WO2013057519A1

WO2013057519A1 - Dental adhesive systems

Info

Publication number: WO2013057519A1
Application number: PCT/GB2012/052619
Authority: WO
Inventors: Romano Mongiorgi; Giovanni Valdre; Boris DE CARLO; Salvatore SAURO; Timothy Watson; Francesco MANNOCCI
Original assignee: King's College London; Alma Mater Studiorum - Universita Di Bologna
Priority date: 2011-10-20
Filing date: 2012-10-22
Publication date: 2013-04-25
Also published as: GB201118138D0

Abstract

There is provided the use of: i) a composition comprising Portland cement and phyllosilicate; and/or ii) a modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement and phyllosilicate, as an additive in a dental adhesive. Also disclosed is a dental adhesive comprising resin monomers and: 1) a composition comprising Portland cement and phyllosilicate; and/or 2) a modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement and phyllosilicate. Additionally, compositions for improving the characteristics of a dental adhesive system are provided, the composition comprising: Portland cement, phyllosilicate and hydrotalcite; or Portland cement and phyllosilicate, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition.

Description

Dental Adhesive Systems

Field of the Invention

The invention relates to additives for adhesive systems and additive-containing adhesive systems for use in dental applications, for example, in conservative/restorative dentistry.

Background to the Invention

The demineralisation of dentine and the exposure of the collagen matrix is a crucial step in adhesive dentistry. The infiltration of demineralised collagen fibers with resin permits formation of hybrid layers with resin tags and adhesive lateral branches, thus creating micromechanical retention of the resin to the demineralised substrate. The micromechanical retention is the chief mechanism for resin bonding to dentine. Simplified etch-and-rinse and/or self -etch adhesive systems currently used in conservative/restorative dentistry are affected by reduced longevity due to the degradation of the hybrid layer. Hybrid layer degradation is mainly due to the incapability of these bonding systems to completely replace the water from the extrafibrillar and intrafibrillar collagen compartments with resin monomers. Moreover, these resin monomers within the hybrid layer are affected by hydrolytic degradation when exposed to fluids over time.

The degradation of contemporary simplified etch-and-rinse and/or self-etch adhesives has been extensively reported by several scientific reports and peer-reviewed papers which showed nanoleakage and micropermeability within hybrid layers as a potential site of where these phenomenon of degradation occur. It has been advocated that one of the most promising methods to face this problem is the remineralisation of free and loosely bound water within the collagen water compartments. The remineralisation or the protection of the free collagen fibrils within the hybrid layer might also silence the collagenolytic enzymes. Indeed, collagenase hydrolysis of dentine confirmed the protective role played by the mineral phase on collagen degradation. In fact, similar to a host of growth factors and signalling molecules, endogenous matrix metalloproteinase (MMPs) "fossilised" within dentine by the apatite minerals may retain their biologic activities after they are incorporated into the demineralised dentine matrix without any degradation action. These molecules assume their biologic activities upon removal of the mineral phase, provided that the demineralisation agent is not strong enough to denature these molecules. In dentine bonding, the mineral phase of dentine is intentionally removed by acids, chelating agents, or acidic resin monomers to expose the collagen for creating micromechanical retention of resins.

WO 2008/096011 discloses a composition containing Portland cement and phyllosilicate. The composition has a weight percentage of phyllosilicate from about 0.4% to about 3.6%. The composition is used for various forms of tooth filling and for dentine hypersensitivity treatment. However, the composition is not described as being useful as an additive for dental adhesive systems. Summary of the Invention

The inventors have devised adhesive systems which have improved characteristics relative to known adhesive systems. For example, the adhesive systems may retain their bond strength for a longer period of time relative to known adhesive systems. In other words, the bond strength does not degrade as quickly due to remineralisation/protection of the hybrid layer. Further, some of the adhesive systems may also provide increased bond strength. Some of the adhesive systems may provide increased dentine desensitisation and general remineralisation. Further, some adhesive systems induce increased mineral precipitation within the interface due to their hydrophilicity. The improved characteristics of the adhesive systems are brought about by incorporating additives into the adhesive systems. Therefore, in a first aspect, the invention provides a composition for improving the characteristics of a dental adhesive system, the composition comprising Portland cement and phyllosilicate, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition.

In another aspect, the invention provides a composition comprising Portland cement, phyllosilicate and hydrotalcite.

Further, in some embodiments, the invention provides a composition comprising Portland cement, a phyllosilicate, hydrotalcite and titanium oxide. In specific embodiments, the invention provides a composition comprising Portland cement, titanium oxide, smectite and hydrotalcite. This composition acts as a desentisitising/remineralising material. Further, this composition is able to induce a more intense mineral precipitation within the interface due to the hydrophilicity of the titanium oxide. The composition may maintain the bond strength of the dental adhesive system. This means that the durability of the adhesion is improved so that the bond strength degrades more slowly over time. A therapeutic effect will be observed due to intense mineral precipitation within the interface.

Portland cement (also referred to as Ordinary Portland Cement (OPC)) is a hydraulic cement which hardens and sets after being mixed with water. ASTM C150 standard specification for Portland cement defines Portland cement as hydraulic cement produced by pulverising clinker consisting essentially of hydraulic calcium silicates, usually containing one or more of the forms of calcium sulphate as an inter-ground addition.

To manufacture Portland cement, an intimate mixture of limestone and clay is ignited in a kiln to form Portland cement clinker. The following four main phases of Portland cement are present in the clinker: tricalcium silicate (3CaO.Si0₂, also referred to as C₃S); dicalcium silicate (2CaO.Si0₂, called C₂S); tricalcium aluminate (3CaO.Al₂0₃ or C₃A); and tetracalcium aluminoferrite (4CaO.Al₂0₃.Fe₂0₃ or C₄AF). The resulting clinker containing the above compounds is inter-ground with calcium sulphates (e.g. gypsum) to desired fineness to produce the Portland cement. Other compounds that may be present in minor amounts in Portland cement include double salts of alkaline sulphates, calcium oxide, and magnesium oxide.

The term "Portland cement" is well known to one skilled in the art and is intended to cover all forms of Portland cement. This includes Type I, Type II, Type III, Type IV and Type V Portland cement as defined by ASTM CI 50. It also includes grey Portland cement, white Portland cement, and other forms of Portland cement.

An alternative standard for Portland cement is EN 197-1 which defines five types (I-V) of cement containing Portland cement. Preferably, the Portland cement of the invention is a Type I cement in accordance with EN 197- 1.

In various embodiments, the Portland cement may contain the following components by weight:

Tri-calcium silicate 50-80%

Di-calcium silicate 10-40%

Tri-calcium aluminate 1-10%

Calcium sulphate 3-10%

Other elements Traces

In certain embodiments, the compositions described above comprise about 62-99.7% by weight Portland cement relative to the total weight of the components of the composition (e.g. the Portland cement and phyllosilicate (such as smectite) combined; or Portland cement, phyllosilicate and hydrotalcite combined). Preferably, the composition comprises about 75-97.5% by weight Portland cement. More preferably, the composition comprises about 77.5-95% by weight Portland cement. In particular, when the composition comprises Portland cement and phyllosilicate, the composition may comprise about 80-96.3% by weight Portland cement relative to the total weight of the Portland cement and phyllosilicate combined. The composition may comprise about 80-96% by weight Portland cement. Further, the composition may comprise about 85-96% by weight Portland cement. The composition can comprise about 87.5-96% by weight Portland cement. Furthermore, the composition may comprise about 90-95% by weight Portland cement.

When the composition comprises Portland cement, phyllosilicate and hydrotalcite, the composition may comprise about 80-99% by weight Portland cement relative to the total weight of the Portland cement, phyllosilicate and hydrotalcite combined. In some embodiments, the composition may comprise about 80-97.5% by weight Portland cement relative to the total weight of the Portland cement, phyllosilicate and hydrotalcite combined. The composition may comprise about 82.5-97.5% by weight Portland cement. Further, the composition can comprise about 85-95% by weight Portland cement. In some embodiments, the composition comprises about 87.5-92.5% by weight Portland cement. In certain embodiments in which the composition comprises Portland cement, titanium oxide, a phyllosilicate and hydrotalcite, the composition comprises about 62-99.7% by weight Portland cement relative to the total weight of the Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite combined. Preferably, the composition comprises about 76-99.7% by weight Portland cement. More preferably, the composition comprises about 76-94% by weight Portland cement. More preferably still, the composition comprises about 76-89% by weight Portland cement. In other embodiments, the composition comprises about 62-98% by weight Portland cement relative to the total weight of the Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite combined. The composition may comprise about 65-95% by weight Portland cement. Further, the composition may comprise about 70-90% by weight Portland cement. In some embodiments, the composition comprises about 75-85% by weight Portland cement. Furthermore, the composition may comprise about 77.5- 82.5% by weight Portland cement.

The composition may comprise titanium oxide. This may be any titanium oxide such as titanium dioxide (titanium (IV) oxide (Ti0₂)), titanium monoxide (titanium (II) oxide (TiO)), titanium (III) oxide (Ti₂0₃), etc. Preferably, the titanium oxide is titanium dioxide.

In some embodiments, in particular those in which the composition comprises Portland cement, titanium oxide, a phyllosilicate (such as smectite) and hydrotalcite, the composition comprises about 0.1-25% by weight titanium oxide relative to the total weight of the components of the composition (e.g. the Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite combined). Preferably, the composition comprises about 0.1-20% by weight titanium oxide. More preferably, the composition comprises about 5-20% by weight titanium oxide. More preferably still, the composition comprises about 10-20% by weight titanium oxide. In particular embodiments, especially those in which the composition comprises Portland cement, titanium oxide, a phyllosilicate (such as smectite) and hydrotalcite, the composition may comprise about 5-15% by weight titanium oxide relative to the total weight of the components of the composition (e.g. the Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite combined). Further, the composition may comprise about 7.5- 12.5% by weight titanium oxide. In some embodiments, the composition can comprise about 8-12% by weight titanium oxide. Furthermore, the composition may comprise about 10% by weight titanium oxide. In various embodiments, the composition may comprise about 1-10% by weight titanium oxide.

The composition comprises phyllosilicate. Phyllosilicates are sheet silicate minerals, formed by parallel sheets of silicate tetrahedra with S1₂O₅ or a 2:5 ratio. Phyllosilicates are well known to those skilled in the art. In some embodiments, the phyllosilicate is a sodium-calcium-aluminum-magnesium silicate hydroxide hydrate such as (Na,Ca)(Al,Mg)6(Si₄Oio)3(OH)6-nH₂0.

In some embodiments, the phyllosilicate is smectite such that the composition comprises smectite. Smectite, which is a subset of phyllosilicates, is a clay mineral which consists of an octahedral sheet sandwiched between two tetrahedral sheets (referred to as a 2: 1 clay). The smectite group includes dioctahedral smectites such as montmorillonite and nontronite, and trioctahedral smectites such as saponite. The term smectite and the minerals falling within the term smectite are well known to those skilled in the art. Any suitable smectite can be used in the composition. In one embodiment, the smectite is montmorillonite.

When the composition comprises just Portland cement and phyllosilicate, the composition comprises about 3.7- 15% by weight phyllosilicate relative to the total weight of the components of the composition. The composition can comprise about 4-15% by weight. The composition may comprise about 4-12.5% by weight. Further, the composition may comprise about 4-10%. The composition can comprise 5-10% phyllosilicate. In some embodiments, the composition may comprise about 7.5% by weight phyllosilicate. However, in embodiments in which the composition comprises further components such as hydrotalcite and optionally titanium oxide, the phyllosilicate may be present at below 3.7%. Further, when the composition is used as an additive (described in more detail below), the phyllosilicate may also be present at below 3.7%. In certain embodiments, the composition comprises about 0.1-15% by weight phyllosilicate relative to the total weight of the components of the composition (e.g. the Portland cement and phyllosilicate combined; the Portland cement, phyllosilicate and hydrotalcite combined; or Portland cement, phyllosilicate (e.g. smectite), hydrotalcite and titanium oxide combined). The composition can comprise about 0.5-15% by weight phyllosilicate. The composition may comprise about 0.5-12.5% by weight phyllosilicate. Further, the composition can comprise about 0.5-10% by weight phyllosilicate. In some embodiments, the composition comprises about 2.5-15% by weight phyllosilicate. The composition may comprise about 2.5-12.5% by weight phyllosilicate. Further, the composition may comprise about 5-10% by weight phyllosilicate. In some embodiments, the composition may comprise about 7.5% by weight phyllosilicate.

In some embodiments in which the composition comprises Portland cement, phyllosilicate (e.g. smectite), hydrotalcite and titanium oxide, the composition may comprise about 0.1-5% by weight phyllosilicate (e.g. smectite) relative to the total weight of the Portland cement, titanium oxide, smectite and hydrotalcite combined. Preferably, the composition comprises about 0.1-2.5% by weight phyllosilicate (e.g. smectite). More preferably, the composition comprises about 0.5-2.5% by weight phyllosilicate (e.g. smectite).

The composition may comprise hydrotalcite. Hydrotalcite is a layered double hydroxide of general formula (Mg₆Al₂(C0₃)(OH)₁₆ · 4(H₂0). In various embodiments, the composition comprises about 0.1-7.5% by weight hydrotalcite relative to the total weight of the components of the composition (e.g. the Portland cement, phyllosilicate and hydrotalcite combined; or Portland cement, phyllosilicate (e.g. smectite), hydrotalcite and titanium oxide combined). The composition may comprise about 0.5-7.5% by weight hydrotalcite. Further, the composition may comprise about 0.5-5% by weight hydrotalcite. The composition can comprise about 0.5-3% by weight hydrotalcite. In some embodiments, the composition may comprise about 1-7.5% by weight hydrotalcite relative to the total weight of the components of the composition. Further, the composition may comprise about 1-5% by weight hydrotalcite. In particular embodiments, the composition comprises about 2-3% by weight hydrotalcite. In various embodiments comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite, the composition comprises about 0.1-5% by weight hydrotalcite relative to the total weight of the Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite combined. Preferably, the composition comprises about 0.1-3% by weight hydrotalcite. More preferably, the composition comprises about 0.1- 1.5% by weight hydrotalcite. Even more preferably, the composition comprises about 0.5-1.5% by weight hydrotalcite.

In preferred embodiments, the components of the composition may make up the following percentages by weight relative to the total weight of the components (i.e. the Portland cement and phyllosilicate combined; Portland cement, phyllosilicate and hydrotalcite combined; or Portland cement, phyllosilicate (e.g. smectite), hydrotalcite and titanium oxide combined):

1) 85-96.3% Portland cement; and

3.7-15% Phyllosilicate (e.g. smectite).

2) 90-95% Portland cement; and

5-10% Phyllosilicate (e.g. smectite).

3) 82.5-97% Portland cement;

2.5-12.5% Phyllosilicate (e.g. smectite); and

0.5-5% Hydrotalcite.

4) 85-94% Portland cement;

5-10% Phyllosilicate (e.g. smectite); and

1 -5% Hydrotalcite.

5) 62.5-92% Portland cement;

5-20% Titanium Oxide;

2.5-12.5% Phyllosilicate (e.g. smectite); and

0.5-5% Hydrotalcite.

