Antibacterial nanosized silver substituted hydroxyapatite:

Synthesis and characterization

N. Rameshbabu,1 T.S. Sampath Kumar,1 T.G. Prabhakar,2 V.S. Sastry,3 K.V.G.K. Murty,4 K. Prasad Rao1
1
Department of Metallurgical and Materials Engineering, Indian Institute of Technology Madras,
Chennai 600036, India
2
Department of Veterinary Microbiology, Madras Veterinary College, Tamilnadu Veterinary and
Animal Sciences University, Chennai 600007, India
3
Materials Science Division, Indira Gandhi Centre for Atomic Research, Kalpakkam 603102, India
4
AU-KBC Research Centre, M.I.T. Campus of Anna University, Chennai 600044, India

Received 25 February 2005; revised 22 May 2006; accepted 5 June 2006

Published online 9 October 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30958

Abstract: The silver (0.5–3 at %) substituted nanosize
hydroxyapatites (AgHAs) were synthesized by microwave
processing. The X-ray diffraction (XRD) peaks are very
broad, indicating that the AgHAs were of nanosize (30 nm).
Transmission electron microscopy analysis shows needle-
like morphology of AgHA, having length 60–70 nm and
width 15–20 nm. The AgHA phase was stable up to
7008C without any secondary phases. The antibacterial
effect of AgHA against Escherichia coli and Staphylococcus
aureus was observed by spread plate method, even for
low concentration of silver ions (0.5%) with 1
105 cells/
mL of respective bacterial culture, after a 48 h incubation
period. However, some colonies of E. coli were seen with
a high dose of 1
108 cells/mL after 24 h. The zone of
inhibition by disc diffusion test method was found to
vary with the amount of silver in the sintered AgHA pel-
lets, for both the bacteria, after 24 h of inoculation. Osteo-
blast cell attachment in varying density was noticed on
AgHA samples with 0.5, 1.0, and 1.5% silver substitution.
However, osteoblast spreading was significantly greater
on 0.5% AgHA compared to 1.0 or 1.5% substituted
AgHA samples. Thus, the low amount of AgHA has a
potential of minimizing the risk of bacterial contamina-
tion, without compromising the bioactivity, and is ex-
pected to display greater biological efficacy in terms of
osseointegration. Ó 2006 Wiley Periodicals, Inc. J Biomed
Mater Res 80A: 581–591, 2007
Key words: hydroxyapatite; nanoparticle; microwave pro-
cessing; antibacterial; osteoblast

