Silver nanoparticles in dentistry

Highlights

  • AgNPs are applied in nanocomposites; implant coatings; anti-caries formulations; and in the treatment of oral cancer and local anesthesia.

  • In vitro results reveal the excellent antimicrobial activity of AgNPs when associated with acrylic resins, resin co-monomers, adhesives, intracanal medication and implant coatings.

  • In vivo results also confirm the potential use of AgNPs against microbial infections, especially caries.

Abstract

Objective

Silver nanoparticles (AgNPs) have been extensively studied for their antimicrobial properties, which provide an extensive applicability in dentistry. Because of this increasing interest in AgNPs, the objective of this paper was to review their use in nanocomposites; implant coatings; pre-formulation with antimicrobial activity against cariogenic pathogens, periodontal biofilm, fungal pathogens and endodontic bacteria; and other applications such as treatment of oral cancer and local anesthesia. Recent achievements in the study of the mechanism of action and the most important toxicological aspects are also presented.

Methods

Systematic searches were carried out in Web of Science (ISI), Google, PubMed, SciFinder and EspaceNet databases with the keywords “silver nano* or AgNP*” and “dentist* or dental* or odontol*”.

Results

A total of 155 peer-reviewed articles were reviewed. Most of them were published in the period of 2012–2017, demonstrating that this topic currently represents an important trend in dentistry research. In vitro studies reveal the excellent antimicrobial activity of AgNPs when associated with dental materials such as nanocomposites, acrylic resins, resin co-monomers, adhesives, intracanal medication, and implant coatings. Moreover, AgNPs were demonstrated to be interesting tools in the treatment of oral cancers due to their antitumor properties.

Significance

The literature indicates that AgNPs are a promising system with important features such as antimicrobial, anti-inflammatory and antitumor activity, and a potential carrier in sustained drug delivery. However, there are some aspects of the mechanisms of action of AgNPs, and some important toxicological aspects arising from the use of this system that must be completely elucidated.

Introduction

The use of silver in oral care has been known for centuries and gained worldwide spread in the 19th century as one the main components in dental amalgams used for tooth restoration. Its use in amalgams has been reduced since 1930 as they were progressively substituted by esthetic polymer-based resins . Since nanoscience has evolved and the outstanding antimicrobial properties of nanostructured silver-based formulations have been demonstrated against microorganisms such as bacteria, viruses, and fungi , the interest in silver has been renewed, and several promising new technologies are currently under development, especially in dental materials. In this context, AgNPs have been demonstrated to be effective antimicrobial components in prosthetic materials , adhesives , and implants , to promote caries arrestment , to prevent biofilm formation , and for osteogenic induction . Fig. 1 shows the increasing interest in AgNPs in the 21st century in dentistry. As a result, it is reasonable to foresee that, in the near future, AgNPs will play an important role in oral healthcare.

Fig. 1
Flow chart of (top) the absolute number of publications enrolling the evaluation of AgNPs in Dentistry and (bottom) the citations associated with these publications. The literature search was based on Web-of-Science™, with the keywords “silver nano* or AgNP*” and “dentist* or dental*”. For 2017 it was considered publications up to May (*).

Silver has a [Kr]4d 10 5s 1 electron configuration. Since the 4d shell does not effectively shield the 5s 1 electrons, they are strongly attracted to the nucleus, and a relatively high standard reduction potential is observed (E 0 (Ag + /Ag) = +0.799 V). Hence, metallic silver is rather unreactive. On the other hand, silver has the lowest first ionization potential in Group 11 and thus, Ag + is the most stable species in aqueous solutions, including physiological fluids, and in solids. Due to the filled 4d orbital, Ag + compounds present a significant covalent character and they do not tend to form organometallic compounds. In addition, they do form complexes with coordination numbers as low as 2 . Silver ions form bonds preferably with sulfur but also with nitrogen and oxygen. So Ag + can bind to enzymes with S-pending groups as well as N atoms from nucleic acids . Besides formulations containing Ag + , other silver-based compounds have also been successfully applied in dentistry. For instance, the linear complex [Ag(NH3)2 + ]F, which was recently cleared for caries arrestment in the US, is used in Japan for more than 80 years . However, with the development of nanotechnologies, AgNPs have gained most of the focus.

It is now possible to produce AgNPs with controlled size and morphology, high homogeneity (i.e. low polydispersity index) , and specific target functions, (i.e. functionalized with molecular capping agents, from small hydrophilic and hydrophobic chemical groups to large biomacromolecules, such as proteins) . The nucleation and growth process of AgNPs can be mediated both by synthetic reagents and by biologically available products from plants and microbes . As the nanoparticle composition, particle size distribution, morphology and surface chemistry can be finely tuned , AgNPs can access different sites in the oral cavity; in such a way they can currently be conceived as multifunctional building blocks for dental materials and dentistry protocols.