6) 72-87% Portland cement;

7-13% Titanium Oxide;

5-10% Phyllosilicate (e.g. smectite); and

1-5% Hydrotalcite.

In some embodiments, the components of the composition may make up the following percentages by weight relative to the total weight of the components (i.e. the total weight of the Portland cement, titanium oxide, smectite and hydrotalcite combined): 1) 62-99.7% Portland cement;

0.1-30% Titanium Oxide;

0.1-5% Phyllosilicate (e.g. smectite); and

0.1-3% Hydrotalcite.

2) 76-99.7% Portland cement;

0.1-20% Titanium Oxide;

0.1-2.5% Phyllosilicate (e.g. smectite); and

0.1-1.5% Hydrotalcite.

3) 76-94% Portland cement;

5-20% Titanium Oxide;

0.5-2.5% Phyllosilicate (e.g. smectite); and

0.5-1.5% Hydrotalcite.

4) 76-89% Portland cement:

10-20% Titanium Oxide;

0.5-2.5% Phyllosilicate (e.g. smectite); and

0.5-1.5% Hydrotalcite.

The composition described above is a hydraulic cementitious composition which reacts and hardens when water is added thereto. When water is added, it is primarily the Portland cement which reacts. Generally, Portland cement forms hexacalcium aluminate trisulfate hydrate (also known as ettringite) upon the addition of water. In a composition which contains titanium oxide, hydrotalcite and smectite in addition to the Portland cement, this produces a titanium oxide/hydrotalcite/smectite-modified hexacalcium aluminate trisulfate hydrate when water is added. The composition can be added to any dental adhesive systems to improve the characteristics of the dental adhesive system. In another aspect of the invention, there is provided a phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement and phyllosilicate (e.g. smectite). Any of the various compositions described above comprising Portland cement and phyllosilicate (e.g. smectite) can be hydrated (i.e. have water added thereto) to form the phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product.

In another aspect of the invention, there is provided a hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement, phyllosilicate (e.g. smectite) and hydrotalcite. Any of the various compositions described above comprising Portland cement, phyllosilicate (e.g. smectite) and hydrotalcite can be hydrated (i.e. have water added thereto) to form the hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product.

In another aspect of the invention, there is provided a titanium oxide/hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite. Any of the various compositions described above comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite can be hydrated (i.e. have water added thereto) to form the titanium oxide/hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product.

These hydrate products can be added to dental adhesive systems in the same way as the compositions described above. In particular, the composition comprising i) Portland cement and phyllosilicate; ii) Portland cement, phyllosilicate and hydrotalcite; or iii) Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite can be first mixed with water, left to set and then ground. The product can also be sieved (e.g. <32um). The product may be dried, for example, for 24h at 40°C in an oven. The product can then be mixed with an adhesive system. For example, it may be mixed with resin adhesives.

The invention also provides a method of preparing a phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product, the method comprising adding water to a composition comprising Portland cement and phyllosilicate, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition..

The invention also provides a method of preparing a hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product, the method comprising adding water to a composition comprising Portland cement, phyllosilicate and hydrotalcite.

The invention also provides a method of preparing a titanium oxide/hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product, the method comprising adding water to a composition comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite.

Preferably, in the above methods, water is added at a ratio of greater than 1 : 1 by weight relative to the composition, i.e. more than 1 weight of water : 1 weight of composition. More preferably, water is added at a ratio of about 2: 1 by weight relative to the composition. Also provided is the use of a composition comprising Portland cement and phyllosilicate as an additive in a dental adhesive. Further, there is provided use of a modified hexacalcium aluminate trisulfate hydrate product as an additive in a dental adhesive, wherein the product is formed by the hydration of a composition comprising Portland cement and phyllosilicate, As mentioned above, since compositions comprising Portland cement and phyllosilicate, and hydrate products formed from these compositions, have not been previously used as additives, the compositions can comprise 0.1- 15% by weight phyllosilicate relative to the total weight of the components of the composition. The composition can comprise about 0.5-15% by weight phyllosilicate. The composition may comprise about 0.5-12.5% by weight phyllosilicate. Further, the composition can comprise about 0.5-10% by weight phyllosilicate. In some embodiments, the composition comprises about 2.5-15% by weight phyllosilicate. The composition may comprise about 2.5-12.5% by weight phyllosilicate. Further, the composition may comprise about 5-10% by weight phyllosilicate. In some embodiments, the composition may comprise about 7.5% by weight phyllosilicate.

The compositions can also comprise hydrotalcite. The percentages as described above for the other compositions are also relevant in this aspect. For example, the composition may comprise about 0.1-7.5% by weight hydrotalcite relative to the total weight of the components of the composition. The composition may comprise about 0.5-7.5% by weight hydrotalcite. Further, the composition may comprise about 0.5-5% by weight hydrotalcite. The composition can comprise about 0.5-3% by weight hydrotalcite. In some embodiments, the composition may comprise about 1-7.5% by weight hydrotalcite relative to the total weight of the components of the composition. Further, the composition may comprise about 1-5% by weight hydrotalcite. In particular embodiments, the composition comprises about 2-3% by weight hydrotalcite. The compositions can also comprise titanium oxide in addition to the hydrotalcite. Again, the percentages as described above are also relevant in this aspect. For example, the composition may comprise about 5-15% by weight titanium oxide relative to the total weight of the components of the composition. Further, the composition may comprise about 7.5-12.5% by weight titanium oxide. In some embodiments, the composition can comprise about 8-12% by weight titanium oxide. Furthermore, the composition may comprise about 10% by weight titanium oxide.

In preferred embodiments, the components of the composition used as an additive make up the following percentages by weight relative to the total weight of the components: 1) 87.5-97.5% Portland cement; and

2.5-12.5% Phyllosilicate (e.g. smectite).

2) 90-95% Portland cement; and

5-10% Phyllosilicate (e.g. smectite).

3) 82.5-97% Portland cement;

2.5-12.5% Phyllosilicate (e.g. smectite); and

0.5-5% Hydrotalcite. 4) 85-94% Portland cement;

5-10% Phyllosilicate (e.g. smectite); and

1-5% Hydrotalcite.

5) 62.5-92% Portland cement;

5-20% Titanium Oxide;

2.5-12.5% Phyllosilicate (e.g. smectite); and

0.5-5% Hydrotalcite.

6) 72-87% Portland cement;

7-13% Titanium Oxide;

5-10% Phyllosilicate (e.g. smectite); and

1-5% Hydrotalcite. All the compositions described above regardless of which aspect they relate to can be used as an additive for dental adhesive systems.

In another aspect, the invention provides a dental adhesive comprising resin monomers and, in addition, a composition comprising Portland cement and phyllosilicate, and/or a phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product. It was previously not known that a composition comprising Portland cement and phyllosilicate could be used as a resin additive. Therefore, the composition is not limited to having about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition. In some embodiments, the dental adhesive comprises resin monomers and either a composition comprising Portland cement and phyllosilicate, or a phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product.

In another aspect, the invention provides a dental adhesive comprising resin monomers and, in addition, a composition comprising Portland cement, phyllosilicate and hydrotalcite, and/or a hydrotalcite/phyllosilicate- modified hexacalcium aluminate trisulfate hydrate product. In some embodiments, the dental adhesive comprises resin monomers and either a composition comprising Portland cement, phyllosilicate and hydrotalcite, or a hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product.

In another aspect, the invention provides a dental adhesive comprising resin monomers and, in addition, a composition comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite, and/or a titanium oxide/hydrotalcite/phyllosilicate (e.g. smectite)-modified hexacalcium aluminate trisulfate hydrate product. In some embodiments, the dental adhesive comprises resin monomers and either a composition comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite, or a titanium oxide/hydrotalcite/phyllosilicate (e.g. smectite)-modified hexacalcium aluminate trisulfate hydrate product. The dental adhesive comprises resin monomers which can react together to form a polymerised resin polymer. Any suitable resin monomers can be used and these are well known to those skilled in the art. In one embodiment, the resin monomers may be methacrylate monomers such as urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)]-phenyl propane (BisGMA), butan-l,2,3,4-tetracarboxylic acid di- 2-hydroxyethylmethacrylate ester (TCB), 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM), triethyleneglycol dimethacrylate (TEGDMA), and/or 2-hydroxyethylmethacrylate (HEMA). Preferably, 50% or more of the resin monomers are hydrophilic. Preferably, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)] -phenyl propane (BisGMA), butan- 1,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester (TCB), and 2-hydroxyethylmethacrylate (HEMA). Alternatively, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), 2,5 -dimethacryloyloxyethyloxycarbony 1-1,4- benzenedicarboxylic acid (PMDM), and 2-hydroxyethylmethacrylate (HEMA). In some embodiments, the resin monomers are 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)]-phenyl propane (BisGMA), 2,5- dimethacryloyloxyethyloxycarbonyl- 1 ,4-benzenedicarboxylic acid (PMDM) and 2-hydroxyethylmethacrylate (HEMA). In some embodiments, the resin monomers are Bis-EMA: ethoxylated bisphenol A glycol dimethacrylates, 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM), and 2- hydroxyethylmethacrylate (HEMA).

The dental adhesive may further comprise an initiator and/or co-initiators to induce the polymerisation reaction of the resin monomers when external energy (e.g. light, heat, etc.) is applied to the adhesive. This may be camphoroquinone or 1.7.7-trimethylbicyclo-[2,2,l]-hepta-2,3-dione (CQ; initiator),2-ethyl-dimethyl-4- aminobenzoate (ETDA: co-initiator) , lucirin (TPO; initiator), N,N-di-(2-hydroxyethyl)-4-toluidine (DHEPT; co- initiator), diphenyliodonium hexafluorophosphate (DPIHP: co-initiator). In some embodiments, the initiator is camphorquinone. A plurality of initiators may be added to the dental adhesive. In such embodiments, the initiator may be camphorquinone (CQ; initiator ),2-ethyl-dimethyl-4-aminobenzoate (EDAB: co-initiator) and diphenyliodonium hexafluorophosphate (DPIHP: tertiary co-initiator).

The dental adhesive may further comprise a catalyst such as ethyl-dimethyl-4-aminobenzoate. The dental adhesive may comprise between about 20% and about 60% by weight of the modified hexacalcium aluminate trisulfate hydrate product described above or of the compositions described above. The dental adhesive may comprise between about 30% and about 50% by weight of the modified hexacalcium aluminate trisulfate hydrate product described above or of the compositions described above. The dental adhesive can comprise about 40% by weight of the modified hexacalcium aluminate trisulfate hydrate product described above or of the compositions described above.

The dental adhesive may comprise between about 40% and about 80% by weight resin monomers. The dental adhesive can comprise between about 50% and about 70% by weight resin monomers. In some embodiments, the dental adhesive comprises about 60% by weight resin monomers. Adhesive systems for dental applications typically fall into two categories: 1) etch-and-rinse adhesives; and 2) self -etching adhesive. Etch-and-rinse adhesives are available as three-step and two-step systems. Typically, etch-and-rinse adhesives produce relatively high and substantive adhesion values for both enamel and dentine. If the surface to which the adhesive will be applied consists of significantly more enamel than dentine, or has a circumference of enamel that will help provide marginal integrity, an etch-and-rinse system is preferred. The basic steps of the three-step etch-and-rinse adhesive systems are as follows:

1) demineralisation of the surface by an acid etchant (i.e. 30-40% ortho-phosphoric acid) including removal of the smear layer;

2) diffusion of adhesive monomers into the spaces created by the etchant (permeability), and curing of the adhesive monomers to form a microscopic mechanical bond to the enamel and dentine, referred to as a hybrid layer. This is created by applying a resin primer to the etched dental tissue surface. The primer contains a solvent which is removed, generally by drying, before the next step; and

3) Applying a more hydrophobic bond layer to the primered dental tissue surface to allow the build up of aesthetic resin composite layers. In a two-step system, steps 2) and 3) above are combined so that the primer and the bond are applied to the etched dental surface consecutively. This can be referred to as a self -priming adhesive system.

The compositions and products described above can be added to the primer and/or the bond resin in a three-step etch and rinse system, or the compositions and products described above can be added to a self -priming adhesive system in a two-step system.

In a further aspect, the invention provides a dental resin primer for a three-step adhesive system, the primer comprising resin monomers and a composition comprising Portland cement and phyllosilicate. In a further aspect, the invention provides a dental resin primer for a three-step adhesive system, the primer comprising resin monomers and a composition comprising Portland cement, phyllosilicate and hydrotalcite.

In a further aspect, the invention provides a dental resin primer for a three-step adhesive system, the primer comprising resin monomers and a composition comprising Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite.

The dental resin primer comprises resin monomers which can polymerise together to form a resin polymer. This forms a hybrid layer on the dental surface of, for example, dentine or enamel, to which further resin can be bonded. Any suitable resin monomers can be used and these are well known to those skilled in the art. In one embodiment, the resin monomers may be methacrylate monomers such as urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)]-phenyl propane (BisGMA), butan-l,2,3,4-tetracarboxylic acid di- 2-hydroxyethylmethacrylate ester (TCB), 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM), triethyleneglycol dimethacrylate (TEGDMA), and/or 2-hydroxyethylmethacrylate (HEMA). Preferably, 50% or more of the resin monomers are hydrophilic. Preferably, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)] -phenyl propane (BisGMA), butan- 1,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester (TCB), and 2-hydroxyethylmethacrylate (HEMA). Alternatively, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), 2,5 -dimethacryloyloxyethyloxycarbony 1-1,4- benzenedicarboxylic acid (PMDM) and 2-hydroxyethylmethacrylate (HEMA). In some embodiments, the resin monomers are Bis-EMA: ethoxylated bisphenol A glycol dimethacrylates, 2,5- dimethacryloyloxyethyloxycarbonyl- 1 ,4-benzenedicarboxylic acid (PMDM), and 2-hydroxyethylmethacrylate (HEMA). In certain embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 32-42% UDMA, about 1-7% BisGMA, about 24-34% TCB, and about 24-34% HEMA.

In other embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 32-42% UDMA, about 1-7% BisGMA, about 24-34% PMDM, and about 24-34% HEMA.

In further embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 35-45% BisGMA, about 24-34% HEMA, and about 35-45% PMDM.

The resin primer may further comprise a solvent such as water, acetone or ethanol. Preferably, the solvent is ethanol. This solvent may be contained in the primer in a ratio of between about 5:4 and 4:5 by weight relative to the resin monomers (i.e. between about 5 weight of solvent : 4 weight of total resin monomers and about 4 weight of solvent : 5 weight of total resin monomers). In some embodiments, the resin primer contains solvent (e.g. ethanol) at a ratio of about 1 : 1 by weight relative to the resin monomers.

