WO1999007326A2

WO1999007326A2 - Antimicrobial cement compositions

Info

Publication number: WO1999007326A2
Application number: PCT/CA1998/000754
Authority: WO
Inventors: Joseph Paul Santerre; Shimon Friedman
Original assignee: The University Of Toronto Innovations Foundation
Priority date: 1997-08-11
Filing date: 1998-08-07
Publication date: 1999-02-18
Also published as: WO1999007326A3; CA2268478A1; AU8846398A

Abstract

Antimicrobial cement compositions are disclosed which contain antimicrobial zeolites. Preferred are antimicrobial cement compositions having dental applications including fillers, adhesives, sealants and restorative materials, and medical applications such as bone cements, implant components and bone substitutes. A particularly preferred cement is an antimicrobial glass ionomer cement composition for dental use comprising a polyelectrolyte, glass ionomer particles, and antimicrobial zeolite particles. When the composition is cured to form a hardened cement, the glass ionomer particles and the zeolite particles form ionic bonds with the polyelectrolyte, the zeolite particles being present in the composition in an amount sufficient to prevent growth of bacteria in the composition and over the hardened cement formed by curing the composition. Preferably, the zeolite contains an antimicrobial metal ion such as silver ion, and is present in the glass ionomer cement composition in an amount of from about 0.2 % to about 20 % by weight of the glass ionomer particles.

Description

Title:

"ANTIMICROBIAL CEMENT COMPOSITIONS"

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/055361, filed August 1 1, 1997.

Field of the Invention

This invention relates to cement compositions having antimicrobial properties, and particularly to cement compositions containing antimicrobial zeolites.

Background of the Invention

Numerous types of cements have been developed for use in medicine and dentistry. Generally, medical and dental cements comprise a powder component comprising finely divided metal oxides, metal hydroxides, calcium hydroxyapatite bioglasses, silicate-cement glazes, or glass ionomer particles, which is induced to react with a liquid medium containing phosphoric acid, polyelectrolytes such as polycarboxylic acid, or salicylic acid derivatives. These cements set through ionic interactions between the various components.

These cements are typically used in medicine as bone cements, implant components and bone substitutes, and in dentistry as adhesives, sealants and restorative (filling) materials.

One cement type used in dentistry and medicine is glass ionomer cement (GIC). glass ionomer cements are preferred for use as dental adhesives and restoratives, for example to restore cavities in dental tissue, and to seal the interfacial area between a filling and surrounding dental tissue, for example as sealers in root fillings. However, due to their limited physical strength, glass inomer cements are less frequently used as restorative materials for surfaces that undergo considerable physical stress. Glass ionomer cements typically comprise at least two components; firstly, a glass ionomer cement powder comprising an acid-soluble calcium fluoroaluminosilicate glass powder; and secondly, a polyanion, also referred to as a "polyelectrolyte", in a water base. When the solid and liquid components are combined to form a paste, the surfaces of the glass particles are attacked by the polyanion. Calcium, aluminum, sodium and fluorine ions are leached into the aqueous medium, and the polyanion chains are cross-linked by the calcium ions to form a solid mass. The aluminum ions also become ionically bound within the cement mix. The unreacted portions of the glass particles become sheathed by silica gel, so that the set cement consists of an agglomeration of unreacted powder particles surrounded by silica gel in an amorphous matrix of hydrated calcium and aluminum polysalts. The polyanion also reacts with calcium ions in the substrate, for example a tooth, forming a chemical bond between the cement and the substrate. Other monomer components may be added in order to carry out a dual cure process, for example where polymer chains are generated by light-curing the monomer components.

Therefore, glass ionomer cement cures primarily by means of ionic reactions in which "ion bridges" are formed between the polyanion, the ions originating from the glass ionomer particles, and the substrate to which the cement is being adhered. As a result of this ion bridging, glass ionomer cements have satisfactory adhesion to hard dental tissue, such as teeth and bones, which are comprised of calcium hydroxyapatite. It is desirable to provide dental cements having antimicrobial properties. One particularly important potential application of antimicrobial cements is in endodontic treatment of root canal systems. The goals of endodontic therapy include disinfection of the root canal system and subsequent bacteria-tight sealing of the root canal system. Disinfection is accomplished during the root canal treatment by instruments, irrigants and medicaments. Sealing is accomplished by root filling materials, such as gutta percha (rubber) and sealer cements. However, the sealing properties of known root filling materials are insufficient to prevent leakage and bacterial ingress into the filled root canal. The interfacial area between the endodontic filling materials and the dentin wall of the root canal is particularly susceptible to bacterial ingress, particularly where the technical quality of the root filling is poor Consequently, bacteria that challenge the endodontically treated tooth may proliferate through the filled root canal system and cause treatment failure