Recent scientific achievements demonstrated the benefits of the use of AgNPs in dentistry as antimicrobial agents in a wide range of applications, preventing the need for infection therapies in several cases . AgNPs have shown a very high antimicrobial effect, in comparison with several antimicrobial molecules, with good biocompatibility . They can also act synergistically with several types of antibiotics . Furthermore, AgNPs have been used as the main agent in inorganic and polymeric-based antimicrobial (nano)composites . Also, important studies have been carried out in order to elucidate the potential risks to human health, and through environmental exposure of AgNPs, thus trying to delimit the safe use of AgNPs-based products and technology . Multiple and broad aspects of AgNP synthesis, processing, applications and toxicology have been well covered in recent reviews . In the last three years, some influential reviews with different focuses on the application of nanotechnology in dentistry have been published . However, aspects involving AgNPs production and nanobiotechnological applications were reviewed only in a general sense, and not specifically for dentistry . Also, their applications in dental biomaterials covered specifically implants and their incorporation into nanocomposites .

In this review, the perspectives related to AgNPs-based technologies aimed at oral care are presented in detail, and organized by their application in (i) nanocomposites, (ii) implant coatings, (iii) pre-formulation with antimicrobial activity against cariogenic pathogens, periodontal biofilm, fungal pathogens and endodontic bacteria, and (iv) other applications (local anesthesia and oral cancer). Recent achievements in the study of the mechanism of action of AgNPs are also presented, as well as the most important toxicological aspects.

Mechanistic aspects of silver acting on bacteria

Although the antibacterial mechanism of AgNPs has not been fully elucidated, some aspects of the antimicrobial action of AgNPs have been recognized. Silver ions are capable of acting on different structures of the bacterial cell. Primarily, these ions seem to adhere to the cell wall and cytoplasmic membrane through electrostatic attraction and affinity to sulfur proteins, thus enhancing the permeability of the membrane and also leading to disruption of these structures . In Gram-negative bacteria, porins in the outer membrane are also involved in the uptake of AgNPs . Bacterial molecules that can be damaged by AgNPs include DNA , proteins and lipids . AgNPs also stimulate oxidative stress response causing bacterial cell destruction and increase dephosphorylation of tyrosine residues on bacterial peptide substrates, inhibiting bacterial growth and viability . Cell membrane disruption and DNA modification via reactive oxygen species (ROS) as the principal agent were also described in the literature . These mechanisms are illustrated in Fig. 2 .

Fig. 2
Mechanisms of action of silver ions, released from AgNPs, against bacteria. These include (i) interaction with the peptidoglycan cell wall and the membrane, thus causing cell lysis, (ii) interaction with bacterial (cytoplasmic) DNA, preventing DNA replication, and (iii) interaction with bacterial proteins and disrupting protein synthesis. From Ref. with permission from Elsevier.

Bacteria in the oral cavity are preferably organized in biofilms, which confer better conditions for growth, immunological evasion and resistance to antibiotics . Specifically in dentistry, the preparation of nanoparticles must take into account the biofilm architecture and the mechanistic aspects of AgNPs. The nanoparticle’s properties may affect its efficiency and interfere with its mechanism of action. Important aspects to be mentioned in this connection: (i) the diffusion of nanoparticles in biofilm exhibits an inverse relationship between effectiveness and size; nanoparticles over 50 nm are not able to penetrate the biofilm due to the relative self-diffusion coefficients in the biofilm, and this decrease exponentially with the square of the nanoparticle diameter . In addition, (ii) charged nanoparticles do not diffuse easily through the biofilm, probably because the presence of phosphoryl and carboxyl groups on the surface of the bacteria, which gives the cell surface an electronegative character .

The concentration in formulations is commonly given by the total amount of Ag (metallic Ag and Ag + ), and this value is provided in μg/mL. The commonly used technique to determine the total amount of Ag (per mL) is ICP (inductively coupled plasma spectrometry) . In some cases, it is differentiated the metallic silver nanoparticles (Ag 0 ) and the silver ions (Ag + ) . However, this concentration is difficult to be compared because the dissolution of the Ag + , and thus, its concentration, depends on several synthetic and processing parameters, such as the medium composition and ionic strength, AgNPs size and morphology, and the AgNPs capping agents . It is currently accepted that the actual antibacterial element is Ag + . But silver nanoparticles can be oxidized inside bacteria. In this case, aspects involving the bacterial uptake (penetrability) of AgNPs become very relevant . It is also important to mention that the penetrability of ions and nanoparticles are different.

Due to necessity of AgNPs dissolution for effective antibacterial action (through Ag + release), the size and shape of the particles can influence their action along with their concentration. Between nanoparticles with spherical or quasi-spherical format, those with smaller size have higher activity once their surface exhibits a larger area, thus being more prone to dissolution when compared to larger AgNPs. This also can explain why aggregated nanoparticles possess lower antibacterial activity, since they had a smaller surface area exposed to solvent than isolated nanoparticles .