The resin primer may further comprise an initiator to initiate polymerisation of the resin monomers when external energy (e.g. light, heat, etc.) is applied to the primer. This may be camphorquinone or 1.7.7-trimethylbicyclo- [2,2,l]-hepta-2,3-dione (CQ; initiator), 2-ethyl-dimethyl-4-aminobenzoate (ETDA: co-initiator), lucirin (TPO; initiator), N,N-di-(2-hydroxyethyl)-4-toluidine (DHEPT; co-initiator), diphenyliodonium hexafluorophosphate (DPIHP: co-initiator). In some embodiments, the initiator is camphorquinone. A plurality of initiators may be added to the dental adhesive. In such embodiments, the initiator may be camphorquinone (CQ; initiator), 2-ethyl- dimethyl-4-aminobenzoate (EDAB: co-initiator) and diphenyliodonium hexafluorophosphate (DPIHP: tertiary co-initiator). The dental resin primer may comprise between about 20% and about 60% by weight of the various compositions described above. This may be a composition comprising Portland cement and phyllosilicate; Portland cement, phyllosilicate and hydrotalcite; or Portland cement, titanium oxide, phyllosilicate (e.g. smectite) and hydrotalcite. Preferably, the dental resin primer comprises between about 30% and about 50% by weight of the composition. More preferably, the dental resin primer comprises about 40% by weight of the composition.

The dental resin primer may comprise between about 40% and about 80% by weight resin monomers. Preferably, the dental resin primer comprises between about 50% and about 70% by weight resin monomers. More preferably, the dental resin primer comprises about 60% by weight resin monomers.

The invention also provides a dental resin primer for a three-step adhesive system, the primer comprising resin monomers and a modified hexacalcium aluminate trisulfate hydrate product. This may be the phyllosilicate - modified hexacalcium aluminate trisulfate hydrate product; the hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product; or the titanium oxide/hydrotalcite/phyllosilicate (e.g. smectite)-modified hexacalcium aluminate trisulfate hydrate product described above. The description above relating to the primer containing the composition is equally applicable to the primer containing the hydrate product. For example, regarding the identity of the resin monomers, and the amounts of the resin monomers (e.g. about 60%) and additive of the composition (e.g. about 40%).

The invention also provides a dental resin bonding agent for a three-step adhesive system, the bonding agent comprising resin monomers and a modified hexacalcium aluminate trisulfate hydrate product. This may be the phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product; the hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product; or the titanium oxide/hydrotalcite/phyllosilicate (e.g. smectite)- modified hexacalcium aluminate trisulfate hydrate product described above.

The dental resin bonding agent comprises resin monomers which can polymerise together to form a resin polymer. This forms a resin layer on top of the primered dental hard tissue. Any suitable resin monomers can be used and these are well known to those skilled in the art. In one embodiment, the resin monomers may be methacrylate monomers such as urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), butan-l,2,3,4-tetracarboxylic acid di-2- hydroxyethylmethacrylate ester (TCB), 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM), triethyleneglycol dimethacrylate (TEGDMA), and/or 2-hydroxyethylmethacrylate (HEMA). Preferably, 50% or more of the resin monomers are hydrophilic. Preferably, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)] -phenyl propane (BisGMA), butan- 1,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester (TCB), and 2-hydroxyethylmethacrylate (HEMA). Alternatively, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), 2,5 -dimethacryloyloxyethyloxycarbony 1-1,4- benzenedicarboxylic acid (PMDM) and 2-hydroxyethylmethacrylate (HEMA). In some embodiments, the resin monomers are Bis-EMA: ethoxylated bisphenol A glycol dimethacrylates, 2,5- dimethacryloyloxyethyloxycarbonyl- 1 ,4-benzenedicarboxylic acid (PMDM), and 2-hydroxyethylmethacrylate (HEMA). In certain embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 30-40% UDMA, about 2-8% BisGMA, about 25-35% TCB, and about 25-35% HEMA.

In other embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 30-40% UDMA, about 2-8% BisGMA, about 25-35% PMDM, and about 25-35% HEMA.

The resin bonding agent may further comprise an initiator to initiate polymerisation of the resin monomers when external energy (e.g. light, heat, etc.) is applied to the bonding agent. This may be camphorquinone or 1.7.7- trimethylbicyclo-[2,2, l]-hepta-2,3-dione (CQ; initiator), 2-ethyl-dimethyl-4-aminobenzoate (ETDA: co-initiator), lucirin (TPO; initiator), N,N-di-(2-hydroxyethyl)-4-toluidine (DHEPT; co-initiator), diphenyliodonium hexafluorophosphate (DPIHP: co-initiator). In some embodiments, the initiator is camphorquinone. A plurality of initiators may be added to the dental adhesive. In such embodiments, the initiator may be camphorquinone (CQ; initiator), 2-ethyl-dimethyl-4-aminobenzoate (EDAB: co-initiator) and diphenyliodonium hexafluorophosphate (DPIHP: tertiary co-initiator). The resin bonding agent may further comprise a catalyst such as ethyl-dimethyl-4-aminobenzoate.

The dental resin bonding agent may comprise between about 20% and about 60% by weight of the modified hexacalcium aluminate trisulfate hydrate product. Preferably, the dental resin bonding agent comprises between about 30% and about 50% by weight of the modified hexacalcium aluminate trisulfate hydrate product. More preferably, the dental resin bonding agent comprises about 40% by weight of the modified hexacalcium aluminate trisulfate hydrate product.

The dental resin bonding agent may comprise between about 40% and about 80% by weight resin monomers. Preferably, the dental resin bonding agent comprises between about 50% and about 70% by weight resin monomers. More preferably, the dental resin bonding agent comprises about 60% by weight resin monomers.

The invention also provides a dental resin self-priming bonding agent for a two-step adhesive system, the bonding agent comprising resin monomers and a modified hexacalcium aluminate trisulfate hydrate product. This may be the phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product; the hydrotalcite/phyllosilicate-modified hexacalcium aluminate trisulfate hydrate product; or the titanium oxide/hydrotalcite/phyllosilicate (e.g. smectite) -modified hexacalcium aluminate trisulfate hydrate product described above. The dental resin self -priming bonding agent comprises resin monomers which can polymerise together to form a resin polymer. This forms a resin layer on top of the hybrid layer previously formed by the primer. Any suitable resin monomers can be used and these are well known to those skilled in the art. In one embodiment, the resin monomers may be methacrylate monomers such as urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), butan-l,2,3,4-tetracarboxylic acid di-2- hydroxyethylmethacrylate ester (TCB), 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM), triethyleneglycol dimethacrylate (TEGDMA), and/or 2-hydroxyethylmethacrylate (HEMA). Preferably, 50% or more of the resin monomers are hydrophilic. Preferably, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)] -phenyl propane (BisGMA), butan- 1,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester (TCB), and 2-hydroxyethylmethacrylate (HEMA).

Alternatively, the resin monomers are urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3- methacryloylpropoxy)] -phenyl propane (BisGMA), 2,5 -dimethacryloyloxyethyloxycarbony 1-1,4- benzenedicarboxylic acid (PMDM) and 2-hydroxyethylmethacrylate (HEMA). In some embodiments, the resin monomers are Bis-EMA: ethoxylated bisphenol A glycol dimethacrylates, 2,5- dimethacryloyloxyethyloxycarbonyl- 1 ,4-benzenedicarboxylic acid (PMDM), and 2-hydroxyethylmethacrylate (HEMA).

In certain embodiments, the resin monomers may make up the following percentages by weight relative to the total weight of the resin monomers: about 30-40% UDMA, about 2-8% BisGMA, about 25-35% TCB, and about 25-35% HEMA.

The resin self-priming bonding agent may further comprise an initiator to initiate polymerisation of the resin monomers when external energy (e.g. light, heat, etc.) is applied to the bonding agent. This may be camphorquinone or 1.7.7-trimethylbicyclo-[2,2, l]-hepta-2,3-dione (CQ; initiator),2-ethyl-dimethyl-4- aminobenzoate (ETDA: co-initiator), lucirin (TPO; initiator), N,N-di-(2-hydroxyethyl)-4-toluidine (DHEPT; co- initiator), diphenyliodonium hexafluorophosphate (DPIHP: co-initiator). In some embodiments, the initiator is camphorquinone. A plurality of initiators may be added to the dental adhesive. In such embodiments, the initiator may be camphorquinone (CQ; initiator),2-ethyl-dimethyl-4-aminobenzoate (EDAB: co-initiator) and diphenyliodonium hexafluorophosphate (DPIHP: tertiary co-initiator).

The resin self-priming bonding agent may further comprise a catalyst such as ethyl-dimethyl-4-aminobenzoate.

The dental resin self-priming bonding agent may comprise between about 20% and about 60% by weight of the modified hexacalcium aluminate trisulfate hydrate product. Preferably, the dental resin self-priming bonding agent comprises between about 20% and about 40% by weight of the modified hexacalcium aluminate trisulfate hydrate product. More preferably, the dental resin self-priming bonding agent comprises about 30% by weight of the modified hexacalcium aluminate trisulfate hydrate product.

The dental resin self-priming bonding agent may comprise between about 40% and about 80% by weight resin monomers. Preferably, the dental resin self-priming bonding agent comprises between about 60% and about 80% by weight resin monomers. More preferably, the dental resin self-priming bonding agent comprises about 70% by weight resin monomers.

Detailed Description of the Invention

The invention will now be described in detail by way of example only with reference to the figures in which: Figure 1 shows the variations of micro-hardness induced by the modification of the range of the experimental additives after storage for 28 days in H₂0 or phosphate buffer solution (PBS).

Figure 2 shows the porosities of cements formed from the tested compositions. Figure 3 is SEM Micrographs showing: a) the presence of acicular crystals of ettringite (ac) of the compact crystal surface of the Filler A; b) The other fillers presented high porosity on the outer surface (Magnification: 1600X, white scale-bar ΙΟμπι).

Figure 4 is a graph showing the mean (S.D.) of μΤΒ8 (MPa) to dentine when the experimental and control adhesives were applied on acid-etched dentine. *This symbol indicates significant statistical differences after PBS storage media (P < 0.05). Adhesive captions: Ctrl: control primer + control bond resin containing only resin monomers and no filler; OPC/HEMA: resin primer + bond containing hexacalcium aluminate trisulfate hydrate; OPC: primer containing ordinary calcium silicate + resin bond containing no filler; HPCMM: resin primer + bond containing Montmorillonite-modified hexacalcium aluminate trisulfate hydrate hexacalcium aluminate trisulfate hydrate; PCMM: primer containing Montmorillonite-modified calcium silicate + resin bond containing no filler; PCTO: primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler; HPCTO/HEMA: resin primer + bond containing titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate hydrate. The 24 hour bar is the left-hand bar for each adhesive and the 6 month bar is the right-hand bar for each adhesive. Figure 5 shows the characterisation and nanoleakage of the resin-dentine interfaces created using the experimental adhesives. (A) Resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) applied on acid-etched dentine showed the penetration of the primer into the demineralised layer and inside the dentinal tubules (pointer) immediately after the bonding procedure (24h). (B) Resin-bonded dentine interfaces created with HPCTO experimental adhesive (resin primer + bond containing titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate) applied on acid-etched dentine showed severe gap (pointer) formation after 6 months of PBS storage due to degradation of the hybrid layer. (C) Resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) applied on acid-etched dentine showed a more intense reflection signal of the penetration of the primer into the demineralised layer and inside the dentinal tubules (pointer) after 6 months of PBS storage (pointer). (D) further image of the resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) showing the presence of an absorptive/reactive layer above the hybridized dentine surface (pointer).

Figure 6 shows SEM failure analysis of debonded specimens. (A) SEM micrograph (lOOOx) of an adhesively fractured stick bonded with Res-HOPC after 24 h of SBS storage. Observe the dentine entirely covered with adhesive resin (ra) with some fillers' lacunas (pointer) and rarely found opened dentinal tubules (dt). (Al) In another specimen, note the presence of resin adhesive (ra) without unprotected collagen fibrils. Some fillers were detached during SEM preparation (pointer) and initial mineral precipitation may be observed (white asterisk). (A2) After 6 months, the debonded dentine surface at higher magnification (2.500X) covered with adhesive resin (ra). Mineral crystals (asterisk) embedded within a preserved collagen network were vastly encountered albeit some fillers were detached (pointer). (B) SEM micrograph of a specimen bonded with Res-HCPMM after 24h. Note the presence of adhesive resin (ra) mostly covering the dentine and dentinal tubules (dt); some fillers' lacunas are also observed (pointer). (B l) In another portion of the same specimen, more lacunas are observed due to filler detaching, but also initial mineral crystallisation was depicted (asterisk). (B2) After 6 months storage, a failure mode observed under higher magnification (2.500X) similar to that found in B l is disclosed showing the very slow bonding degradation. More mineral precipitation was observed (asterisk) and the fillers' lacunas (pointer) are wider probably due to the expansion of the fillers when exposed to water. (C) Micrograph of a debonded stick from group Res -HPCTO after 24h showing dentine completely covered with rare filler detachment (pointer). (CI) In other region, note the presence of few exposed dentinal tubules as well as intact resin tags (rt). (C2) After 6 months, the fractured dentine surface bonded with Res-HPCTO showed a de-bonding within the hybrid layer and/or at its bottom. A lot of resin tags (rt) were observed well-hybridized with peritubular dentine (black pointer) suggesting potential remineralisation albeit some funneled dentinal tubules were encountered without resin tags (black asterisk). (D) SEM micrograph from a specimen bonded with Res-Ctr and presenting a perfectly hybridized and resin covered dentine. (Dl) A fractured stick from the same group showed an adhesive failure at the bottom of hybrid layer with remnants of resin adhesive (ar) and some resin tags (rt). (D2) The control adhesive after 6 months showed many signs of degradation since most collagen fibrils were degraded, the funneled dentinal tubules (asterisk) were often found along with poorly hybridized resin tags (pointer). White finger - filler lacuna; white asterisk - mineral precipitation; black asterisk - funneled degraded peritubular dentine; black finger - hybridisation between tags and peritubular. Figure 7 shows confocal laser scanning microscopy (CLSM) single-projection images showing the interfacial characterization and nanoleakage, after 24 h of storage in SBS. Images (1) indicate the projection of fluorescein dye whereas the images (2) disclose the projection of rhodamine B dye. The images (3) are depicting the projections of both dyes. (Al, A2, A3) CLSM images showing the interfacial characteristics of the bonded-dentin interface created using Res-HOPC. It is possible to observe a clear hybrid layer (hi) with long resin tags (rt) penetrating the dentinal tubules (dt) underneath an adhesive layer (ad) characterized by evident mineral fillers (FL). Intense fluorescein uptake was observed within the entire resin-dentin interface as well as the adhesive layer. (B l, B2, B3): Micrographs showing the interfacial characteristics of the bonded-dentin interface created using Res-HCPMM. Similarly to images from Res-HOPC, these images presented high dye uptake throughout the entire resin-dentin interface as well as the adhesive layer. (CI, C2, C3) The resin-dentin interface created using the Res-HPCTO bonding system was characterized by a clear hybrid layer (hi) located underneath the adhesive layer (ad) containing the experimental filler (FL). Long resin tags (rt) penetrating the dentinal tubules (dt) were observed as well as evident nanoleakage and dye uptake along the entire interface and adhesive layer. (Dl, D2, D3) CLSM images showing the bonded-dentin control interface (RES-Ctr) characterized by a thick and fluorescent hybrid layer (hi) (approximately 8 μπι thickness) located underneath an adhesive layer (ad) devoid of fillers.

