TECHNICAL FIELD

The invention relates to a glass ionomer cement containing silver nanoparticles. In particular, the invention relates to a glass ionomer cement containing silver nanoparticles bound to a polymer backbone. The cement exhibits desirable antibacterial and mechanical properties making it useful a for a varierty of dental applications.

BACKGROUND OF THE INVENTION

Dental caries is caused by bacterial processes that lead to demineralisation of dental hard tissues resulting from the acid produced by bacteria as a by-product of carbohydrate metabolism. Dental caries is a biofilm-initiated process involving over 700 species of bacteria and archea possibilities which form on the tooth surface, with the colonisation community becoming more complex and the bacterial proportions continually changing as the disease progresses and cavitation develops. Historically, the dental profession has used a surgical "drill and fill" approach to 'surgically excise' or remove all demineralised, softened dentine. Gradually thinking has shifted towards removing less tissue; with only the more severely damaged carious tissue (infected dentine) requiring removal. Subjacent to this, a further layer of affected dentine is usually retained where bacteria have invariably invaded the dentine tubules. Current philosophy has shifted to even more minimally interventive approaches, where enamel lesion tissue is completely removed, but even quite severely affected demineralised dentine may be left remaining, and sealed off by a covering restoration. Sealing the lesion off from the oral environment effectively deprives the biofilm of substrate, causing bacteria to become quiescent. However, success is heavily dependent on persistence of the seal. Unfortunately, all restorative materials, and in particular composite resin bonded restorations, have finite life-spans such that effectiveness of the resin bond seal eventually becomes compromised.

Prior to placement of composite resin fillings, acid treatment with 37% phosphoric acid is used to remove the smear layer and to etch (partially demineralise) the enamel, creating micro-porosities for subsequent attachment of adhesive resin micro-tags. However, to achieve bonding of dentine, where there is much greater organic content, mild acid treatment is used to remove the smear layer, open the dentine tubules, and to partially demineralise the peri-tubular dentine, exposing the collagen matrix. This is followed by application of bifunctional primer molecules, such as hydroxyethyl methacrylate (HEMA), to encourage resin infiltration allowing the formation of a hybrid layer within the uncollapsed collagen-mineral matrix. The hydrophilic end of the HEMA molecule penetrates the aqueous dentine tubule environment facilitating the formation of large resin macro-tags, and facilitating the open porous partially demineralised peri-tubular collagen to form an interlocking network. The hydrophobic end of the HEMA molecule interacts with the non-polar composite resin filling material to chemically bind the resin to the tooth. An alternative to composite, amalgam, does not require acid treatment or other sophisticated chemistry as it is merely placed as a space filler in the cavity and is only retained mechanically.

Treating the symptoms of dental caries by merely cutting away the demineralised tissue, however, does not address the multifactorial aetiology of this disease process, leaving the dentition vulnerable to further acid attack resulting from bacterial activity. Conventional filling materials do not target the bacterial source of the disease either. Instead, they simply seal the remaining bacteria dormant within the tooth. This prevents decay until the seal provided by the filling is breached, allowing re-activation of the bacteria and leading to a recurrence of the infection. Thus, in order to reduce the risk of disease recurrence, removal of all remaining bacteria is desirable.

Various approaches for disinfecting tooth surfaces are currently available include chemical regimes (chlorhexidine, fluoride, iodine, calcium hydroxide, zinc oxide eugenol (ZnOE), hypochlorites, ethylenediaminetetraacetic acid (EDTA), peroxide bleaching agents, enzymatic digestion with Carisolv™, ozone application) and/or photo-activated disinfection (PAD) by low power laser irradiation. All in some way are ineffective, are unable to penetrate tooth tissue, have undesirable side effects, or are not cost effective.

While the antibacterial effects of silver species (in particular, ionic silver) have been known for centuries, in recent years there has been renewed interest in silver in the form of silver nanoparticles (Ag NPs) for applications in health care and medicine. This interest is due in part to increasing bacterial resistance to classical antibiotics (Rai, M. K., et a/., J. Appl. Microbiol., 2012, 112, 841-852). Ag NPs offer novel modes of action and target different cellular structures compared with existing antibiotics, and have vastly increased reactivity over ionic silver, based on equivalent silver mass content, as a result of their large surface area to volume ratios. Several areas of medical care have already benefitted from the ongoing development of Ag NP-based materials. Applications include Ag NP-based wound dressings (Fong, J. and Wood, F., Int. J. Nanomedicine, 2006, 1, 441-449), Ag NP-based biomaterials for orthopaedics, such as use in artificial joint replacement and bone prostheses (Ren, N., et a/., J. Mater. Chem., 2012, 22, 19151-19160), Ag NPs as bactericidal coatings for medical devices (Roe, D., et a/., J. Antimicrob. Chemotherapy, 2008, 61, 869-876), and Ag NP incorporation into dental materials (US 2007/0213460).

Silver has a long history of use in preventative dentistry. For instance, silver nitrate (AgNC ) and diamine silver fluoride (Ag(NH3)2F), often referred to simply as AgF, have been used to prevent or arrest carious lesions. However, a recognised undesirable side effect of these products is that they stain tooth structure and tooth-coloured restorations (Knight, G. M., et a/., Aust. Dent. J., 2005, 50, 242-245). Suspensions of Ag NP-based materials may offer a unique solution to this problem, as they are non-staining, but have the potential to deliver enhanced antibacterial effects.

The antimicrobial activity of Ag NPs is known to be critically dependent on the dimensions of the particles. Specifically, many studies have revealed that smaller sized particles impart greater antimicrobial activity, on the basis of equivalent silver mass content (Morones, J. R., et a/., Nanotechnology, 2005, 16, 2346-2353, and Guzman, M., et a/., Nanomed. Nanotech. Biol. Med., 2012, 8, 37-45). The origin of this apparent size-dependent effect has been the subject of much investigation, and there are several commonly cited explanations. The first is that under aerobic conditions, Ag NPs of smaller size exert increased bacterial toxicity as a result of increased availability of Ag ⁺ ions on the surface of the particles, due to their higher specific surface areas when compared to larger sized particles. While the specific mechanism of bactericidal action of Ag ⁺ ions is currently not fully understood, it is thought to be related to the inactivation of critical thiol-containing enzymes upon cellular interaction. Additionally, Ag ⁺ is believed to detrimentally affect the replication of DNA in cells treated with AgNC . Furthermore, experimental evidence has also shown that ionic silver from both Ag NP and AgNC sources causes structural and morphological changes in treated cells. The second explanation for the observed particle size dependence of Ag NP antibacterial activity is based on known size-dependent interactions of Ag NPs with bacteria.

Glass ionomer cements (GICs) are restorative materials used for a wide range of applications in dentistry, primarily distinguishable by variations in glass filler particle content. This includes application as a lining, luting/bonding agent, temporary filling material for provisionalisation, or as a definitive restorative replacement for deciduous teeth or permanent teeth in non-load bearing situations. In contrast to enamel which is brittle in nature, glass ionomer has a relatively low modulus of elasticity, similar to dentine, such that GICs make suitable materials for dentine replacement. GICs are often applied as part of a 'sandwich' restoration involving dentine replacement by glass ionomer and an outer protective layer of composite resin acting as enamel substitute.