It was recently presented that the activity of AgNPs with sizes less than 10 nm is due mainly to the nanoparticle itself, while for those larger than 10 nm, the predominant mechanism occurs through the silver ions . In this way, the precise range of size in which AgNPs have antimicrobial effects is yet to be determined. Furthermore, it is important to research how the AgNPs are oxidized to Ag + ions in the cells. Finally, but not less important, we still do not have an historical report on the long-term bacterial response towards AgNPs. These studies are imperative to assess the possible bacterial resistance to AgNPs. Despite all these issues, studies have shown that AgNPs have considerable potential as alternatives in antibacterial applications in the future.

The time necessary to completely release Ag + from AgNPs depends on their dissolution process, which is attributed to oxidation of the metallic Ag . The dissolution depends on the medium ionic strength, pH, concentration of the dissolved O 2 , temperature, presence of complexing ligands, surface coatings, and AgNPs shape and size . For instance, Peretyazhko et al. examined size-dependent dissolution of AgNP with different sizes (6, 9, 13, and 70 nm) coated with thiol-functionalized ligands (methoxyl polyethylene glycol (PEGSH)) at neutral and acidic pHs by measuring the Ag + concentration up to 80 days . They demonstrated a size dependent dissolution (increased as the size decreased) in both solutions and a faster dissolution in the acidic solution ( Fig. 3 ). Moreover, TEM images demonstrated that the particle morphology was not affected by dissolution and no aggregation was observed. In addition, AgNPs increased in size after dissolution likely due to Ostwald ripening, where in suspension, dissolved Ag + redeposits on AgNPs surface. In the case of citrate-stabilized AgNPs, the oxidation process is slow (6–125 days to completion), and the particle shape can persist for a long time. Along this period, these AgNPs can be submitted to dissolution, transport, aggregation, and/or endocytic/phagocytic uptake into cells to reach different biological targets .

Fig. 3
(A) Kinetics of dissolution of Ag + (in wt%) from AgNPs as a function of time (in days) and as a function of the AgNPs sizes. The analysis was performed for AgNPs with sizes of 6 (AgNP_6), 9 (AgNP_9), 13 (AgNP_13) and 70 nm (AgNP_70), in water (top) and acetic acid (bottom). (B) Normalized size distribution functions of AgNPs prior to and after ∼30 days of dissolution in water. Adapted from Ref. with permission from American Chemical Society.

It is important to consider that the effect of AgNPs on Gram-negative bacteria was different from other studied bacteria, suggesting that the antimicrobial activity cannot be explained only by the release of Ag + from AgNPs. These latter enter into bacteria more effectively than Ag + , depending on the bacterial strains analyzed. Therefore, taken together, Ag + is important on the antibacterial activity, but bacterial internalization/uptake of AgNPs needs to be considered .

Mechanistic aspects of silver acting on bacteria

Although the antibacterial mechanism of AgNPs has not been fully elucidated, some aspects of the antimicrobial action of AgNPs have been recognized. Silver ions are capable of acting on different structures of the bacterial cell. Primarily, these ions seem to adhere to the cell wall and cytoplasmic membrane through electrostatic attraction and affinity to sulfur proteins, thus enhancing the permeability of the membrane and also leading to disruption of these structures . In Gram-negative bacteria, porins in the outer membrane are also involved in the uptake of AgNPs . Bacterial molecules that can be damaged by AgNPs include DNA , proteins and lipids . AgNPs also stimulate oxidative stress response causing bacterial cell destruction and increase dephosphorylation of tyrosine residues on bacterial peptide substrates, inhibiting bacterial growth and viability . Cell membrane disruption and DNA modification via reactive oxygen species (ROS) as the principal agent were also described in the literature . These mechanisms are illustrated in Fig. 2 .

Fig. 2
Mechanisms of action of silver ions, released from AgNPs, against bacteria. These include (i) interaction with the peptidoglycan cell wall and the membrane, thus causing cell lysis, (ii) interaction with bacterial (cytoplasmic) DNA, preventing DNA replication, and (iii) interaction with bacterial proteins and disrupting protein synthesis. From Ref. with permission from Elsevier.

Bacteria in the oral cavity are preferably organized in biofilms, which confer better conditions for growth, immunological evasion and resistance to antibiotics . Specifically in dentistry, the preparation of nanoparticles must take into account the biofilm architecture and the mechanistic aspects of AgNPs. The nanoparticle’s properties may affect its efficiency and interfere with its mechanism of action. Important aspects to be mentioned in this connection: (i) the diffusion of nanoparticles in biofilm exhibits an inverse relationship between effectiveness and size; nanoparticles over 50 nm are not able to penetrate the biofilm due to the relative self-diffusion coefficients in the biofilm, and this decrease exponentially with the square of the nanoparticle diameter . In addition, (ii) charged nanoparticles do not diffuse easily through the biofilm, probably because the presence of phosphoryl and carboxyl groups on the surface of the bacteria, which gives the cell surface an electronegative character .