Glass ionomer cements have shown some promise as antimicrobial endodontic fillers These cements release fluoride ions over a period of time and are believed to provide an antimicrobial effect However, fluoride-releasing glass ionomer cements have several disadvantages Firstly, fluoride ions may participate in ionic bridging between dental tissue and the glass ionomer cement, and thus form part of the cement matrix When the fluoride is released from the cement into the oral cavity and is replaced by other anions, structural changes occur in the cement which may compromise its mechanical strength Secondly, the quick release rate of fluoride from the cement provides a high initial dose, which rapidly decreases within a few days or weeks The fluoride ions must be regenerated regularly in order to provide a continuous effect against bacteria

Therefore, the disadvantage exists that no cements have yet been developed for medicine and dentistry which provide a long term antimicrobial effect

A further, more specific, disadvantage exists in that no cements have been developed for use as endodontic filling materials which provide a long term antimicrobial effect

Other types of materials having antimicrobial properties have been developed For example, U S Patent No 4,938,958, issued on July 3, 1990 to Niira et al , which is incorporated herein by reference, discloses zeolites having antimicrobial properties Zeolites are aluminosilicates having a three dimensional skeletal structure and represented by the following formula

XM2/nO-Al₂O -YSiO₂-ZH₂O

In the general formula, M represents an ion-exchangeable ion and in general a monovalent or divalent metal ion, n represents atomic valency of the (metal) ion, X and Y represent coefficients of metal oxide and silica respectively, and Z represents the number of water of crystallization In antimicrobial zeolites, such as those taught by Niira et al., ion-exchangeable ions present in the zeolite are completely or partially replaced by ammonium and antimicrobial metal ions such as silver, copper, zinc, mercury, tin, lead, bismuth, cadmium, chromium and thallium.

Numerous materials have been prepared containing antimicrobial zeolites. For example, U.S. Patent No. 4,91 1,898, issued March 27, 1990 to Hagiwara et al.; U.S. Patent No. 5,556,699, issued September 17, 1996 to Niira et al.; and U.S. Patent No. 4,937,273, issued June 26, 1990 to Okuyama et al. disclose blending antimicrobial zeolites with polymers to form antimicrobial fibres for fabrics, films for packaging or laminating, and flexible foams.

Composite materials are known comprising a polymerizable binder reinforced with inert organic or inorganic filler particles. Such materials are commonly used as dental restorative materials such as fillings. It would be desirable to provide such materials having antimicrobial properties, and antimicrobial zeolites are known to be incorporated into polymeric materials. However, the inventors are not aware of any commercially available composite materials incorporating antimicrobial zeolites for use as dental restorative materials. Several disadvantages exist with respect to incorporation of antimicrobial zeolites in polymeric materials, which may explain their lack of use. Firstly, the exchangeable ions contained in the zeolite diffuse poorly through polymeric materials. Therefore, a high concentration of antimicrobial zeolite is required to produce an antimicrobial composite material. Secondly, antimicrobial zeolites have been known to cause staining of the polymeric materials in which they are incorporated. This is particularly disadvantageous in composites for dental restorative materials, which must be colour-stable. The staining problem is aggravated by the need to incorporate large amounts of zeolites in the polymeric material. Thirdly, it is believed by some that zeolites do not incorporate antimicrobial metal ions in amounts sufficient to provide an antimicrobial effect, for example in U.S. Patent No. 5,009,898, issued April 23, 1991 to Sakuma et al., at column 2, lines 29 to 3 1.

