WikiJournal Preprints/Mapping the Dental Applications of Nano Silver Fluoride

Introduction
Dental caries is a sugar-biofilm influenced disease giving rise to alterations in tooth tissue hardness. It is still a major health concern despite advances in dental care. Fluoride has been widely used as a remineralizing agent but, due to fluoride toxicity and other drawbacks, seeking alternatives could be valuable. Newer alternatives for preventing dental caries are metal ions. Silver has different applications in medicine and dentistry due to its anti-caries, antimicrobial and anti-rheumatic potentials. Silver has come a long way along with dentistry. Perhaps the earliest use of silver in dentistry was in the form of dental amalgam, which is a liquid mercury and metal alloy comprising mercury (50%), silver (22– 32%), tin (14%), copper (8%) and other trace metals.

Another silver-containing compound, silver diamine fluoride (SDF) has powerfully prevented and ceased caries in primary teeth and also root caries in permanent teeth. The main advantage of SDF is its efficacy, relative affordability and accessibility. The procedure of treatment is fast and does not need any exorbitant equipment or infrastructure support. It is a noninvasive approach due to which there is a very low likelihood of transmitting diseases. The Federal Drug Administration (FDA), USA has authorized the use of SDF for dental and associated purposes since 2014. SDF is formed from three ingredients: silver (Ag), diammonia (2NH3) and fluoride. It extends the synergistic antimicrobial effect of both silver along with fluoride hindering the cariogenic bacteria, offering the benefit of fluoride as re-mineralizing agent to the de-mineralized tooth surface and converting hydroxyapatite crystal to fluorapatite making the surface less susceptible to demineralization. The ammonia is added to minimize oxidation potential and thus yielding a more stable compound with extended shelf life.

Nevertheless, the application of SDF pigments the lesions black due to the oxidation of silver ions which may fade within 48 hours, which can be a consequential pitfall of its use, particularly on anterior teeth. [3–6] SDF solution has a metallic taste and occasionally creates reversible white lesions while it comes into contact with the oral tissues.

An experimental study on the pharmacokinetics of silver fluoride has been described by Vasquez et al. Results give the impression that serum concentrations of fluoride and silver after topical application of SDF should give rise to little toxicity risk when used in adults and impede the demineralisation and safeguard the collagen from degradation in demineralized dentine.

Contemporary advances in emerging nanotechnology have led to the evolution of ‘second generation’ of silver products, and these include nanomolecules and nano-scaled silver nanoparticles. Nanoscaled silver particle formulations is undergoing expeditious growth with several uses in dentistry and is perhaps the most modern breakthrough in cariology.

Nano Silver Fluoride (NSF), a recently developed innovative preparation comprising of silver nanoparticles, chitosan and fluoride blends preventive and antimicrobial properties and was developed to be an effective anti-caries agent without unaesthetically staining the dental tissues black, as does SDF and amalgam. This novel substance is safe to be used in humans and has exceptional antimicrobial properties against Mutans streptococci and Lactobacilli, the principal pathogens accountable for dental caries. NSF is available as a yellow solution and is stable for three years. It is eco-friendly and economical. The silver  nanoparticles of NSF is a promising aid to combat residual bacteria in tooth cavity and invading bacteria at the margins of restorations.

Nano Silver Fluoride
The caries arresting property of NSF was described in a controlled clinical trial. The effectiveness of NSF in the arrestment of caries is attributed to the synergistic actions of both silver nanoparticles and fluoride. However, the mechanism of arrestment is not clear.

NSF was developed to overcome the staining problems of SDF as the material doesn’t undergo oxidation, and has proved to be ecofriendly. It combines the antimicrobial effect of chitosan and Nano silver ions, plus the preventive action of its fluoride content.

Composition of NSF:


 * Chitosan (28,585 μg/ml)
 * Silver (376.5 μg/ml)
 * Sodium fluoride (5028.3 μg/ml)

Chitosan is a polysaccharide substance comprising glucosamine copolymers and N-acetyl glucosamine. Chitosan is employed as a carrier to stabilize Silver nano particles. Chitosan is used widely in the medical field for its antimicrobial and anti-inflammatory action, and as a stabilizing agent. Chemically, chitosan is synthesized through the regulated N-deacetylation of chitin, a linear polysaccharide obtained primarily from arthropod exoskeletons such as marine crustaceans (crabs). The resulting polycation is soluble in aqueous solutions of small organic acids such as acetic acid and lactic acids and can be linked in the presence of polyvalent anions like phosphates. During the adhesion process, chitosan can inhibit Streptococcus mutans and Lactobacilli and show significant antibacterial and plaque reduction effect at successive stages of accumulation. Also, in vitro studies have shown that chitosan interferes with demineralization of the tooth enamel inhibiting the release of mineral elements.