GICs are formed from a reaction between an acid-decomposable glass powder (e.g. calcium fluoroaluminosilicate) and water soluble polymers containing carboxylic acid (-COOH) functional groups. The glass powder-containing phase is referred to as the solid phase, and the polymer-containing phase is referred to as the liquid phase. In a commercial GIC, these two phases are initially separated from each other by a membrane within a small capsule. Before use, the membrane is pierced and the two phases come into contact with each other, thus initiating a setting reaction. As the glass particles are attacked by the acid from the liquid phase (H ⁺ ions donated by the -COOH groups), Ca ²⁺ , Al ³⁺ , Na ⁺ and F ^" ions are released by the glass into the surrounding aqueous medium. The cations form intermolecular salt bridges with the now-formed carboxylate anions (COO ^" ) on the polyelectrolyte chains, forming a bridged hydrogel around the glass particles. Gel formation involves a two-stage set, with Ca ²⁺ ions predominantly involved in formation of the initial relatively weakly cross-linked silica gel, with subsequent more slowly leached Al ³⁺ ions involved in substitution of a significant proportion of the bound Ca ²⁺ ions by Al ³⁺ ions, thus increasing the extent and stability of cross linking. Upon hardening, a cement is produced which continues to mature over a period of months to years following initial setting. The final GIC structure, now a composite material, consists of porous glass particles, surrounded by a silica gel, and bound together by a cross-linked network of hydrated calcium and aluminium polymer salts. However, due to the glass filler content, these materials have a slightly roughened surface texture, abrasive against dentine.

The applicant has now found that silver nanoparticles can be incorporated into GICs thereby providing novel GICs that exhibit an antibacterial effect with no negative effect on mechanical strength of the GIC nor any unwanted discolouration of teeth due to contact with silver. The applicant has found a method for incorporating silver nanoparticles into GICs avoiding problems associated with agglomeration of silver particles and hence has developed a novel class of antibacterial GICs for use in various dental applications.

It is therefore an object of the invention to provide a glass ionomer cement containing silver nanoparticles, or to at least provide a useful alternative to existing materials.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a glass ionomer cement comprising :

(i) nanoparticles of metallic silver (Ag);

(ii) a polymer comprising carboxylate groups;

(iii) bridging molecules comprising at least one group capable of binding to Ag;

(iv) metal ions; and

(v) aluminosilicate particles;

wherein at least some of the aluminosilicate particles are bound to the polymer through a linking structure of the general formula (I) :

polymer-M-B-Ag-B-M-aluminosilicate (I)

where M is a metal ion, B is a bridging molecule, and Ag is a silver nanoparticle. In some embodiments of the invention the polymer is a homopolymer or copolymer prepared from any of acrylic acid, 2-chloroacrylic acid, 3-chloroacrylic acid, 2-bromoacrylic acid, 3-bromoacrylic acid, methacrylic acid, itaconic acid, maleic acid, glutaconic acid, aconitic acid, citraconic acid, mesaconic acid, fumaric acid and tiglicinic acid, or a copolymer prepared from any of acrylamide, acrylonitrile, vinyl chloride, allyl chloride, vinyl acetate, and 2- hydroxyethyl methacrylate. Preferably the polymer is a homopolymer or copolymer prepared from acrylic acid, for example polyacrylic acid.

In some embodiments of the invention the bridging molecule comprises one or more thiol, disulfide or amine groups. In some embodiments of the invention the bridging molecule is an alkylcarboxylate molecule. Examples include 6-mercaptohexanoic acid, 8-mercaptooctanoic acid, mercaptosuccinic acid, 4-mercaptobenzoic acid, 4-mercaptophenylacetic acid, lipoic acid (thioctic acid), dihydrolipoic acid, glutathione, penicillamine, 5-(4-amino-6-hydroxy-2- mercapto-5-pyrimidinyl)pentanoic acid, and 2-mercapto-4-methyl-5-thiazoleacetic acid. Preferably the bridging molecule is lipoic acid.

In some embodiments of the invention the metal ions are calcium, aluminium, zinc, sodium, or strontium ions, or any combination thereof.

In some embodiments of the invention the aluminosilicate compound is calcium fluoroaluminosilicate.

In some embodiments of the invention the glass ionomer cement has been modified by incorporation of a resin. In preferred embodiments the resin is bisphenol A glycidyl methacrylate. The glass ionomer cement may further comprise a bifunctional molecule mediator such as hydroxyethyl methacrylate. In some embodiments the glass ionomer cement further comprises a photoinitiator to enable polymerisation of the resin on application of light.

In preferred embodiments of the invention the glass ionomer cement is capable of disrupting or preventing the formation of a biofilm. Preferably the glass ionomer cement is capable of inhibiting the growth of one or more bacteria selected from the grou p comprising Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, and Pseudomonas aeruginosa.

In a second aspect of the invention there is provided a dental preparation product comprising a liquid phase and a solid phase separated from each other, wherein the liquid phase comprises:

(i) nanoparticles of metallic silver;

(ii) a polymer comprising carboxylate groups; and

(iii) bridging molecules comprising at least one group capable of binding to Ag; wherein the solid phase comprises particles of an aluminosilicate compound.

In some embodiments of the invention a glass ionomer cement of the invention forms upon mixing of the liquid phase and the solid phase.

In a further aspect of the invention there is provided the use of the glass ionomer cement of the invention, or the dental preparation of the invention, for filling cavities or fissures in teeth, or as a protective coating for teeth.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic representation of Ag NP incorporation into a GIC matrix.

Figure 2 is a photograph of a deconstructed GIC capsule. Figure 3 is a photograph of Fuji IX GICs containing : a) 0 μg Ag NP (Fuji IX control), b) 6.4 μg Ag NP, c) 32 μg Ag NP, and d) 64 μg Ag NP.

Figure 4 shows optical density measurements performed at a wavelength of 630 nm (O. D630nm) for bacterial cultures grown in the presence of non-modified Fuji IX control GICs A and Ag NP-modified Fuji IX GICs containing□ 6.4 μg and ø 64 μg of silver, and · a bacterial control, (a) P. aeruginosa, (b) S. mutans, (c) S. mitis, (d) S. sanguis.

Figure 5 shows SEM images of P. aeruginosa biofilm growth on : (a) and b) non- modified Fuji IX control GIC, (c) Ag NP-GIC modified with 6.4 μg silver, (d) GIC modified with 64 μg silver.

Figure 6 shows SEM images of S. sanguis biofilm growth on : a) and (b) non-modified

Fuji IX control GIC, (c) Ag NP-modified GIC with 6.4 μg silver, (d) GIC modified with 64 μg silver.

Figure 7 shows expanded SEM images of S. sanguis biofilm growth on : (a), (b) and (c) Ag NP-modified GIC with 6.4 μg silver, (a) shows a GIC defect area absent of bacterial growth, (b) highlighted area of bacteria with disrupted morphology that were present on the Ag NP-modified GIC and, (c) view of lysed or partially lysed bacteria on the Ag NP-modified GIC surface, (d) Ag NP-modified GIC with 64 μg silver showing absence of bacterial growth .

Figure 8 shows SEM images of S. mitis biofilm growth on : (a) and (b) non-modified Fuji IX control GIC, (c) and (d) Ag NP-modified GIC with 6.4 μg silver, (e) and (f) Ag NP- modified GIC with 64 μg silver.

Figure 9 shows SEM images of S. mutans biofilm growth on : (a) and (b) non-modified Fuji IX control GIC, (c) Ag NP-modified GIC with 6.4 μg silver, (d) Ag NP-modified GIC with 64 μg silver.

Figure 1 shows expanded SEM images of S. mutans on the surface of an Ag NP- modified GIC with 64 μg silver, (a) image showing some bacteria found within defect areas of the GIC, (b) intracellular lysed materials on the surface of the GIC.

Figure 11 shows CLSM images of 48 h formed monospecies biofilms on the surface of a non-modified Fuji IX control GIC (Ag content = 0 μg) and Ag NP-modified Fuji IX GICs (Ag content = 6.4 and 64 μg) . Grey represents live biofilm biomass on the GIC surface.