Therefore, the disadvantage exists that antimicrobial zeolites have not successfully been incorporated in dental restorative materials. Although the above discussion has focussed on composite polymer systems and glass ionomer cements used in dentistry, similar problems exist with cements in general Specifically, there is a need for antimicrobial cements in other fields, such as bone cements for use in medicine, as well as in building materials in industrial, institutional and residential applications For example, tiles are frequently used as a floor or wall covering in wet or humid areas Grouts which are used to fill joints between tiles are frequently subject to deterioration and discolouration by molds and mildew, as well as contamination by bacteria To date, there has been no completely satisfactory solution to this problem

Summary of the Invention

To at least partially overcome the above disadvantages, the inventors have developed antimicrobial cement compositions containing antimicrobial zeolites The cement compositions of the invention generally comprise a cement containing an antimicrobial zeolite in an amount sufficient to prevent growth of bacteria in the composition and over the hardened cement formed when the composition is cured

The cement compositions of the invention have a wide variety of possible applications, including dentistry, medicine, as well as in building materials for institutional, industrial and domestic use Preferably, the antimicrobial cement compositions according to the present invention are used in dentistry or medicine For these applications, the antimicrobial dental cement compositions according to the present invention preferably comprise an anionic component, an inorganic particulate component, and an antimicrobial zeolite The anionic component preferably comprises a liquid medium containing an acid such as phosphoric acid, a polyelectrolyte such as a polycarboxylic acid, or a salicylic acid derivative The inorganic particulate component preferably comprises finely divided metal oxides, metal hydroxides, calcium hydroxyapatite bioglasses, silicate-cement glazes, or glass ionomer particles The antimicrobial zeolite is preferably as described in Niira et al '958, discussed above, and most preferably contains silver ions Most preferably, the antimicrobial cement composition according to the invention is an antimicrobial glass ionomer cement composition comprising a polyelectrolyte, glass ionomer particles and antimicrobial zeolite particles.

The antimicrobial zeolite is preferably contained in the cement composition of the invention in an amount of from about 0.1 to about 30 percent by weight of the powder component, more preferably from about 0.2 to about 20 percent by weight of the powder component.

The most preferred dental uses of the antimicrobial cement compositions of the invention include endodontic filling materials, dental cements, dental lining and restorative materials, dental sealers, and dental luting adhesives, for example to adhere metal brackets to dental tissue. The most preferred medical uses include bone cements, implant components and bone substitutes.

Most preferably, antimicrobial glass ionomer cement compositions of the invention are used in endodontic therapy to provide a bacteria-tight seal in a root canal system. The antimicrobial glass ionomer cement composition of the invention may preferably be used to fill the root canal or may preferably be used as a sealer in combination with a core root canal filling material such as gutta percha (rubber).

The cement compositions of the present invention overcome many of the difficulties experienced with previously known antimicrobial cement compositions. It is believed that when zeolites are incorporated into a cement composition according to the invention, for example a glass ionomer cement composition, it is the zeolites, and not the exchangeable metal ions contained therein, which form ionic bridges with the polyacrylic acid, the glass particles and the substrate. Therefore, when antimicrobial ions are released from within the zeolites, the structures of the zeolites and the cement matrix as a whole are not significantly altered.

Furthermore, the inventors have found that antimicrobial zeolites contained in cement compositions of the invention release antimicrobial metal ions in a highly controlled fashion, providing a substantially constant release of such ions over an extended period of time. Therefore, the inventors expect that antimicrobial cement compositions according to the invention will retain their mechanical stability as well as their antimicrobial activity over a relatively long period of time.

Because the structure of the antimicrobial cement matrix of the invention differs significantly from that of a polymeric composite material, the cement compositions of the invention overcome many of the problems experienced with polymer resins containing antimicrobial zeolites. For example, the inventors have found that antimicrobial ions contained in the zeolites of the cement compositions of the invention may more easily diffuse through the cement matrix to be released in the oral cavity or at the interface between the root filling and the dental tissue This permits the amount of zeolite in the cement to be relatively low, yet sustain the antimicrobial activity of the cement The use of smaller amounts of zeolites permits cements of the invention to be produced economically, and also avoids problems such as staining which becomes more severe when zeolite content is increased Furthermore, because antimicrobial metal ions may diffuse to the surface from the interior of the cement, more antimicrobial metal ions are available for release, and therefore the cement composition of the present invention is expected to maintain its antimicrobial activity for a longer period of time

Accordingly, it is one object of the present invention to provide an antimicrobial cement composition containing antimicrobial zeolites.