Silver has a high chemical affinity for compounds containing nitrogen, sulfur and phosphorus, so it has been proposed that the inhibitory power of silver ions is due to its interaction with the thiol groups of proteins and the phospholipid portion of the bacterial membrane. When formulated into nanoparticles, silver interacts more strongly with other organic and inorganic molecules due to its extensive surface area and on the bacterial membrane to alter its permeability, thus triggering its rupture. Inside the cell, silver reacts with nucleic acids to prevent the cell replication action.

Advantages of NSF
NSF does not stain the dental tissue black, which is a preferred effect for not stigmatizing patients. Also, it does not form oxides when coming into contact with oxygen in the medium and has no metallic taste. The treatment procedure does not require a full dental equipment or a clinical setting. Because this process is uncomplicated and non-invasive, the risk of cross-infection is significantly reduced. NSF (600 and 1500 ppm) brings about less dentin staining than 35% silver fluoride and silver diamine fluoride at 30% and 38%. NSF application is inexpensive and thus can be afforded by most communities. 5% NSF is eight times economical compared to 38% SDF. NSF can be a global asset in treating mass population, uncooperative patients and children with special health care needs (CHSCN).

Mechanism of action of silver nano particles in SNF
Silver nanoparticles bring about an antibacterial effect for silver nanomaterials. Although the exact mechanism of silver nanoparticles’ antibacterial effects has not been precisely elucidated, numerous probable ways by which it may cause microbial destruction have been postulated. SNPs are both bacteriostatic and bactericidal. Furthermore, it has been reported that the antibacterial efficacy of SNPs increases with smaller particle size and spherical shape by providing a larger surface area for contact with bacteria. Most studies used silver nanoparticles smaller than 50 nm, as it is harder for larger silver nanoparticles to penetrate biofilm.

Action of SNP on Bacteria
Electrostatic bonding of the positively charged silver ions to the anionic cell membrane of the S. mutans. The positively charged silver nanoparticles show strong bactericidal activity against Streptococcus mutans. SNPs show effective inhibition on gram-positive and gram-negative bacteria. Gram-negative bacteria are more resistant to antibiotics than gram-positive bacteria. The outer cell membrane of gram-negative bacteria acts as a unique barrier to prevent many antibiotics from entering cells.

SNP penetrate the cell membrane followed by a burst in release of Silver ions which leads to:


 * Enhanced permeability and denaturation of the cell membrane
 * change the organization of the cell membrane due to their nanoscale size
 * Deactivation of respiratory enzymes
 * Halts ATP production
 * Inhibits the respiratory protein
 * Results in direct and indirect lipid peroxidation
 * Inhibits protein synthesis, DNA and RNA replication via yet unknown pathways
 * Disruption of bacterial envelope and destruction of cell membrane
 * cell content leakage
 * abrogation of cell motility
 * Interacts with cell inclusion
 * disrupts the respiratory chain reactions
 * inhibition of cell division
 * establish pits in the cytoplasmic membrane through reactive oxygen species and
 * Ultimately causes cell death

In addition, SNPs can kill bacteria by themselves. Silver nanoparticles can accumulate in the pits that form on the cell wall after they anchor to the cell surface. Silver ions seem to be toxic and


 * poison the metabolic enzymes
 * obstruct electron transport systems

Also, capping agents affect SNP’s ability to inhibit cariogenic bacteria. Capping agents modify the surface of silver nanoparticles and then change nanoparticles’ dissolution efficiency.

Action of SNP on tooth at molecular level- A study using a chemical model (i.e., no bacteria) found that silver nanoparticles increased the microhardness of enamel caries. SNP can penetrate into carious lesions and attach to hydroxyapatite crystals. In addition, silver ions released from silver nanoparticles can generate insoluble silver chloride on dental hard tissue. The precipitated SNP and insoluble silver chloride increase the mineral density of dental hard tissue.