INTRODUCTION ical devices.3 Silver and silver-based compounds are

The biomaterial-centered infection is one of the
main causes of revision surgery.1 The presence of
implant materials inside the body will interfere with
the host defense mechanism and influence the clini-
cal dose of antibiotics that is needed to protect
against infection. The concentration of antibiotic
loaded in implant materials should be high enough
to prevent bacterial infections at the biomaterial tis-
sue interface.2 However, the antibiotic released from
the implant materials in the serum levels should be
below the threshold required for systemic toxicity,
so that no side effects are expected. The use of silver
has recently become one of the preferred methods to
confer microbial resiliency on biomaterials and med-
Correspondence to: T.S.S. Kumar; e-mail: tssk@iitm.ac.in
' 2006 Wiley Periodicals, Inc.
highly antimicrobial by virtue of their antiseptic
properties to as many as 16 kinds of bacteria, includ-
ing Escherichia coli and Staphylococcus aureus.4 An
antimicrobial agent is an agent that has antiviral,
antibacterial, and/or antifungal properties. Antivi-
rals and antifungals are capable of killing or sup-
pressing the replication of viruses and fungi, res-
pectively. Antibacterials may be bacteriostatic or
bactericidal. Bacteriostat inhibits the growth of mi-
croorganisms, while bactericide kills the microor-
ganisms. Silver-based antimicrobials capture much
attention, because of the low toxicity of the active
Ag ion to human cells,5,6 as well as being a long last-
ing biocide with high thermal stability and low vola-
tility. Bioactive glass doped with 3 wt % Ag2O con-
ferred antimicrobial properties of the glass, without
compromising the glass bioactivity.3
Hydroxyapatite [HA, Ca10(PO4)6(OH)2] has been
extensively used as an implant material, because of
582
its similarity with human bone composition and
thereby its ability to form a strong bond to the
human hard tissue.7 However proteins, amino acids,
and other organic substances are adsorbed easily on
HA, which in turn favors the adsorption and replica-
tion of the bacteria in HA. Silver-loaded HA powder
has shown antibacterial effects, both in nutrient-rich
and poor environments.8 The substitution of Agþ
(1.28 Å) ions takes place for Ca2þ (0.99 Å) preferen-
tially in the Ca(1) site of HA, and this leads to an
increase in the lattice parameters linearly with the
amount of silver added in the range of atomic ratio
Ag/(AgþCa) between 0 and 0.055.9 The silver con-
taining HA coating prepared through ion exchange
reaction in solution also exhibited excellent antimi-
crobial activity.2,10 The metallic silver has been used
to reinforce the sintered HA pellets by taking the
advantage of the ductility of the silver, according to
the crack-bridging mechanism operated by the elas-
toplastic stretching of unbroken silver ligaments
along the crack wake.11 However, the extent of silver
substitution for calcium and its effect on the struc-
tural stability of the nano HA has not been clearly
established.
In recent years, much attention has been paid to
the synthesis and characterization of nanomaterials
because of their interesting properties, which mainly
come from the high surface/volume ratio. Nanocrys-
talline HA has proved to be of greater biological effi-
cacy in terms of osteoblast adhesion, proliferation,
osseointegration, and formation of new bone on its
surface.12 Recently, nanocrystalline silver has shown
excellent antimicrobial activity compared to other
available silver antimicrobial agents such as silver
nitrate or silver sulfadiazine, not only due to the
rapid release of silver cation (Agþ), but also due to
the release of other silver species such as Ag0 as
well.13 Nanosized HA is densified with minimal or
no sintering additives at substantially lower temper-
atures and demonstrates improved strength and
fracture toughness compared to micron-sized HA.14
The microwave synthesis is a fast, simple, and effi-
cient method to prepare nanosized materials,15 with
narrow particle size distribution due to fast homoge-
nous nucleation.16 Microwave plays an important
role in reactions in aqueous media and has been
used for preparing HA in 25 min.17 Precipitation of
nanosized HA and calcium phosphate nanowhiskers
using microwave irradiation has been recently re-
ported.18 The objective of the present study was to
synthesize nanosized silver substituted HA (AgHA)
using microwaves and to demonstrate its usefulness
as a new bactericidal material against two important
bacteria, gram negative E. coli and gram positive
S. aureus. The effect of Ag substitution in HA to func-
tion satisfactorily as a bone substitute material will
also be evaluated by osteoblast cell culture method.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
RAMESHBABU ET AL.
MATERIALS AND METHODS
Synthesis
Analytical grade calcium hydroxide [Ca(OH)2, E. Merck,
Germany], diammonium hydrogen phosphate [DAP,
(NH4)2HPO4, E. Merck], and silver nitrate [Ag(NO)3, S.R.L.,
India] were used for the preparation of silver substi-
tuted hydroxyapatite (AgHA) nanoparticles. The selected
precursor materials will maintain the necessary pH condi-
tions (pH >> 9.0) for the synthesis without any ammonium
hydroxide addition, which is generally added to maintain
the pH condition for the synthesis of HA with other precur-
sor materials. A total of eight compositions were prepared
with different silver contents of x ¼ 0.05, 0.10, 0.15, 0.2, 0.3,
0.4, 0.5, and 0.6, to check the extent of silver substitution for
calcium in the HA [Ca10xAgx(PO4)6(OH)2]. These composi-
tions can be further referred to as 0.5AgHA, 1.0AgHA,