It is another object of the present invention to provide an antimicrobial cement composition containing antimicrobial zeolites which is useful in dentistry and medicine

It is yet another object of the present invention to provide an antimicrobial cement composition containing antimicrobial zeolites for use as a dental sealer, adhesive and filler, which provides controlled, long-term release of antimicrobial metal ions.

It is yet another object of the present invention to provide an antimicrobial glass ionomer cement composition containing antimicrobial zeolites for use as a dental sealer, adhesive and filler, the cement composition providing controlled, long-term release of antimicrobial metal ions

It is yet another object of the present invention to provide an antimicrobial glass ionomer cement composition for use as an endodontic filling and sealing material which provides long-term protection against ingress of bacteria into a filled root canal Brief Description of the Drawings

Further aspects and advantages of the present invention will become apparent from the following description, taken together with the accompanying drawings, in which"

Figure 1 is a plot of optical density versus incubation times, comparing the antimicrobial activity of an antimicrobial GIC composition according to Example 1 of the invention with a conventional GIC composition,

Figure 2 is a flow chart description of the microbiology study of Example 2,

Figure 3 shows bacterial colony counts from substrates of Example 2 following exposure to bacteria for 1, 3, 7, 10, 15, 30 hours Prior to inoculation the substrates were incubated in a bacteria free medium for 84 days Averages of three measurements were taken for bacterial colony counts (1000 colonies were labelled as having complete growth),

Figure 4 shows the change in the average optical density values measured for each group from Example 2 at 84 days as a function of bacteria culture time

Figure 5 shows the statistical significance of interactions between the different ZUT materials of Example 2 as measured by the lumped optical density values (over the entire 84 day experiment) versus the bacteria culture time Statistical differences were found between the ZUT materials at 7 and 10 hours (p < 0 0001)

Figure 6 shows the cumulative silver ion release (ppm) for ZUT 2% and ZUT 2% of Example 2 over a period of 84 days

Detailed Description of Preferred Embodiments

Preferred antimicrobial cement compositions according to the present invention comprise a cement and an antimicrobial zeolite

The type of cement used in the preferred antimicrobial cement compositions of the invention is at least partially dependent on the intended use of the cement composition For example, antimicrobial cements for building materials used in industrial, institutional and domestic applications preferably comprise a hydraulic cement and an antimicrobial zeolite

The term "hydraulic cement" is to be understood as including any mixture of fine- ground lime, alumna and silica that will set to a hard product by admixture of water which combines chemically with the other ingredients to form hydrate A preferred hydraulic cement is Portland cement

Preferred types of building materials into which antimicrobial cement compositions according to the present invention may be incorporated include grout, mortar and concrete, which are preferably formed by admixture of the antimicrobial cement composition with an aggregate selected from the group comprising sand, gravel and crushed stone, and optionally with other conventional ingredients For example, a cement composition of the invention may be used as a component in a mildew and bacterial resistant tile grout for use in bathrooms, hospitals or other areas where cleanliness is important

The present invention also includes within its scope tiles and similar building materials incorporating antimicrobial zeolites, whether or not such tiles or other materials are made from or contain cement

More preferably, the antimicrobial cement composition of the invention contains a cement for medical or dental use Preferred medical cements include bone cements, implant components and bone substitutes, and preferred dental cements include cements for use as dental adhesives, sealers, fillers and restorative materials Most preferably, the cement is an endodontic filler or an endodontic sealer to be used in combination with a core filling material of different composition, for endodontic treatment of root canal systems

Preferred types of cements for medical and dental use preferably comprise an anionic component, an inorganic particulate component, and an antimicrobial zeolite The anionic component preferably comprises a liquid medium containing an acid such as phosphoric acid, a polyelectrolyte such as a polycarboxylic acid, or a salicylic acid derivative The inorganic particulate component preferably comprises a finely divided metal oxides, metal hydroxides, calcium hydroxyapatite bioglasses, silicate-cement glazes, or glass ionomer particles

Most preferably, the antimicrobial cement composition according to the invention is an antimicrobial glass ionomer cement composition comprising a polyelectrolyte, glass ionomer particles and antimicrobial zeolite particles This antimicrobial cement composition is particularly preferred as an endodontic filling and sealing material for use in endodontic treatment of root canal systems.