The concentration in formulations is commonly given by the total amount of Ag (metallic Ag and Ag + ), and this value is provided in μg/mL. The commonly used technique to determine the total amount of Ag (per mL) is ICP (inductively coupled plasma spectrometry) . In some cases, it is differentiated the metallic silver nanoparticles (Ag 0 ) and the silver ions (Ag + ) . However, this concentration is difficult to be compared because the dissolution of the Ag + , and thus, its concentration, depends on several synthetic and processing parameters, such as the medium composition and ionic strength, AgNPs size and morphology, and the AgNPs capping agents . It is currently accepted that the actual antibacterial element is Ag + . But silver nanoparticles can be oxidized inside bacteria. In this case, aspects involving the bacterial uptake (penetrability) of AgNPs become very relevant . It is also important to mention that the penetrability of ions and nanoparticles are different.

Due to necessity of AgNPs dissolution for effective antibacterial action (through Ag + release), the size and shape of the particles can influence their action along with their concentration. Between nanoparticles with spherical or quasi-spherical format, those with smaller size have higher activity once their surface exhibits a larger area, thus being more prone to dissolution when compared to larger AgNPs. This also can explain why aggregated nanoparticles possess lower antibacterial activity, since they had a smaller surface area exposed to solvent than isolated nanoparticles .

It was recently presented that the activity of AgNPs with sizes less than 10 nm is due mainly to the nanoparticle itself, while for those larger than 10 nm, the predominant mechanism occurs through the silver ions . In this way, the precise range of size in which AgNPs have antimicrobial effects is yet to be determined. Furthermore, it is important to research how the AgNPs are oxidized to Ag + ions in the cells. Finally, but not less important, we still do not have an historical report on the long-term bacterial response towards AgNPs. These studies are imperative to assess the possible bacterial resistance to AgNPs. Despite all these issues, studies have shown that AgNPs have considerable potential as alternatives in antibacterial applications in the future.

The time necessary to completely release Ag + from AgNPs depends on their dissolution process, which is attributed to oxidation of the metallic Ag . The dissolution depends on the medium ionic strength, pH, concentration of the dissolved O 2 , temperature, presence of complexing ligands, surface coatings, and AgNPs shape and size . For instance, Peretyazhko et al. examined size-dependent dissolution of AgNP with different sizes (6, 9, 13, and 70 nm) coated with thiol-functionalized ligands (methoxyl polyethylene glycol (PEGSH)) at neutral and acidic pHs by measuring the Ag + concentration up to 80 days . They demonstrated a size dependent dissolution (increased as the size decreased) in both solutions and a faster dissolution in the acidic solution ( Fig. 3 ). Moreover, TEM images demonstrated that the particle morphology was not affected by dissolution and no aggregation was observed. In addition, AgNPs increased in size after dissolution likely due to Ostwald ripening, where in suspension, dissolved Ag + redeposits on AgNPs surface. In the case of citrate-stabilized AgNPs, the oxidation process is slow (6–125 days to completion), and the particle shape can persist for a long time. Along this period, these AgNPs can be submitted to dissolution, transport, aggregation, and/or endocytic/phagocytic uptake into cells to reach different biological targets .

Fig. 3
(A) Kinetics of dissolution of Ag + (in wt%) from AgNPs as a function of time (in days) and as a function of the AgNPs sizes. The analysis was performed for AgNPs with sizes of 6 (AgNP_6), 9 (AgNP_9), 13 (AgNP_13) and 70 nm (AgNP_70), in water (top) and acetic acid (bottom). (B) Normalized size distribution functions of AgNPs prior to and after ∼30 days of dissolution in water. Adapted from Ref. with permission from American Chemical Society.

It is important to consider that the effect of AgNPs on Gram-negative bacteria was different from other studied bacteria, suggesting that the antimicrobial activity cannot be explained only by the release of Ag + from AgNPs. These latter enter into bacteria more effectively than Ag + , depending on the bacterial strains analyzed. Therefore, taken together, Ag + is important on the antibacterial activity, but bacterial internalization/uptake of AgNPs needs to be considered .

Application of AgNPs in nanocomposites

AgNPs have been incorporated into tissue conditioner, denture resins, and other biomaterials . Antifungal effect against Candida albicans has been demonstrated when AgNPs were added to poly(methyl methacrylate) (PMMA) resins for dentures and silicone-based soft liners . This microorganism may cause denture stomatitis and mucosal infections. Nanocomposites of acrylic resins and AgNP (∼38 nm) have also shown a strong antimicrobial effect against Escherichia coli together with improved flexural strength and modulus . In addition, it is an interesting approach to introduce AgNPs into new experimental PMMA formulations to decrease the microbial adherence and colonization in prosthetic devices in general. For instance, AgNPs (∼60 nm) were incorporated in PMMA denture resins to conjugate antimicrobial properties for complete denture wearers for controlling infections in oral mucosal tissues . It was shown that AgNPs were stably incorporated in the acrylic resin of which the denture was composed, and no nanoparticle release was observed during 120 days of denture storage in deionized water. In another work, AgNPs (∼60 nm) incorporated in acrylic resin denture base material led to an enhancement of storage modulus E’ and loss tangent Tan δ values in concentrations equal to or higher than 2 wt% . This modification led to resins with greater mechanical energy dissipation properties, thus enhancing the material durability.

