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Nanomedicine: Nanotechnology, Biology, and Medicine

11 (2015) 1331 1344

Regenerative Nanomedicine (Ed. A. Seifalian)


Development of nanosized silver-substituted apatite for biomedical

applications: A review
Poon Nian Lim, PhD 1 , Lei Chang, PhD 1 , Eng San Thian, PhD
Department of Mechanical Engineering, National University of Singapore, Singapore
Received 1 July 2014; accepted 23 March 2015

The favorable biocompatibility of hydroxyapatite (HA) makes it a popular bone graft material as well as a coating layer on metallic
implant. To reduce implant-related infections, silver ions were either incorporated into the apatite during co-precipitation process
(AgHA-CP) or underwent ion-exchange with the calcium ions in the apatite (AgHA-IE). However, the distribution of silver ions in
AgHA-CP and AgHA-IE was different, thus affecting the antibacterial action. Several studies reported that nanosized AgHA-CP containing
0.5 wt.% of silver provided an optimal trade-off between antibacterial properties and cytotoxicity. Nevertheless, nanosized AgHA and
AgHA nanocoatings could not function ideally due to the compromise in the bone differentiation of mesenchymal stem cells, as evidenced
in the reduced alkaline phosphatase, type I collagen and osteocalcin. Preliminary studies showed that biological responses of nanosized
AgHA and AgHA nanocoatings could be improved with the addition of silicon. This review will discuss on nanosized AgHA and
AgHA nanocoatings.
From the Clinical Editor: In many patients needing bone graft material, hydroxyapatite (HA) has proven to be a popular choice. Nonetheless,
implant-related infections remain a major concern. Hence, effective preventive measures are needed. In this review article, the authors discussed the
application of incorporating silver nanoparticles in HA and its use as bone graft biomaterials together with the addition of silica.
2015 Elsevier Inc. All rights reserved.
Key words: Antibacterial; Apatite; Nanosized; Silver

Hydroxyapatite (HA) is a synthetic bone alternative, which

possesses a chemical similarity to the bone mineral. Since HA is
biocompatible when implanted in-vivo, it exhibits bioactive
behavior by forming a direct bond between the implants and
bones (osseointegration). Therefore, HA is commonly used as a
bone graft to fill the defects or deposited as a coating layer on
orthopedic implant, to promote bone regeneration in order to
facilitate bone healing process. However, due to the lack of
protection from the body immune system, HA is susceptible to
immediate and delayed infections, leading to implant-related
infections. Despite the use of perioperative antimicrobial
prophylaxis and laminar flow operating rooms, implant-related
infections were common. 1 According to the U.S. Centers for
Disease Control and Prevention (CDC), the risk of acquiring

This work was supported by the Ministry of Education Academic

Research Fund (Singapore) Project Number MOE2013-T2-1-074.
Corresponding author.
E-mail address: mpetes@nus.edu.sg (E.S. Thian).
These authors contributed equally to this work.

serious infections during medical treatments had risen over 35%

in the last 20 years. 2 In 1990s, implant-related infections
accounted for about half of all hospital-acquired infections. 3
Today, with the increasing use of implants, the cases of
implant-related infections are expected to increase rapidly, particularly in sub-populations comprising of immune-compromised,
chronically ill, and elderly patients. Besides pain and suffering,
implant-related infections often incurred huge medical costs. 4
For example, an estimated average direct cost associated with an
infected case was reported to range between US$15 K and
US$30 K, which was approximately three times of the initial
intervention. 5 Although the rate of infections was observed to
be only 5% for the primary cases, it could be greatly increased
to 43% for previously infected cases. 6 Thus, the issue of
implant-related infections is a major problem affecting the
service life of the medical implants. As such, it is equally
important to reduce the intrinsic vulnerability of the biomaterial
so as to minimize bacterial colonization.
Upon implantation, a competition exists between the
integration of material into the surrounding tissues and the
adhesion of bacteria onto the implant surfaces. 7 For a successful
implantation, tissue integration must occur prior to appreciable
bacterial adhesion as host defenses are often not capable of

1549-9634/ 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Lim PN, et al, Development of nanosized silver-substituted apatite for biomedical applications: A review. Nanomedicine: NBM
2015;11:1331-1344, http://dx.doi.org/10.1016/j.nano.2015.03.016


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Table 1
Various proposed mechanism of antibacterial action by silver ions.
Proposed mechanism


Silver inhibited the uptake of phosphate and caused the

efflux of intracellular phosphate
Silver ions bound to sulfhydryl groups of the many important
metabolic enzymes of the bacterial electron transport and
respiration. For example, silver bound to the thiol groups
(sulfhydryl, S-H) presented in the cysteine residues of the
transport proteins, which induced a massive proton leakage
though the bacterial membrane, resulting complete
de-energization, and ultimately leading to cell death.
Silver ions entered into the bacterial cells by penetrating
through the cell wall, and turned the DNA into
condensed form. As a result, DNA lost its replication
ability and led to cell death.
Silver ions bound to microbial DNA by interacting with
nucleic acids, and changed DNA structure, which
consequently prevented bacterial replication.
Silver ions were observed to increase the DNA mutation
frequencies during polymerase chain reactions.
Silver ions generated reactive oxygen species (ROS) and
damaged the cell membrane.
Bacterial cell suffered from morphological changes such as
cytoplasm shrinkage and detachment contents such as
potassium ion.

1, 35, 99-102




Figure 1. Photograph of antibacterial test results of AgHA-IE samples against

E. coli. 28 Reprinted from applied surface science, 257, Stanic V., Janackovic
D., Dimitrijevic S., Tanaskovic S. B., Mitric M., Pavlovic M. S., Krstic A.,
Jovanovic D., Raicevic S., Synthesis of antimicrobial monophase silverdoped hydroxyapatite nanopowders for bone tissue engineering, p. 4510,
Copyright (2011), with permission from Elsevier.