The preferred antimicrobial glass ionomer cement compositions according to the present invention include resin modified glass ionomer cements which include a small amount of a resin monomer to provide the cement with improved compliance, as well as metal-modified glass ionomer cements.

Zeolitic structures are comprised primarily of an aluminosilicate framework of alkali or alkali earth metals, with a regular three-dimensional skeleton consisting of a methane-type tetrahedron of linked SiO and AJO , the oxygen atoms being shared. This framework can house exchangeable ions, typically cations.

Antimicrobial zeolites which are preferably used in the antimicrobial cement compositions of the present invention include any zeolites in which the exchangeable ions have antimicrobial activity. Preferred ions include ammonium ion and antimicrobial metal ions. Preferably, the antimicrobial metal ions are selected from one or more members of the group comprising silver, copper, zinc, mercury, tin, lead, bismuth, cadmium, chromium and thallium ions; more preferably silver, copper and zinc ions; and most preferably silver ions. The content of the antimicrobial metal ions in the zeolite is preferably from about 0.1% to about 15% by weight of the zeolite.

Particularly preferred zeolites used in the cement compositions of the invention contain silver ions and ammonium ions, and may preferably also contain copper and/or zinc ions. Silver ions are preferably contained in the zeolite in an amount of from about 0.1% to about 15% by weight of the zeolite. In zeolites which contain copper and/or zinc ions, the preferred total content of copper and/or zinc ions is from about 0.1% to about 8% by weight of the zeolite. Ammonium ion is preferably contained in the zeolite in an amount of less than about 20% by weight of the zeolite, more preferably from about 0.5% to about 15% of the zeolite, and most preferably from about 1.5% to about 5% by weight of the zeolite.

The most preferred zeolites for use in the cement composition according to the invention are zeolites sold under the trade mark Zeomic™ by Shinagawa Fuel Co., Ltd. The particle size of the zeolite particles is preferably from about 20 to about 50 microns. Cement compositions according to the present invention preferably contain from about 10%) to about 95% filler, with the antimicrobial zeolite being present in amounts of up to about 30%) by weight of the filler. More preferred cements according to the present invention are antimicrobial glass ionomer cement compositions containing antimicrobial zeolite particles in amounts of up to about 20%> by weight of the glass ionomer particles, and more preferably from about 0.2% to about 20%> by weight of the glass ionomer particles.

In particularly preferred antimicrobial cement compositions of the invention, the cement compositions contain antimicrobial zeolites in amounts which substantially do not cause staining of the cement composition, but which provide the cement composition with acceptable antimicrobial activity. The preferred amount of antimicrobial zeolite to accomplish these objects is from about 0.2%> to about 2%o by weight of the powder component of the antimicrobial cement composition.

The present invention also includes within its scope the incorporation of antimicrobial zeolites in dental cements selected from the group comprising zinc phosphate, zinc oxide eugenol (ZOE), silicophosphate and polycarboxylate dental cements.

The present invention also includes within its scope the use of antimicrobial zeolite cements in combination with conventional, non-cementitious dental restorative materials including polymeric materials such as gutta percha, polymeric composite materials, calcium hydroxide, and pulp canal sealers, as well as in combination with porcelain for crowns.

The inventors have found that antimicrobial cement compositions according to the present invention are capable of preventing, or reducing by at least 50%>, the ability of bacteria to penetrate through filled root canals for periods of at least 90 days. The ions leach out of the antimicrobial zeolite to the surface of the root canal filler or sealer of the invention, and are believed to disrupt bacterial activities such as cellular respiration, enzyme activation and active transport from the cell wall, with subsequent inhibition of the bacterial cell function and proliferation. The antimicrobial cement compositions of the present invention are also expected to have efficacy against a wide range of bacteria, including the at least fifty strains of bacteria which have been isolated from root canals, such as Enterococcus faecalis, Actinomyces, Lactobacillus, black-pigmented Bacteroides, Peptostreptococcus, non- pigmented Bacteroides, Veillonetia, Fusobacterium nucleatiim, and Streptococcus mutans. The cement compositions of the invention preferably also have activity against molds, fungi and algae.