Inclusion of silver and amorphous calcium phosphate nanoparticles of sizes ∼3 nm and ∼100 nm, respectively, into a resin co-monomer blend represents an important method to produce antibacterial nanocomposites . The authors used two raw mixtures: (i) silver 2-ethylhexanoate salt dissolved in 2-( tert -butylamino)ethyl methacrylate (Ag-TBAEMA); and (ii) bis(2-methacryloyloxyethyl) dimethylammonium bromide (QADM) mixed with photo-activated BisGMA–TEGDMA resin, and also incorporated with amorphous calcium phosphate (NACP) and barium boroaluminosilicate glass. The final composition of the resulting cohesive paste was 30 wt% NACP, 35 wt% glass, 27.972 wt% BisGMA-TEGDMA, 7 wt% QADM, and 0.028 wt% Ag. The AgNPs were formed in the resin by simultaneous reduction of the silver ions and photopolymerization of the dimethacrylates (see Fig. 4 ). Composites were submitted to a water-ageing process, and mechanical and biofilm tests. The composite containing both QADM and AgNPs (NAg) showed better antibacterial effects than that with QADM or AgNPs (NAg) alone. Twelve months of water ageing did not affect the mechanical and anti-biofilm properties. In addition, Melo et al. demonstrated that the presence of AgNPs in bonding agents had no impact on the dentin shear bond strength, but led to a substantial reduction in the viability and metabolic activity of the biofilms. A decrease in colony-forming units was observed (total microorganisms, streptococci, and Streptococcus mutans ), as well as a reduction in the production of lactic acid when AgNPs were added to the adhesive.

Fig. 4
An important achievement in silver nanocomposites was obtained with the incorporation of AgNPs (NAg) and amorphous calcium phosphate (NACP) in mixtures of BisGMA–TEGDMA resins, also containing quaternary ammonium dimethacrylate (QADM). Transmission electron microscopy (TEM; first column) in (a) low and (b) high magnifications confirmed the formation of 3 nm AgNPs in the nanocomposite (indicated by arrows). These experimental materials were compared to commercially available restorative composites with (Ivoclar Heliomolar™; CompositeF) or without (Cosmedent Renamel™; CompositeNoF) fluorine. Confocal laser scanning microscopy (CLSM; second and third columns) indicated live (green) and dead (red; indicated by arrows) bacteria in S. mutans biofilms formed after 1 day on (c) CompositeNoF, (d) CompositeF, and BisGMA–TEGDMA resin incorporated with (e) NACP, (f) NACP + QADM, (g) NACP + NAg, and (h) NACP+ QADM + NAg. Adapted from Ref. with permission from Elsevier. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

AgNPs obtained from Fusarium oxysporum (20–40 nm) were incorporated in heat-activated acrylic resin discs. The surface roughness and hardness did not change significantly, and the C. albicans biofilm formation showed a decrease of up to 79% when compared to the control group . Furthermore, it was reported for composite resin discs that AgNP incorporation (0.35%) led to an inhibition of S. mutans and Lactobacillus acidophilus biofilms growth, with no effect on the strength in compression and surface roughness . Akhavan et al. demonstrated that incorporating AgNPs into orthodontic adhesives could increase or maintain the shear bond strength of an orthodontic adhesive . In addition, a recent study by Chladek and co-workers found that incorporating 22 nm colloidal AgNPs into dental composites could reduce the colonization of microorganism of lining materials, enhancing the antifungal efficiency against C. albicans . Finally, when AgNPs were incorporated into the commercially available resinous cement Vitrebond™, the resulting nanocomposite displayed an increased antibacterial effect against S. mutans .

More recently, glass ionomer cements were modified by the incorporation (up to 2% weight ratio) of biogenically produced AgNPs, which synthesis was mediated with the extract from Mangifera indica (Mango leaves). AgNPs crystallite size calculated from the X-ray diffractograms was of 32 ± 2 nm, and the maximum band energy observed from UV–vis absorption spectroscopy was of 392 nm . Monsanto hardness increased in the nanocomposite by about 20% with the AgNPs incorporation, while Vickers hardness increased about 35%. Both results indicated a reinforcement effect led by the presence of AgNPs. Finally, the authors also observed an increase in the inhibition zone induced by the AgNPs-modified glass ionomer cements (nanocomposite) against Escherichia coli and Staphylococcus aureus .