103, 110

preventing further colonization if bacterial adhesion occurs

before tissue integration. Implant-related infections are caused
by the consequence of bacterial adhesion and subsequent biofilm
formation at the implantation site. Biofilm formation proceeds as
a four-step process: (1) initial attachment of bacterial cells; (2)
accumulation in multiple cell layers; (3) biofilm maturation; and
(4) detachment of cells from the biofilm into a planktonic state to
initiate a new cycle of biofilm formation elsewhere. 8 Bacteria
living in a biofilm are highly resistant to antibacterial agents, and
biofilm shields them from the influence of hosts defense. 9 Thus,
even antibiotic therapies remain ineffective against biofilms.
Previous works on loading biomaterials with antibiotics for
orthopedic applications were carried out using bone-filler
materials and bone cements. 10 -12 However, the removal of
pathogen in the open fracture injury and periapical lesions of
jaws (dental) was not easily resolved by systematic antibiotic.
Moreover, bacteria would develop resistance against antibiotic
over time. In some worst cases, when biomaterial responded
poorly to the antibiotic therapy, the removal of the infected
implant was necessary to cure it. 13 Thus, implant-related
infections complicate bone healing, and can also lead to the
failure of orthopedic surgery. Nevertheless, effective results of
antibacterial activities of implant materials designated with
antibacterial properties were reported in recent commercially
available silver-impregnated dressing and catheters. 14 -16 Hence,
this shows the potential of incorporating antibacterial properties
to the implant material, which will become the upcoming
strategies to treat implant-related infections.
Therefore, it will be beneficial if HA-based bone grafts or HA
coatings are incorporated with antibacterial properties as this will
aid in reducing the occurrence of implant-related infections. To
overcome the infection issues encountered in HA, substitution of
functional ion such as silver has emerged as a preventative

approach. Silver ions were reported to interfere with the integrity

of the bacterial cell, or bind to the enzymes and proteins within
the bacteria, which severely damaged the cell and its major
functions such as permeability, regulation of enzymatic signaling
activity, cellular oxidation and respiratory processes, resulting in
the bacteria death. Hence, this review paper will firstly discuss
on the antibacterial action of silver. Nanosized silver-substituted
HA (AgHA) will be featured in two forms bulk and
nanocoatings. Silver ions were either incorporated into the
apatite structure during the co-precipitation process (AgHA-CP)
or underwent ion-exchange with calcium ions in the apatite
(AgHA-IE), which would be discussed in detail in the synthesis
section. However, the distribution of the silver ions in AgHA-CP
and AgHA-IE was different, which in turn affected the
antibacterial action. This would be discussed through summarizing the chemical and physical characterization, antibacterial
properties and biological responses of AgHA-CP and AgHA-IE,
which were reported from numerous experimental studies. For
the second part of the review, AgHA-CP and AgHA-IE were
deposited using various techniques onto the substrate to form
nanocoatings. Similarly, the different distribution of the silver
ions in AgHA-CP and AgHA-IE nanocoatings affected the
chemical and physical characterization, antibacterial properties
and biological responses, and these properties would also be
discussed. Lastly, preliminary work on the introduction of silicon
to improve the biological responses of bulk AgHA and AgHA
nanocoatings will be presented.

Silver as an antibacterial agent

One common and accepted strategy to prevent implantrelated infections is to create antibacterial properties for the
implant. Implants with antibacterial properties reduce the
treatment duration, 17 and decrease the side effects of systemic
treatments, 18 thereby improving its efficacy. Inorganic antibacterial agents generally have a low resistance against bacteria, and
are comparatively stable than organic ones, thus they become an

P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344



Figure 2. Antibacterial effect of the treated (A) HA and (B) AgHA-IE

nanocoatings on titanium against E. coli. 97 Reprinted from surface coating
technology, 204, Chen Y., Zheng X., Xie Y., Ji H., Ding C., Antibacterial
properties of vacuum plasma sprayed titanium coatings after chemical
treatment, p. 685, Copyright (2009), with permission from Elsevier.

ideal choice for local antibacterial treatment. Among the

inorganic antibacterial agents, silver and its ions show
oligodynamic effect with a broad spectrum of antibacterial
activities against bacteria, viruses, algae, and fungi. 19 -24 Both
metallic and ionic silver have been widely incorporated into
biomaterials and medical devices such as bone cements, catheter,
orthopedic fixation pins, dental implants, cardiac prostheses, and
burn wound treatments. 25 -27 Although the antibacterial activity
of AgHA has been demonstrated in several works, 28 -33 the exact
mechanism of antibacterial action in which AgHA exerts this
antibacterial activity is not fully known and understood. Possible
mechanisms of the antibacterial action of silver ions have been
suggested according to the morphological and structural changes
found in the bacterial cells. The common proposed mechanism in
the literature suggested that silver ions could interact with
bacterial cells in several ways (Table 1).
Bulk AgHA
Synthesis of bulk AgHA
A number of studies considered the synthesis of AgHA
through a precipitation route 28,29,33,34 while others engaged
various spraying methods 35 -39 and coating techniques 35,37,40 to
synthesize AgHA. The incorporation of silver ions into apatite is
based on Eq. (1).

10yCa2 aq yAg aq 6PO4 3 aq

2yOH aq Ca10y Agy PO4 6 OH2y aq

where y is the molar amount of silver to be incorporated.

Silver precursor was firstly added into the calcium precursor
as silver would substitute at the calcium site of the apatite
structure. During the precipitation method, phosphate precursor
was added drop-wise into the calcium and silver precursor
mixture, and stirred for several hours for the reaction to occur
before undergoing aging. Since silver precursor was added
during the co-precipitation (CP) process, it would be termed as
AgHA-CP. Although precipitation method was the common
route to synthesize nanosized AgHA, a variety of different
combinations of precursors was engaged for the synthesis of