Figure 8 shows CLSM single-projection images disclosing the fluorescent calcium-chelators dye xylenol orange. All images were obtained from specimens immersed in simulated body-fluid solution for 24h or 6 months. A: CLSM image of the resin-dentin interface created with Res-HOPC after 24h of SBS storage. Mineral deposition can be visualized within the adhesive layer (ad), the hybrid layer (hi) along the walls of dentinal tubules (dt) and the filler inside the resin tags (rt). B: CLMS image of the resin-dentin interface created with Res-HCPMM and immersed in SBS for 6 months where it is possible to observe a clear fluorescence signal due to a consistent presence of Ca-deposits within the adhesive layer (ad), hybrid layer, walls of the dentinal tubules (dt) and resin tags (rt). C: Image of the resin-dentin interface created with Res-HPCTO and immersed in SBS for 24h. Xylenol Orange was able to stain the Ca- minerals within adhesive layer, hybrid layer and dentinal tubule (dt). Note the intense calcium deposition at bottom of hybrid layer. D: Image of the resin-dentin interface created with Res- HPCTO and immersed in SBS for 6 months showing also in this case Ca-mineral presence at the bottom and within the hybrid layer, dt and rt. E: Image of the resin-dentin interface created with Res-Ctr (no filler) in which one may note absence of calcium deposition both within the hybrid (hi) and adhesive layer (ad). Only the walls of the dentinal tubule tubules (dt) were stained by the fluorescent dye.

Figure 9 shows confocal laser scanning microscopy (CLSM) single-projection images showing the interfacial characterization and nanoleakage after 6 months of SBS storage. (A) Image showing the resin-dentin interface created using Res-HOPC characterized by reduced nanoleakage within the hybrid layer (hi). Note the absence of fluorescein uptake within the adhesive layer. (B) Image showing the interfacial features of the bonded-dentin interface created using the Res-HCPMM. Note the low overall nanoleakage with very little fluorescein uptake in hybrid and adhesive layers. (C) Image of the resin-dentin interface created using Res-HPCTO. Despite the mineral deposition and reduced nanoleakage within adhesive layer, the weak bond strength created gaps in which the fluorescein was deposited. The gaps may be induced by the cutting procedures as the resin degradation was replaced by mineral precipitation creating an interface with low elasticity (high stiffness properties). The nanoleakage was only observed within the hybrid layer (hi). (D) Micrograph of the resin-dentin interface created using the control adhesive system (RES-Ctr). Note the presence of intense dye uptake (nanoleakage) within the hybrid layer and at the bottom of adhesive layer. In this case the presence of gaps frequently observed between hybrid and adhesive was very likely due to hybrid layer degradation (reduced thickness). Dt - dentinal tubules; rt - resin tags; ad - adhesive layer; c - composite; hi - hybrid layer.

Figure 10 shows optical images obtained during the micro-hardness test along the resin-dentine interface. A) Picture illustrating how the five measurements (indentations) were taken along each line every 30 μπι up to 115 μπι in depth. B) At high magnification it is possible to observe that the first indentation was performed exactly on a hybrid layer (arrow) located between the adhesive resins (a) and the dentine surface (d). 91x50mm (300 x 300 DPI).

Overview

The inventors have devised bioactive/therapeutic bonding agents containing modified calcium-silicate cements. These bonding agents are more durable and provide an increased bonding strength over a longer period of time compared to previous adhesive systems. It is thought that this may be accomplished by replacing water from resin-sparse regions of the hybrid layer with apatite crystallites that are small enough to occupy the extrafibrillar and intrafibrillar compartments of the collagen matrix and restoring the enzyme exclusion and fossilisation properties of mineralised dentine preserving the longevity of resin dentine bonds.

Calcium-silicate cements demonstrate a wide range of clinical applications including root-end filling material, root perforation repair, pulp capping and dentine hypersensitivity. Type I ordinary Portland cement is the active ingredient and it is a hydraulic material mainly composed of di-tricalcium silicate (2CaO- Si0₂ belite and 3CaO- Si0₂ alite), tricalcium aluminate (3CaO- A1₂0₃) and gypsum (CaS0₄-2H₂0) hydrophilic powders.

When hydrated, the silicate phases of Portland cements undergo a series of physicochemical reactions resulting in the formation of a nanoporous matrix/gel of calcium silicate hydrates ("C-S-H phases"), Ettringite (hexacalcium aluminate trisulfate with formula: Ca₆Al₂(SO _t) 3(ΟΗ)ι₂·26Η₂0) and a soluble fraction of calcium hydroxide Ca(OH)₂ or portlandite. When the hexacalcium aluminate trisulfate is stored in physiological-like phosphate solutions (PBS), it produces a superficial layer characterised by the presence of carbonated apatite and calcite due to the continuous replenishment of phosphate ions from fluids. Several in vitro investigations have shown apatite layer formation in a ground Portland cement-PBS system, formation of calcium deficient apatite precipitates in a Portland cement-PBS system, apatite-like and calcite crystals on set Portland cement exposed to phosphate solution, and apatite-like phase formation on carbonated substrates. Calcium ions supplied by the hydration-derived basic calcium hydroxide (portlandite) react with the environmental carbonate ions forming calcium carbonate (calcite). The crystalline form of calcium carbonate as calcite formed on cement surface by the carbonation process showed good biological activity and positively affected the cytocompatibility. Soluble portlandite crystals allow the nucleation of calcium carbonate polymorphs and/or meta-stable calcium salt crystals that readily transform into the stable calcite phase in water. Calcium carbonate may precipitate at the surface and in the cement paste porosity forming a protective layer of calcium carbonate on the outer zone of cement.

Moreover, in the case of the guided tissue remineralisation scheme, the calcium-silicates represent an innovative potential recipe for extending the longevity of resin-dentine bonds. In this scheme, Tricalcium Silicates and Ettringite are slow-releasing calcium source, and two biomimetic analogs are used to simulate the function of noncollagenous dentine proteins such as DMP-1 during dentineogenesis. The biomimetic analog mimics the binding of phosphoproteins to dentine collagen, so that the doped collagen can guide the dimension and hierarchy of remineralised apatite crystallites within the collagen matrix.

Example 1

1st AIM: Evaluation of the appropriate composition of bioactive fillers for dental adhesive systems.

The aim of the present study was to identify the structural and physical properties of an ordinary calcium silicate (Portland cement*) in relation to their potential in the development of new dental filling materials after the addition of different concentration ranges of smectite, hydrotalcite and titanium oxide. This aim was accomplished by monitoring the porosity, micro-hardness and scanning electron microscope (SEM) surface analysis of the cements during the hydration process in distilled water and phosphate buffered saline (PBS) storage at 37°C for 28 days.

MATERIALS AND METHODS

Ordinary calcium silicates and experimental additives

The ordinary calcium silicate (Portland cement*) used in this study contained by weight:

Tri-calcium silicate 70%

Di-calcium silicate 29%

Tri-calcium aluminate 1 %

Other elements Traces

The precise composition of the Portland cement by weight used in the following examples is as follows:

Si0₂ (%) 18.76

Ti0₂ (%) 0.37 A1₂0₃ (%) 5.04

Fe₂0₃ (%) 4.42

FeO (%) 0.00

MnO (%) 0.02

MgO (%) 2.16

CaO (%) 59.07

Na₂0 (%) 0.27

K₂0 (%) 1.01

P₂0₅ (%) 0.08

LOI (%) 8.81

The Portland cement also contained calcium sulphate in the form of gypsum in addition to the above components. The gypsum was present at about 5% by weight. The smectite/titanium oxide/hydrotalcite-modified calcium silicate compositions which were tested contained the following components (percentage by weight):

Filler A:

76-89% Ordinary Calcium Silicate (Portland cement*)

10-20% Titanium Oxide

0.5-2.5% Smectite

0.5-1.5% Hydrotalcite

Filler B:

89-100% Ordinary Calcium Silicate (Portland cement*)

<10% Titanium Oxide

<0.5% Smectite

<0.5% Hydrotalcite Filler C:

50-76% Ordinary Calcium Silicate (Portland cement*)

>20% Titanium Oxide

>2.5% Smectite

>1.5% Hydrotalcite

Filler control:

100% Ordinary Calcium Silicate (Portland cement*)

The components of the compositions were obtained from: Ordinary Calcium Silicate (Portland cement*) - EN 197-1 Italbianco (Type I) - 52.5 R ("Italcementi Group", Italy);

Titanium Oxide - Titanium IV Oxide; Sigma- Aldrich; cod. 14027; CAS Number 13463-67-7;

Smectite - Fluka; codice 69866; Cas Number 1318-93-0;

Hydrotalcite - Hydrotalcite, synthetic; Aldrich; cod. 652288; CAS Number 11097-59-9.

Apparent volumetric mass and porosity evaluation

The specimens were weighed in the dry condition (md) and subsequently inserted in a vacuum container (2 kPa) for 24 hours. Distilled water was then added and the vacuum was maintained for a further 24 hours.

The apparent volumetric mass (pb) was calculated as the ratio between the mass of the dry specimen and its apparent volume (volume of the specimen as outlined by its external surface). The open porosities (effective porosity) of the specimen corresponded to the percentage ratio between the volume of the open pores and the apparent volume of the specimen. The hydrostatic weighing (mh) and the saturated weight (ms) were evaluated by using a Hydrostatic Scale (Exacta Optech Labcenter, San Prospero, Italy) to analyse the modification of the volumetric mass and porosity during the maturation and setting of the cements.

The value of the apparent volumetric mass (Kg/m³) was calculated using the following formula: p_b =——* p*

m s - m n,

pb = apparent volumetric mass

prh (20 °C) = 998 kg/m³

md = dry weight

mh = hydrostatic weighing

ms = saturated weighing in air

Whereas the value of the open porosity was expressed as a percentage and was calculated with the following formula:

_{P0 =} m_s - m_h

md = dry weight

ms = saturated weighing in air

Micro-hardness measurement

The measurement of the micro-hardness was performed using a static Knoop HK testing device and a micro- durometer (Leitz Durimet, Leitz Rockleigh, NJ, USA). In brief, each sample of the two principal groups was impressed with loads of 50 g for 5 seconds using a Knoop indenter. Each test condition with the same load and time was conducted 3 times per each sample. Data were averaged and differences in Koop hardness values were statistically analysed.

Statistical analysis

Micro-hardness and volumetric mass/porosity data were statistically compared using a multiple way ANOVA, including interactions between factors: 1) different formulation of calcium silicates (modified or ordinary Portland cement); 2) Immersion solution (H₂0 or PBS): 3) storage time (7, 14 or 28 days).

Post hoc multiple comparisons were performed using the Tukey's test. (p<0.01). Statistical significance level was set at a = 0.01.

SEM Surface analysis

The surface analysis was performed for the experimental cements at different intervals of time (3, 7, 14 and 28 days).

The specimens were initially washed in 0.2M phosphate buffer pH 7.3 and fixed in Karnovsky's fixative (1 % paraformaldehyde, 1.5% glutaraldehyde, and 0.1M sodium cacodylate buffer, pH 7.4) for 1 h and then dehydrated using ascending grades of ethanol at room temperature and air-dried. All specimens were coated with palladium- gold and examined using a SEM (Philips 515, Eindhoven, The Netherlands).

RESULTS

Figure 1 shows the variations of micro-hardness induced by the modification of the range of the experimental additives after storage for 28 days in H₂0 or PBS. The highest micro-hardness values were obtained with the Filler A: 76-89% Ordinary Calcium Silicate (Portland cement*); 10-20% Titanium bi-oxide; 0.5-2.5% smectite; 0.5-1.5% Hydrotalcite. The fillers B,C,D and control had lower micro-hardness values after 14 days and 28 days.

Figure 2 shows the porosities of cements formed from the tested compositions. The lowest porosity values were obtained with the Filler A: 76-89% Ordinary Calcium Silicate (Portland cement*); 10-20% Titanium bi-oxide; 0.5-2.5% smectite; 0.5-1.5% Hydrotalcite. The fillers B and C, and the control showed higher porosities.

Figure 3 is SEM Micrographs showing: a) the presence of acicular crystals of ettringite (ac) of the compact crystal surface of the Filler A; b) The other fillers presented high porosity on the outer surface (Magnification: 1600X, white scale-bar ΙΟμπι). DISCUSSION AND CONCLUSION

The significant differences in porosities and micro-hardness observed in this study allowed the inventors to develop new dental filling/adhesive materials to be used for conservative and restorative purposes. The highest micro-hardness was observed when the ordinary silicate cement was modified with the following additives in specific ranges: 10-20% Titanium bi-oxide; 0.5-2.5% Smectite; and 0.5-1.5% Hydrotalcite. Further, the same range of the additives tested in this study showed the lowest porosity. These characteristics are extremely important for a filler to be included in resin bonding system.

The presence of Smectite produces a negative charge on the surface of mineral particles, imparting a high cation exchange capacity (CEC). This substance is distributed evenly throughout the cement mass and consists of strips of about 10 A in thickness that is packaged before mixing, but then tend to exfoliate and orientate in all directions during the mixing procedures with water and polymer improving the mechanical performance of the final product.

There may also be an improved resistance to solvents and a clear decrease in permeability and fluid filtration, mainly due to the ability of these materials to bind water between the slats. In fact, in contact with liquid, smectite forms a thixotropic gel with very low permeability.

The presence of a specific range of a mineral anion such as the hydrotalcite may favour the interaction between substances containing anions:

• type of inorganic anions: F, CI^", Br^", T, (C10₄)^", (N0₃)^", (C10₃)^", (I0₃)^",OH^", (C0₃)^2", (S0₄)^2", (S₂0₃)^2", (WO₄)^2", (G-O4)²-, [Fe(CN)₆]^3",[Fe(CN)₆]^4";

· ISO or eteropolyanions (PMO₂O₄₀)^3", (PWi₂O₄₀)^3";

• organic acid anions: oxalate, succinate, malonate and others;

• anionic organometallic compounds; and

• anionic resin monomer. Indeed, hydrotalcite helps the intercalation reaction and the adhesion with polymeric resinous in nature. Concentrations of Hydrotalcite between 0.5-1.5% may induce an homogeneous distribution of the entire cement with the polymer without altering the mechanical properties of the final product.

The presence of a specific concentration of Titanium Dioxide (10-20%) increases the mechanical characteristic of the final cement due to its high degree of resistance against various stresses. Moreover, due to its high hydrophilicity, it may tolerate better the humidity of the dental tissue and absorb biological fluids which are necessary for the bioactive phenomenon of mineral deposition and dental remineralisation.

The final product obtained by mixing 80-90% Ordinary Calcium Silicate, 10-20% Titanium bi-oxide, 0.5-2.5% Smectite, 0.5-1.5% Hydrotalcite and H₂0 (1 :2) is smectite/titanium-oxide/hydrotalcite-modified hexacalcium aluminate trisulfate.