Examples Example 1

A variation of the direct contact test (DCT) was used to compare the bactericidal ability of a preferred antimicrobial glass ionomer cement composition of the invention with that of a conventional glass ionomer cement. The DCT test is suitable to test insoluble materials such as cements since it relies on direct and close contact between the test microorganism and the tested material and is virtually independent of the diffusion properties of both the tested material and the media.

The results of Example 1 are shown in Figure 1. The antimicrobial cement composition tested comprised a glass ionomer cement sold under the trade mark ChemFill II™, containing 2% by weight of a silver-containing zeolite sold under the trade mark ZEOMIC by Shinagawa Fuel Co., Ltd., which is identified in figure 1 as ZUT2. The antimicrobial glass ionomer cement ZUT2 was placed in a vial containing brain heart infusion (BFfl) culture media and was inoculated with Enterococcus faecalis. The optical density of the vial was observed over a period of 30 hours, turbidity in the culture media being indicative of bacterial proliferation. The optical density of antimicrobial glass ionomer cement sample ZUT2 was compared with samples containing inoculated media, inoculated ChemFill II without zeolite (CF), and a paper disc control (PD).

Optical density is a means by which to measure the clarity of a solution containing particulate, in this case bacterial, growth. Optical density is measured by determining the degree of light that can be transmitted through the test specimen. A low value indicates low bacterial growth activity, while a high value indicates the presence of elevated bacterial growth activity.

The results of this test showed that the antimicrobial glass ionomer cement composition ZUT2 effectively prevented all growth of Enterococcus faecalis from its surface within 10 hours of inoculation. On the other hand, the samples of the unmodified glass ionomer cement CF and the paper disc control PD showed elevated growth of bacteria as indicated by high turbidity in the culture vessels.

Example 2

A study was undertaken to characterize the anti-bacterial properties of various antimicrobial glass ionomer cement compositions of the present invention (ZUT formulations) and to compare them to a standard glass ionomer formulation. Materials were assessed for their physical characteristics and their ability to suppress adherent E. faecalis and sustain anti-bacterial function over a period of 3 months.

The objective of this study was to assess the efficacy of a modified endodontic filler (ZUT), consisting of an experimental glass ionomer (GI), KT-308 (GC Corp.,

Japan), and an anti-microbial agent (AA). Five experimental groups were studied: Group

1- KT-308, Group 2- ZUT-.2%, Group 3- ZUT-2%, Group 4- ZUT-20%, Group 5- paper disc (blank control), where .2, 2, and 20%> refer to the respective wt % concentration of

AA in the ZUT materials. In order to evaluate the ability of each material to eliminate bacterial growth, the following experiment was carried out. Discs were incubated in BHI

(Brain Heart Infusion) at 37°C for a period of 84 days. At 14-day intervals, discs were inoculated with a suspension of Enterococcus faecalis. The discs were incubated at

37°C and removed at intervals of 1,3,7, 10, 15 and 30 hours. At each time interval the discs were washed in fresh BHI media to remove non-adherent bacteria, and then transferred to fresh media. Following further incubation for 8 hours, 200 μl aliquots of the medium were pippeted onto agar plates, which were then incubated for 8 hours and assessed for colonies. The remaining medium was used to measure its optical density in a spectrophotometer. The three ZUT materials displayed bactericidal effects throughout the 84-day experiment, as expressed by complete elimination of bacteria within 15 hours of inoculation. Groups 3, 4 and 5 displayed their bactericidal effects at 7, 10 and 15 hours respectively. Groups 1 and 2 were positive for growth during the entire 84 days. It was concluded that ZUT provided a significant bactericidal effect relative to glass ionomer alone and that this effect was maintained for approximately 3 months during exposure to an aqueous environment.

Materials and Methods

The materials were arranged into five study groups, comprised of the following: Group 1 - KT-308 (a GIC); Group 2 - ZUT 0.2%; Group 3 - ZUT 2%; Group 4 - ZUT 20%; Group 5 - a paper disc (blank control). KT-308 is an experimental cement/sealer glass ionomer provided in kind by GC Corp., Japan. ZUT formulations were prepared with the GIC and Zeomic AJ 10D (Shinagawa Fuel Co., Nagoya, Japan), which is a silver containing zeolitic agent. ZUT formulations were prepared by blending KT with Zeomic® in percentages of 20%, 2%>, and .2%) with respect to the ceramic content. Using a custom made Teflon® mold, the materials were prepared to form discs (n=108 for each group) with dimensions of 0.25 cm x 0.08 cm. A slight overfill of the mold was introduced in order to compensate for material shrinkage. During setting, the molds were covered with Mylar sheets and glass slides to render the disc surfaces optically smooth. After being fully set, discs were placed into sterile 6-ml vials containing Brain Heart Infusion (BHI; Difco, Detroit, MI) medium, and incubated at 37°C for up to 84 days. This is referred to as the incubation stock.