AgHA. Oh et al used silver nitrate, calcium nitrate and

ammonium phosphates for the synthesis of nanosized AgHA
while Kim et al 33 used silver nitrate, calcium hydroxide and
orthophosphoric acid. As ammonia could react readily with silver
ions in the basic conditions to form diamminesilver(I) ion, which
would then reduce the amount of available silver ions to be
incorporated into the apatite structure, 34,41 Rameshbabu et al 29
thus used silver nitrate, calcium hydroxide and diammonium
hydrogen phosphate, while Stanic et al 28 used silver oxide, calcium
hydroxide and orthophosphoric acid, as the starting materials for
the synthesis of AgHA-CP.
Likewise, AgHA could also be synthesized by immersing the
apatite into a silver precursor solution. 30,35-38,40,42-44 Silver ions
underwent ion-exchange (IE) with the calcium ions in the apatite,
and would be termed as AgHA-IE. Apatite was firstly formed via
precipitation method, and subsequently dipped into a silver
nitrate solution to allow ion-exchange. This method was more
commonly used when AgHA was being produced in a form of
coating. 37 -39,42,45-62
However, the addition of silver precursor to produce
AgHA-CP or AgHA-IE will affect the distribution of the silver
ions in AgHA, which in turn influence its antibacterial properties
and biological responses. This will be further discussed in the
following sections.
Chemical and physical characterization of bulk AgHA
Obtaining a phase-pure AgHA is desirable for biocompatibility, but has met with many difficulties. Kim et al 33 detected
the production of apatite along with nitrate-apatite in AgHA-CP
incorporated with 1 wt.% of silver. Rameshbabu et al 29 managed
to produce a single-phase nanosized apatite (~ 60 nm ~ 15 nm)
at a low silver content (b 1.63 wt.% of silver) heated below
700 C. However, silver phosphate and -tricalcium phosphate
were produced when heated above 700 C, and metallic silver
was observed when heated to 800 C for AgHA-CP at higher
silver content (N 1.63 wt.% of silver), thus indicating that the
phase stability of AgHA-CP was not maintained with the
increase of silver content. Similarly, Singh et al 63 demonstrated a
phase-pure apatite at silver content up to 2 wt.%. However,
-tricalcium phosphate was produced when silver content was
increased to 3 and 5 wt.%. The lack of phase stability in
AgHA-CP at high temperature might be explained by the production
of vacancy at the hydroxyl site, which was formed to compensate for
the charge imbalance when silver partially substituted for calcium.
Recently, our group reported on a range of phase-pure nanosized
AgHA-CP (~50 nm ~20 nm) containing of 0.2-1.1 wt.% of
silver, which were stable up to 1150 C. 64
The presence of silver in the phase-pure AgHA-CP was
revealed when the lattice parameters namely a- and c-axes
increased with increasing amount of incorporated silver ions in
the XRD refinement analysis. 29,65 Alteration in the lattice
parameters implied that silver substituted for calcium in the HA
lattice since silver ion (0.128 nm) was larger than calcium ion
(0.099 nm), thus corresponding to an increase in the lattice
parameters. It was also noted that there was a significant loss in
the amount of incorporated silver in nanosized AgHA-CP. 34 Due
to a bigger ionic size, silver ions had difficulty in occupying the


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Figure 3. Scanning electron microscope images of adherent E. coli treated by (A) HA, and (B) AgHA-CP particles. 63 Reprinted from material science
engineering: C, 31, Singh B., Dubey A. K., Kumar S., Saha N., Basu B., Gupta R., In vitro biocompatibility and antimicrobial activity of wet chemically
prepared Ca10 xAgx(PO4)6(OH)2 (0.0 b x b 0.5) hydroxyapatites, p. 1320, Copyright (2011), with permission from Elsevier.

calcium sites. With the formation of diamminesilver(I) ion and

the physical effect of washing during the synthesis process, the
amount of silver that could be incorporated into the apatite
structure was further reduced. Oh et al 34 attempted to minimize
the loss of incorporated silver in nanosized AgHA-CP by
synthesizing nanosized AgHA-CP with a higher Ca/P molar ratio
(e.g. 2.0) while Stanic et al 28 avoided the use of ammonia and
nitrate precursors for the synthesis of nanosized AgHA-CP
(~ 70 nm ~ 15 nm).
Past results indicated that the presence of silver ions to a
certain amount (N 10 mg/l) in the human body could be cytotoxic
as it could affect the basic metabolic cellular function of the
mammalian cells. 66 Silver ions were reported to deplete the
intracellular adenosine-5-triphosphate (ATP) content, which
could compromise the cell energy charge and precede to cell
death. 67 As far as toxicity was concerned, toxicity from silver
was observed in the form of argyria, which would only occurred
when a large amount of silver ions was used for dressing a large
open wound. So far, there was no regular report of silver
allergy. 68 Nevertheless, it is important to consider the amount of
released silver ions for a long period as it may lead to the
accumulation of silver in the organs. Therefore, it is prudent to
incorporate optimum amount of silver in HA to reduce bacterial
adhesion adequately, with minimum silver release for tissue
cytotoxicity during the treatment of implant-related infections.
However, the rapid release of silver ions is a major drawback for
AgHA-IE. AgHA-IE would yield silver ion-rich surface, where
premature release of silver ions was anticipated, which greatly
compromised the sustainability of antibacterial effect. 69
AgHA-IE was reported to release more than 50% of the loaded
silver ions within the first 24 h. 57,69 Furthermore, Wu et al 70
observed the release of silver ions from AgHA-IE scaffold
increased with longer immersion period, and AgHA-IE scaffold
with higher silver content would release more silver ions.
AgHA-IE released silver ions to interact with the surrounding
bacteria. However, rapid release of silver ions would accumulate
at a localized site and this could result in local cytotoxicity.
AgHA-IE released large amount of silver ions in the initial stage,
which gave rise to immediate antibacterial efficacy and this
effect was considered to be beneficial in preventing implantrelated infections as higher risk of infections often occurred in
the early period of the surgery. 71 However, at the later stage,
minute and steady release of silver ions was the key to provide

long-term antibacterial property for implantation. Therefore, the

antibacterial efficacy of AgHA-IE was not long-lasting.
Antibacterial properties of bulk AgHA
Several antibacterial studies demonstrated that AgHA (-CP
and -IE) exhibited excellent antibacterial activity in-vitro, with a
reduction of more than 99% against the following pathogens:
Staphylococcus aureus (S. aureus), Escherichia coli (E. coli),
Streptococcus mutans (S. mutans), Staphylococcus epidermidis
(S. epidermidis), Pseudomonas aeruginosa (P. aeruginosa),
Scaphirhynchus albus (S. albus), methicillin-resistant S. aureus
(MRSA), Bacillus subtilis (B. subtilis) and Candida albicans
(C. albicans). 28,29,33,35,37,39,63,69,72 The antibacterial effect
against the bacteria in the medium was suggested to be
dependent on the release of silver ions. 28,29,33,37 The antibacterial effects were demonstrated either by the visibility of
inhibition zones around samples or quantified by the number
of colony forming unit (CFU). Zone of inhibition 28 and
reduction in the number of bacteria in the growth medium
were demonstrated in the spread plate method as represented in
Figures 1 and 2. On the other hand, reduction of the adherent
bacteria 63 was mostly observed using scanning electron
microscopy (Figure 3) as an indicative of antibacterial effect.
Rameshbabu et al 29 observed a complete inhibition of the
growth of S. aureus with an initial bacterial population of
10 8 CFU/ml after 24 h in nanosized AgHA-CP containing 0.5, 1
and 1.5 wt.% of silver. Among these nanosized AgHA-CP
samples, 0.5 wt.% of silver was suggested as the optimum silver
content since greater osteoblast spreading was demonstrated.
Similarly, Stanic et al 28 observed pronounced reduction of S.
aureus in nanosized AgHA-CP with 0.4 wt.% after 4 h. Singh
et al 63 reported a comparable antibacterial effect against E. coli
among AgHA-CP containing silver content of 2, 3 and 5 wt.%.
However, AgHA-CP with silver content greater than 3 wt.% was
reported to be cytotoxic toward mouse fibroblast cells. It was
suggested that a silver content between 0.5 and 2 wt.% in
AgHA-CP could achieve effective antibacterial effect. 28,29,34,63
Until recently, a range of nanosized AgHA-CP containing 0.2,
0.3, 0.5, 0.9, 1 and 1.1 wt.% of silver were investigated
(Figures 4). A 3-log reduction of the S. aureus population was
observed in the nanosized AgHA-CP containing 0.5, 0.9, 1 and
1.1 wt.% of silver as compared to HA. 64 However, there was no