Example 2

This example was conducted as a pilot study to produce preliminary results. 2nd AIM: Evaluation of an appropriate resin mixture to create adhesive systems containing the smectite/titanium- oxide/hydrotalcite-modified hexacalcium aluminate trisulfate filler. The aim of the experimental project was to evaluate the micro-tensile bond strength (μΤΒ8) of two different resin mixtures containing the smectite/titanium-oxide/hydrotalcite-modified hexacalcium aluminate trisulfate filler after 24 hours and 6 months PBS storage in order to understand which of them is the most appropriate for the formulation of dental bonding system. MATERIALS AND METHODS

Formulation of the experimental adhesives

Resin 1 :

PRIMER = 18 wt% UDMA, 2% BisGMA, 14.4 wt% TCB, 14.35% HEMA, 50 wt% absolute ethanol, 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate.

BOND = 60% (35 wt% UDMA, 5 wt% BisGMA, 30 wt% TCB, 28.75% HEMA, 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate) + 40% filler (smectite/titanium-oxide/hydrotalcite-modified hexacalcium aluminate trisulfate).

Resin 2:

PRIMER = 18% wt% UDMA, 2% BisGMA, 14.4 wt% TEGDMA, 14.35% HEMA, 50 wt% absolute ethanol, 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate.

BOND = 60% (35 wt% UDMA, 5 wt% BisGMA, 30 wt% TEGDMA, 28.75% HEMA, 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate) + 40% filler (smectite/titanium-oxide/hydrotalcite-modified hexacalcium aluminate trisulfate).

Abbreviations:

UDMA: urethane dimethacrylates;

BisGMA = 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)]-phenyl propane;

TCB: butan-l,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester;

TEGDMA = triethyleneglycol dimethacrylate;

HEMA = 2-hydroxyethylmethacrylate;

Absolute ethanol = 100% ethylic ethanol.

Micro-tensile bond strength test

The microtensile bond tests were performed using a customized microtensile jig on a linear actuator (SMAC Europe Ltd., Horsham, West Sussex, UK) with LAC-1 (high speed controller single axis with built-in amplifier) and LAL300 linear actuator that has a stroke length of 50 mm with peak force of 250 N, and a displacement resolution of 0.5 mm. Bond strength data were statistically analyzed by Two-way ANOVA including interactions between factors, using μΤΒ8 as a dependent variable and experimental adhesive system and PBS storage were considered as independent variables. Post hoc multiple comparisons were performed using the Student-Newman- Keuls test. Statistical significance level was set at a = 0.05. Modes of failure were classified as percentage of adhesive (A) or mixed (M) or cohesive (C) failures when the failed bonds were examined at 30X by stereoscopic microscopy.

RESULTS

The above table (Table 1) shows microtensile bond strength values (MPa) mean and SD of dentine bonded specimens created with the experimental resin adhesives applied on acid-etched dentine (37% H₃PO₄). Number of beams (intact stick/pre-failed sticks) and percent of mode of failures [Adhesive/Mix/Cohesive] are also shown in the table.

Same letter indicates no differences in columns with different adhesive system maintained in the same time storage media. Same number indicates no differences in rows for different PBS storage time (P > 0.05).

It was also found that the functional monomer 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM) could be used in place of TCB in resin 1 in both the primer and bond parts. This was found to give very similar results. DISCUSSION AND CONCLUSIONS

The significant reduction in μΤΒ8 observed between resin 1 and resin 2, in particular after PBS aging must be attributed to the level of hydrophilic and hydrophobic monomers used in each resin. Indeed, resin 2 contains three hydrophobic monomers representing 2/3 of the total weight of the blend. On the contrary, resin 1 contains one hydrophilic (HEMA) and one functional hydrophilic -acidic monomer. These two hydrophilic monomers contained in resin 1 have probably induced the solubility parameters of the monomers mixture to be closer to those of the etched-dentine permitting a greater diffusion of the hydrophilic as well as the hydrophobic monomers into the demineralised dentine. That is why the resin degradation within the interface of the resin- 1 -bonded dentine was less pronounced then that attained within the resin-bonded interface created with resin 2. In fact, this latter interface may have been affected by phase separation caused by the diffusion of the HEMA into the demineralised dentine and all the hydrophobic part of the resin blend only slightly infiltrated the dentine and most of it remained laid on the dentine surface. If the demineralised dentine is well infiltrated by hydrophobic monomers it is likely to achieve a higher monomer conversion during the polymerisation. Moreover, the presence of a functional monomer within the composition of resin 1 may have also influenced the chemical bonding between the filler and the resin matrix and between the resin adhesive and the dentine substrate (i.e. Ca²⁺/COO^" bonds). In conclusion, it is possible to affirm that the composition of resin 1 is a suitable candidate for the creation of the new experimental Bioactive filler-containing resin bonding systems.

Example 3

This example was conducted as a pilot study to produce preliminary results.

3rd AIM: Comparison between the experimental bioactive adhesive systems containing the smectite/titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate and different calcium silicate containing adhesives applied to dentine using two applicative bonding procedures.

The aim of the present work was to perform a series of experiments based on the confocal microscopy characterisation and micro-tensile bond strength (μΤΒ8) evaluation after 24 hours and 6-months storage in PBS solution of three experimental etch-and-rinse-rinse adhesives containing different modified calcium silicate cements and a control calcium-silicate-free adhesive system.

MATERIALS AND METHODS

Experimental and control adhesives

Control adhesive system (Ctrl):

resin primer (ctrl) + resin bond containing no filler (ctrl).

Adhesive 1 (OPC/HEMA):

ctrl-primer + ctrl-bond containing hexacalcium aluminate trisulfate hydrate.

Adhesive 2 (OPC):

primer containing ordinary Portland cement + ctrl-bond.

Adhesive 3 (HPCMM):

ctrl-primer + bond containing smectite/hydrotalcite -modified hexacalcium aluminate trisulfate hydrate. Adhesive 4 (PCMM): primer containing smectite/hydrotalcite -modified calcium silicate + ctrl-bond.

Adhesive 5 (PCTO): Primer containing smectite/titanium oxide/hydrotalcite-modified calcium silicate + ctrl- bond.

Adhesive 6 (HPCTO):

ctrl-primer +bond containing smectite/titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate hydrate*. *smecfite/fitanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate is the result of mixing the smectite/titanium oxide/hydrotalcite-modified calcium silicate with water (1 :2).

The control resin (control adhesive system (Ctrl)) was formulated using the following components:

BOND = 35 wt% UDMA, 5 wt% BisGMA, 30 wt% TCB, 28.75% HEMA, 0.25 wt% camphoquinone and 1.0 wt% ethyl-dimethyl-4-aminobenzoate.

Bonding procedures

A series of in vitro studies was designed according the bonding procedures.

Bonding procedure:

It was designed to test the experimental bonding systems made up of a resin-based bond incorporating hydrated calcium silicate-based micro-filler (smectite/titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate hydrate) or experimental modified-calcium silicates primers. Two consecutive coats each adhesive component (i.e. primer and bond) were applied on etched dentine (37% H₃PO₄; 15 seconds). Excess of solvent was gently air-dried from the primer/dentine for 3 seconds and light-cured for 20 seconds (Translux EC halogen light-curing unit, Kulzer GmBh, Bereich Dental, Werheim, Germany). The output intensity was monitored with a Demetron Radiometer (Model 100, Demetron Rsearch, danbury, CT, USA). A minimal light output intensity of 600 mW/cm² was employed for the entire experiment.

Composite build-ups (6 mm) were constructed with a flow resin-composite in four 1-mm-thick increments. The resin-bonded teeth were stored in deionised water (24 h) and then serially sectioned after 24 h of water storage using a hard tissue microtome (Isomet 11/1180, Buehler, Coventry, UK) in both x and y directions across the adhesive interface to obtain beams with cross sectional areas of approx. 0.9 mm². The specimens were divided in two groups and tested for micro-tensile bond strength (μΤΒ8) after 24 hr of PBS immersion or after aging in a phosphate buffered solution (PBS) for 6 months at 37°C. Modes of failure were examined by stereoscopic light microscopy. The functional monomer 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM) can also be used in place of TCB in both the primer and bond parts. This was found to give very similar results to TCB. Micro-tensile bond strength test

The microtensile bond tests were performed using a customized microtensile jig on a linear actuator (SMAC Europe Ltd., Horsham, West Sussex, UK) with LAC-1 (high speed controller single axis with built-in amplifier) and LAL300 linear actuator that has a stroke length of 50 mm with peak force of 250 N, and a displacement resolution of 0.5 mm. Bond strength data were statistically analyzed by multiple ANOVA test, using μΤΒ8 as a dependent variable and experimental adhesive system, dentine surface treatment and PBS storage were considered as independent variables. Post hoc multiple comparisons were performed using the Student-Newman- Keuls test. Statistical significance level was set at a = 0.05. Modes of failure were classified as percentage of adhesive (A) or mixed (M) or cohesive (C) failures when the failed bonds were examined at 30X by stereoscopic microscopy. The μΤΒ8 (mean-MPa) data for each group were subjected to a repeated measures ANOVA and Tukey's post-hoc test for pair- wise comparisons (a = 0.05). Fisher's least significant difference (LSD) test was used to isolate and compare the significant differences (P < 0.05) between the groups. Premature failures were included in the statistical analysis as zero values.

Confocal microscopy evaluation

Specimen preparation for Confocal nanoleakage evaluation was performed and then serially sectioned across the adhesive interface to obtain resin-dentine slabs with sectional thickness of approx. 0.9 mm. The resin dentine- slabs were divided into 2 groups based on the period of storage in PBS (24h and 6-months). Subsequent to the storage period, the specimens were immersed in 1 wt% aqueous Rhodamine-B solution for 24h and left undisturbed in a dark room. The specimens were then slightly polished from 1200 to 4000 grit silicon carbide paper and ultra-sonicated for 2 min at each step. The Confocal nanoleakage evaluation of the dentine/adhesive interfaces was examined using a tandem scanning confocal microscope (TSM: Noran Instruments, Middleton, Wisconsin, USA) in the reflection and fluorescence mode (546 nm excitation and 600 nm long-pass filter), using a lOOx 1.4/NA oil immersion objective. Reflection and fluorescence images were recorded using digital camera in conjunction with lOx ocular and phototube. Three representative images were taken from resin-bonded dentine surfaces located 1mm from the dentine-enamel junction. One image from the centre of the interface and two in proximity of the pulpal horns were obtained from each site after a complete investigation of the entire resin- dentine interface. These images were intended to be representative of the most common features observed in each specimen.

Two specimens of each subgroup were bonded with the resin adhesives previously mixed with 0.1 wt% rhodamine-B in order to image monomer diffusion into differently pre-treated dentine and delineated important morphologic details of the resin-dentine interface such as the thickness of the adhesive, hybrid layer and resin tags. RESULTS

The above table (Table 2) shows microtensile bond strength values (MPa) mean and SD of dentine-bonded specimens created with the experimental resin adhesives applied on acid-etched dentine (37% H₃PO₄). Number of beams (intact stick/pre-failed sticks) and percent of mode of failures [Adhesive/Mix/Cohesive] are also shown in the table. In each row, same numbers indicate no differences (p>0.05) after 24 h and 6 m of SBS storage. In columns, same capital letter indicates no statistically significant differences between each group (p>0.05). Figure 4 is a graph showing the mean (S.D.) of μΤΒ8 (MPa) to dentine when the experimental and control adhesives were applied on acid-etched dentine.

Figure 5 shows the characterisation and nanoleakage of the resin-dentine interfaces created using the experimental adhesives. (A) Resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) applied on acid-etched dentine showed the penetration of the primer into the demineralised layer and inside the dentinal tubules (pointer) immediately after the bonding procedure (24h). (B) Resin-bonded dentine interfaces created with HPCTO experimental adhesive (resin primer + bond containing titanium oxide/hydrotalcite-modified hexacalcium aluminate trisulfate) applied on acid -etched dentine showed severe gap (pointer) formation after 6 months of PBS storage due to degradation of the hybrid layer. (C) Resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) applied on acid-etched dentine showed a more intense reflection signal of the penetration of the primer into the demineralised layer and inside the dentinal tubules (pointer) after 6 months of PBS storage (pointer). (D) further image of the resin-bonded dentine interfaces created with PCTO experimental adhesive (primer containing titanium oxide/hydrotalcite-modified calcium silicate + resin bond containing no filler) showing the presence of an absorptive/reactive layer above the hybridized dentine surface (pointer).

DISCUSSION AND CONCLUSIONS

This series of experiments has shown that the experimental adhesive with the most evident bioactivity and protective/therapeutic effects was the PCTO (ctrl-primer containing smectite/titanium oxide/hydrotalcite- modified calcium silicate + ctrl-bond). This adhesive has been formulated as an active primer containing smectite/titanium oxide/hydrotalcite-modified calcium silicate and a bond resin containing no mineral filler. The microtensile results showed an increase in bond strength after 6-month storage in PBS (Table 2) and an evident increase in reflective signal, indicating the mineral presence (possible bioactivity), within the resin-dentine interface (Fig. 5C). The presence of an absorptive/reactive layer above the PCTO primer within the resin-dentine interface (Fig 5D) seems to confirm the bioactivity of this experimental bonding procedure. These results may be attributed to the high bioactivity of calcium silicates to induce Ca₃(P0₄)₂ precipitation as well as to the high hydrophilicity of the titanium oxide which may have contributed to the absorption of water containing Ca²⁺ and P0₄. The presence of the hydrotalcite contributed to create an interaction between the resin matrix and the modified calcium silicates used in this type of adhesive system. On the other hand, the hydrotalcite may have been responsible for the poor performance of the HPTCO adhesive due to the strong interaction between the hydrotalcite-modified calcium silicates which may have reduced the possibility of the in-and-out ion exchange with the PBS solution. Indeed, this strong reduction of the bond strength was not observed in the specimens prepared using the ordinary and the montmorillonite (smectite)-modified calcium silicates fillers used in HOPC and HPCMM.

Based on the results of these experiments, there are three potential etch-and-rinse modified calcium-silicates containing adhesives: 1) Etch-and -rinse three-step adhesive system:

resin primer containing titanium oxide/hydrotalcite/smectite-modified calcium silicate (cement not hydrated); resin bond containing resin monomers only (no fillers).

2) Etch-and-rinse three-step adhesive system:

pure resin primer (no filler); resin bond containing titanium oxide/hydrotalcite/smectite-modified hexacalcium aluminate trisulfate hydrate. 3) Etch-and-rinse self priming adhesive system:

single bond resin adhesive containing titanium oxide/hydrotalcite/smectite-modified hexacalcium aluminate trisulfate hydrate.

Example 4

INTRODUCTION

The durability of resin-dentine interface represents one of the main concerns in adhesive dentistry as it is affected by severe degradation processes. Bond degradation occurs maily via water sorption [1], hydrolysis of monomer methacrylates ester bonds caused by salivary esterases [2], and hydrolysis of collagen fibrils which may be enhanced by activation of endogenous dentine matrix metalloproteinases (MMPs) [3]. Regarding these different mechanisms of degradation, strategies to preserve the hybrid layer such as ethanol-wet bonding [4, 5] and the use of MMP inhibitors [6] have been proposed. Nevertheless, current attempts to extend the longevity of resin- dentine bonds via incorporation of more hydrolytically stable resin monomers [7] and/or the use of matrix metalloproteinase inhibitors [8] fail to address two fundamental issues: 1) replacement of the mineral phase within the demineralised dentine collagen; 2) protection of the collagen from biodegradation [9].