Figure 12 shows CLSM images of S. mutans biofilms grown on the surface of (a) unmodified Fuji IX control GIC, and (b) Ag NP-modified Fuji IX GIC (Ag = 6.4 μg) . The black image indicates a lack of biofilm for CLSM detection .

Figure 13 shows CLSM images of S. mutans biofilms grown on the surface of (a) unmodified SDI Riva Self cure GIC, (b) Ag NP-modified SDI Riva Self cure GIC (Ag = 10 μg), and (c) Ag NP-modified SDI Riva Self cure GIC (Ag = 24 μg) .

Figure 14 shows CLSM images of S. mutans biofilms grown on the surface of (a) unmodified 3M Ketac Molar GIC, (b) Ag NP-modified 3M Ketac Molar GIC, (Ag = 10 μg), and (c) Ag NP-modified 3M Ketac Molar GIC (Ag = 24 μg) . Figure 15 shows S. mutans viability of bacterial biofilm grown on the surface of Ag NP- modified GICs, compared to a non-modified Fuji IX control GIC upon weekly specimen aging.

Figure 16 shows a Box and Whisker plot of the flexural strength values for: control = non-modified Fuji IX control GIC, Add 100% = Ag NP-modified Fuji IX GIC with addition of 5 μΙ_ of an Ag NP suspension containing a [Ag] of 1280 μg mL ^"1 , Subl00% = Ag NP-modified Fuji IX GIC with addition of 5 μΙ_ of an Ag NP suspension containing a [Ag] of 1280 μg mL ^"1 , Add50% = Ag NP-modified Fuji IX GIC with addition of 10 pL of an Ag NP suspension containing a [Ag] of 640 μg mL ^"1 , and Sub50% = Ag NP-modified Fuji IX GIC with substitution of 10 pL of an Ag NP suspension containing a [Ag] of 640 μg mL ^"1 .

Figure 17 Flexural strength of SDI Riva, SDI Riva modified with 10 μg of AgNPs and

24 μg of AgNPs, and SDI Riva Silver.

Figure 18 shows flexural strength of 3M Ketac Molar, 3M Ketac Molar modified with 10 μg of AgNPs and 24 μg of AgNPs, and 3M Ketac Silver.

Figure 19 shows flexural strength of GC Fuji IX, GC Fuji IX modified with 6.4 μg of AgNPs and 10 μg of AgNPs, and GC Miracle Mix.

Figure 20 shows elastic modulus values obtained for Ag NP-modified GICs and Fuji IX control GICs at day one and day seven.

Figure 21 shows compressive strength of 3M Ketac Molar, 3M Ketac Molar modified with 10 μg of AgNPs and 24 μg of AgNPs, and 3M Ketac Silver.

Figure 22 shows compressive strength of SDI Riva Control, SDI Riva modified with 10 μg of AgNPs and 24 μg of AgNPs, and SDI Riva Silver.

Figure 23 shows compressive strength of GC Fuji IX, GC Fuji IX modified with 10 μg of AgNPs and 24 μg of AgNPs, and GC Miracle Mix.

Figure 24 shows luminescence values for unmodified GIC and AgNP-modified GIC specimens (n=3) at 0 h, 24 h, and 2 weeks. L = 0 yields black and L* = 100 indicates diffuse white.

Figures 25a, 25b and 25c show a*b* values for unmodified GIC and AgNP-modified GIC specimens (n=3) . Figure 25a shows values recorded at Time = 0 h. Figure 25b shows values recorded at Time = 24 h. Figure 25c shows values recorded at Time = 2 weeks. Colour channels, a* and b*, represent true neutral grey values at a* = 0 and b ^~ * = 0, The red/green opponent colours are represented along the a* axis, with green at negative a* values and red at positive a* values. The yellow/blue opponent colours are represented along the b~* axis, with blue at negative d* values and yellow at positive b* values, DETAILED DESCRIPTION

The invention is based on the applicant's finding that a GIC incorporating silver nanoparticles (Ag NPs) bound to a polymer backbone in a specific manner exhibits antibacterial activity and has good mechanical properties. The cement is therefore useful a for a varierty of dental applications.

The applicant determined that modifying the surface of size-controlled Ag NPs so that they contain terminal -COOH groups enabled them to participate in the formation of intermolecular salt bridges with the polymer backbone. In this way, discrete Ag NPs are distributed throughout the polymer matrix and held in place through ionic interactions, thus preventing their aggregation and the subsequent discolouration known to occur upon Ag NP aggregation. Furthermore, by distributing the Ag NPs, or small Ag NP assemblies, uniformly throughout the polymer, mechanical failure caused by localised regions of high Ag NP concentration is avoided.

The term "nano-" or "nano-sized" means having at least one size, dimension or scale in the nanometre range, typically several nanometres to several hundred nanometres. A nanoparticle (NP) is therefore any particle having at least one dimension, e.g. diameter, in the range of several nanometres to several hundred nanometres.

The term "polymer" means a synthetic or natural macromolecule comprising multiple repeated subunits or monomers.

The term "homopolymer" means a polymer formed from a single type of monomer.

The term "copolymer" means a polymer formed from two or more different types of monomers.

The term "ionomer" means a polymer containing a proportion (usually 5-10%) of substituted ionic groups.

The term "glass ionomer cement" means a cement material formed by reaction between a glass material (usually calcium fluoroaluminosilicate) and an ionomer.

The term "bridging molecule" means a molecule having at least one functional group able to bind to a Ag nanoparticle and at least one functional group able to bind to metal ions such that a bridge forms between the Ag nanoparticle and a metal ion bound to the polymer and/or a metal ion bound to an aluminosilicate particle.

The GIC of the invention comprises nanoparticles of metallic silver, a polymer comprising carboxyiate groups, bridging molecules comprising at least one group capable of binding to Ag, metal ions, and aluminosilicate particles. At least some of the aluminosilicate particles are bound to the polymer through a linking structure of the general formula : polymer-M-B-Ag-B-M-aluminosilicate. In this formula, M is a metal ion, B is a bridging molecule, and Ag is a silver nanoparticle.

The longstanding problem of incorporating silver particles into polymer matrices has been the leaching of silver causing discoloration of the polymeric material and any surface to which it is applied, particularly in the presence of light where a photochemical reaction occurs. Even though the antimicrobial effects of silver have been known for a long time, polymers incorporating silver are unsuited to most dental applications due to the undeniable cosmetic effect of discolouration of teeth. Leaching of silver is prevalent where the silver particles are not tightly held in the polymer matrix. This occurs when the silver particles are not well - dispersed in the matrix and instead agglomerate into groups or clusters.

The bridging molecule of the GIC of the invention is important for preventing agglomeration of the silver nanoparticles. By binding to silver nanoparticles and to metal ions that are bound to aluminosilicate particles or to polymer chains, the bridging molecule stabilises the silver nanoparticles in the cement matrix and prevents or minimises leaching.

Any bridging molecule able to carry out this function is contemplated as part of the invention. However, preferred bridging molecules comprises one or more thiol or amine groups. Thiol groups in particular tend to bind well to metallic silver. Some examples of suitable bridging molecules are 6-mercaptohexanoic acid, 8-mercaptooctanoic acid, mercaptosuccinic acid, 4-mercaptobenzoic acid, 4-mercaptophenylacetic acid, lipoic acid (thioctic acid), dihydrolipoic acid, glutathione, penicillamine, 5-(4-amino-6-hydroxy-2- mercapto-5-pyrimidinyl)pentanoic acid, and 2-mercapto-4-methyl-5-thiazoleacetic acid.