1.5AgHA, 2AgHA, 3AgHA, 4AgHA, 5AgHA, and 6AgHA,
according to their silver content with HA. The amount of
the reactants was calculated based on the calcium þ silver:
phosphorus molar ratio of 10:6. The silver nitrate was first
dissolved in deionized distilled water (0.25M solution), and
it was added to 0.3M calcium hydroxide suspension in
deionized distilled water under stirring conditions. The
0.3M DAP solution was added to the silver nitrate dis-
solved calcium hydroxide aqueous suspension for 5 min
under vigorous stirring conditions. The amount of silver ni-
trate and calcium hydroxide has been varied, in order to
increase silver content from 0.5AgHA to 6AgHA. However,
calcium þ silver:phosphorus molar ratio of 10:6 has been
maintained in all the compositions. For comparison, nano-
sized pure HA was also prepared with calcium:phosphorus
molar ratio of 10:6. However, silver nitrate was not used for
pure HA preparation, keeping the remaining procedure
being the same. For all preparations, the precursor solution
in an uncovered glass beaker was immediately subjected to
the microwave irradiation for about 30 min in a domestic
microwave oven (BPL India, 2.45 GHz, 800 W power). The
crystallization of AgHA and its aging occurs under the
microwave irradiation in a short time of about 30 min.
Microwave irradiation time was optimized based on the lit-
erature17 and the author’s experience in the synthesis of
nanosized HA using Ca(OH)2 and DAP as calcium and
phosphorus precursors.19 The precipitate was thoroughly
washed with distilled water to remove impurity ions (NH4þ,
NO3). The product obtained after filtration was oven-dried
at 908C for overnight, and the flakes were powdered using
an agate mortar and pestle. Roughly, about 25 g of AgHA
was prepared in each batch. The characterization was carried
out for the powder in the oven-dried condition (further
referred to as synthesized condition). A small amount of
powder of each composition was heat-treated at 9008C for
2 h in box furnace at a ramp speed of 108C/min and fur-
nace-cooled (further referred as heat-treated condition).
Compaction
Compacts were produced by pressing uniaxially 0.6 g of
AgHA powder, in a 12-mm diameter stainless steel die, at
ANTIBACTERIAL NANOSIZED HYDROXYAPATITE
200 MPa. The powder compacts were sintered in a box
furnace at 9008C for 2 h at a ramp speed of 108C/min, and
furnace-cooled (further referred as sintered pellets).
Characterization
The AgHA powder samples were characterized by X-
ray powder diffraction (XRD) method (Shimadzu, XD-D1
diffractometer, Japan), with Cu Ka radiation (l ¼ 0.154056
nm) for phase analysis, crystal size measurement, and cell
parameter calculation. Data were collected over the 2y
range of 20–608, using a step size of 0.028 and step time of
2 s. The average crystallite size of the samples was calcu-
lated by using the Scherrer’s formula. The cell parameters
of AgHA samples were calculated by least-squares fit
method. The input data were 2y-values and corresponding
indices (hkl) of the diffraction lines in the range from 208
2y to 608 2y, with relative intensities above 5. The func-
tional groups present in HA were ascertained by Fourier
transform infrared spectroscopy (FTIR, Perkin Elmer, Spec-
trum One, USA) over the region 450–4000 cm1 in pellet
form for the powder samples of 1 mg mixed with spectro-
scopic grade KBr (Merck) of 200 mg. Spectra were
recorded at 4 cm1 resolution, averaging 80 scans. The
powder size and morphology were examined using a
transmission electron microscopy (TEM, Philips, CM12
STEM, Netherlands). For TEM analysis, the powder sam-
ple was ultrasonically dispersed in ethanol to form very
dilute suspensions, and then few drops were deposited on
the carbon-coated copper grids. The AgHA sintered pellets
were also analyzed for crystallite size with a atomic force
microscope (AFM, Digital Instruments, Nanoscope IV,
USA) in a contact mode, with a silicon nitride probe.
Antibacterial assessment
Depending on the application, the HA can be used in
the powder form as well as the sintered compacts, and so
the antibacterial action of AgHA was studied for both the
powder and pellet forms. The E. coli (strain 025) and
S. aureus (Cowan strain) bacteria were used for the present
study (Tamil Nadu Veterinary and Animal Sciences Uni-
versity, Chennai, India). Antibacterial action of AgHA
powder was studied by the spread plate method. One
milligram of each AgHA (0.5, 1.0, and 1.5AgHA) was
mixed individually with 1 mL of phosphate buffer solution
(pH 7.4), containing 1
105 cells/mL E. coli and S. aureus
separately, in flat-bottom test tubes. Each tube was shaken
at 200 rpm, at 308C, for 24 h. One hundred microliters of
shaken AgHA solutions were placed into petri plates in
duplicate and 15 mL of molten tryptose soy agar was over-
laid onto the inoculum, spread evenly in clockwise–anti-
clockwise directions, and left undisturbed for the agar to
solidify. These plates after solidification were incubated at
378C for 24 and 48 h. The colony formation was examined
and photographed. These tests were also done at higher
bacterial concentration of 1
108 cells/mL of E. coli and
S. aureus. Antibacterial action of AgHA pellets were
studied by the disc diffusion test method (commonly
known as the Kirby–Bauer disc diffusion method or anti-
583
biotic sensitivity test). Mueller–Hinton agar was cast into
the petri plates, and the plates containing the nutrient me-
dium were evenly inoculated with the test organisms of
E. coli and S. aureus separately. The AgHA disc specimens
with different Ag concentrations, sintered at 9008C for 2 h,
were planted onto the agar plates. All the plates were