The use of bioactive materials which promptly interact with dental hard tissues through therapeutic/protective effects may provide a feasible means to extend the longevity of resin-dentine bonds [10]. Furthermore, experimental resin-based calcium-phosphate cements have been advocated as potential therapeutic restorative base-liner materials due to their ability to induce remineralisation of caries -affected dentine-bonded interfaces [11]. Nonetheless, alternative strategies are being developed in order to enhance calcium (Ca²⁺), hydroxyl (OH ), and phosphate (P0₄ ^~3) ions delivery within and beneath the hybrid layer. Calcium-silicate cements are Portland- derived cements able to release calcium and hydroxyl ions, so creating favorable conditions for the remineralisation of dental hard tissues (i.e. dentine and enamel) [12, 13]. These materials possess a bioactive behavior since they are able to induce the formation of apatite on their surface in a short induction period [14] eliciting a positive response at the interface from the biological environment [15]. However, the use of the Portland cement-based materials in operative dentistry is still debated due to clinical limitations related to their long setting time [14, 16], high dissolution rate and "poor" mechanical properties [17]. In contrast, the incorporation of resin specific monomers such as 2-hydroxyethyl methacrylate (HEMA), triethyleneglycol dimethacrylates (TEGDMA) and urethane dimethacrylates (UDMA) in silicate-based materials has been proposed to improve the mechanical properties, bond strength to dental tissues and reduce the setting time [14, 18].

Since there is little information concerning the use of such "hybrid" resin-base light-curable adhesive materials, this study was purposed to assess the therapeutic/bioactive effects of three newly developed experimental bonding agents containing modified Portland cement-based micro-fillers on resin-dentine interface. This aim was accomplished by evaluating the microtensile bond strength (μΤΒ8) and micro-hardness after simulated body fluid solution (SBS) storage (24h or 6 months). Scanning electron microscopy (SEM) fractography on the de-bonded specimens and confocal microscopy (CLSM) analysis of the ultramorphology and nanoleakage of the resin- dentine interface were executed. The null hypotheses to be tested were that the inclusion of tested micro-fillers within the composition of the experimental bonding agent induces: (i) no effect on the bond strength durability; (ii) no mineral precipitation and nanoleakage reduction within the demineralised 'poorly resin-infiltrated' areas within the resin-dentine interface. MATERIALS AND METHODS

Preparation of the experimental bioactive resin-base bonding agents

A type I ordinary Portland cement (92.5wt%), (OPC: Italcementi Group, Cesena, Italy) mainly consisting of tri- calcium silicate (Alite: 3CaO x Si0₂), di-calcium silicate (Belite: 2CaO x Si0₂), tri-calcium aluminate (3CaO x AI₂O₃) and gypsum (CaSC^ x 2H₂0) was mixed with 7.5 wt% of phyllosilicate consisting of sodium-calcium- aluminum-magnesium silicate hydroxide hydrate [(Na,Ca)(Al,Mg)6(Si₄0io)3(OH)6-nH₂0; Acros Organics, Fair Lawn, NJ, USA] in deionized water (Ratio 2: 1) to create the first experimental filler modified hexacalcium aluminate trisulfate (HOPC). The second experimental modified hexacalcium aluminate trisulfate filler (HCPMM) was created by mixing 90wt% of type I OPC, 7.5 wt% phyllosilicate and 2.5wt% of hydrotalcite consisting of aluminum-magnesium-carbonate hydroxide hydrate [(Mg₆Al₂ (C0₃)(OH)16-4(H₂0); Sigma- Aldrich]. The third modified hexacalcium aluminate trisulfate filler (HPCTO) used in this study was created by mixing OPC (80wt%), phyllosilicate (7.5wt%), hydrotalcite (2.5wt%) and 10 wt% titanium oxide (Ti0₂: Sigma- Aldrich, GiUingham, UK). The three modified Portland-base silicates were mixed with deionised water (Ratio 2: 1) and allowed to set in incubator at 37°C for 24h. Subsequently, they were ground in an agate jar and sieved to obtain <30 μπι-sized micro-filler particles.

A resin co-monomer blend was prepared as a typical three-step, etch-and-rinse bonding agent including a neat resin blend as bond and a 50 wt% ethanol-solvated resin mixture as primer (Res-Ctr - no filler). The neat resin blend was formulated by using 40 wt% of a hydrophobic cross-linking dimethacrylate 2,2-bis[4(2-hydroxy-3- methacryloyloxy-propyloxy)-phenyl]-propane (Bis-GMA; Esstech, Essington, PA, USA) and 28.75 wt% of hydrophilic 2-hydroxy ethyl methacrylate (HEM A; Aldrich Chemical, GiUingham, UK). An acidic functional monomer 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM: Esstech Essington) was also added (30 wt%) to the blend solution to obtain a dental bonding system with chemical affinity to Calcium (Ca²⁺) present in the micro-fillers. The neat resin was made light-curable by adding 0.25 wt% of camphoroquinone (CQ; Aldrich), 0.5 wt% of 2-ethyl-dimethyl-4-aminobenzoate (EDAB; Aldrich) and 0.5% diphenyliodonium hexafluorophosphate.

The resin co-monomer blend was used as control filler-free or mixed with each micro-filler in order to formulate three experimental resin-base agents: i) Res-HOPC: 60wt% of neat resin and 40wt% of HOPC; ii) Res- HCPMM: 60wt% of neat resin and 40wt% of HCPMM; iii) Res-HPCTO: 60wt% of neat resin and 40wt% of HPCTO filler (Table 3). The hybrid calcium silicate-based adhesives systems were prepared by mixing the neat resin and the fillers for 30 seconds on a glass plate to form a homogeneous paste prior the bonding procedures. Specimen preparation and bonding procedures

Caries-free human molars (age 20-40 y), extracted for periodontal reasons were used in this study. The treatment plan of any of the involved patients, who had given informed consent that their extracted teeth could be used for research purposes, was not altered by this investigation. This study was conducted in accordance with the ethical guidelines of the Research Ethics Committee (REC) for medical investigations.

The teeth were stored in deionized water (pH 7.1) at 4° C and used within 1 month after extraction. A flat mid- coronal dentine surface was exposed using a hard tissue microtome (Accutom-50; Struers, Copenhagem, Denmark) equipped with a slow-speed, water-cooled diamond wafering saw (330-CA RS-70300; Struers). The roots were sectioned 1 mm beneath the cemento-enamel junction (CEJ) using the slow-speed diamond saw. A 180-grit silicon carbide (SiC) abrasive paper mounted on a water-cooled rotating polishing machine (Buehler Meta-Serv 3000; Grinder-Polisher, Dusseldorf, Germany) was used (30 s) to remove the diamond saw smear layer and to replace it with a standard and more clinically related smear layer [19]. The specimens were divided into four groups (n=5/group) based on the tested materials (Table 3). The specimens were etched using a 37% phosphoric acid solution (H₃PO₄; Aldrich Chemical) for 15s followed by a copious water rinse. The etched-dentine surfaces were gently air-dried for 2 s to remove the excess of water. The control (Res-Ctr) and experimental adhesives (Res-HOPC; Res-HCPMM; Res-HPCTO) were applied within a period of 20 s. The specimens were immediately light-cured for 30 s using a quartz-tungsten-halogen (QTH) lamp (>600mW/cm ², Optilux VLC; Demetron, CT, USA). Five 1 -mm-thick incremental build-up were performed using a resin composite (Filtek Z250; 3M-ESPE, St Paul, MN, US) light-activated for 20 s each step with a final curing of 60 s (Figure 2). The specimens were finally stored in SBS solutions (Oxoid, Basingstoke, Hampshire, UK) for 24 h and 6 months at 37° C. μΤΒ8 test and SEM observations of the failed bonds

The specimen of each group were sectioned perpendicular to the adhesive interface with a slow speed water- cooled diamond wafering blade (Accutom-50; Struers) mounted on a hard tissue microtome (Isomet 11/1180; Buehler). Subsequently, match-sticks with cross-sectional adhesive area of 0.9 mm² were created. As each tooth yielded 16 beams, there were a total of 80 match-sticks in each group. Half of these match-sticks (n = 40) were tested after 24 h and the remaining half (n = 40) after 6 months of SBS storage (37 °C). Each resin-dentine match-stick was attached to a testing apparatus with a cyanoacrylate adhesive (Zapit; Dental Ventures, CA, USA). A tensile load was applied with a customized micro-tensile jig in a LAL300 linear actuator (SMAC Europe; Horsham, West Sussex, UK) with LAC-1 high speed controller single axis with built-in amplifier, that has a stroke length of 50 mm, peak force of 250 N, displacement resolution of 0.5 mm and crosshead speed of 1 mm^"1 [20]. The load (N) at failure and the cross-sectional area of each failed beam (Digital micrometer Mitutoyo CD15; Mitutoyo, Kawasaki, Japan) permitted calculation of the μΤΒ8 that in MPa. The μΤΒ8 (mean-MPa) data for each group were subjected to a repeated measures ANOVA and Tukey's post-hoc test for pair-wise comparisons (a = 0.05). Fisher's least significant difference (LSD) test was used to isolate and compare the significant differences (P < 0.05) between the groups. Premature failures were included in the statistical analysis as zero values.

Modes of failure were classified as percentage of adhesive (A), mixed (M), or cohesive (C) when the failed bonds were examined at x 30 magnification with a stereoscopic microscope (Leica M205A; Leica Microsystems, Wetzlar, Germany). For each group, five representative de-bonded specimens, depicting the most frequent failure modes, were chosen for SEM ultra-morphology analysis of the fractured surfaces. They were dried overnight and mounted on aluminum stubs with carbon cement. They were sputter-coated with gold (SCD 004 Sputter Coater; Bal-Tec, Vaduz, Liechtenstein) and examined using an SEM (S-3500; Hitachi, Wokingham, UK) with an accelerating voltage of 15 kV and a working distance of 25 mm at increasing magnifications from x 60 to x 5000. Knoop Hardness Analysis ( KHN)

Three further dentine samples for each group were bonded as previously described. Each resin-bonded specimen was longitudinally sectioned in four 2 mm-thick slabs and sequentially polished with #500, #1200, #2400 and #4000 grit SiC papers (Versocit, Struers A/S) as previously described. At each polishing step the slabs were treated in an ultrasonic bath containing deionised water for 5 min. The Knoop microhardness evaluation (Duramin-5, Struers A/S, DK-2750 Ballerup, Denmark) was performed using a 25g load and 15s dwell time to produce indentations with a size suitable for accurate measurements according to the thickness of the hybrid layer and for minimising surface damage. Five indentations were executed in straight lines starting from the hybrid layer along three different positions of the resin dentine interfaces. The five measurements along each line were taken every 30 μπι up to 115 μπι in depth (Figure 2 and 3); each slab was considered as a sample unit [17].

The dentine surface was covered with a wet tissue paper for 1 min after each indentation to avoid dehydration of the surface [18]. As a result, interference of deformation areas caused by nearby indentations was avoided. The length of the long diagonal of each indentation was measured to a resolution of 0.1 μπι and values were converted into Knoop Hardness Numbers according to the following formula: KHN = 14,229 P/d² where P = applied load,(g), and d = length of the longest diagonal (μπι).

Mean (±SD) KHN were calculated and a multiple ANOVA analysis was performed including interactions between factors, using surface microhardness as a dependent variable. Adhesive system (RES-Ctr/BAG- AD/B AG-PR), position along the interface (1^st to 5th) and storage time (24h/6 months) were considered as independent variables. Multiple comparisons were performed using the Student-Newman-Keuls test. Statistical analysis was performed at a significance level of a = 0.05.

Dye-assisted CLSM evaluation

Three further dentine-bonded specimens were prepared as previously described for each group with the primer/bond resins doped with 0.05 wt% Rhodamine-B (Rh-B: Sigma- Aldrich) and then serially sectioned across the adhesive interface to obtain resin-dentine slabs (n = 12 per group) with a thickness of approx. 1 mm. The resin-dentine slabs were then allocated to two subgroups (n=6/group) based on the period of storage in SBS (24 h or 6 months). Following each aging period, the specimens were coated with two layers of fast-setting nail varnish applied 1 mm away from the resin-dentine interfaces. Three specimens from each subgroup were immersed in 1 wt% aqueous fluorescein (Sigma-Aldrich) and the other three specimens in 0.5wt% Xylenol Orange solution (XO: Sigma-Aldrich) for 24h at 37°C (pH: 7.2). The latter is a calcium-chelator fluorophore commonly used in bone remineralisation studies [21], due to its ability to form complexes with divalent Ca²⁺ ions. The specimens were then treated in an ultrasonic water bath for 2 min and polished using ascending (#1200 to #4000) grit SiC abrasive papers (Versocit; Struers) on a water-cooled polishing device (Buehler Meta-Serv 3000 Grinder- Polisher; Buehler). A final ultrasonic treatment (5 min) concluded the specimen preparation for the confocal microscopy analysis which was immediately performed using a confocal laser scanning microscope (DM-IRE2 CLSM; Leica, Heidelberg, Germany) equipped with a 63x / 1.4 NA oil immersion lens. The fluorescein was excited at 488-nm, while xylenol orange at 514-nm using an argon laser. The ultramorphology evaluation (resin- diffusion) was executed using a 568-nm krypton (rhodamine excitation) laser. CLSM images were obtained with a 1 μπι z-step to optically section the specimens to a depth up to 20 μπι below the surface [22]. The z-axis scans of the interface surface were arbitrarily pseudo-colored by the same operator for better exposure and compiled into single projections using the Leica image-processing software (Leica). The configuration of the system was standardized and used at the same settings for the entire investigation. Each resin-dentine interface was completely investigated and then five optical images were randomly captured. Micrographs representing the most common features of nanoleakage observed along the bonded interfaces were captured and recorded [10, 19].

RESULTS

μΤΒ8 test and SEM observations of the failed bonds

The interaction bonding system vs SBS storage was statistically significant only for the Res-HOPC and Res- HCPMM groups (P = 0.001); no significant reduction of the μΤΒ8 values was observed after 6 months of SBS aging (P > 0.05). Conversely, significant drops in μΤΒ8 values were observed in both Res-HPCTO and Res-Ctr groups (P < 0.05) after prolonged storage in SBS (6 months). The μΤΒ8 results (expressed as Mean and SD) and modes of failures obtained for each group are summarized in Table 4.