Most of previous reports indicate that incorporation of AgNPs into experimental restorative materials did not affect their mechanical properties in comparison to commercial materials and other controls . However, some recent studies concerning the impact of the addition of nanoparticles in dental materials have focused their efforts specifically on the evaluation of the degree of polymerization by quantifying free monomers or by measuring shear bond strength of their materials. Durner et al. added from 0.0125 to 0.3% of AgNPs into a commercial composite. The specimens were light-cured and they were stored up to 7 days in methanol. The analysis of the eluate was performed through chromatography/mass spectrometry. When AgNPs were added to the composites, higher levels of camphorquinone, BisEMA and TEGDMA were found within the methanol elution in groups containing AgNPs than in controls. The authors concluded that AgNPs might impair the polymerization process of dental materials, as non-polymerized substances were easily eluted in methanol, and therefore found in higher concentrations. In another study, shear bond strength to dentin of different dental materials (dentin bonding systems, glass ionomer cement and nanohybrid composite filling material) alone and combined with silver and gold nanoparticles was evaluated . The addition of AgNPs apparently did not have impact on the adhesives to non-carious dentin, as no significant differences among experimental materials in comparison to controls were found.

As discussed previously , AgNPs might interfere in polymerization, chemically or physically, by reflecting or scattering the light from the curing lamp or even by reacting with the unpaired electrons from the photoinitiator system. Therefore, it is still controversial whether and to which extent AgNPs may impact polymerization process, and further studies are necessary to elucidate these mechanisms and hypothesis.

Implants modified with AgNPs

The coating of implants has been a strategy to hamper bacterial adhesion to their surfaces and also to stimulate osseointegration and fibroblast proliferation. AgNPs together with other antibiotics have been tested in coating formulations, showing favorable results regarding antimicrobial activity. AgNPs were used in combination with tantalum nitride for the coating of titanium substrates. The composites with a silver concentration of 21.4 wt% presented significant antibacterial effect against Staphylococcus aureus . When biocompatibility tests were performed by evaluating the growth of human gingival fibroblasts (HGF) exposed to AgNPs, the coated samples exhibited greater cell viability and proliferation than did the uncoated samples . In addition, AgNPs (size not provided) prevented contamination of the internal surface of the implant by C. albicans, produced by the implant/abutment microgap infiltration. The abutments were screwed with 10 N torque for all groups studied: (G1) implants that received application of AgNPs suspension on their internal screw surface (abutment); (G2) a positive control group with implants that received the application of sterile phosphate buffered saline (PBS) on the abutment; and (G3) a negative control group that also received the application of AgNPs. Both G1 and G2 were immersed in a suspension of C. albicans and G3 was immersed in sterile Sabouraud dextrose broth (SDB). No C. albicans contamination was observed in the negative control group (after 72 h of immersion in the microorganism suspension) and when analyzed the positive control group was found to contain statistically higher values of colony forming units of C. albicans as compared with the experimental group that contained AgNPs .

In a similar study, citrate-capped AgNPs had their antimicrobial and biocompatibility properties tested for application in titanium coating. AgNPs were tested in two different approaches: (i) the first aimed to evaluate their activity against planktonic bacteria, in which AgNPs were applied dispersed in the liquid medium, and (ii) the second evaluated their activity against sessile bacteria, in which AgNPs were immobilized on Ti implants. As a result, the bactericidal effects against both S. aureus and Pseudomonas aeruginosa (<4 μM) were observed at a much lower concentration than that at which cytotoxicity effects manifested against UMR-106 osteoblastic cells in in vitro assays (>50 μM) . Furthermore, a tiny amount of AgNPs on the surface of Ti, attached by immersing the Ti substrate in a 293 μmol L −1 suspension of AgNPs, had an antimicrobial effect similar to that of a pure AgNPs surface.

AgNPs have also been deposited by anodic spark deposition in combination with a solution containing silicon, calcium, phosphorus and sodium for a biomimetic coating on titanium substrates that could provide osteointegrative properties. The biomimetic coating was able to reduce bacterial adhesion up to 50% ( S. epidermidis, S. mutans and E. coli were tested) after 3 h and 24 h of incubation and also allowed optimal spreading and proliferation of human osteoblast-like cells (SAOS-2) due to the enhancement of surface roughness by AgNPs . Another work has also employed anodic spark deposition to graft AgNPs onto titanium disks. Cytotoxic effects for the modified Ti surface were evaluated in vitro by using primary human gingival fibroblasts (HGF). Antibacterial effects were also tested in an in vivo assay in which seven individuals wore appliances with polyvinyl chloride (PVC) molds containing the Ti substrates for 24 h, and in an in vitro assay through incubation of the substrates with S. mutans for 72 h. Silver coating was able to reduce the presence of bacterial colonies by about 30% as observed in the in vivo assay, and to completely inhibit the presence of biofilm or viable bacterial colonies in vitro . No cytotoxic effects were detected and no adverse effects were observed on antibacterial in vivo assay .