The carboxylate groups of the polymer bind to metal ions present in the matrix which in turn bind to bridging molecules typically, although not necessarily, through carboxylic acid functionalities of the bridging molecule. The polymer may be a homopolymer or copolymer. The preferred polymer is a polyacrylic acid. However, the polymer may be any suitable polymer including 2-chloroacrylic acid, 3-chloroacrylic acid, 2-bromoacrylic acid, 3- bromoacrylic acid, methacrylic acid, itaconic acid, maleic acid, glutaconic acid, aconitic acid, citraconic acid, mesaconic acid, fumaric acid and tiglicinic acid, or a copolymer prepared from any of acrylamide, acrylonitrile, vinyl chloride, allyl chloride, vinyl acetate, or 2- hydroxyethyl methacrylate.

Any aluminosilicate compound suitable as a glass material may be used. The aluminosilicate is preferably a calcium fluoroaluminosilicate which is the commonly used type of material for preparing a GIC. The metal ions involved in the cement are typically calcium, aluminium, strontium, zinc or sodium ions. All or some may be present depending on the aluminosilicate material and how it is formed.

GICs typically form by reaction between polyacrylic acid material in aqueous solution and an aluminosilicate in the form of a powder. The components are usually mixed just prior to application to teeth. The reaction occurs slowly allowing the cement to set and harden over several hours.

The setting reaction begins with the mixing of the components. The first phase of the reaction involves dissolution. The acid begins to attack the surface of the aluminosilicate glass particles, as well as the adjacent tooth substrate. As the pH of the aqueous solution rises, the carboxylic acid groups begin to ionise which sets up a diffusion gradient and helps draw cations out of the glass and dentine, The alkalinity also induces the polymers to dissociate, increasing the viscosity of the aqueous solution, The second phase is gelation . As the pH continues to rise and the concentration of ions in solution increases, a critical point is reached and insoluble polyacrylates begin to precipitate. Cations bind to the carboxylate groups, especially Ca ²⁺ in this early phase, crosslinking into calcium polyacrylate chains that begin to form a gel matrix, resulting in the initial hard set, within five minutes. Over the next 4-20 hours maturation occurs with replacement of divalent Ca ^2÷ ions involved in crosslinking, and trivalent Ai ³⁺ ions forming more stable crosslinks within the gel structure. Tartaric acid can be added to the solid phase to extend the working time and to promote a snap set. Tartaric acid reacts preferentially with Ca ^2"1" to form a calcium tartrate complex. This enables longer working time until the tartaric acid is completely consumed, or until the A! ³⁺ is able to react with poiyalkenoate carboxyl groups. Other factors affecting setting time are the temperature of the cement, and the powder to liquid ratio.

In a typical embodiment of the invention, a dental preparation product for application to teeth will comprise a liquid phase and a solid phase which are held separately from each other so that they do not come into contact until the time of use. Many existing products require the two components to be mixed together on a plate using a small spatula before applying to teeth . Figure 2 shows one example of a product where the liquid phase (a solution of the polymer) and the powdered solid phase are contained within a plastic vessel with a thin membrane separating them. When the plunger shown is depressed, the membrane is disrupted enabling the solid and liquid phases to come into contact.

In the dental preparation of the invention, the liquid phase comprises the nanoparticles of metallic silver, the polymer comprising carboxylate groups, and the bridging molecules, and the solid phase comprises particles of the aluminosilicate compound . The GIC of the invention forms upon mixing of the liquid phase and the solid phase.

The GIC of the invention may be used for any suita ble dental application. Such applications include, but are not limited to, filling cavities or fissures in teeth, or use as a protective coating for teeth .

Resin-modified GICs (RM-GICs) are also contemplated as part of the invention . Resin modification of GICs by incorporation of a polymer, such as bisphenol A glycidyl methacrylate (BIS-GMA), and a bifunctional molecule mediator such as hydroxyethyl methacrylate (HEMA), improves the physical properties of the resulting RM-GIC, offering better load bearing capacity, polishability and aesthetics, increased modulus of elasticity, and wear resistance. In addition, incorporation of organic peroxide photo initiators into resin systems, which can be activated by blue light (~470 nm), allows the polymerisation reaction to be controlled allowing 'command setting' of the restoration at the convenience of the clinician, rather than having to wait longer for the GIC setting reactions to occur. Incorporation of resin has the effect of reducing the capacity for free ion exchange and passage of ions throughout the resulting restorative material . Despite this, the advantages of resin -modification make RM- GICs popular restorative materials. They are most typically used as dentine replacements or lining materials beneath other materials, capitalising on the greater propensity for free ion exchange throughout the final silicate hydrogel structure persisting well beyond final setting, maximising the ability for interaction of silver nanoparticles and consequent antimicrobial effects. RM-GICs also have adequate strength for application as provisional or 'temporary' fillings, often used in clinical situations involving active dental caries in attempts to stabilise oral health, whereby enhancing the material by addition of silver nanoparticles may offer a distinct therapeutic advantage.

Examples 1 and 2 describe a preparation of GICs incorporating thioctic acid-capped Ag NPs. The Ag NP-modified GICs were all within acceptable tooth shades according to the VitaPlan 3D master chart. The cement colour ranged from an off-white (Ag content of 6.4 μg) to dark brown (Ag content of 64 μg) with increasing Ag NP incorporation. The range of colours produced could potentially influence the type of clinical application for which the GIC is used.

Example 3 investigates the antibacterial effects of GICs incorporating thioctic acid- capped Ag NPs. The utilisation of optical density as an indication of bacterial cell density is a long-standing method typically used to determine the minimal inhibitory and bactericidal concentration of antibiotics or other antibacterial agents. When a broth culture is inoculated with a bacterium, usually an exponential increase of optical density is observed parallel to the increase in bacterial density. The liquid culture assay was used in this study to evaluate any effect on optical density, and therefore bacterial growth profiles, when bacteria were grown in the presence of the non-modified and Ag NP-modified GICs. Both the non-modified Fuji IX control GIC and the Ag NP-modified GICs demonstrated no apparent antibacterial effect on the surrounding planktonic cultures, as evidenced by the increase in optical density over the monitored time equivalent to the bacterial control in all cases. Furthermore, the bacterial cultures grown in the presence of non-modified Fuji IX control GICs had similar growth profiles to those grown in the absence of GICs (bacterial controls) . Therefore, it can be concluded that the GICs have no apparent influence on the rate of surrounding microbial growth compared to the positive control, as evaluated by multivariate ANOVA (Ag content = 6.4 μg, p = 0.130; 64 μg, p = 0.445; and Fuji IX control GIC, p = 0.585). P. aeruginosa demonstrated increased growth on the non-modified Fuji IX control GIC, and on the Ag NP-modified Fuji IX GIC (Ag content = 6.4 μg) compared to the bacterial control, (p < 0.001). These results suggest that the non-modified Fuji IX GIC does not provide a high enough quantity of leached F ^" ions to affect the surrounding media to a measurable effect. Additionally, the results obtained for the Ag NP-modified GICs indicate that the Ag NPs incorporated within the GIC were probably not able to migrate from the cement matrix (or not at a high enough concentration) to induce antimicrobial effects into the medium. The Ag NP-modified and non-modified GICs subjected to biofilm surface growth were analysed by Scanning Electron Microscopy (SEM) in order to investigate the morphologies of the bacterial cells grown on the GIC surfaces (Example 4). Representative SEM images of the biofilms are shown in Figures 5-10. Control Fuji IX GICs possessed a higher abundance of complex bacterial communities on their surfaces with some evidence of extracellular polymeric substance (EPS) present, observed as stringy connections/network between bacterial cells (Figures 5(b), 6(b), 8(a) and 9(b)). There was a silver concentration- dependent effect on the bacterial communities observed as a visual decrease of surface-bound bacteria when a higher concentration of silver was incorporated into the GIC material. The antibacterial effects of the Ag NPs were consistent across the species tested. There were some examples of bacteria present on the Ag NP-modified GICs. However, typically the bacteria were found in low numbers only in the vicinity of material defects. Furthermore, the bacteria were scattered sporadically in non-regular patterns. Additionally, the morphology of the bacteria in contact with the Ag NP-modified GIC surfaces were irregular, and EPS could not be observed. It was frequently challenging to find bacteria present on the Ag NP-modified GIC containing 64 μg of silver, which possessed a smooth surface with some evidence of lysed material. With an increase in Ag NP inclusion, there was a decrease in the presence of bacteria, particularly in biofilm form. When observing the surface, only scarce instances of single bacteria were found. Of these, the morphological structure was often disrupted and the bacterium appeared to be "melted" to the surface of the GIC. Initial colonisation of dental restoratives is reported to be affected by the physico-chemical properties of the restoration. When bacteria are in contact with the Ag NP-modified GIC, their adherence is believed to be hampered by the bactericidal effects of the NPs. This has the potential to reduce and delay the biofilm development that leads to plaque formation.