In details, all the tested materials showed high μΤΒ8 values after 24 h of SBS storage with failures occurring mainly in cohesive mode. However, only the resin-dentine specimens of the Res-HOPC and Res-HCPMM groups maintained high μΤΒ8 values (P > 0.05) after 6 months of storage in SBS (31.3 + 1 1.5 and 24 + 12.7 MPa, respectively); the failure mode was prevalently mixed (57% and 54%, respectively). The SEM analysis of the fractured surfaces at 24h of SBS storage revealed the absence of both exposed dentine tubules and collagen fibrils indicating a good hybridisation of dentine (Figures 6A and 6B, respectively). After 6 months of SBS storage, the dentine surfaces were characterized by embedded mineral crystals and remnant resin presenting filler lacunas (Res-HQPC: figures 6A1 and 6A2; Res-HCPMM: figures 6B 1 and 6B2). In contrast, a significant drop (P < 0.05) in μΤΒ8 was observed after 6 months of storage in SBS, with the specimens created using the Res-Ctr group (filler-free) and with those created with the Res-HTCPO. These latter specimens showed, after 24h, a well- hybridized de-bonded surface embedding several micro-fillers (Figure 6C). On the contrary, the specimens de- bonded after 6 months SBS aging showed a de-bonded surface with a few dentinal tubules but with no sign of clear degradation and well-hybridized peritubular dentine with resin tags (Figures 6C1 and 6C2). The Res-Ctr specimens tested after 24 h of SBS storage and analyzed with SEM presented few exposed dentinal tubules but mostly were obliterated by resin tags or covered by resin remnants (Figure 6D). Conversely, the surface of the specimens de-bonded after 6 months of SBS exhibited no collagen fibrils on the dentine surface with rare resin tags and degraded funneled dentinal tubules (Figures 6D1 and 6D2). Knoop Hardness (KHN) Analyses

The ANOVA analysis revealed a significant effect of the following factors: position along the interface (F: 54.215; p=0.0005), bonding approach (F: 34.321 ; p=0.0005) and storage time (F: 46.543; p=0.0005). Interactions between factors were also significant (p < 0.05).

The control group (RES-Ctr) showed a statistical reduction of KHN values after prolonged PBS storage solely in the hybrid layer (24h: 24.4 + 2.5 KHN; 6m: 15.9 + 4.2 KHN), (p < 0.05); no significant KHN decrease was observed between all the indentations at different depths away from the hybrid layer (p > 0.05).

All the experimental resin materials applied on H3P04-etched dentine, maintained high KHN values with no statistical difference after the aging period both in the hybrid layer and in all the indentations at different depths along the dentine surface.

Dye-assisted CLSM evaluation

CLSM imaging of the bonded-dentine interfaces subsequent to 24 h of SBS storage showed relevant ultramorphology and nanoleakage information for all groups. It was observed that all tested materials were able to diffuse within the demineralised dentine, creating a hybrid layer 7-10 μπι thick, with a multitude of resin tags penetrating the dentinal tubules. Nevertheless, all these interfaces were affected by conspicuous fluorescein penetration (nanoleakage) through dentinal tubules into a porous hybrid layer. Furthermore, the resin-dentine interface created using the experimental bonding agents containing the micro-fillers showed presence of calcium- chelator dye (XO-dye: Xylenol orange) within the hybrid, adhesive layers and inside the dentinal tubules. On the contrary, the acid-etched dentine bonded using the resin control (Res-Ctr, filler-free) showed no presence of XO along the interface.

Significant ultramorphological changes were observed subsequent prolonged SBS storage. For instance, the CLSM analysis revealed no gap and limited fluorescein penetration (nanoleakage) within the resin-dentine interfaces created using the Res-HOPC and Res-HCPMM. In addition, OX-dye produced a clearly outlined fluorescence due to a consistent Ca-minerals deposited within the resin-dentine interface and inside the dentinal tubules. The resin-dentine interfaces created using Res-HPCTO showed less nanoleakage within the hybrid layer; evident resin degradation of the adhesive layer was also observed. Intense nanoleakage and constant gaps affected the resin-dentine interfaces created using the Res-Ctr. When the same interfaces were investigated employing OX-dye, only the walls of the dentinal tubules were highlighted.

TABLE 3 - Chemical composition (wt%) and application mode of the experimental adhesive system used in this study.

Bis-GMA: bisphenyl A glycidyl methacrylate; HEMA: hydrophilic 2-hydroxyethyl methacrylate; PMDM: 2,5- dimethacryloyloxyethyloxycarbonyl- 1 ,4-benzenedicarboxylic acid; HOPC: set Portland cement and smectite; HPCMM: Portland cement, Smectite and Hydrotalcite; HPTCO: set Portland cement, Smectite , Hydrotalcite and Titanium Oxide.

* Three discs for each experimental resin-base material (6 mm in diameter and 1 mm thick) and were light-cured for 30s immersed in 25ml of H₂0 (pH 6.7) at 37 °C and maintained for 30 days; the pH/alkalinizing activity was evaluated using a professional pH electrode (Mettler-Toledo, Leicester, UK) at room temperature (-24 °C).

TABLE 4 - Mean and standard deviation (SD) of the μΤΒ8 (MPa) to dentine.

Values are mean + SD in MPa. In each row, same numbers indicate no differences (p>0.05) after 24 h and 6 m of SBS storage. In columns, same capital letter indicates no statistically significant differences between each group (p>0.05). Premature failures were included in the statistical analysis as zero values and are indicated in parentheses (for instance 5/35 means that there were 5 premature failures and 35 testable beams). The modes of failure are expressed in percentage in the brackets [adhesive/mix/cohesive]. TABLE 5 - Mean and standard deviation (SD) of the microharness (MPa) to dentine

In each row, same numbers indicate no differences (p>0.05) after 24 h and 6 m of SBS storage. In columns, same capital letter indicates no statistically significant differences along the dentine interface position (p>0.05).

DISCUSSION

The resin-dentine interfaces created using contemporary "simplified" etch-and-rinse bonding agents are affected by bond strength reduction subsequent to prolonged water aging [23]. This phenomenon occurs due to the inability of such materials to completely replace loosely bound and bulk-free water from the apatite- depleted dentine collagen matrix during bonding procedures which cause hygroscopic swelling effects and hydrolytic degradation of polymer networks and favors metalloproteinases (MMPs)-mediated collagenolytic degradation [23-27]. Nevertheless, the presence of water may be essential to facilitate apatite nucleation within the gap zones of collagen fibrils and fossilisation of the host-derived, collagen-bound enzymes (MMPs) [24, 25, 28]. Recent investigations have demonstrated that it is possible to reduce the nanoleakage and micropermeability within the resin-dentine interface and maintain the bond strength [10] of bioactive resin-base materials applied to H₃PO₄ acid-etched dentine subsequent to simulated body fluid storage for 3-6 months [18, 22]. Ryou et al, [29] demonstrated that using a biomimetic remineralisation approach it is feasible to remineralise the dentine collagen within the resin-dentine interface via slow release of calcium ions from set white Portland cement and subsequent interaction of these ions with phosphate species from SBS or dentine substrate. Portland cements designed for dental applications, also called hydraulic silicate cements or MTA, exhibit outstanding biological properties and high bioactivity when immersed in PBS [18, 29, 30].

In the present study, modified Portland cement-based micro-fillers (<20 μπι) were included within the composition of a representative three-step/etch-and-rinse bonding agent in order to create a material with therapeutic remineralizing effects on the mineral-deficient areas along the bonding interface. Based on the results obtained in this study, the first null hypothesis that the inclusion of bioactive micro-fillers within the composition of the experimental bonding agent has no effect on the bond strength durability must be must be rejected as the use of Res-HOPC and Res-HCPMM bonding agents preserved the bond strength durability. The second null that no mineral precipitation and nanoleakage reduction will be observed within the demineralised 'poorly resin- infiltrated' areas within the resin-dentine interface must be rejected.

In detail, the three experimental bonding agents containing experimental micro-fillers (Res-HOPC, Res-HCPMM and Res-HPCTO) and the control co-monomer blend (RES-Ctr) used to bond the acid-etched dentine produced comparably high μΤΒ8 values (P > 0.05) following 24 h of storage in SBS (Table 4). Conversely, after 6 months of storage in SBS a significant decrease in μΤΒ8 (P < 0.05) was observed for the RES-Ctr and Res-HPCTO groups, while the specimens bonded using Res-HOPC or Res-HCPMM maintained consistent long-term bond strength values (P > 0.05) compared to the control group (24h SBS storage). The specimens of the Res-HOPC and Res-HCPMM groups de-bonded after 6 months of SBS storage showed, during SEM fractography examination, residual resin presence and mineral -bodies on the fractured surface (Figures 6A1 and 6A2, 6B 1 and 6B2). Important morphological differences were observed in the specimens of the Res-HPCTO group which presented a de-bonded surface characterized by very few partially exposed dentinal tubules and an important presence of mineral crystals after 6 months of SBS storage (Figures 6C1 and 6C2). The SEM analysis revealed that the de-bonded dentine surface of the specimens in RES-Ctr group was well resin-hybridized and characterized by no exposed collagen fibrils after 24 h of SBS storage (Figure 6D). In contrast, the prolonged SBS storage (6 months) induced radical changes; the dentine surface presented funneled dentinal tubules as essential sign of degradation of the "poorly resin-infiltrated" demineralised and peritubular dentine (Figures 6D1 and 6D2). These results were also supported by the CLSM analysis performed to evaluate the nanoleakage and the presence of calcium-compounds within the resin-dentine interface subsequent to SBS storage (24h or 6 months). Indeed, further evidences of the therapeutic bioactivity of the experimental bonding agents containing the modified Portland cement-based micro-fillers were attained; reduced fluorescent-dye uptake (nanoleakage) was observed along the entire resin-dentine interface after 6 months of storage in SBS. These latter observations along with the strong xylenol orange (XO-dye) signal from the hybrid layer and the dentinal tubules, clearly indicated the remineralisation of those areas which were previously detected within the resin-dentine interface as mineral-deficient/poor-resin infiltrated zones.

The inventors hypothesize that the therapeutic remineralizing effects observed within the mineral-depleted resin- dentine interface were essentially due to the bioactivity of the experimental micro-fillers. Indeed, the reaction mechanism of the Portland cement-based micro-fillers may have involved the reaction of the polymerized calcium-silicate hydrate gel with water to release calcium hydroxide and to the consequent increase of the alkalinity of the surrounding environment [31]; this increase of pH was confirmed in this study (Table 4). This localised increase in pH within the resin-dentine interface may have also interfered with the activity of MMPs [3, 23]. Furthermore, the interaction between the phosphate ions present in the aging solution (PBS) or in the dentine substrate, and the calcium released from the Portland-based micro-fillers may have enhanced the formation of new apatite deposits upon existing mineral constituents within the dentine matrix (biocatalysation) [32]. However, it is well known that the increase in environmental pH and the presence of free OH^~ may facilitate apatite nucleation and reduce the solubility of intermediate Ca/P species formed during apatite formation [33]. The most appropriate pH to support the formation of stoichiometric hydroxyapatite (HA) in vitro [34] and in-vivo [35] falls in a range between 8 and 9. At higher pH it is common to obtain a Ca-deficient HA (lower solubility then stoichiometric hydroxyapatite) characterized by higher concentrations of P0₄ ^3" and lower Ca²⁺ ions [36]. Furthermore, the presence of carboxylic species (R-COO ) within the acidic functional monomer (PMDM) used in this study may have acted as sequestering agent for Ca/P cluster favoring the precipitation of nano-apatite within the polymer network and dentine collagen [28, 29]. Moreover, the R-COO^" species of PMDM may have interacted with the remnant calcium present along the front of demineralisation at the bottom of the hybrid layer acting as a sort of biomimetic template primer which promoted precipitation of Ca-compounds [18, 22]. During all these processes for Ca/P nucleation, apatite precipitation may have reduced the distribution of water-rich regions within the interface [18, 37] and absorbed the water responsible for the hydrolytic and hygroscopic mechanisms involved in the degradation of dental polymers [38]. In addition to the formation of apatite crystals, the nanostructure of the calcium-silicate hydrate may also have contributed to seal the dentinal tubules due to the small-scale volume of the forming gels, along with a slight expansion of the calcium silicate-based materials once immersed in SBS [39]. In particular, the phyllosilicates (i.e. Smectite) and hydrotalcite, which were contained in the micro-fillers used in this study, have the ability to expand considerably following water sorption into the interlayer molecular spaces [40]. The amount of expansion is due largely to the type of exchangeable cation contained in the micro-filler; the uptake kinetics of cation exchange is fast and the presence of Na⁺, as the predominant exchangeable cation, can result in material swelling. In this condition, the exceeding water is removed, thereby preventing hygroscopic effects and hydrolytic degradation of the polymer chains [41]. Also, it is reasonable to expect that the metallic ions intercalated on phyllosilicate were easily released by ion-exchange with cations present in the surrounding solutions and acted as effective antibacterial substances in the long term [38]. In contrast, the bond strength reduction observed in the resin-dentine interfaces created using the Res- HPCTO bonding agent after prolonged storage in SBS (Table 4) may be due to the high hydrophilicity of the T1O₂. Micro-fine titanium oxide (T1O₂) have been used as inorganic additive of resin composites to match the opaque properties of teeth [42] and as nano-particles to increase the microhardness and flexural strength of dental composites [40]. However, T1O₂ has been advocated as a super-hydrophilic component, in particular under ultraviolet (UV) light irradiation [43-45]. Therefore, a possible explanation for the μΤΒ8 reduction may be attributed to this high hydrophilicity which may have permitted excessive water adsorption which induced severe hydrolytic resin and collagen degradation as well as the extraction of water-soluble un-reacted monomers or oligomers from the resin-matrix [46]. Moreover, the replacement of the degraded resin by mineral crystallisation within the Res-HPCTO bonded-dentine interface over prolonged SBS storage may have conferred mechanical characteristics related to bond strength comparable to those created by conventional glass-ionomer cements (GICs) applied onto polyacrylic acid-etched dentine and submitted to tensile tests [47, 48]. Indeed, several studies indicated that the bond strength of GICs when tested using tensile or shear methods was approximately 5 MPa; these values results do not reflect the true adhesive strength to dentine [49]. These factors may have been also responsible for the formation of gaps within the resin-dentine interface created by the Res-HPTCO during the cutting/sample preparation [10].

Knoop microhardness testing may provide valuable information towards predicting the behaviour of dentine/restoration interfaces [50]. The micro-hardness measurements results can also be correlated with mechanical properties such as the modulus of elasticity, fracture resistance [51], and yield strength [52]. A close correlation exists between the microhardness of dentine and the respective bond strength [53]. Accordingly, the chief characteristic of the Knoop hardness test is its sensitivity to surface effects and textures and the ability to provide an average hardness which may reflect the integrity of the structure under examination [54, 55]. However, many factors may influence the results of KHN measurements such as the dentine depth, the relative quantities of the tubular, peritubular and intertubular areas [56-58]. For this reason, in this work the Knoop test measurements along the interfaces were accomplished in the same specimens and close to previously assessed locations after 6 m of PBS storage in order to minimise the effect of the structural variations within the same tooth, and to establish a reasonable baseline for evaluation (Figure 10A and 10B). CONCLUSION

In conclusion, as the results of this study demonstrated that the resin-dentine bond may be maintained overtime by inducing a therapeutic remineralisation of the bonding interface, specific experimental resin bonding systems containing bioactive micro-fillers, such as Res-HOPC, Res-HCPMM or Res-HPCTO may offer the possibility to improve the durability of the resin-dentine interfaces. The characteristic of promoting bioactivity should also open up the potential to create therapeutic restorative materials able to reduce the incidence of secondary caries. Indeed, it is important to consider that restorative materials containing bioactive fillers may be effective in killing a wide selection of aerobic bacteria due to the increase of the local pH and concentration of alkali ions [59, 60]. The antibacterial properties are potentially of great importance as the infiltration of microorganisms may cause secondary caries which jeopardize the longevity of resin-dentine interface leading to the replacement of dental restorations [61]. Further studies are ongoing in order to evaluate the species-specific antibacterial effects and biocompatibility of the materials tested in this study.