Furthermore, a mussel-inspired AgNP/calcium phosphate (CaP) composite coating was synthesized on anodized Ti, with the prospect of its surface maintaining suitable biological performance, leading to long-term antibacterial activity. This approach involves three possible steps: (i) Ti anodic oxidation to enable it to exhibit a TiO 2 nanotubular (TNT) surface structure, (ii) dopamine self-polymerization on TNT and the reduction of Ag + ions, and (iii) AgNP modification by using polydopamine and further immersion in SBF for the biomimetic mineralization of CaP (see Fig. 5 ). The AgNPs/CaP coatings exhibited antibacterial effects against S. aureus and a reasonable good in vitro cytocompatibility with MG63 cells. Pristine Ti exhibited less alkaline phosphatase (ALP) activities than the cells cultured on the coated Ti .

Fig. 5
(a) A nanostructured Ti implant with antimicrobial and osteointegrative properties was produced with (i) the generation of titania nanotubes (sample TNT) on the implant surface, (ii) adsorption of dopamine (sample TNT-D), (iii) attachment of AgNPs, and finally with (iv) the formation of calcium phosphate on the surface (sample Ag-D-1CaP) after 1 day of immersion in simulated body fluid (SBF). (b) Scanning electron microscopy in low (top-left) and high (top-right) magnifications showing the morphology of the calcium phosphate (CaP) coating formed after 1 day of immersion in SBF. Ag, Ca and P elemental maps acquired from energy dispersive X-ray spectroscopy (EDS) confirm the presence of CaP and AgNPs. (c) CLSM (Confocal laser scanning microscopy of stained S. aureus present on the surface of sample Ag-D-1CaP indicated the anti-adherent property of the nanostructured implant. (d) CLSM also indicated the capacity of the nanostructured Ti implant to support and induce proliferation of MG-63 osteoblast-like cells. Reproduced from Ref. with permission from The Royal Society of Chemistry.

AgNPs with narrow particle size distribution were electrochemically deposited onto titanium coated with hydroxyapatite (HA) and the composite coating tested for antibacterial activity against Staphylococcus aureus . Calcium deposition from simulated body fluid was evaluated. The AgNPs/HA surface was able to inhibit S. aureus growth and promote in vitro biomineralization .

Starch-capped AgNPs were incorporated into ordered nanoporous silica coatings on the surface of titanium disks by a combined sol–gel and evaporation-induced self-assembly method. The antibacterial effect was evaluated against Aggregatibacter actinomycetemcomitans by viable counting, epifluorescence microscopy and colorimetric assay. The coated surface was able to kill adherent bacteria and inhibit biofilm formation for up to 7 days of incubation . In addition, AgNPs have been impregnated into a sol–gel-derived nanobioglass system. Different silver concentrations were tested, resulting in different nanoparticle morphologies on the bioglass surface. The antibacterial activity was tested against Bacillus subtilis , S. aureus , P. aeruginosa and E. coli , which are typically found in oral biofilms. The bioglass doped with AgNPs with the lowest Ag load (1 mM) was able to deliver a similar response regarding antibacterial activity when compared to the rest of the treatments. Furthermore, as this sample presented a nanospike morphology, the authors considered it possibly to increase bone integration .

Very few studies were performed on in vivo osseointegration. A noble metal coating consisting of Pt, Au and Ag was deposited in a commercially available coating system onto titanium implants and an in vivo evaluation was performed in rabbits. Coated implants were inserted into the femurs and the tibiae of 16 adult female New Zealand White rabbits and control implants were also inserted into one leg of each of the rabbits with no adverse effects. Bacterial adhesion properties were also evaluated in an in vitro assay against S. aureus , and a reduction of 99% of adhesion was observed for coated implants compared to non-coated .

AgNPs were embedded into dental titanium implants, which were sandblasted and acid-etched prior to the embedding to produce a micro-rough surface. The AgNP incorporation was performed by a plasma immersion/ion-implantation technique, and the implants inserted into the jaw of six Labrador dogs. Using this technique, it was possible to minimize silver release to less than a 10 ppb Ag/cm 2 after 90 days immersed in water. Samples embedded with AgNPs were able to enhance bone mineral density, bone formation, and trabecular pattern, with no harm to tissues adjacent to dental implants, when compared to sandblasted-only and acid-etched-only control groups. It was concluded that the hierarchical micro/nanotopography related to the size and distribution of embedded AgNPs was able to promote osseoconductivity by mimicking natural extracellular matrix structure . Zhu et al. have also applied this AgNPs embedding technique to titanium disks for in vitro evaluation of antibacterial properties and osteogenic functions. No significant difference in the growth of rat bone marrow mesenchymal stem cells was observed between control and AgNPs treatments, thus indicating that there was no apparent toxic effect on the proliferation, viability and differentiation of this cell line. On the other hand, the immobilized AgNPs were able to inhibit proliferation of both tested microorganisms, Fusobacterium nucleatum and Staphylococcus aureus , which are related to peri-implant disorders .