P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Figure 4. Log reduction assays of various AgHA-CP disks incubated for

24 h. [a] p b 0.05 when comparing bacteria to 0.5AgHA, [b] p b 0.05 when
comparing bacteria to 0.9AgHA, [c] p b 0.05 when comparing bacteria to
1.0AgHA, [d] p b 0.05 when comparing bacteria to 1.1AgHA, [e] p b 0.05
when comparing bacteria to AgNO3; [f] p b 0.05 when comparing HA to
0.5AgHA, [g] p b 0.05 when comparing HA to 0.9AgHA, [h] p b 0.05 when
comparing HA to 1.0AgHA, [i] p b 0.05 when comparing HA to 1.1AgHA,
[j] p b 0.05 when comparing HA to AgNO3. 64 Adapted from effect of silver
content on the antibacterial and bioactive properties of silver-substituted
hydroxyapatite/Lim P. N., Teo E. Y., Ho B., Tay B. Y., Thian E. S/Journal of
biomedical material research and 101A. Copyright (c) [2013] [John Wiley
and Sons].

significant difference in the bacterial inhibition rate between the

nanosized AgHA-CP of 0.5, 0.9, 1 and 1.1 wt.% of silver. The
increase of silver content up to 1.1 wt.% did not have a
significant impact on the retardation of bacterial growth. It was
essential to minimize the presence of silver ions in the body as
silver ions could deplete the intracellular ATP content, which
could affect the basic metabolic cellular functions of the
mammalian cells. 67 Therefore, substitution of 0.5 wt.% of silver
into HA was reported to be sufficient to retard and inhibit
bacterial growth.
Oh et al 69 compared the durability of the antibacterial effect
of nanosized AgHA-CP with AgHA-IE. Despite the rapid
reduction of E. coli for the first 10 min, proliferation of E. coli
was noticed after 100 h for the nanosized AgHA-IE samples. In
contrast, nanosized AgHA-CP could suppress effectively the
proliferation of E. coli until 1000 h. The difference in the
durability of the antibacterial effect was explained by the amount
of silver ions released during the initial 10 min. 69 Despite a
lower silver content, Oh et al 69 demonstrated that AgHA-CP
containing 0.53 wt.% of silver had a more durable antibacterial
effect than nanosized AgHA-IE containing 1.5 wt.% of silver.
Regardless of the low amount of released silver ions in nanosized
AgHA-CP, it was able to achieve excellent antibacterial
properties. These results signaled that nanosized AgHA-CP
was not dependent on the released silver ions for the antibacterial
action. Furthermore, Stanic et al 28 observed a reduction of the
bacterial growth in nanosized AgHA-CP, with no detectable
silver ions released in the dissolution test. Recently, our group
compared the growth of adherent S. aureus on nanosized HA
with AgHA-CP containing 0.5 wt.% of silver (Figure 5). A 7-log
reduction of adherent S. aureus was observed for nanosized
AgHA-CP as compared to nanosized HA over a culture period of
120 h. When comparing the inhibition rate of the adherent
S. aureus and S. aureus suspended in the medium, 64 it was


Figure 5. Log reduction assays of adherent S. aureus on the surface HA and

AgHA-CP disks incubated for 120 h.

interesting to note that there was greater inhibition in the

population of adherent S. aureus. All these findings suggested
that the antibacterial action of nanosized AgHA-CP against
bacteria might not be entirely attributed by the released
silver ions.
For bacteria to exert its lethality, the bacteria have to be
attached onto the surface, thus surface-bound silver ions of
nanosized AgHA-CP might play a role in the antibacterial action
and, this was further investigated by determining the surface
content. 64 XPS studies revealed increasing silver content on the
surface of nanosized AgHA-CP with longer immersion period,
which implied that silver ions diffused toward the crystal surface
of nanosized AgHA-CP. Therefore, it was postulated that the
antibacterial action of AgHA-IE was dependent on the released
silver ions while AgHA-CP was dependent on the surface-bound
silver ions (Figure 6). AgHA-IE released silver ions to attack the
surrounding bacteria. However, it might not be very effective as
there might be a possibility that some of the bacteria were not
killed by the released silver ions and got attached onto AgHA. In
contrast, AgHA-CP killed all adherent bacteria. Although the
remaining bacteria could continue to adhere on AgHA-CP, the
sustained inhibition of the growth of the adherent bacteria till
120 h (Figure 6) demonstrated the antibacterial efficacy of
AgHA-CP. Unlike AgHA-IE, AgHA-CP tended to be more
beneficial as local cytotoxicity at the implant site could be
potentially minimized based on the proposed hypothesis that
antibacterial action only took place when surface-bound silver
ions interacted with the adherent bacteria. As a result, the
antibacterial efficacy of AgHA-CP could be maintained for a
longer period of time.
Biological characterization of bulk AgHA
Upon optimizing the antibacterial properties, it will be
essential to determine the biological responses of AgHA (-CP
and -IE). Human osteoblast cell attachment was observed on
nanosized AgHA-CP samples containing 0.5, 1.0, and 1.5 wt.%
silver, 29 and mouse fibroblast cells were also seen attaching on
nanosized AgHA (-CP and -IE) samples with up to 5 wt.%


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Figure 6. Schematic diagram illustrating the antibacterial action of (A) AgHA-CP and (B) AgHA-IE.