Biofilm adhesion was studied via live/dead fluorescence staining of the GICs with subsequent CLSM analysis (Example 4), the results of which are presented in Figure 11. The areas of grey represent the original green fluorescence intensity originating from bacterial biofilms growing on the non-modified and Ag NP-modified Fuji IX control. Biofilm growth was significantly higher on the non-modified GICs than on the Ag NP-modified Fuji IX GICs. This indicates that there was an antibacterial effect produced at the surface of the Ag NP-modified GICs that was higher than that of the non-modified Fuji IX control GIC, which caused a reduction of bacterial adhesion. Extensive biofilm coverage of S. mutans, S. sanguis and S. mitis was observed on the non-modified Fuji IX control GIC, while a significantly reduced population of P. aeruginosa bacteria was adhered to the Fuji IX control. Typically, biofilms with a high presence of green fluorescence (indicated as grey in the black/white images of Figure 11) were detected on the surface of non-modified Fuji IX control GICs, resembling healthy live biofilms. The Ag NP-modified Fuji IX GICs showed greatly reduced bacterial populations, and this was enhanced further with an increase in silver content for all bacterial species tested. Very limited bacterial growth was observed for the Ag NP-modified Fuji IX containing 64 μg silver. However, if there were cracks present in the GIC, residual bacteria could be seen within these areas.

To examine the substantivity of the anti-biofilm effects of Ag NP-modified GICs previously observed with CLSM and SEM imaging, the same biofilm growth techniques were utilised, but the experiment was repeated over a two-month period at weekly intervals where the GIC samples were routinely and artificially aged in DI H2O for 7 days prior to biofilm surface growth. See Example 5. The biofilms adhered to the surface of the GICs were removed using vortex mixing, and fluorescent intensities of live and dead bacteria were recorded and viability quantified using known techniques. S. mutans was the microorganism selected for this study due to its pathogenesis in the production of caries. The viability of the biofilms grown on the surface of Ag NP-modified and non-modified GICs, measured over the two-month period, are shown in Figure 12.

The biofilm viability of the Fuji IX biofilm at day 0 was represented as 100% biofilm quantity throughout the experiment. Therefore, changes in the growth rates of biofilm on the Fuji IX itself could be monitored and comparisons to the Ag NP-modified GICs made. Ag NP- modified GICs recorded consistently lower numbers of biofilm present on the GIC surface. Typically, there was no significant difference in biofilm quantities between the incorporated mass of silver, except for at day 0, where the 6.4 μg-Ag NP-modified GIC demonstrated a higher antibacterial effect, and day 41 where the 6.4 μg Ag NP-modified GIC demonstrated a lesser antibacterial effect (a significant effect of biofilm growth changes were observed over time p<0.001). All GICs tested had highest antibacterial effects observed at day fourteen. A time-dependent antimicrobial effect was observed, with decreased levels of antibacterial activity observed with increasing time. It was found that there was not a statistically significant effect over the range of silver mass incorporated into the GIC materials when compared to the non-modified Fuji IX (Greenhouse-Geisser F(6.653,17.741) = 1.819, p = 0.148). However, when the 6.4, 32, and 64 μg Ag mass variables were combined, to compare silver modification against the non-modified Fuji IX, the increase in repeat values demonstrated a higher correlated significance (Greenhouse-Geisser F(2.138,21.381) = 2.844, p = 0.077) . The results indicate that incorporation of the Ag NPs within the GICs provides a better anti-biofilm adherence effect over time, compared to GICs that contain fluoride only, and therefore Ag NP-containing GICs are able to reduce the bacterial accumulation that leads to the formation of caries.

From the collation of the results from the bacterial growth assays in relation to Ag NP- modified GICs, it can be seen that the Ag NPs delay and reduce the overall colonisation of, and subsequent biofilm formation on, the surface of GICs. Biofilm presence was significantly reduced on Ag NP-modified GIC surfaces, as visualised by confocal microscopy after 48 h incubation, and reduced live/dead ratios were demonstrated over the course of 8 week GIC aging. SEM imaging indicated stochastic dispersal of planktonic bacteria was typical of the Ag NP-modified GICs, with morphological disturbance to some bacteria. It is thought that the Ag NPs prevented high level adhesion of the tested bacteria due to direct contact with the Ag NP surface rather than through leaching of Ag ⁺ ions. A GIC material that possesses an antibacterial effect with non-releasing properties is considered superior to releasing materials in which the bond strengths are reduced due to vacuolation of the matrix and induced weakening of the cement.

Mechanical testing was conducted to assess the usefulness of the GICs of the invention for a range of dental applications. Where appropriate, "addition" refers to the added incorporation of an Ag NP suspension to the GIC polymeric phase, without alteration of the polymeric phase volume prior to the addition. The term "substitution" on the other hand refers to the removal of an equivalent volume of polymeric phase to that of the Ag NP suspension which is being incorporated.

Flexural strength values (Example 6) were calculated using BlueHill 3 software. The characteristic strength is defined as the strength of the material below which not more than 5% of the test results are expected to fall. A simple mean or characteristic strength represents a symmetric Gaussian distribution. However, sub-populations of values commonly exist. To address this, the Weibull Modulus can be calculated which describes the variability in the measured material strength of brittle materials. Materials which record low variation of measured flexural strength produce higher Weibull modulus values. A minimum sample size of 30 was used in the Weibull Modulus calculation (excluding the discarded specimens containing visible defects).

A Kruskal-Wallis one-way analysis of variance was performed on the data, as the data did not adhere to normal distributions. From this analysis, a statistically significant difference (p = 0.0065) was observed. Dunns post-hoc analysis determined that only an addition of 5 μΙ_ of Ag NP suspension, incorporating an Ag mass of 6.4 μg, possessed a significantly higher strength than that of the control group (p = 0.0149). The strength test therefore demonstrated that the addition of Ag NPs within the GIC matrix at the silver contents tested does not affect the flexural strength and therefore brittleness of the cement material. In fact, the results suggest that the Ag NPs can improve the mechanical properties of the cement, which may be due to increased cross-linking within the matrix, provided by surface-bound molecules.