incubated at 378C for 24 h and were examined for a zone
of inhibition of bacterial growth around the discs.
Cell adhesion test
To study the biocompatibility of the AgHA samples, an
in vitro cell adhesion test was performed on AgHA (0.5, 1,
and 1.5AgHA) samples, using well-characterized human
osteoblast cell line (HOS, National Centre for Cell Science,
Pune, India). These cells were cultured in MEM supple-
mented with 10% fetal bovine serum (FBS), streptomycin
(S, 100 mg per mL), and penicillin (P, 100 mL per mL),
under standard cell culture conditions (sterile chamber
maintained at 378C and a humidified environment: 5%
CO2/95% air). All substrates were autoclaved for steriliza-
tion purposes at 1208C for 30 min. The sintered pellets
were placed in each well of the 24-well tissue culture plate,
before seeding with 1
104 cells/well (3500 cells/cm2), in
DMEM supplemented with 10% FBS and 1% P/S. HOS
cells seeded on conditioned material (tissue culture poly-
styrene) were used as a test control. The cells were then
allowed to adhere in standard cell culture conditions for
24 h at 378C in a humidified atmosphere of 95% air and
5% carbon dioxide. After the prescribed time (24 h), non-
adherent cells were removed by rinsing three times with
phosphate buffered saline (PBS), while adherent cells were
fixed with 3% glutaraldehyde. Cell-seeded AgHA samples
were processed for scanning electron microscopy analysis
(SEM, Hitachi, S2400, Japan) by dehydrating through a se-
ries of ethanol concentrations (30%, 55%, 70%, 100% (v/v)
in distilled water), followed by isoamylacetate and critical
point drying. Dried scaffolds were sputter-coated with
gold, prior to analysis.
RESULTS AND DISCUSSION
The XRD patterns of the AgHA samples in as-syn-
thesized condition are shown in Figure 1. The XRD
peaks were markedly broader, which suggested that
AgHA particles were nanosized. All the patterns are
found to be nearly similar and correspond to the
hexagonal HA crystal (JCPDS 9-432). However, the
samples with silver content x  0.4 clearly shows
the presence of silver phosphate (Ag3PO4, JCPDS 6-
505) peak at 2y * 36.68. The peak broadening of the
XRD reflection was used to estimate the average
crystallite size in a direction perpendicular to the
crystallographic plane, based on Scherrer’s formula
Xs ¼ Kl/b cosy, where Xs is the average crystallite
size (nm); K is the shape factor (K ¼ 0.9); l is the
wavelength of the X-rays (l ¼ 0.154056 nm for Cu
Ka radiation); b is the broadening of the diffraction
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
584
Figure 1. The XRD patterns of the 0.5AgHA (a), 1.0AgHA
(b), 1.5AgHA (c), 2AgHA (d), 3AgHA (e), 4AgHA (f),
5AgHA (g), 6AgHA (h) samples in as synthesized condi-
tion. ‘‘*’’ marked peaks are Ag3PO4 and all others are HA
peaks.
peak measured at half of its maximum intensity (in
radians), and y is the Bragg’s diffraction angle (8).
The diffraction peak at 25.98 was chosen for calcula-
tion of the crystallite size; since it is sharper and iso-
lated from others. This peak assigns to (002) Miller’s
plane family and shows the crystal growth along the
c-axis of the HA crystalline structure. The estimated
crystallite sizes were listed in Table I.
The thermal stability of the AgHA samples were
studied by heating a small amount of the sample to
9008C for 2 h and cooling in air to room tempera-
ture. The XRD patterns of the 9008C heat-treated
samples are shown in Figure 2. All the heat-treated
samples show marked improvement in crystallinity,
with the separation of (211) and (112) diffraction
peaks. The presence of b-TCP phase (JCPDS 9-169)
was noticed for AgHA samples with x  0.15. The
heat-treated AgHA samples with x ¼ 0.05 and 0.10
are also free of calcium oxide phase, characterized
typically by peak at 2y of *37.58. However, the me-
tallic silver with a XRD peak at 2y * 38.18 (JCPDS
TABLE I
Crystal Size and Unit Cell Parameters of the HA Phase in AgHA Samples
Xs(002) in Xs(002) of
As-Synthesis Powders at 9008C
Sample Condition (nm) for 2 h (nm)
0.5AgHA 30 56
1.0AgHA 32 50
1.5AgHA 31 53
2AgHA 31 51
3AgHA 31 52
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
RAMESHBABU ET AL.
Figure 2. The XRD patterns of the 0.5AgHA (a), 1.0AgHA
(b), 1.5AgHA (c), 2AgHA (d), 3AgHA (e), 4AgHA (f),
5AgHA (g), 6AgHA (h) samples in 9008C heat-treated con-
dition. HA peaks are indexed and ‘‘*’’ marked peaks are
b-TCP and ‘‘þ’’ marked peaks are Ag.
4-783) was observed for all the 9008C heat-treated
samples. To elucidate further the role of Ag substitu-
tion in lowering the thermal stability of HA, both
0.5AgHA and 1.5AgHA samples were heated to 700,
750, and 8008C for 4 h and furnace-cooled to room
temperature in air. The XRD pattern of the 7008C
heated samples of 0.5AgHA and 1.5AgHA (Fig. 3) is
found to be without any impurity phases. Presence
of metallic silver was observed in both the AgHA
samples heated to 8008C (Fig. 3). In Table II, the var-
ious phases observed along with AgHA samples in
as-synthesized condition as well as at selected heat-
treated conditions (700, 750, and 8008C for 4 h and
9008C for 2 h) are summarized. Since there are no
impurity phases in 0.5AgHA, 1.0AgHA, 1.5AgHA,
2AgHA, and 3AgHA powders in as-synthesis condi-
tion and Ag3PO4 impurity phase has been observed
in 4AgHA, 5AgHA, and 6AgHA powders in as-syn-
thesis condition, the XRD results, thus, indicate sil-
ver substitution for calcium in HA below 4 at %.
The XRD results also indicate that the AgHA phase
Cell Parameters
Xs(002) of Pellets (As-Synthesis Condition)
at 9008C for
2 h (nm) a-Axis (Å) c-Axis (Å)
50 9.393 6.879
49 9.398 6.882
49 9.403 6.887
51 9.410 6.891
50 9.419 6.899
ANTIBACTERIAL NANOSIZED HYDROXYAPATITE 585