Action on exposed collagen - Silver nanomaterials can preserve exposed collagen in carious teeth. In the oral environment, the exposed collagen can be degraded by bacterial collagenases and proteinases in saliva and the dentin matrix, such as activated matrix metalloproteinases and cysteine cathepsins. Silver nanoparticles can inhibit and deactivate these enzymes. Then, the preserved collagen can act as a scaffold for the deposition of a mineral crystal and for the prevention of further diffusion of calcium and phosphate.

Anti-adhesion property of SNF- Bacterial adhesion plays an important role in biofilm formation and the pathogenesis of caries since bacterial glucosyltransferase enzymes produce glucans which promote adhesion between cell-cell and cell-surfaces. Silver ions have shown the ability to prevent bacterial biofilm formation by disrupting this adhesion.

1. NSF containing toothpaste
Various nanoparticles have been incorporated into toothpastes, mouthwashes and even on bristles of toothbrushes. Teixeira et al formulated a novel dentifrice composed of NSF which has the potential of preventing the pH decrease caused by the production of acids that results from S. mutans metabolism of sucrose and showed ability to evade demineralization. Also, the NSF-containing dentifrices outperformed the NaF-containing dentifrices in preventing bacterial adhesion. NSF had a better antibacterial activity and potential effectiveness to prevent caries. The dentifrice with NSF depicted bacteriostatic but not bactericidal actions, this is a positive factor in an ecological perspective, given that successful antimicrobial agents would be able to avoid the disruption of the natural and beneficial inhabitant oral bacteria, which are vitally important for sound oral health.

2. Remineralising solution with NSF
These formulations can prevent and arrest demineralization of tooth enamel. Considering the fact that deciduous tooth is more sensitive to remineralization treatments, because deciduous enamel has around 150 times more permeability than permanent tooth. Since this enamel is more sensitive to acid action, the bactericidal potential of silver will be able to increase the effect of fluoride while preserving the remineralization potential. Currently, the use of these formulations with silver nanoparticles and fluoride in different concentrations display a positive effect on dental hard tissue for enamel remineralization in deciduous and permanent teeth, increasing the remineralizing, and enhance the performance of fluoride with antimicrobial action of silver added to these formulations, preventing and arresting the carious lesion without causing staining.

Ata MS et al investigated the influence of nano-silver fluoride (NSF), nano-hydroxyapatite (N-HAP), and casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste treatments on the enamel surface microhardness (SMH) after dental bleaching in vitro. NSF paste showed the highest SMH values and % SMHR.

Zhi et al demonstrated that, silver and fluoride ions were responsible for enamel remineralization. In addition, silver ions could infiltrate into areas of demineralized enamel and precipitate there, which result in enamel hardening. Furthermore, the small size of the NSF nanoparticles (3.2–1.2 nm) and their spherical shapes potentiate the antimicrobial effect by increasing the contact surface.

Esilva et al in 2018 reported that Nano Silver Fluoride was found to be simpler and more effective than conventional fluorides in treating incipient caries lesions due to its remineralizing potential. NSF had greater effectiveness compared to Sodium Fluoride (NaF) in preventing lowering of pH and adhesion of S. mutans to the enamel surface. Higher interference on S. mutans adhesion to the enamel surface and higher inhibition on S. mutans acidogenicity than NaF has been reported.

3. Nano silver incoorporated Sodium Fluoride varnish (NSSF)
Tirupathi et al compared the clinical efficacy of annual application of 5% Nano-silver incorporated Sodium fluoride varnish (5% NSSF) with commercially available 38% SDF in preventing the progression of dentine caries lesion in primary molars. It was concluded that NSSF is equivalent to SDF in inhibiting dentine caries progression in primary teeth.

According to Ali Nozari et al., NSF varnish could have the greatest remineralization capability in comparison to n-HAP serum NaF varnish.

NSF and PPF fluoride-based varnishes show clear antibacterial effects that are comparable to those obtained with SDF fluoride-based varnish, the present study effectiveness of NSF on E. faecalis also, which is known to be more resistant than S. mutans. This research also confirmed that NSF was more effective than SDF (38%), due to both the concentration and the components of NSF. The present research showed that NSF was better than SDF at inhibiting E. faecalis biofilm formation.