The elastic modulus of Ag NP-modified GICs and non-modified Fuji IX control GIC were investigated to determine the influence of Ag NP incorporation on the compressive and elastic behaviour of the material (Example 7). This material property is particularly important for dental materials, such as GICs, that undergo continual force loading from mastication. Elastic modulus values were measured for GIC specimens at one day and seven day maturation (40 measurements were performed per sample). The elastic modulus results are shown in Figure 14.

The results indicate that GIC groups had similar elastic moduli at day 1. However, the substitution groups, especially substitution of 10 μΙ_ with 6.4 μg Ag mass, had the lowest elastic moduli. A comparison of the mean moduli using a one-way ANOVA analysis revealed a significant difference between the groups (p < 0.0001). Post-hoc pairwise multiple comparisons with a Tukey's test (p <0.05) with adjusted p values also revealed statistically significant differences between the groups.

At 7 days, the elastic modulus of the non-modified Fuji IX control GIC had reduced significantly. Similarly, the GIC groups containing an Ag mass of 6.4 μg incorporated as a volume of 10 μΙ_, both addition and substitution groups had significantly reduced elastic moduli values. In contrast, groups containing an Ag mass of 6.4 μg incorporated as a volume of 5 μΙ_ showed little variance in elastic moduli upon seven day specimen maturation. A comparison of the mean moduli using a one-way ANOVA analysis, across all GIC groups, revealed a significant difference between the groups (p<0.0001). Post-hoc pairwise multiple comparisons with a Tukey's test (p<0.05) with adjusted p values also revealed statistically significant differences between the groups. All groups were distinguished as being significantly different from one another.

Elastic modulus is the measure of the resistance of a material when deformed elastically as a force is applied, and is effectively defined by the slope of the stress-strain curve. A stiffer material will have a higher elastic modulus. In theory, GICs require a high modulus of elasticity to provide sufficient load bearing capacity in order to resist masticatory and parafunctional stresses. However, they also need to maintain integrity of binding to either dentine or prosthodontic devices. Therefore GICs should possess an elastic moduli intermediate between that of dentine and other restoratives in order to decrease interfacial stress and physical flexural strain.

Add 100% and Subl00% groups demonstrated a 6.4% increase in elastic moduli, with the addition group demonstrating a higher value than the substitution group. It was theorised that within the samples of these groups, the Ag NPs were involved in the chelation of metal cations and formation of additional polysalt bridges with the polyacrylic acid carboxylate groups. Such Ag NP involvement within the matrix may have produced a higher viscosity material due to increased cross-linking. Therefore a higher cross-linked Ag NP matrix could potentially produce GICs of improved elastic modulus for clinical application. When a 5 μΙ_ volume of AgNP suspension was added to the GIC liquid phase, this appeared to have no detrimental effect to the GIC elasticity, as seen for Add 100% and Subl00%. Substitution of the liquid phase changed the polymer content, thus producing a slight decrease in elastic modulus. It was evident that a high Ag NP content delivered by a small volume of H ₂ 0, without changing the existing polymer:water:glass ratios, increased the elastic moduli of the GIC. Finalised flexural strength values of tested GICs (GC, 3M, and SDI) indicated either an equivalent or an increase in flexural strength values of the AgNP-modified GICs when compared to the non-modified GICs. See Example 7.

Compression tests (Example 9) indicated that the AgNP-modified GICs did not significantly impact on the compressive strength of the GICs when compared to the non- modified GIC controls. For 3M Ketac Molar and GC Fuji IX, the deviation in compressive strength values was reduced when the GICs were modified with 24 μg and 10 μg of AgNPs, respectively. The AgNP-modified GICs demonstrated significantly higher compressive strength values when compared to 3M Ketac Silver, SDI Riva Silver and GC Miracle Mix.

At high Ag NP concentrations, a colour shift affecting the GIC material is noticeable, but the change observed falls within the VITA classic or VITA 3D Master shade spectra. See Example 10.

Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field.

As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to".

The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

EXAMPLES

Example 1: Synthesis of th iodic acid capped silver nanoparticles

A solution of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in heptane (40 ml_, 0.33 M) was placed in two separate flasks. To the first solution, an aqueous solution of AgNC (1.6 ml_, 0.13 M) was added dropwise with stirring, forming microemulsion 1. To the second solution, an aqueous solution of NaBH ₄ (1.6 ml_, 1.84 M) was added dropwise with stirring, forming microemulsion 2. The flasks were placed in separate ice baths, and microemulsion 1 was covered with aluminium foil. Microemulsion 2 was added dropwise to microemulsion 1, with continuous stirring. Upon addition, a colour change from light yellow to dark yellow- brown was observed, suggesting the production of Ag NPs. This mixture was allowed to stir in the dark for up to 24 h. Subsequently, l,2-dithiolane-3-pentanoic acid (thioctic acid) (1 mM, dissolved in 0.25 mL ethanol) was introduced and the microemulsion was stirred for an additional 1-60 min. Upon discontinuation of the stirring, a 1 : 1 methanol/acetone mixture was added at an equivalent half volume of the combined microemulsion (40 mL). Phase separation was clearly observed, and a dark-coloured interface formed between the two phases, where the particles resided.

The nanoparticles at the interface were carefully collected. Subsequently, the particles were washed 3 times with ethanol, which included centrifugation (8000 rpm for 5 min) then resuspended. The final resuspension of the particles took place in 1 -6 mL of DI H2O (depending on the [Ag] desired) which was pre-adjusted to pH 9 with anhydrous ammonia. The resulting yellow-brown coloured colloidal suspension was centrifuged twice at 13,000 rpm for 45 min, with the final supernatant collected and retained for characterisation and further work.

Example 2: Preparation of GICs incorporating thioctic acid capped Ag NPs

A commercially-available GIC (Fuji IX, GC; Japan) was modified with thioctic capped- Ag NPs by incorporating them within the liquid phase of the GIC. The GIC capsule (see Figure 2) was deconstructed by careful removal of the plunger with pliers. The liquid phase is contained within a thin plastic vessel . When the plunger is depressed flush with the container, the separating membrane is pierced and the solid and liquid phases come into contact. A specific volume of a Ag NP suspension was added to the liquid polymeric phase and the mixture was gently stirred with a pipette tip. The plunger was then carefully replaced into its original position within the receptacle.

Various silver quantities were incorporated into Fuji IX GICs. Photographs of the modified GICs are shown in Figure 3. With increasing concentration of Ag NPs incorporated into the GIC structure, an increasing yellow/brown colour was produced (as seen with increasing intensities of grey in the black and white version of Figure 3). The yellow/brown colour is characteristic of non-aggregated Ag NPs and appeared to be well-dispersed throughout the GIC. The Fuji IX control (Figure 3a) was creamy white in colour. With increasing Ag NP content, the cement material transitioned from a light beige (Ag content of 6.4 μg, Figure 3b), to a slightly darker beige (Ag content of 32 μg, Figure 3c) and finally to a warm brown (Ag content of 64 μg, Figure 3d). All of the GIC colours observed following Ag NP addition were consistent with restorative tooth shades as described by the Vita Plan 3D- master tooth guide in terms of lightness, chroma and hue. Therefore, incorporation of Ag NPs into the GICs could produce modified materials still applicable for clinical use in terms of colour and appearance.

Example 3: Antibacterial liquid culture assays of non-modified and Ag NP-modified GICs

Stock cultures of Streptococcus mutans (UA159), Streptococcus mitis (IL8), Streptococcus sanguis and Pseudomonas aeruginosa (ΌΤΙ5) were obtained from the Department of Oral Sciences, University of Otago, New Zealand. S. mutans, S. mitis, and S. sanguis were grown aerobically in brain heart infusion media. P. aeruginosa was grown aerobically in tryptic soy media . Incubations were performed at 37 °C for 10 h, unless otherwise indicated.