of the HA, may also influence the substitution.

Figure 3. The XRD patterns of the 700, 750, and 8008C
heated 0.5AgHA and 1.5AgHA samples.
was stable up to 7008C without any secondary phase
formation. The nanosized pure HA prepared by
microwave processing using calcium hydroxide and
DAP was thermally stable at 9008C.19
The cell parameters of AgHA samples, calculated
by least-squares fit method, were found to increase
with the amount of silver substitution, as shown in
Table I. The increase in the cell parameters may be
due to the substitutions of larger size Agþ (1.28 Å)
ions for Ca2þ (0.99 Å) ions. The amount of silver
substituted was found to be less compared to the
value of 0.55 reported.9 As silver is a noble element,
apart from the Agþ size effect, polarizabilty, charge,
and chemical nature of silver, as well as crystal size
Hence, the smaller amount of silver substitution in
nanosize AgHA was synthesized in the present
study. Although the Ca10xAgx(PO4)6(OH)2x&x sa-
mples were prepared by wet synthesis, the role of
silver in the thermal stability of HA was not
reported.9 The vacancy at the hydroxyl site due to
charge imbalance caused by Agþ for Ca2þ ions
seems to lower the thermal stability of the AgHA
samples.
The TEM morphology of the HA and 0.5, 2, and
5AgHA samples in as-synthesized condition is
shown in Figure 4(a–d). All the particles were found
to be of nanosize. The HA and 0.5AgHA particles
were of nanosize with needle-like morphology, with
width ranging from 15 to 20 nm and length around
60–70 nm, the size comparable to that of bone apa-
tite.20 They were a bit thinner and longer (aspect ra-
tio * 4), with more irregular and less clear contour.
In addition, the particles showed high tendency to
agglomerate. The 2AgHA and 5AgHA particles are
also of needle-like morphology with particle size
(70 nm length and 15–20 nm width), without being
much dependent on the amount of silver content.
The 5AgHA particles were more regular with clear
contours and less agglomeration. This may be due to
the effect of surrounding silver phosphate phase.
Both the XRD and TEM results show that the AgHA
particle synthesized by microwave processing was
nanosized and has a narrow size distribution. This is
because, microwave irradiation of the reaction mix-
tures results in rapid heating, particularly those con-
taining microwave active polar molecules such as
water, acetonitrile, CH2Cl2, ethanol, and DMF.21 As
a consequence, the precipitation of particles from
such solutions tends to be rapid and nearly simulta-
neous. This leads to very small particle sizes and
narrow particle size distributions, in the final prod-
ucts.16 The TEM morphology of the 9008C heat-
treated HA and 1.5AgHA powder samples are
shown in Figure 5(a,b). The powder morphology of
the heat-treated samples has been completely
changed from needle to plate shape and results in
an increase in particle size (80–100 nm).

