Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 1331 – 1344 nanomedjournal.com

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

Abstract 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. © 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: [email protected] (E.S. Thian). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.nano.2015.03.016 1549-9634/© 2015 Elsevier Inc. All rights reserved.

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

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

Ref.

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.

98 1, 35, 99-102

103

104

105

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.

106-109 103, 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 host’s 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

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34

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). þ

ð10−yÞCa2 ðaqÞ þ yAgþ ðaqÞ þ 6PO4 3− ðaqÞ þ ð2−yÞOH− ðaqÞ→ Ca10−y Agy ðPO4 Þ6 ðOHÞ2−y ðaqÞ

ð1Þ

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 β-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, α-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

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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

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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

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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.%

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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 sol–gel 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 sol–gel 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

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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 substrate’s surface due to electrostatic forces. Using CoBlast technology, AgHA-IE was deposited onto substrate’s 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 substrate’s 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 Hank’s 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 sol–gel deposition. Similarly, phase-purity of AgHA-IE nanocoatings was difficult to retain in

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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.

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

Materials

Conc. of Silver

One-step

Magnetron Co-Sputtering

HA, silver clip

2 wt.%

Radio Frequency Magnetron Sputtering

AgHA

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/Sol–gel HA precipitated from saturated 8 wt.% solution SCS, silver nitrate Ion Beam Assisted Deposition/Sol–gel

HA, silver nitrate

5-100 ppm

Electrophoretic Deposition/ Sol–gel

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

Ref. 35 48

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.

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

Conc. of Silver

Release of Silver ions

Ref.

Electrophoretic Deposition/Sol–Gel

20.87 wt.%

79, 84

Electrospraying

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.

59 39, 57, 58 42, 55, 56

46, 47 50 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

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

Bacterial Strain

Effects

Ref.

Micro-Arc Oxidation Biomimetic Deposition/Sol–Gel Ion Beam Assisted Deposition/Sol–Gel Electrophoretic Deposition/Sol–Gel Sol–Gel

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.

77 80, 81 85

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.

79 87

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 74 35 76 39, 57, 58

42, 55, 56 36, 90 46, 47 50 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.%

Effects

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.%. MG63 Electrophoretic Deposition/ 20.87 wt.% Cells didn’t reach confluence on AgHA coatings after Sol–Gel 5 days. Osteogenic activity of MG63 was inhibited on AgHA coatings. Sol–Gel 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. Osteoblast Sol–Gel 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; Technique 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

Ref. 37, 38, 53, 54 36, 90 39, 57 79

40

35 88, 89 56 48

61

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.

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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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