Thioctic acid-coated Ag NPs were added to the liquid phase of separate Fuji IX GICs, so that the GICs contained an Ag content of 6.4 pg, 32 pg, or 64 pg. Each sample was mixed at high speed for 10 s on a Caulk Vari-Mix III amalgamator (Dentsply, Pennsylvania, USA) then extruded into a 1 cm diameter x 2 mm steel disk mould. Specimens were polished on a TegraPol-21 polisher (Struers, Copenhagen, Denmark) with silicon carbide paper (1,200- 4,000 grit) for 30 s on each side, and sonicated in an ultrasonic cleaner for 5 min in between polishing sessions. Samples were autoclaved at 121 °C for 15 min.

A 30 μΙ_ volume of each bacterial culture was placed onto the surface of separate GIC discs (performed in triplicate for each bacterium) . The GICs contained a n Ag content of 0 pg, 6.4 pg and 64 pg. The GIC discs with inoculated surfaces were incubated at 37 °C for 30 min to allow for the bacterial suspension to dry. Samples were then placed in 1 ml_ brain heart infusion (BHI) or tryptic soy broth (TSB) and were incubated within a rotary shaker at 60 rpm, 37 °C for 20 h. The O. D630 nm of the medium was monitored hourly using a microplate reader (Synergy 2 BioTek® (Winooski, VT, USA) for 20 h. At each 1 h interval, the GICs were aseptically removed and placed into a sterile, 24 well plate whilst optical density measurements were recorded . The GICs were subsequently replaced into the original medium . The results are shown in Figure 4.

Example 4: Biofilm growth and adherence on non-modified andAg NP-modified GICs

Sterile GIC specimens were placed into separate wells of a 24 well plate containing 1 ml_ of BHI or TSB supplemented with 1% sucrose. Each well was inoculated with a 10 pL volume of bacterial culture, deriving an absorba nce value of 0.01600- The plate was placed in a rotary shaker incubator (37 °C at 60 rpm) for 24 h. After 24 h, the GIC specimens were aseptically removed and placed into a new well containing 1 ml_ of un-inoculated broth (BHI or TSB with 1% sucrose). The specimens were incubated (37 °C at 60 rpm) for 24 h. This method cultured 48 hr biofilms on the surface of the GIC specimens. The GIC specimens were then dip washed three times with sterile PBS buffer to remove non-adherent bacteria . The Ag NP-modified and non-modified GICs subjected to biofilm surface growth were analysed by SEM in order to investigate the morphologies of the bacterial cells grown on the GIC surfaces. SEM images are shown in Figures 5-10.

The Ag NP-modified and non-modified GICs were also analysed by CLSM. Stock solutions of SacLight™ dye were prepared by mixing equal volumes (6 pL) of SYTO ^® 9 and propidium iodide (PI) thoroughly. This preparation was made to 2 mL with sterilised DI H ₂ 0. The biofilm/GIC specimens were treated with Live/dead ^® SacLight™ stain (200 μΙ_) and were maintained in the dark for 15 min. The resulting stained biofilms were gently washed with PBS, placed on a microscope slide and the excess liquid was wicked away. Stained biofilms were examined with a Zeiss LSM 710 confocal laser-scanning microscope (Carl Zeiss; Jena, Germany) and recorded with Zeiss ZEN software 2009. Ag NP-modified and control GIC specimens were analysed under CLSM without biofilms or staining and w ith staining in the absence of a biofilm . No residual fluorescent materia l was present in the samples. The results are shown in Figure 11. Example 5: Quantitative biofilm growth study utilising Live/ dead BacLight™ stain over time on non-modified and Ag NP-modified GICs

Non-modified Fuji IX GIC and Ag NP-modified GICs containing an Ag content of 6.4, 32 and 64 pg, (performed in triplicate for each type of bacterium), were each placed into separate wells in a 24 well plate. A volume (1 mL) of BHI contain ing 1% sucrose was placed over each GIC and the BHI broth was subsequently inoculated with S. mutans (20 μΙ_). The plate was incubated within a rotary shake incubator (37 °C at 60 rpm) for 24 h. The GICs were then removed and placed into a fresh well containing BHI with 1% sucrose (1 mL). The plate was returned to the rotary shake incubator for another 24 h at 60 rpm, 37 °C. Following the second incubation period, each GIC was aseptically removed and gently dip-washed three times in PBS buffer to remove non-adherent bacteria. The GICs were placed in sterile 15 mL falcon tubes containing tris-buffered saline-peptone (1 mL, pH 7.4) and were vortexed for 1 min to remove adherent bacteria. A volume (100 pL) of each of the GlC/biofilm supernatants was then placed into a sterile well of a 96 microplate well, mixed with the prepared live/dead dye (100 pL) and the microplate was then placed in the dark for 15 min. The fluorescence intensities were monitored using a microplate reader (Synergy 2 BioTek®; Winooski, VT, USA); λ excitation = 485 nm; λ emission (green) = 530 nm for the live stain (SYTO® 9), λ emission (red) = 630 nm for the dead stain (PI) and were used to calculate cell viability. The GIC specimens were then superficially aged by storing the GICs in sterile DI H ₂ 0 for 7, 14, 21, and 28 days followed by direct 48 h incubation with S. mutans for anti-biofilm testing. The results are shown in Figure 12.

Example 6: Mechanical testing - flexural strength concentration estimation

Mechanical testing was conducted on liquid addition GIC specimens containing 6.4 pg of Ag (5 pL) and 6.4 pg of Ag (10 pL). Addition of Ag NPs was performed by adding the Ag NP suspension to the polymeric phase of the GIC capsule and consisted of the addition of 6.4 pg of Ag mass (5 pL) and 6.4 pg of Ag mass (10 pL), Add 100% and Add50% respectively. Substitution of Ag NPs to the liquid phase was also performed by removal of 5 pL and 10 pL of the polymeric liquid phase prior to the addition of 6.4 pg of Ag (5 pL) and 6.4 pg of Ag (10 μΙ_), Subl00% and Sub50% respectively. The GIC specimens tested, and their corresponding sample group names, are shown in Table 1. Each Fuji IX capsule, prior to modification, contained 400 μg of powder and 120 μg (100 μΙ_) of liquid, hence by adding a small volume of an aqueous Ag NP suspension to the liquid phase, the modification only constituted a 5- 10% change in the total liquid phase volume.

Table 1: Composition of control and Ag NP-modified GICs of the different GIC groups evaluated using mechanical testing

GIC group Polymeric liquid Solid phase Ag content Ag NP suspension volume (μί) mass (pg) (M9) volume (μί)

Control 100 400 0 0

Add 100% 100 400 6.4 5

Sub 100% 95 400 6.4 5

Add 50% 100 400 6.4 10

Sub 50% 90 400 6.4 10

A four part, split Teflon mould, with a central cuboid cavity of 2.5 mm x 2.0 mm x 2.0 mm, was designed to shape the GIC specimens into bars for flexural strength testing. The mould was coated with 3% paraffin in hexane prior to placing GICs within the mould. The GICs were mixed according to manufacturer instructions. Briefly, the GIC was agitated to loosen the powder, the plunger was then depressed until it became flush with the main body. The capsule was immediately placed and triturated on an amalgamator (Silamat, Vivadent; Auckland, New Zealand) for 10 s. The mould was placed onto an Ultrasonic vibrator (Ceramosonic II, Shofu; Ratingen, Germany) where the contents of the capsule were extruded into the central cavity of the mould. The mould was tightly screwed closed to ensure a full seal. The vibrator ensured consistency of mixing and prevented defects from forming across the material. Subsequently, the mould was placed in a water bath within an incubator (Labec Laboratory Equipment; New South Wales, Australia) at 37 °C for 1 h and the GIC was left to set. The GIC specimen was removed from the mould and was placed in a small, plastic, clip- lock bag filled with DI H2O (pre-warmed to 37 °C). This was then returned to the incubator (37 °C) for 24 h. Post 24 h GIC maturation, each specimen was removed and visually inspected. Specimens with visible surface defects and specimens that deviated from the mould dimensions (± 0.01 mm) were not used within the study. Quality assured specimens were returned to the water-filled, clip-lock bags within the 37 °C incubator for an additional 24 h.