silver. 39,63 However, it was noted that there was lesser adhesion
of cells on the surface of the nanosized AgHA-CP with greater
amount of silver (Figure 7). Through the examination of
hemolysis ratio, Oh et al, 30 Chen et al 39 and Stanic et al 28
reported that hemolysis ratio of nanosized AgHA-CP with a
silver content of less than 5 wt.% showed good blood
compatibility. Likewise, the cytotoxicity of nanosized AgHA-CP
also increased with increasing silver content, as indicated by the
increasing hemolysis ratio.
Recently, well-stretched human adipose-derived mesenchymal stem cell (hASCs) on nanosized AgHA-CP containing an
optimized amount of 0.5 wt.% silver was also demonstrated. In
addition, cell proliferation was observed on nanosized AgHA-CP
containing 0.5 wt.% of silver over a period of 7 days (Figure 8).
Furthermore, bone differentiation of nanosized AgHA-CP was
evaluated. It was observed that the bone differentiation markers
(Figure 9) such as alkaline phosphatase (ALP), type I collagen
(type I COL) and osteocalcin (OCN) were significantly lower on
nanosized AgHA-CP containing 0.5 wt.% of silver than
nanosized HA. Although the substitution of low silver content
(0.5 wt.%) did not affect the proliferation of hASCs, its bone
differentiation was affected.
In-vivo experiment was carried out at the periapical area of
both mandibular 1st molar of rats. 44 The wound implanted with
AgHA-IE generally healed in 1 week, and showed minimum
signs of inflammation in-vivo after 3 weeks. However, AgHA-IE
containing 4.3 wt.% of silver showed mild delayed in the
organization of fibrinoid materials in the center of defects, with
mild inflammatory reaction at the early healing phase as
compared to AgHA-IE containing 0.15 and 1.5 wt.% of silver.
Both in-vivo and in-vitro results demonstrated that the presence
of silver in apatite did not induce cytotoxicity, but affected
bone growth.
Despite the obvious antibacterial benefit, few studies have
reported on achieving antibacterial properties with excellent
biological responses in the nanosized AgHA-CP. Thus, there
was a compromise between the antibacterial properties and
favorable biological responses of nanosized AgHA-CP. In order

to enhance the biological responses of nanosized AgHA-CP,

substitution of silicon into nanosized AgHA-CP was proposed
recently. 73 A phase-pure apatite was achieved for nanosized
Ag,Si-HA, and in-vitro results demonstrated that there was an
enhancement in the biological response of nanosized Ag,Si-HA
as compared to nanosized AgHA-CP as well as HA. Moreover,
the presence of silicon did not affect the antibacterial properties
of nanosized Ag,Si-HA, whereby the bacterial growth of
nanosized AgHA-CP and Ag,Si-HA was inhibited till day 7.
AgHA Nanocoatings
Deposition of AgHA nanocoatings
AgHA nanocoatings can be formed by depositing nanosized
AgHA (-CP or -IE) onto the substrate as nanocoatings. A variety
of deposition techniques have been studied, and these include
solgel deposition, drop-on-demand (DoD) micro-dispensing
technique, electrospraying, electrophoretic deposition, thermal
substrate method, micro-arc oxidation and CoBlast technology.
For AgHA-CP nanocoatings, AgHA-CP was firstly synthesized using wet precipitation, and was deposited onto the
substrate using various techniques. For example in solgel
deposition, calcium precursor was prepared by reacting calcium
nitrate tetrahydrate with methyl alcohol while phosphorus
precursor was prepared by reacting triethyl phosphite in acetic
acid. Silver nitrate and the two precursors were mixed together,
and a drying control chemical additive was added to the mixture.
All reactions were carried out in an argon atmosphere. The mixed
solution was filtered and aged. Finally, the prepared silver-doped
HA sol was then spin-coated onto the substrates. 40 Alternatively,
silver nitrate, calcium nitrate tetrahydrate and triethyl phosphate
were mixed in anhydrous ethanol. Subsequently, the precursor
mixture was aged at 80 C for 16 h, followed by gelation for
24 h and dried at 80 C. 74,75 DoD micro-dispensing technique
created droplet matrix on the substrate by jetting out AgHA-CP
droplets of ink through the nozzle orifice on specific locations
of the substrate. 61 With electrospraying technique, AgHA-CP

P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344


Figure 7. SEM images of cell morphology on (A) tissue culture plate, (B) 0.5 wt.% AgHA, (C) 1 wt.% AgHA and (D) 1.5 wt.% AgHA pellets. 29 Adapted
from antibacterial nanosized silver substituted hydroxyapatite: Synthesis and characterization/Rameshbabu N., Kumar T. S. S., Prabhakar T. G., Sastry V. S.,
Murty K. V. G. K., Rao K. P./Journal of biomedical material research and 80A. Copyright (c) [2006] [John Wiley and Sons].

suspension was pumped into a nozzle to achieve a stable cone jet

by applying a high voltage of 8 kV to form AgHA-CP
nanocoatings. 59
Electrophoretic deposition, 46,47 thermal substrate method 76
and micro-arc oxidation 77 utilized substrates as working
electrode and applied current to the AgHA-IE suspension.
Particles were then deposited onto the substrates surface due to
electrostatic forces. Using CoBlast technology, AgHA-IE was
deposited onto substrates surface under a high pressure of
620 kPa. 50 Furthermore, AgHA-IE nanocoatings can be deposited directly by using high energy source to bombard target
materials such as HA and silver precursors, allowing them to
interact and form AgHA-IE nanocoatings. This method of
coating technique can be classified into one-step coating and
two-step coating techniques. An overview of the one-step and
two-step coating technologies that are used to deposit AgHA-IE
nanocoatings is illustrated in Table 2.
One-step coating technique refers to technique that deposits
AgHA-IE nanocoatings with the simultaneous addition of silver
and apatite during the coating process. 36,76-90 These include
plasma spraying, 37 magnetron sputtering, 35 ion beam assisted
deposition (IBAD) 55,56 and pulsed laser deposition. 91 Using
plasma spraying technique, HA and silver oxide powders were
injected into a high-temperature plasma, where powders were
melted and then accelerated at a high velocity toward the
substrate, which eventually solidified as AgHA-IE nanocoatings.
In magnetron sputtering, atoms or molecules of silver and HA
targets were ejected in a vacuum chamber by bombardment with
high-energy ions and subsequently, condensed on substrates as
AgHA-IE nanocoatings. Silver could be incorporated into HA

prior to the coating process 48 or silver and HA could be utilized

as two separate coating targets. 35 In the process of IBAD, HA
and silver targets were vaporized to generate an elemental cloud
of ions moving toward the substrates surface. Subsequently,
these ions penetrated into the near surface of the substrate,
thereby forming AgHA-IE nanocoatings. In pulsed laser
deposition, laser beam was focused on HA and silver targets
under high vacuum to vaporize the targets which deposited as a
thin film on a substrate which was subsequently heated up to
350-600 C to decrease nucleation density. 35,36
On the other hand, two-step coating technique refers to the
technique that firstly deposits HA onto the substrate to form HA
nanocoatings, which then followed by the introduction of silver
ions to form AgHA-IE nanocoatings. 37 -39,42,45-62 Apatite
layers were introduced by the one-step coating technologies
such as ion beam assisted deposition 83,85 and electrophoretic
deposition. 79,84 Alternatively, biomimetic deposition was also
used to create apatite layers on substrates. Substrates were
immersed into supersaturated ionic solutions such as Hanks
solution 82 and supersaturated calcification solution (SCS) 80,81 at
37 C for up to 7 days, and calcium phosphates were
precipitated onto the surface. Subsequently, the substrates with
apatite layers were dipped into a silver nitrate solution to induce
silver substitution. 79,83-85
Chemical and physical characterization of AgHA nanocoatings
Phase-pure apatite was not achieved for AgHA-CP nanocoatings that were produced using solgel deposition. Similarly,
phase-purity of AgHA-IE nanocoatings was difficult to retain in