TABLE II
List of Phases Present in AgHA Powder Samples in As-Synthesized Condition and Heat-Treated Conditions
Sample As-Synthesis Condition 9008C 7008C 7508C 8008C

0.5AgHA HA HA, Ag HA HA HA, Ag

1.0AgHA HA HA, Ag – – –
1.5AgHA HA HA, Ag, bTCP HA HA, bTCP HA, bTCP, Ag
2AgHA HA HA, Ag, bTCP – – –
3AgHA HA HA, Ag, bTCP – – –
4AgHA HA, Ag3PO4 HA, Ag, bTCP – – –
5AgHA HA, Ag3PO4 HA, Ag, bTCP – – –
6AgHA HA, Ag3PO4 HA, Ag, bTCP – – –

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

586
Figure 4. The TEM morphology of the HA (a), 0.5 AgHA (b), 2 AgHA (c) and 5 AgHA (d) samples in as synthesized condition.
The XRD patterns of the sintered pellets are
shown in Figure 6. The b-TCP and metallic silver
phases were observed in AgHA sintered pellets,
with x  1.5. In sintered pellets, the relative inten-
sities of the peaks are different compared to pow-
ders heated at same temperature and time because
of preferred orientation. The relative intensity of the
diffraction peak, at 25.98 assigns to (002) Miller’s
plane, chosen for calculation of the crystallite size
was less in sintered pellets compared to the heat-
treated powders. In 0.5AgHA and 1.0AgHA sintered
pellets, the silver peak was not observed, compared
to powders. The preferred orientation of peaks in sin-
tered pellets compared to powders and the very low
intensity of the silver peaks even in 0.5AgHA and
1.0AgHA heat-treated powders might be the reason
for the disappearance of the silver peaks in 0.5AgHA
and 1.0AgHA pellets. The average grain size of the
sintered pellets measured by Scherrer’s formula was
50 nm. The AFM images of the 0.5AgHA, 1.0AgHA,
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
RAMESHBABU ET AL.
and 1.5AgHA sintered pellets are shown in Figure
7(a–c). The grain sizes of the sintered pellets are in
the range of 80–100 nm, as observed by the AFM
images [Fig. 7(a–c)]. Instrumental broadening and
the broadening due to strain, which were not taken
into account for crystallite size measurement by
XRD using Scherrer’s formula, might be the reason
for the smaller average crystallite sizes in XRD, com-
pared to AFM images. However, the TEM confirmed
that the powders in as-synthesized condition are of
nanosize (60–70 nm length and 15–20 nm width) as
shown in Figure 4.
The FTIR spectroscopy further details the silver
substitution in HA and the impurity phase forma-
tion. Figures 8 and 9 show the FTIR spectra of the
oven-dried and 9008C heated AgHA powders, re-
spectively. The characteristic bands for HA are
exhibited in both the spectra: 900–1200 cm1 for
phosphate bending and stretching, 602 cm1 for
phosphate bending, 632 and 3571 cm1 for vibra-
ANTIBACTERIAL NANOSIZED HYDROXYAPATITE 587