Flexural strength measurements were performed on an Instron 3369 Dual Column

Tabletop testing system, following ISO protocol 9917 :2: 2010. Each GIC specimen was centred to the testing fixture (Model No. WTF-CF-43, Wyoming Test Fixtures; Utah, USA). Force loading was performed at 1 mm min ^"1 until the material fractured. A minimum of 32 specimens from each GIC group were tested. The flexural strength values recorded for the non-modified Fuji IX control GICs and Ag NP-modified Fuji IX GICs are shown in Figure 13.

The average three-point flexural strength, characteristic strength and Weibull Modulus are shown in Table 2.

Table 2: Flexural strength values and Weibull Modulus for GIC groups

Group Number of Mean Strength Characteristic j Weibull i

samples/group (Mpa) Strength (MPa) Modulus i

Control 38 14.60 16.31 2.995

Add 100% 37 15.65 17.40 j 3.2677 i

Sub 100% 37 15.00 16.49 4.5914 i

Add 50% 36 16.20 17.86 j 4.5835 I

Sub 50% 32 17.44 19.72 j 3.5661 I

Example 7: Mechanical testing - finalised flexural length testing

Flexural strength testing was conducted on liquid addition GIC specimens containing 6.4 μg of Ag (1.5 μΙ_), 10 μg of Ag (2.35 μΙ_), and 24 μg Ag (5.63 μΙ_) depending on the GIC brand. Addition of Ag NPs was performed by adding the Ag NP suspension to the polymeric phase of the GIC capsule. The GIC specimens tested, and their corresponding sample group names, are shown in Table 3 and Figures 14-16. Each GIC capsule, prior to modification, contained 400 μg of powder and 120 μg (100 μΙ_) of liquid . Hence, by adding a small volume of an aqueous Ag NP suspension to the liquid phase, the modification only constituted a 5- 10% change in the total liquid phase volume as described in Example 6. The percentage reduction in the total biovolume formed on the surface of Ag NP-modified GICs compared to unmodified GICs was determined using CLSM images and Comstatl . The results are shown in Table 3. Table 3: Reduction in biovolume formed on surface of GICs

Example 8: Mechanical testing - nanoindentation

Nanoindentation was performed in an aqueous environment. Specimens were attached to a magnetic mount and were submerged in DI H2O. The IBIS nanoindentation system (Fischer-Cripps laboratories; New South Wales, Australia) was used with a three-sided pyramidal Berkovich indenter to obtain the load displacement curves, from which the hardness and elastic modulus of the specimens were calculated using the software, BlueHill 3, Instron; Massachusetts, USA. A maximum load of 300 mN was used with a hold time of 1 s. The Poisson's ratio used was 0.3. The compliance was fixed at 0.0002 μιη mN ^"1 . A 10 x 10 array of indents was performed. The elastic modulus results are shown in Figure 17.

Example 9: Mechanical testing - compressive strength

15 cylindrical specimens were made for compressive strength (CS) testing for each GIC group. The cylinder dimensions were 2.0 mm diameter x 3.0 mm height for the CS test. The specimens were made at room temperature (23±2 °C) and relative air humidity of 50± 10%. A teflon mould was used to produce the cylindrical GIC specimens of the required dimensions. The mould was coated with 3% paraffin in hexane prior to placing the GICs within the mould. The GICs were mixed according to manufacturers' instructions. Briefly, the GIC was agitated to loosen the powder, the plunger was then depressed until it became flush with the main body. The capsule was immediately placed and triturated on an amalgamator (Silamat, Vivadent; Auckland, New Zealand) for 10 s. The mould was pre- placed onto an Ultrasonic vibrator (Ceramosonic II, Shofu; Ratingen, Germany) where the contents of the capsule were extruded into the central cavity of the mould. The mould was tightly screwed closed to ensure a complete seal. The vibrator ensured consistency of mixing and prevented defects from forming across the material. Subsequently, the mould was placed in a water bath within an incubator (Labec Laboratory Equipment; New South Wales, Australia) at 37 °C for 1 h and the GIC was left to set. The GIC specimen was removed from the mould, placed in a small, plastic, clip-lock bag filled with DI H2O (pre-warmed to 37 °C) and returned to the incubator (37 °C) for 24 h. Post 24 h GIC maturation, each specimen was removed and visually inspected. Specimens with clear surface defects and specimens that deviated from the mould dimensions (± 0.01 mm) were not used within the study. Quality assured specimens were returned to the water-filled, clip lock-bags within the 37 °C incubator for an additional 24 h. Tests were made using an Instron 3369 Dual Column Tabletop testing system at a crosshead speed of 1.0 mm/min.

The specimens were placed in vertical position, with force incident on the long axis. The CS was calculated by the following formula : P/pr ² , where P= load at fracture, r= the radius of sample cylinder, and p= (constant) 3.14. CS values [kgf/cm ² ] were converted into MPa as follows: CS [MPa]=CS[Kgf/cm ² ] x 0.09807. A Kruskal-Wallis one-way analysis of variance was performed on the data, as the data did not adhere to normal distributions. From this analysis, if a statistically significant difference was observed, Dunns post-hoc analysis was used. The compressive strength results are shown in Figures 18-20.

Example 10 - Aesthetics

GIC colour shades using Lab values (L = lightness or luminescence, and a and b for the colour components green-red and blue-yellow), and both master and classical shade guides, were recorded at 0 h, 24 h and 2 weeks using a VITA Easyshade V instrument (VITA Zahnfabrik H . Rauter GmbH & Co, KG, Bad Sackingen, Germany) . GIC specimens (n=3) were stored at 37 °C in H2O in between measurements. The results are shown in Table 4.

Table 4: Assigned VITA Classic shade and VITA 3D Master shade

O h Assigned VITA Assigned VITA 3D

Classic shade Delta E Master shade

Fuji IX control A3.5 16.95 4M3

Fuji IX 6.4 pg AgNP C4 24.38 5M3

Fuji IX 10 pg AgNP C4 30.95 5M3

Ketac Control A4 27.50 5M3

Ketac 10 pg AgNP C4 33.83 5M3

Ketac 24 pg AgNP C4 44.45 5M3

Riva control C4 28.10 5M3

Riva 10 pg AgNP C4 32.83 5M3

Riva 24 pg AgNP C4 29.08 5M3 24 h Assigned VITA Assigned VITA

Classic shade Delta E 3D Master shade

Fuji IX control A3.5 18.98 3.5M3

Fuji IX 6.4 μς AgNP C4 22.03 5M3

Fuji IX 10 μς AgNP C4 25.98 5M3

Ketac Control A4 25.90 5M3

Ketac 10 μς AgNP C4 27.63 5M3

Ketac 24 μg AgNP C4 36.88 5M3

Riva control A3.5 30.85 5M3

Riva 10 μg AgNP A4 26.25 5M3

Riva 24 μg AgNP C4 29.08 5M3

Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.