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Figure 8. Cell growth versus culture period. 64 Adapted from effect of silver
content on the antibacterial and bioactive properties of silver-substituted
hydroxyapatite/Lim P. N., Teo E. Y., Ho B., Tay B. Y., Thian E. S/Journal of
biomedical material research and 101A. Copyright (c) [2013] [John Wiley
and Sons].

the plasma spraying, magnetron sputtering, pulsed laser

deposition and IBAD processes. Either one or more of the
secondary phases such as silver, silver oxide, silver phosphate,
calcium oxide or tricalcium phosphate were observed to be
accompanied with the production of apatite at all silver content
of AgHA-IE nanocoatings. 35,38,39,42,43,74,75 High processing
temperature used in plasma spraying process was the key reason
in causing decomposition of the apatite into tricalcium
phosphate, tetracalcium phosphate, calcium oxide and silver
oxide. The inert atmosphere in magnetron sputtering and IBAD
processes led to the formation of oxyapatite, owing to
dehydroxylation of AgHA-IE nanocoatings, which affected the
coating integrity. 92 -95 In addition, for AgHA-IE nanocoatings
produced by the two-step coating technique, silver was only
adsorbed onto the surface of HA, and did not form chemical bonds
within the apatite structure. Furthermore, the elemental distributions of calcium, phosphorus and silver ions were not uniform on
the surface of AgHA-IE nanocoatings. Non-homogenous distribution of silver ions of AgHA-IE nanocoatings could result in
cytotoxicity, and the dissolution rate of silver ions on the coating
surface would vary from various positions. Most coating
techniques lack the capability to control material distribution,
and thus it is difficult to achieve AgHA-IE nanocoatings with
uniform distributed silver ions.
Table 3 summarizes the various dissolution studies of
AgHA-IE nanocoatings. Ionita et al 79 and Shirkhanzadeh et
al 84 observed the release of 50% of the silver content of
AgHA-IE nanocoatings into stimulated body fluid during the
first 24 h. Similarly to the bulk AgHA-IE, the increasing release
of silver ions in AgHA-IE nanocoatings with longer immersion
time could produce undesirable consequences such as local
cytotoxicity and short-term antibacterial properties. Chen et al 57
compared the release of silver ions between AgHA-CP and
AgHA-IE nanocoatings in the buffering fluid and simulated
body fluid (Figure 10). For the first 50 h, the release of silver
ions from AgHA-CP nanocoatings was greater than AgHA-IE
nanocoatings in both body fluid medium and stimulated body
fluid. However, the release of silver ions from AgHA-IE
nanocoatings greatly increased and exceeded the amount of

Figure 9. Quantitative measurement of protein expressions, (A) ALP, (B) type

I COL and (C) OCN expressed by hMSCs cultured on HA and AgHA-CP.

silver ions released from AgHA-CP nanocoatings after 50 h in

both body fluid medium and stimulated body fluid. Approximately 1.4 and 0.4 ppm of silver ions were released from
AgHA-CP nanocoatings into the body medium and stimulated
body fluid, respectively, and this amount of released silver ions
was maintained till 336 h. The sustained release of silver ions
suggested that AgHA-CP nanocoatings were more homogenously distributed within the apatite structure.

P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344


Table 2
Various coating technologies to deposit AgHA-IE nanocoatings and their characteristics.
Coating Techniques


Conc. of


Magnetron Co-Sputtering

HA, silver clip

2 wt.%

Radio Frequency Magnetron



Radio Frequency
Plasma Spraying
Thermal Spraying

HA, silver oxide

Vacuum Plasma Spraying

HA, silver

Ion Beam Assisted Deposition

HA, silver

HA, silver oxide

Sputtering took 3 h and coatings were

heat-treated at 400 C for 4 h.
0.7-1.6 wt.% Coatings were produced at an radio
frequency power level of 250 W in
argon atmosphere for 180 min.
2-6 wt.%
Coatings were prepared at 25 kW plate
power using a supersonic plasma nozzle.
3-50 wt.%
Powders were melted at 2700 C and
then sprayed onto the substrates.
1-5 wt.%
Silver and HA powers were ball milled
for 2 h prior to the coating process.
0.5-3 wt.%
Prior to coating process, both substrates
and raw materials were required to undergo
high temperature treatment.

Two-step Biomimetic Deposition/Solgel HA precipitated from saturated 8 wt.%

solution SCS, silver nitrate
Ion Beam Assisted

HA, silver nitrate

5-100 ppm

Electrophoretic Deposition/

Calcium nitrate tetrahydrate,

ammonium phosphate dibasic,
silver nitrate

20.87 wt.%

Antibacterial properties of AgHA nanocoatings

Table 4 summarizes the in-vitro antibacterial effects of
AgHA (-CP and -IE) nanocoatings produced by different
techniques. Most of the AgHA-IE nanocoatings presented
immediate bacteria inhibition after 24 h due to the presence of
silver or silver nitrate phases in the nanocoatings. Inhibition of
bacterial growth could reach up to 99%. However, for some
AgHA-IE nanocoatings, antibacterial efficacy was not effective
as viable bacteria colonies were still observed. 10 4 CFU/ml of S.
aureus population was still found adhering on the nanocoatings
despite a five time reduction on S. aureus population on
magnetron-sputtered AgHA-IE nanocoatings. 35 Similarly, 57% of
S. aureus population was killed on AgHA-IE nanocoatings deposited
by CoBlast technology after 24 h, but 9% of the initial S. aureus
population was still observed after 30 days of incubation. 50
Thus far, only a few in-vivo antibacterial studies of AgHA-IE
coated implants were examined. 53,54,59 Kose et al 59 produced
AgHA-IE nanocoatings via electrospray technique with a silver
content of 5.5 wt.%. S. aureus and methicillin-resistant S.
aureus (MRSA) were used to create infections on the left knee of
27 rabbits. Radiographic results demonstrated that all implants
had bacteria colonization, but AgHA-IE coated implants
presented a lower infection rate accompanied with a lower
osteomyelitis rate after 6 weeks. Shimazaki et al 54 evaluated the
antibacterial effects of AgHA-IE nanocoatings produced by
thermal spraying technique in a rat model. MRSA was
pre-cultured and inoculated together with implants in 10 rats.
2-log reduction of the bacterial growth was observed on
AgHA-IE coated implants without detecting inflammation after
72 h. However, the conflicting in-vitro and in-vivo antibacterial