4. NSF containing sealants
In an experimental study, the antibacterial properties of two sealants with fluoride and without fluoride was investigated adding Nano-silver. Results reported sealants with fluoride comparing to non-fluoride ones had significant effect on inhibition of S. mutans growth.

However, according to another study, the invitro remineralization potential of NSF was lower than SDF and NaF. The NSF varnish had more irregularities and more mineral deposits than SDF and NaF varnish and it had lower surface micro hardness too.

5. NSF for arresting dental caries / arresting caries treatment
In an investigation conducted by NagiReddy et al, selected tooth received two drops of NSF with a micro brush applicator tip, equivalent to a dose of 10 mg of the solution 7 days, 5 months, and 12 months. In the NSF batch, majority of the single surface carious lesions demonstrated higher caries arrest status after 12 months while multiple surface exhibited less caries arrest.

Santos et al reported that NSF was effective in arresting dental caries. Upon attempts to remove dentine treated with NSF, the tissue turned hard and presented with a crumbly consistency.

6. NSF for fissure pre-treatment
The objective of pretreating fissures with SDF or NSF in preparation for sealant application would combine the antimicrobial/remineralizing action of these agents and the physical sealing property of FS, this would be of great benefit in caries prevention principally in high risk patients. In a study by El-Tekeya et al, fissures pretreated with SDF or NSF before sealant placement enhanced the prevention of microleakage reducing the possibility of caries under sealant when clinically applied.

7. Disinfection and sanitisation of deep dentinal lesions
Due to the intense penetrative capacity and the microbicidal efficacy of SNP, they appear to be good candidates for sanitization and disinfection of deep dentinal lesions, mainly to destroy the residual bacterial burden, after conservative caries removal.

8. NSF for restoration pre-treatment
Nanda et al reported that pre-treatment with NSF is beneficial in increasing the resistance of GIC and composite resin restoration to secondary caries formation, solving the most common cause of failure of restorations.

i) NSF incoorporated into GIC

Owing to the increasing interest in the use of NSF, SNPs were incorporated into a conventional GIC. This modification showed improved mechanical and bond strength properties. It may be suggested for use in higher stress-bearing site restorations.

9. NSF incoorporated in orthodontic brackets
An adverse-effect of fixed orthodontic therapy is white spot lesions (WSLs) formed as a result of enamel demineralization, dissolution of hydroxyapatite due to acidic by-products of bacteria in the dental plaque. Nanocoated orthodontic bracket is effective in the inhibition of S. mutans resulting in reduced white spot lesion after fixed orthodontic treatment.

10. Modifications of NSF
i) NSF-GTE

Chemical synthesis of Nano-Silver particles has raised controversial arguments since the process contains hazardous substances. A biological approach using Green tea extract was used by Al Nerabieah et al to synthesize SNP in lieu of the chemical approach. This eco-friendly “green” method offers a simple, fast and biogenic approach. Green tea extract acts as a reducing and capping agent for the production of SNPs without adding hazardous chemicals.

Drawbacks
Although silver nanomaterials possess antibacterial and remineralizing properties, they are easily oxidized and aggregative. The toxicity of silver nanomaterials is influenced by the presence of free silver ions released from silver nanoparticles which can enter mammalian cells and produce of reactive oxygen species. This escalates the oxidative stress and cause deleterious effects. But, Silver nanoparticles present lower cytotoxicity to human oral cells. The severity of cytotoxicity of NSF can be associated to their nanoscale size, shape, surface area per unit mass, surface charge and nonspecific oxidative damage.

Secondly, there is a concern regarding the perilous chemical agents used as a reducing agent such as acetic acid and borohydride sodium highlighting the risk of this product. However, Targino et al, evaluated the cytotoxicity of different concentrations of Nano-Silver solutions from 1% to 5%, and they found no toxic effect for erythrocyte. Thus, NSF is more biocompatible than SDF.

No experimentation has declared the long-term stability and antibacterial effect of silver nanoparticles and fluoride. Evidence on the safety of Nano silver in the oral cavity is not extensive. Hence, further studies elucidating the efficacy and safety of NSF are warranted to unequivocally ascertain the status of SNF as a promising formulation worthy of day to day clinical use.

Conclusion
The applications of NSF for management of tooth sensitivity and root caries need to be evaluated. To conclude, with the increasing popularity and progress in nanoparticle technology, it is anticipated that in the fullness of time, the popularity of nano chemicals will increase as a main stream in dental practice.