the increase in the intensities of the hydroxyl bands

at 632 and 3571 cm1 and the phosphate band at
962 cm1, as shown in Figure 9. These results are
coincident with the XRD results, as shown in Figures 1
and 2. Even though the XRD patterns of the AgHA
samples, with x ˜ 4, show the presence of Ag3PO4
in as-synthesized condition, the vibration band cor-
responding to the silver phosphate (bulk Ag3PO4
frequency 975 cm1 and nano Ag3PO4 frequency
1017 cm1)23 was not observed in FTIR spectrum.
The intense phosphate band of HA (1034 cm1) might
be responsible for totally obscuring the Ag3PO4 band.
The absorption bands at 1421 and 1455 cm1 of oven-
dried samples are attributed to the carbonate substi-
tution for the phosphate ions in the HA lattice. The
carbonate ion may have come from a reaction
between carbon dioxide and high solution pH. Since
carbonate containing HA is well known to have a bet-
ter bioactivity due to its similarity in the chemical
composition of biological apatite in natural bone, the
nanosized HA obtained in the present study is
expected to demonstrate good biocompatibility.
The XRD and FTIR analysis of AgHA samples
indicate incorporation of low concentration of silver
in the HA lattice in as-synthesized condition. So the
bactericidal study by spread plate method and the
osteoblast cell culture study were carried out for
AgHA samples, with silver content x ¼ 0.05, 0.10,
and 0.15, to characterize the effect of Ag substitution
in HA. The spread plate results of AgHA samples
after 24 and 48 h with 105 cells/mL of E. coli and
S. aureus are examined. The AgHA samples showed
complete inhibition of the growth of both the bacte-

Figure 5. The TEM morphology of the HA (a) and

1.5AgHA (b) powders heated at 9008C for 2h.

tional and stretching modes of hydroxyl vibrations.22

In addition, two broad bands are observed at 1630
and 3400 cm1 in oven-dried samples, corresponding
to the bending and stretching modes of the H2O
molecules,22 which decreased drastically with heat-
ing to 9008C. In addition, the characteristic bands
corresponding to b-TCP phase was observed at 988
Figure 6. The XRD patterns of the 0.5AgHA (a), 1.0AgHA
and 1123 cm1 for the 9008C heated AgHA powders, (b), 1.5AgHA (c), 2AgHA (d), 3AgHA (e), 5AgHA (f) pellets
with silver content x  0.15. The increase of crystal- sintered at 9008C for 2 h. HA peaks are indexed and ‘‘*’’
linity of the heated (9008C) samples was noticed by marked peaks are b-TCP and ‘‘þ’’ marked peaks are Ag.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

588 RAMESHBABU ET AL.

Figure 8. The FTIR spectra of the 0.5AgHA (a), 1.0AgHA

(b), 1.5AgHA (c), 2AgHA (d), 4AgHA (e), 5AgHA (f),
6AgHA (g) samples in as synthesized condition.

0.10. The numbers of E. coli colonies were found to

decrease with increasing silver content, and when
x ¼ 0.15, there is no colony formation (Fig. 11). All
three samples (0.5, 1.0, and 1.5AgHA) showed com-
plete inhibition of S. aureus at high cell concentration
(108 cells/mL), as shown in Figure 11. The bacterial
concentration used in this study, namely, 108 cells,
may be much higher than the realistic value encoun-
tered by the biomaterials at surgical sites. For exam-
ple, 103 cells of methicillin-resistant S. aureus bacteria
in surgical sites containing biomaterials in a spinal
implant model have shown 100% infection rates.24

Figure 7. The AFM images of 0.5AgHA (a), 1.0AgHA (b),

1.5AgHA (c) pellets sintered at 9008C for 2 h. [Color figure
can be viewed in the online issue, which is available at
www.interscience.wiley.com.]

ria under the present incubation conditions at 24 h

and even after 48 h (Fig. 10). However, when the cell
concentration was increased to 108 cell/mL, E. coli Figure 9. The FTIR spectra of the 0.5AgHA (a), 1.0AgHA
colonies were seen after 24 h in the plates with (b), 1.5AgHA (c), 2AgHA (d), 4AgHA (e), 5AgHA (f),
AgHA samples, with silver content x ¼ 0.05 and 6AgHA (g) samples in 9008C heat-treated condition.