Process Description


51, 52
37, 38, 53, 54
39, 57, 58
55, 56

Substrates were immersed into saturated

80, 81
solution for at least 7 days and then dipped
into silver nitrate for 1 or 2 days.
83, 85
HA layers were first formed via ion beam
assisted deposition; then dipped into
silver nitrate.
HA layers were first formed via electrophoretic 79, 84
deposition; then dipped into silver nitrate.

results could not confirm the antibacterial efficacy of AgHA-IE

nanocoatings. Therefore, further intensive studies were needed.
Biological characterization of AgHA nanocoatings
Table 5 summarizes the biological responses of AgHA (-CP
and -IE) nanocoatings toward various cells. Similarly to the bulk
AgHA (-CP and -IE), adipose-derived stem cells, murine macrophages and osteoblast attachment were observed in the low silver
content (less than 0.5 wt.%) of AgHA (-CP and -IE) nanocoatings.
With a higher silver content in the AgHA-IE nanocoatings, fewer
cells were observed to attach and spread onto the nanocoatings. 56,96
Titanium implants electrosprayed with AgHA-IE nanocoatings
containing 5.5 wt.% of silver were inserted into rabbits. 59 Healthy
cortical osteons were observed on AgHA-IE coated implants without
cellular inflammation after six weeks. On the other hand, bone
damage as well as inflammation occurred on the surfaces of
uncoated implants. Yonekura et al 53 compared the in-vivo
performances of HA and AgHA-IE coated implants with two
different silver contents (3 and 50 wt.%). After implanted for
12 weeks into rat tibia, the mean affinity of bone formation of
HA-coated, 3 wt.% AgHA-IE coated and 50 wt.% AgHA-IE coated
implants was 84.9%, 83.7% and 40.5%, respectively. The indices of
bone formation and bone contact around 3 wt.% AgHA-IE coated
implants were similar to that of HA coated implants, but superior to
50 wt.% AgHA-IE coated implant. Moreover, an inhibition zone
around 50 wt.% AgHA-IE coated implants was observed. These
results were in agreement with the in-vitro study indicating that high
silver content in the nanocoatings would increase the cytotoxicity.
Although preliminary in-vivo results were promising, further in-vivo
results were necessary to conclude the clinical relevance.


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

Table 3
Silver release of AgHA (-CP and -IE) nanocoatings produced by various coating techniques.
Coating Techniques

Conc. of Silver

Release of Silver ions


Electrophoretic Deposition/SolGel

20.87 wt.%

79, 84


1 wt.%

Vacuum Plasma Spraying

1-5 wt.%

Ion Beam Assisted Deposition

0.5-3 wt.%

Electrophoretic Deposition

0.4 wt.%

CoBlast Technology

0.6 at.%

Radio Frequency Plasma Spraying

2-6 wt.%

Thermal Spraying

3-50 wt.%

50% of the incorporated silver ions were released into SBF solution
within first 24 h.
Silver content of AgHA was reduced from 5.03 wt.% to 4.21 wt.%
after immersing in stimulated body fluid for 21 days.
Silver ions were released quickly in the few days and then slowed
down after day 14. After 49 days, 0.9 ppm silver ions were measurable.
Silver content was reduced from 0.63 wt.% to 0.15 wt.% after 28 days
in phosphate buffer saline solution. Silver ions were rapidly released
within the first few hours and the release rate decreased after 4 h in
ultrapure water.
Initial silver ions release was 56 ppb and accumulated to 1.704 ppm
up to 10 days in stimulated body fluid solution.
90% of incorporated silver was released into phosphate buffer saline
solution after 30 days, with 4% silver remained in coatings.
Silver release rate was slower over the first 12 h and reached near
steady-state afterwards till 168 h in phosphate buffer saline.
At 168 h, 373 ppb silver ions were measurable.

39, 57, 58
42, 55, 56

46, 47
51, 52
37, 38, 53, 54

Figure 10. Concentrations of silver in (I) body fluid and (II) stimulated body fluid after the immersion of the (A) AgHA-CP and (B) AgHA-IE coating for
different times. 57 Reprinted from surface and coatings technology, 205, Chen Y, Zheng X, Xie Y, Ji H, Ding C, Li H, Dai K., Silver release from
silver-containing hydroxyapatite coatings, p. 1892, Copyright (2010), with permission from Elsevier.

As discussed in the bulk AgHA and AgHA nanocoatings,

AgHA could not provide antibacterial properties with excellent
biological response. Chang et al 61 constructed dual-layer nanocoatings using silicon-substituted HA (SiHA) and AgHA-CP via
DoD micro-dispensing technique, to improve the biological
responses of the nanocoatings. Results showed an enhanced
bone differentiation in the dual-layer nanocoatings of SiHA and
AgHA-CP as compared to single layer nanocoatings of AgHA-CP.
On the whole, the studies on bulk Ag,Si-HA and dual-layer SiHA
and AgHA nanocoatings demonstrated that the addition of silicon
to AgHA-CP helped to mitigate the reduced bone differentiation
attributed by silver, giving rise to bi-functional properties of
antibacterial and enhanced biological responses.

Conclusions and future outlook

In summary, to minimize implant-related infections, synthetic
implants designed with antibacterial properties are desirable.