Pre-formulations containing AgNPs as antimicrobials

Antimicrobial activity against cariogenic pathogens

A healthy human oral microbiota has around 600 microorganisms living in different habitats such as teeth, tongue, cheeks and gingival sulcus . They are mainly bacteria, viruses and fungi and many of them co-evolved with our species through mutualism . Tooth decay and gingivitis are two of the most prevalent bacterial diseases. Dental caries is a condition caused by a specific biofilm that produces acid, leading to tooth enamel and dentin demineralization. It is a global and costly oral disease which compromises the health and quality of life of children and adults alike . S. mutans is the main bacteria in cariogenic biofilms on tooth surfaces, followed by other saccharolytic bacteria, including other streptococci, Actinomyces , and Lactobacillus that also participate in caries development . Although good oral hygiene and access to professional dental care are determinant for preventing caries, new antimicrobial agents for the control, prevention and elimination of these pathogens are still important, and AgNPs have been tested as promising candidates as oral antimicrobial agents.

Recently, flower-like silver nanocolloids biosynthesized using the extract of a marine sponge ( Haliclona exigua ) were produced and tested against important primary colonizers of oral biofilm ( Streptococcus oralis , Streptococcus salivarius and Streptococcus mitis ). The silver nanocolloids of size 100–120 nm promoted growth inhibition at applications of 10 μg .

AgNPs (10–20 nm) produced by biogenic synthesis employing the leaf extract of Justicia glauca were tested alone and also conjugated with other antimicrobials (Azithromycin, AZM; or Clarithromycin, CLR) against S. mutans and Lactobacillus acidophilus . Both nanomaterials showed an important antibacterial and antifungal activity. AgNP minimum inhibitory concentration (MIC) value determined against the several microorganisms were in the range of 25–75 μg/mL . In addition, AgNPs produced from plant chewing sticks of Azadirachta indica , Ficus bengalensis and Salvadora persica (biogenic nanoparticles, ∼100 nm) had their antibacterial activity tested on Lactobacillus acidophilus , L. lactis , and S. mutans . The antibacterial effect of these biogenic AgNPs from chewing sticks demonstrated that S. persica AgNPs are extremely effective against oral pathogens, followed by A. indica and F. bengalensis, when compared to silver nitrate (AgNO 3 ) as control .

S. mutans is the main cariogenic bacteria for which the antibacterial and size-dependent activity of AgNPs has been investigated. The antibacterial activity of AgNPs (chemical synthesis, 9–15 nm) associated with chitosan was evaluated based on signs of vascular change on the chorioallantoic membrane using the hen’s egg test (HET-CAM). These nanoparticles were non-irritating and showed an effective bactericidal effect against S. mutans dynamic biofilm. In this study, particle size and morphology did influence antibiofilm activity .

It is known that nanomaterials based on AgNPs are effective against biofilms , as they can attack multiple sites within the cell at a low concentration (0.5–1.0%) to prevent bacterial growth . In this way, the influence of size on the antimicrobial activity of chemically synthesized AgNPs was demonstrated against S. mutans . AgNPs of 5 and 15 nm present lower minimum inhibitory concentration (MIC = 50 μg/mL) than 55 nm-AgNPs (MIC = 200 μg/mL) . Microscopic analysis showed that smaller AgNPs (9.3 and 21.3 nm) were more efficient than larger AgNPs (93 nm) in reducing S. mutans adherence on bovine enamel blocks in vitro . Also, 9.3 and 21.3 nm-AgNPs showed similar effects to 0.12% chlorhexidine, a gold standard for antimicrobial activity against oral bacteria . Chlorhexidine was also compared to AgNPs (<100 nm) and to titanium dioxide and silica nanoparticles (chemical synthesis, ∼60 nm). In this study, the authors also compared the antibacterial effects of the nanoparticles with metal salts and bulk powders of the same composition (i.e. AgNO 3 , TiO 2 and SiO 2 ). It was demonstrated that AgNO 3 showed the most efficient bactericidal effect against S. mutans , followed by AgNPs. AgNPs reduced bacterial growth 25-fold higher than chlorhexidine, while silica and titanium dioxide NPs had limited effects .

A formulation containing AgNPs, chitosan, and fluoride (chemical synthesis, 6 nm) was tested against S. mutans (see Fig. 6 A) and compared to chlorhexidine and silver diamine fluoride. The MIC and MBC were, respectively, around 34 μg/mL and 50 μg/mL. Silver diamine fluoride showed similar values. Chlorhexidine, on the other hand, resulted in values of MIC and MBC of 3 and 6 μg/mL, respectively. The formulation containing AgNPs proved to be an antimicrobial agent similar to silver diamine fluoride, but less cytotoxic and with a potential advantage of not staining the teeth, as does chlorhexidine .

Nov 22, 2017 | Posted by in Dental Materials | Comments Off on Silver nanoparticles in dentistry
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