ions
the glass network structure at the leached layer solution and water molecules, the PAOAP bonds in hydrated layer
interface. This type is addressed by a linear relation break up and result in the destruction of the glass network
between 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 in
their structure. According to their polymeric structural the dissolution process of phosphate glass is the Na-H ion
model, the basic unit in the network is the tetrahedral PO4 exchange reaction and divided the dissolution process into
group that can be bonded to a maximum of three neighbor- two kinetic periods according to the profiles of dissolved
ing 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 linear
lowers 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 function
ear 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 of
Moreover, the dissolution of phosphate glasses is minimized the bulk glass. On the other hand, Liu et al.19 proposed that
between 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 change
The reaction is highly complex and involves many processes. of solution and FTIR analysis of phosphate glass with the
For instance, water penetration and subsequent decomposi- composition of 50P2O5–25CaO–25Na2O and its solution
tion of a complex mixture leading to the formation of sub- before and after the dissolution process. They concluded
stances completely different from the original glasses, and that the Na-H ion exchange reaction plays a role no more
moreover, 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 in
in the following two interdependent steps that are similar aqueous media and their dissolution rates are dependent on
to 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. 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 test
micro-organisms. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
138 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
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 that
FIGURE 11. Variation of IZD with Ag2O content 60P2O5–30CaO–(10 – x) became concentrated enough to form a buffer solution. The
Na2O–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 higher
some P2O5–CaO–Na2O glasses in water decreases gradually
decrease in pH than that showed by the glasses having low
as Ag2O replaces Na2O. In general, an explanation of the role
dissolution rates. This may be due to that the high soluble
of Ag2O in minimizing the dissolution rate in water is indi-
glasses showed the breakdown of more PO4 groups, and
cated by the solubility of Ag2O and Na2O in water. Ag2O has
thus creating more acidic species in the solution than that
very low solubility, even in hot water (0.0054 g per 100 cm3
showed by the low soluble glasses. Glasses containing
of water), while Na2O react violently with water to form
xAg2O showed similar trend to that of P2O5–CaO–Na2O
NaOH. NaOH has solubility in hot water of 347 g per 100
glasses, but the pH drop shifted to slightly higher values
cm3 of water.21 Also Agþ ions retard the chemical attack by
than that of P2O5–CaO–Na2O glasses. This observation can
water due to its large size and greater polarizability. Agþ is
be explained by the gradual improvement in the chemical
similar to Pb2þ, has a large polarizability, hence the PAOAAg
durability of P2O5–CaO–Na2O glasses due to the replacement
group like the PAOAPb group has some covalent character,
of Na2O by small amounts of Ag2O. As shown in Figures 6
which 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 changes
When glass reacts with an aqueous solution, both chemical Silver ions release
and 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. As
change.22 In general, during the dissolution of P2O5–CaO– expected, the highest levels of silver ions release were
Na2O glasses in distilled water, the pH of the solution is observed for the glass compositions having high dissolution
affected by the relative dominance of one of the following
reactions 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 or
alkalinity can be attributed to the relative dominance of one
of these reactions over the other. In the present work,
P2O5–CaO–Na2O glasses I5, and I7 showed a sharp drop in
pH of water after the first hour of glass dissolution (the so-
lution became acidic quickly), and then a gradual slow
decrease in pH of water with time of dissolution was
observed (Figs. 3 and 4). The contents of basic and acidic
oxides in the glass composition play a significant role in
controlling 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. rates. Also, glass compositions having high Ag2O content of pH or an increase of the phosphate ion concentration of
released 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 antibacterial
Antibacterial activity effect of the silver free-glasses may be attributed to other
It was thought that these silver-free glasses would not show factors,27,28 for example, the ionic strength of the medium
any 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 the
and 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 to
of 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 that
the antibacterial activity of gallium free 45P2O5–16CaO– the antibacterial effect observed with these glasses can be
39Na2O glass. They attributed the antibacterial effect of this explained by the dramatic changes in the physicochemical
gallium 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 the
ities 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 been
in pH as the glass dissolves or by reduced water activity as found to potentate its antibacterial activity. Silver-doped
ions leach out. The glass compositions investigated contain glasses showed increased antibacterial activities (depending
60 mol % of P2O5 and due to their high P2O5 contents, upon the Ag2O content) more than silver-free glasses as
these glasses have acidic composition and this means that shown in Figure 10. This increase is attributed to the release
the dissolution of such glasses change the pH and produce of the Agþ ions that are well-known as antibacterial metal
acidic media (since more acidic phosphate ions are ions.29 An increase in the antibacterial activity as represented
released). The pH value of the medium (in which the glass by the increase in the IZD was seen with increasing the Ag2O
dissolves) was found to be related to the glass dissolution content in the glass as displayed in Figures 11 and 12. This
rate. The dissolution rate of P2O5–CaO–Na2O glasses was in good agreement with the results of Agþ ions release
depends on the contents of P2O5, CaO, and Na2O in the seen in water, whereas an increase in concentrations of silver
glass. From the results of dissolution of such glasses, it was ions released from glass into water was seen with the
found that the glass dissolution rate is directly proportional increase in Ag2O content. Generally, in an aqueous medium
to either P2O5 or Na2O contents and inversely proportional or in presence of moisture, the silver-doped glass gradually
to 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 medium
bacteria. The pH affects the rate of enzyme action and plays and inhibit the growth of bacteria. Thus, the mechanism for
a role in determining the ability of bacteria to grow or sur- antibacterial action of silver-doped glasses is bacterial growth
vive 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 of
range of pH between 6.7 and 7.5. Thus, the decrease in pH Ag2O-doped bioactive glasses on S. aureus, E. coli, and
during 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 ions
glasses. 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 viable
produced by glass dissolution was certainly a critical factor count and growth rate studies. They concluded that Agþ ions
for glass antibacterial effect. Overall, the antibacterial effect interfered with the metabolism of the micro-organism, thus
of silver-free glasses was influenced by glass composition, inhibiting its growth. The exact mechanism of antibacterial
glass dissolution rate, and the dissolution conditions in the action of silver ions is still unknown. Antibacterial mechanisms
glass 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 antibacterial
chemical 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 protein
the glasses because high dissolution rates cause a decrease synthesis, inhibition of synthesis of bacterial RNA and DNA, or
140 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES
10. ORIGINAL ARTICLE
inhibition 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 dissolution
in proteins, and attach to sulphydryl, amino, imidazole, phos- of the glass network with no selective leaching of cations
phate, and carboxyl groups of membranes or enzymes, result- from glass is the predominating mechanism of
ing 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 glasses
genase,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 of
Bard37 examined the interaction of silver ions with the silver-free and silver-doped glasses in water revealed a
respiratory 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 drop
in 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-free
tom of the interaction of Agþ with enzymes of the respiratory glasses and this was attributed to the Ag2O addition that
chain. 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 and
rial mechanism was due to DNA not being able to replicate, silver-doped glasses demonstrated different antibacterial
and proteins becoming inactivated after contact with effects (depending on the glass composition and the type
Agþ 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 the
low concentrations, appears to be the Naþ-translocating clear zone (zone of no bacterial growth) around each
NADH: 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 growth
plasm 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 was
reinforced 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 most
ton leakage through the bacterial membrane, resulting in susceptible micro-organism to the tested antibacterial
complete 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 found
membrane 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 promise
ver-free and silver-doped glasses exhibited different antibac- as antibacterial glasses and could offer many advantages
terial 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-
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142 AHMED ET AL. ANTIBACTERIAL P2O5–CaO–Na2O–Ag2O GLASSES