Antibacterial properties could be created in apatite through the

substitution of silver. Silver ions were either incorporated during
the co-precipitation of apatite (AgHA-CP) or underwent
ion-exchange with the calcium ions in the apatite (AgHA-IE).
However, it was found that antibacterial action of AgHA-IE and
AgHA-CP tended to differ. The antibacterial action of AgHA-IE
was dependent on the released silver ions. Although the rapid
release of silver ions from AgHA-IE was beneficial for
immediate antibacterial efficacy, this effect was not long-lasting
as viable bacteria were still observed. Moreover, the accumulation of the released silver ions at a localized site could lead to
local cytotoxicity. On the other hand, the antibacterial action of
AgHA-CP was dependent on the surface-bound silver ions. The
antibacterial efficacy would be more sustainable as antibacterial
action only took place when surface-bound silver ions interacted
with the adherent bacteria, and thus the effect of local
cytotoxicity could be minimized. Several studies reported that
nanosized AgHA-CP containing 0.5 wt.% of silver, provided an
optimal trade-off between antibacterial properties and

P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344


Table 4
In-vitro antibacterial properties of AgHA (-CP and -IE) nanocoatings.
Coating Techniques

Bacterial Strain



Micro-Arc Oxidation
Biomimetic Deposition/SolGel
Ion Beam Assisted
Electrophoretic Deposition/SolGel

S. aureus, E. coli
S. aureus, E. coli
S. aureus, E. coli, S.
epidermidis, P. aeruginosa
E. coli
S. aureus, E. coli

N99% bacteria were killed after 24 h.

N92% bacteria were killed after 24 h.
~ 100% bacteria were killed after 24 h.

80, 81

98% of bacterial growth was inhibited after 24 h.

4-log reduction of E. coli, 5-log reduction of S. aureus
after 12 h, N 95%.
S. albus and E. coli were killed after 12 h; 1 log
reduction of MRSA was observed after 1 h.
Bacteria colonies were found near the corner of coatings
but inhibition zone was visible.
1-log reduction of bacteria was observed after 24 h.
2-log reduction of bacteria were observed after 48 h.
95% of bacteria were killed after 24 h;
Sparse bacteria were detected after 48 h; 96 h later,
complete confluence of bacterial layer was found.
Dead bacteria were observed after 8 h but no significant
inhibition effect was detected.
Inhibited N99% bacteria growth after 24 h.
N98% reduction of bacteria were killed immediately after
1 h and the inhibition effect maintained till 24 h later.
57% of bacteria were killed after 24 h and 9% of initial
bacteria amount was observed after 30 days incubation.
42 times reduction of colonies were observed on dual-layer
AgHA coatings after 24 h.
Bacterial growth was inhibited after 24 h but scarce colonies
were still noticeable on surface with highest Ag concentration.
Colony on AgHA coatings reduced to less than 10 but less
effective for MRSA in 24 h.


S .albus, MRSA, E. coli

S. mutans
Magnetron Co-Sputtering
Thermal Substrate Method
Vacuum Plasma Spraying

S. aureus, S. epidermidis
E. coli
S. aureus E. coli, P. aeruginosa

Ion Beam Assisted Deposition

S. epidermidis, P. aeruginosa

Pulsed Laser Deposition

Electrophoretic Deposition

B. subtilis, E. coli
S. aureus

CoBlast Technology

S. aureus

DoD Micro-Dispensing Technique

S. aureus

Radio Frequency Plasma Spraying

P. aeruginosa

Thermal Spraying

MRSA, S. aureus, E. coli

88, 89
39, 57, 58

42, 55, 56
36, 90
46, 47
61, 62
51, 52
37, 38, 53, 54

Table 5
Biological properties of AgHA (-CP and -IE) nanocoatings in in-vitro cell studies.
Cell Type

Coating Techniques

Conc. of Ag

V79 Chinese hamster lung cells

NIH 3T3 fibroblast

Thermal Spraying
Pulsed Laser Deposition

3 wt.%
1.2/4.4 wt.%


No cytotoxicity.
Mild cytotoxic effect was observed on AgHA coatings
with cell morphology destruction and decolonization.
L929 fibroblast
Vacuum Plasma Spraying
1/3/5 wt.%
No cytotoxicity and hemolysis effects of AgHA
coatings when Ag content is less than 5 wt.%.
Electrophoretic Deposition/
20.87 wt.%
Cells didnt reach confluence on AgHA coatings after
5 days. Osteogenic activity of MG63 was inhibited
on AgHA coatings.
1.0, 1.5 wt.% Osteoconductivity of AgHA coatings was proved. A
Human embryonic palatal
mesenchyme (HEPM) cells
significant less ALP activity was observed on
1.5 wt.% AgHA.
HEPM cells
Magnetron Co-Sputtering
2 wt.%
No cytotoxicity.
0.3-1.6 wt.% Cell adhesion, spreading and ALP expression was
depressed on more concentrated AgHA coatings.
MC3T3 osteoblast
Ion Beam Assisted Deposition 1/3/6.6 wt.% Silver concentration within 1 to 3 wt.% gave good
biocompatibility and antibacterial properties.
Human osteoblast cell line (hFOB) Radio Frequency
0.7-1.6 wt.% Less cell proliferated on AgHA coatings as compared
Magnetron Sputtering
to HA coatings; poor cell attachment and almost no ALP
activity on AgHA coatings indicated cytotoxicity.
Adipose-derived stem cells
DoD Micro-Dispensing
0.5 wt.%
No cytotoxicity in cell proliferation and differentiation;
but was not superior to HA coatings in terms of
cellular response.
HEp2 cell line
Pulsed Laser Deposition
0.53 wt.%
No cytotoxicity.

cytotoxicity. Nevertheless, with the substitution of silver, bone

differentiation markers such as alkaline phosphatase, type I
collagen and osteocalcin cultured on nanosized AgHA-CP were

37, 38, 53, 54
36, 90
39, 57


88, 89


49, 60

compromised. Thus, nanosized AgHA-CP could not achieve a

balance between antibacterial properties and favorable biological
responses, hindering its usage in the biomedical applications.


P.N. Lim et al / Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 13311344

This greatly motivated the addition of silicon to AgHA. The

initial research of nanosized Ag,Si-HA demonstrated promising
results of having antibacterial properties and enhanced biological
response as compared to nanosized AgHA-CP and HA. However
a better understanding in the mechanism of the antibacterial
action of surface-bound silver ions of Ag,Si-HA was required. It
was not clear how surface-bound silver ions of the nanosized
Ag,Si-HA interacted with bacteria, leading to bacteria death.
Furthermore, in order to work toward clinical applications, more
experiments should be conducted in the in-vivo antibacterial
activity and in-vivo bioactivity of the nanosized Ag,Si-HA. For
nanocoatings, AgHA-IE nanocoatings also suffered the problem of
attaining sustained antibacterial efficacy and local cytotoxicity.
The need for better depositing techniques would be required to
deposit silver ions homogenously in the coating layer. The
preliminary results of dual-layer SiHA and AgHA nanocoatings
also showed enhanced biological responses as compared to single
layer of AgHA nanocoatings. The strategy of combining silver
with silicon is a potential way to promote bone-implant integration,
with reduced bacterial infection and enhanced bioactivity.

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