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

ANTIBACTERIAL NANOSIZED HYDROXYAPATITE 589

Figure 10. The spread plate results of 0.5AgHA (a), 1.0AgHA (b) and 1.5AgHA (c) samples after 48 h with 105 cells/mL
of E. coli (1) and S. aureus (2). [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]

Figure 11. The spread plate results of 0.5AgHA (a), 1.0AgHA (b) and 1.5AgHA (c) samples after 24 h with 108 cells/mL
of E. coli (1) and S. aureus (2). [Color figure can be viewed in the online issue, which is available at www.interscience.
wiley.com.]

Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a

590
Figure 12. Disc diffusion test results of sintered pellets:
pure HA (a), 1.0AgHA (b), 2AgHA (c), 4AgHA (d),
5AgHA (e), and 6AgHA (f) in E. coli. [Color figure can be
viewed in the online issue, which is available at www.
interscience.wiley.com.]
The direct effect of silver ions leaching out of the
AgHA samples in the culture media was studied by
the disc test method, with Mueller–Hinton agar me-
dium containing E. coli and S. aureus bacteria sepa-
Figure 14. SEM photographs of osteoblast cell attachment on tissue culture plate (a), 0.5AgHA (b), 1.0AgHA (c), and
1.5AgHA (d) pellets.
Journal of Biomedical Materials Research Part A DOI 10.1002/jbm.a
RAMESHBABU ET AL.
Figure 13. Disc diffusion test results of sintered pellets:
pure HA (a), 0.5AgHA (b), 1.0AgHA (c), 2AgHA (d),
4AgHA (e), 5AgHA (f), and 6AgHA (g) in S. aureus. [Color
figure can be viewed in the online issue, which is available
at www.interscience.wiley.com.]
rately, after 24 h of inoculation. The AgHA samples
with silver content x > 0.2 shows the zone of inhibi-
tion, which increases with the increased silver con-
tent in the sample as shown in Figures 12 and 13 for
ANTIBACTERIAL NANOSIZED HYDROXYAPATITE
E. coli and S. aureus, respectively. Although AgHA
samples with silver content x ¼ 0.05 and x ¼ 0.10 do
not exhibit any appreciable zone of inhibition, the
growth of bacterial colonies was not observed in the
same silver concentrations, with 105 cells/mL of
E. coli and S. aureus in spread plate test. This sug-
gests the bacteriostatic effect of the silver substituted
HA samples. Interestingly, the AgHA nanosized par-
ticles show the same antibacterial properties even af-
ter 2 years of storing at room temperature, indicating
its long-standing stability of antibacterial nature.
Though the silver loaded HA samples showed
good antibacterial properties, the present work is
aimed at the use of nano HA for hard tissue applica-
tion with optimal silver concentration, to avoid any
bacterial infection. As a first attempt to investigate
how osteoblasts would interact with the various
AgHA samples, cell adhesion assays were per-
formed. Figure 14 shows the cell growth on various
specimens. Control cells grew well on the tissue cul-
ture plate [Fig. 14(a)]. Osteoblast cell attachment in
varying density was noticed on 0.5AgHA, 1.0AgHA,
and 1.5AgHA samples [Fig. 14(b–d)]. All the surfa-
ces were able to support cell growth as the metabolic
activity of HOS cells. However, osteoblast function
was significantly greater on 0.5AgHA pellet, with
the osteoblast cells being well adhered and spread
on the pellet. On 1.0AgHA and 1.5AgHA samples,
osteoblast cells were attached but could not spread
as on 0.5AgHA, suggesting that AgHA samples with
low amount of silver are more favorable for implant
applications. The integration of nanotechnology and
bacteriology seems to lead to the formulation of new
types of antibacterial nanomaterials for orthopedic
and dental applications.
CONCLUSIONS
Microwave processing was found to be effective in
synthesizing AgHA particles with narrow size distri-
bution in a shorter time of 30 min. AgHA phase
seems to be stable till 7008C. The AgHA particles
were found to be nanosize with needle-like morphol-
ogy, comparable to that of the bone apatite. Bacteri-
cidal activity of AgHA samples was observed even
with low content of silver, which also exhibited
excellent osteoblast adherence and spreading. Pres-
ent study suggests a new class of nanomaterials for
orthopedic and dental applications, with incorpo-
rated bactericidal effects.
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