REFERENCES FOR EXAMPLE 4

1. Ito S, Hashimoto M, Wadgaonkar B, Svizero N, Carvalho RM, Yiu CK, et al. Biomaterials,

2005;26:6449-59.

2. Moszner N, Salz U and Zimmermann J. Dent Mater, 2005;21 :895-910.

3. Pashley DH, Tay FR, Yiu CK, Hashimoto M, Breschi L, Carvalho RM, et al. J Dent Res, 2004;83:216- 21.

4. Sadek FT, Pashley DH, Ferrari M and Tay FR. J Dent Res, 2007;86:524-528.

5. Tay FR, Pashley DH, Kapur RR, Carrilho MR, Hur YB, Garrett LV, et al. J Dent Res, 2007;86: 1034-9.

6. Carrilho MR, Carvalho RM, de Goes MF, di Hipolito V, Geraldeli S, Tay FR, et al. J Dent Res, 2007;86:90-94.

7. Moszner N, Fischer UK, Angermann J and Rheinberger V. Dent Mater, 2006;22: 1157-62.

8. Carrilho MR, Carvalho RM, Sousa EN, Nicolau J, Breschi L, Mazzoni A, et al. Dent Mater,

2010;26:779-85.

9. Brackett MG, Li N, Brackett WW, Sword RJ, Qi YP, Niu LN, et al. J Dent, 2011 ;39:238-248.

10. Prof eta AC, Mannocci F, Foxton RM, Thompson I, Watson TF and Sauro S. Eur J Oral Sci, 2012;120:353-62.

11. Peters MC, Bresciani E, Barata TJ, Fagundes TC, Navarro RL, Navarro MF, et al. J Dent Res,

2010;89:286-91.

12. Huan Z, Chang J, and Huang XH. J Biomed Mater Res B: Appl Biomater, 2008;87:387-94.

13. Coleman NJ, Awosanya K, and Nicholson JW. J Biomed Mater Res A, 2009;90: 166-74.

14. Taddei P, Modena E, Tinti A, Siboni F, Prati C and Gandolfi MG. J Mol Struct, 2011 ;993:367-75. 15. Parirokh, M. and Torabinejad M. J Endod, 2010;36:400-13.

16. Camilleri J. J Endod, 2009;35: 1412-7.

17. Bogen G and Kuttler S. J Endod, 2009;35:777-790.

18. Sauro S, Osorio R, Watson TF, Osorio E and Toledano M. J Biomater Sci. Polym Ed, (in press) doi:

10.1080/09205063.2012.727377.

19. Koibuchi H, Yasuda N and Nakabayashi N. Dent Mater, 2001 ; 17: 122-6.

20. Sauro S, Watson TF, Thompson I, Toledano M, Nucci C and Banerjee A. Eur J Oral Sci, 2012; 120: 168- 177.

21. Rahn BA and Perren SM. Stain Technol, 1971 ;46: 125-129.

22. Sauro S, Osorio R, Watson TF and Toledano M. J Mater Sci Mater Med, 2012;23: 1521-32.

23. Breschi L, Mazzoni A, Ruggeri A, Cadenaro M, Di Lenarda R and De Stefano Dorigo E. Dent Mater,

2008;24:90-101.

24. Kim J, Gu L, Breschi L, Tjaderhane L, Choi KK, Pashley DH, et al. J Dent Res, 2010;89:575-80.

25. Pashley DH, Tay FR, Breschi L, Tjaderhane L, Carvalho RM, Carrilho MR, et al. Dent Mater, 2011 ;27: 1-16. Breschi L, Mazzoni A, Nato F, Carrilho MR, Visintini E, Tjaderhane L, et al. Dent Mater, 2010;26:320- 325

Liu Y, Li N, Qi Y, Niu LN, Elshafiy S, Mao J, et al. Dent Mater, 2011 ;27:465-77.

Tay FR and Pashley DH. J Dent Res, 2009;88:719-24.

Ryou H, Niu LN, Dai L, Pucci CR, Arola DD, Pashley DH, et al. J Dent Res, 2011 ;90: 1122-8.

Darvell BW and Wu RC. Dent Mater, 2011 ;27:407^122.

Lawrence CD. in LEA's Chemistry of Cement and Concrete, P. C.Hewlett, Ed.,Woburn,Mass, USA, Butterworth & Heinemann, 4th edition, 1998:343^120.

Gandolfi MG, Van Landuyt KL, Taddei P, Modena E, Van Meerbeek B and Prati C. J Endod, 2010;36:851-857.

Somasundaran P, Ofori Amankonah J and Ananthapadmabhan KP. Colloids Surf, 1985;15:309-33. Kikuchi M, Itoh S, Ichinose S, Shinomiya K and Tanaka J. Biomaterials, 2001 ;22: 1705-1711.

ReciUas S, Rodriguez-Lugo V, Montero ML, Viquez-Cano S, Hernandez L and Castano VM. J Ceramic Process Res, 2012;13:5-10.

Durucan C and Brown PW. J Mater Sci Mater Med, 2000; 11 :365-371.

Qi YP, Li N, Niu LN, Primus CM, Ling JQ, Pashley DH, et al. Acta Biomater, 2012;8:836-42.

Ferracane JL. Dent Mater, 2006;22:211-22.

Skinner LB, Chae SR, Benmore CJ, Wenk HR and Monteiro PJM. Phys Rev Lett, 2010; 104: 1955021-4. Bhattacharyya KG and Gupta SS. Adv Colloid Interface Sci, 2008;140: 114-31.

Malachova K, Praus P, Pavlickova Z and Turicova M. Appl Clay Sci, 2009;43:364-368.

Yu B, Ahn JS, Lim JI and Lee YK. Dent Mater, 2009;25: 1142-7.

Xia Y, Zhang F, Xie H and Gu N. J Dent, 2008;36:450-455.

Chadwick RG, Traynor N, Moseley H and Gibbs N. Brit Dent J, 1994;176: 17-21.

Bonding N, Graem N, Rygaard J and Dabelsteen E. Scand J Dent Res, 1987;95:463-6.

Karuppuchamy S and Jeong JM. Mater Chem Phys, 2005;93:251-254.

Spencer P, Ye Q, Park J, Topp EM, Misra A, Marangos O, et al. Ann Biomed Eng, 2010;38: 1989-2003 Hewlett ER, Caputo AA and Wrobel DC. J Prosthet Dent, 1991 ;66:767-772.

Berry EA 3rd and Powers JM. Oper Dent, 1994;19: 122-6.

Perinka L, Sano H, Hosoda H.. Dent Mater 1992;8:229-233.

Currey JD, Brear K. J Mater Sci Mater Med 1990;1 : 14-20.

Mahoney E, Holt A, Swain M, Kilpatrick N.. J Dent Res 2000;28:589-594.

Panighi M, G'Sell C. J Biomed Mat Res 1993;27:975-981.

Knoop F, Peters CG, Emersoon WB. J Res Nat Bur Stand 1939;23:39-61.

Miyauchi H, Iwaku M, Fusayama T. Bull Tokyo Med Dent Univ 1978;25: 169-179.

Fuentes V, Toledano M, Osorio R, Carvalho RM. J Biomed Mater Res A. 2003;66:850-853.

Hosoya Y, Marshall SJ, Watanabe LG, Marshall GW. Oper Dent 2000;25:81-89.

Pashley D, Okabe A, Parham P. Endod Dent Traumatol 1985;1 : 176-179.

Yip HK, Tay FR, Ngo HC, Smales RJ and Pashley DH. Dent Mater, 2001 ; 17:456-70.

Hu S, Chang J, Liu M and Ning C. J Mater Sci Mater Med, 2009;20:281-286. 61. Boeckh C, Schumacher E, Podbielski A and Haller B. Caries Res, 2002;36: 101-107.

Claims

1. Use of: i) a composition comprising Portland cement and phyllosilicate; and/or ii) a modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement and phyllosilicate, as an additive in a dental adhesive.

2. The use of claim 1, wherein the composition comprises about 0.5-15% by weight phyllosilicate relative to the total weight of the components of the composition.

3. The use of claim 1, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition.

4. The use of claim 1, wherein the composition comprises, by weight, about 85-99.5% Portland cement and 0.5-15% phyllosilicate.

5. The use of any preceding claim, wherein the composition further comprises hydrotalcite.

6. The use of claim 5, wherein the composition comprises about 0.5-5% by weight hydrotalcite relative to the total weight of the components of the composition.

7. The use of claim 5, wherein the composition comprises, by weight, about 80-99% Portland cement; 0.5- 15% phyllosilicate; and 0.5-5% hydrotalcite.

8. The use of claim 5, wherein the composition further comprises titanium oxide.

9. The use of claim 8, wherein the composition comprises about 5-20% by weight titanium oxide relative to the total weight of the components of the composition.

10. The use of claim 8, wherein the composition comprises, by weight, about 60-94% Portland cement; about 0.5-15% phyllosilicate; about 0.5-5% hydrotalcite; and about 5-20% titanium oxide.

11. A composition for improving the characteristics of a dental adhesive system, the composition comprising Portland cement, phyllosilicate and hydrotalcite.

12. The composition of claim 11, wherein the composition comprises about 0.5-5% by weight hydrotalcite relative to the total weight of the components of the composition.

13. The composition of claim 11 or claim 12, wherein the composition comprises about 0.5-15% by weight phyllosilicate relative to the total weight of the components of the composition.

14. The composition of claim 13, wherein the composition comprises about 5-10% by weight phyllosilicate relative to the total weight of the components of the composition.

15. The composition of claim 11, wherein the composition comprises, by weight, about 80-99% Portland cement; 0.5-15% phyllosilicate; and 0.5-5% hydrotalcite.

16. The composition of claim 11, wherein the composition comprises, by weight, about 86-94% Portland cement; 5-10% phyllosilicate; and 1-4% hydrotalcite.

17. The composition of claim 11, further comprising titanium oxide.

18. The composition of claim 17, wherein the composition comprises about 5-20% by weight titanium oxide relative to the total weight of the components of the composition.

19. The composition of claim 17, wherein the composition comprises, by weight, about 60-94% Portland cement; about 0.5-15% phyllosilicate; about 0.5-5% hydrotalcite; and about 5-20% titanium oxide.

20. The composition of claim 17, wherein the composition comprises, by weight, about 71-89% Portland cement; about 5-10% phyllosilicate; about 1-4% hydrotalcite; and about 5-15% titanium oxide.

21. The composition of claim 1 1, wherein the composition comprises Portland cement, titanium oxide, smectite and hydrotalcite.

22. The composition of any one of claims 17 to 21, wherein the titanium oxide is titanium dioxide.

23. The composition of any one of claims 11 to 22, wherein the phyllosilicate is montmorillonite.

24. The composition of claim 21 comprising 62-99.7% Portland cement; 0.1-30% Titanium Oxide; 0.1-5% Smectite; and 0.1-3% Hydrotalcite.

25. The composition of claim 21 comprising: 76-89% Portland cement: 10-20% Titanium Oxide; 0.5-2.5% Smectite; and 0.5-1.5% Hydrotalcite.

26. A composition for improving the characteristics of a dental adhesive system, the composition comprising Portland cement and phyllosilicate, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition.

27. The composition of claim 26, wherein the composition comprises about 85-96% by weight Portland cement relative to the total weight of the Portland cement and phyllosilicate combined.

28. The composition of claim 26, wherein the composition comprises about 5-10% by weight phyllosilicate relative to the total weight of the components of the composition.

29. The composition of claim 26, wherein the composition comprises 90-95% Portland cement and 5-10% phyllosilicate.

30. The composition of claim 26, further comprising hydrotalcite.

31. The composition of claim 30, wherein the composition comprises about 0.5-5% by weight hydrotalcite relative to the total weight of the components of the composition.

32. The composition of claim 30, wherein the composition comprises about 85-94.5% Portland cement; 5-10% phyllosilicate; and 0.5-5% hydrotalcite.

33. The composition of claim 30, further comprising titanium oxide.

34. The composition of claim 33, wherein the composition comprises about 5-20% by weight titanium oxide relative to the total weight of the components of the composition.

35. The composition of claim 33, wherein the composition comprises about 65-89.5% Portland cement; about 5-10% phyllosilicate; about 0.5-5% hydrotalcite; and about 5-20% titanium oxide by weight.

36. A modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition according to any one of claims 11 to 35.

37. A method of preparing a modified hexacalcium aluminate trisulfate hydrate product, the method comprising adding water to a composition according to any one of claims 11 to 35.

38. A dental adhesive comprising resin monomers and:

1) a composition comprising Portland cement and phyllosilicate; and/or

2) a modified hexacalcium aluminate trisulfate hydrate product formed by the hydration of a composition comprising Portland cement and phyllosilicate.

39. The dental adhesive of claim 38, wherein the composition comprises about 0.5-15% by weight phyllosilicate relative to the total weight of the components of the composition.

40. The dental adhesive of claim 38, wherein the composition comprises about 3.7-15% by weight phyllosilicate relative to the total weight of the components of the composition.

41. The dental adhesive of claim 38, wherein the composition comprises, by weight, about 85-99.5% Portland cement and 0.5-15% phyllosilicate.

42. The dental adhesive of any one of claims 38 to 41, wherein the composition further comprises hydrotalcite.

43. The dental adhesive of claim 42, wherein the composition comprises about 0.5-5% by weight hydrotalcite relative to the total weight of the components of the composition.

44. The dental adhesive of claim 42, wherein the composition comprises, by weight, about 80-99% Portland cement; 0.5-15% phyllosilicate; and 0.5-5% hydrotalcite.

45. The dental adhesive of claim 42, wherein the composition further comprises titanium oxide.

46. The dental adhesive of claim 45, wherein the composition comprises about 5-20% by weight titanium oxide relative to the total weight of the components of the composition.

47. The dental adhesive of claim 45, wherein the composition comprises, by weight, about 60-94% Portland cement; about 0.5-15% phyllosilicate; about 0.5-5% hydrotalcite; and about 5-20% titanium oxide.

48. The dental adhesive of any one of claims 38 to 47, wherein the resin monomers are selected from: urethane dimethacrylates (UDMA), 2,2-bis[4-(2-hydroxy-3-methacryloylpropoxy)] -phenyl propane (BisGMA), butan-

1,2,3,4-tetracarboxylic acid di-2-hydroxyethylmethacrylate ester (TCB), and 2-hydroxyethylmethacrylate (HEMA), 2,5-dimethacryloyloxyethyloxycarbonyl-l,4-benzenedicarboxylic acid (PMDM).

49. The dental adhesive of any one of claims 38 to 48, wherein the resin monomers make up the following percentages by weight relative to the total weight of the resin monomers: about 32-42% UDMA; about 1-7%

BisGMA; about 24-34% TCB or PMDM; and about 24-34% HEMA.

50. The dental adhesive of any one of claims 38 to 48, wherein the resin monomers make up the following percentages by weight relative to the total weight of the resin monomers: about 35-45% BisGMA; about 24-34% HEMA; and about 35-45% PMDM.