A modification of the direct contact test that was reported by Palenik et al (Inhibition of microbial adherence and growth by various glass ionomers in-vitro, Dental Materials. 1992; 8: 16-20) was used to evaluate the antibacterial efficacy of the materials. A flowchart of the complete protocol is given in Figure 2 and is described as follows. At 14-day intervals, eighteen discs from the experimental groups were removed from the incubated stock and inoculated with a 200μl aliquot of E. faecalis. E. faecalis ATCC 29212 was grown in BHI ( 18g/ 500ml) from frozen stock culture (American Type Culture Collection, Rockville, MD). The purity of the culture was maintained by quadrant streaking on a weekly basis. Discs were incubated in a humid environment at 37°C to allow for bacterial adherence. At pre-defined incubation periods of 1, 3, 7, 10, 15 and 30 hours, three discs from each group were removed and gently rinsed with 3 ml of fresh BHI in sterile petridishes in order to discard non-adherent bacteria. Each disc was then transferred to a test tube containing 0.5 ml of fresh BHI, and incubated for 8 hours. It was then washed twice for 15 min with 3 ml of fresh BHI in a sterile petridish to again remove any non-adherent bacteria from its surface, transferred to a sterile test tube with 3 ml BHI, and incubated for a final 8 hours. An aliquot of 200 μl from the latter incubation medium was then pippeted onto an agar plate (BHI agar 26 g/500 ml) and spread evenly on the surface. The agar plates, one for each disc, were incubated for 8 hours and assessed for growth of bacterial colonies. The remaining incubation solution was pippeted into individual plastic curettes, and the optical density was measured with a spectrophotometer (LKB Biochrome, Cambridge, U.K.) at a wavelength of 560 nm. The absorbency of pure BHI was measured as a reference sample. Each disc that showed a significant reduction in growth, in the fore-mentioned procedures, was subjected to a direct sampling of its surface to assess for cell viability. The surface of each disc was gently scraped with a sterile scalpel blade (Lance, Sheffield, U.K.) and the shavings washed with 3 ml fresh BHI into a sterile petridish. The resulting medium was pippeted onto an agar plate, incubated for 8 hours, and assessed for growth of bacterial colonies.

Discs from groups 2 to 4 were characterized for changes in hardness using a Knoop value hardness test (Leitz Wetzlar, Germany). A weight of 300 ponds was used for this test and ten disc specimens were measured from each group. Three measurements were taken and averaged for each disc. Measurements were obtained for post incubation periods of 28 days and 84 days.

The incubation stock from 28, 56 and 84 days was analyzed for silver ions that diffused from ZUT materials into the incubation stock. Ten samples each of ZUT .2% and ZUT 2% from each of the above time periods were diluted 1 : 121 with 1% Triton X- 100. All samples were measured by a graphite furnace atomic absorption spectrophotometry (GFAAS) located in Dr Stanley Lugowski's laboratory at the Centre of Biomaterials, using Varian AA-875 spectrophotometer and Varian GTA-95 (Melbourne, Australia).

Colony counts and optical density data were statistically analyzed by a factor analysis of variance (Statistical Analytical Systems, Cary, NC). Interactions among the three factors (materials, days and hours) were tested using the same statistical method, ANOVA. The least square means comparison was applied to examine interactions. Correlation coefficients were calculated between optical density and colonies for the data combining interactions among the three factors.

Results

Bacterial colony counts for the 84-day samples are shown in Figure 3 and typically represent the observed performance of all materials for the different time points over the 84-day experiment. The data reflect the measure of each material's ability to sustain adhesion and eventual growth of E. faecalis. While all materials showed evidence of growth after three hours of incubation with the bacteria, ZUT materials exhibited a complete elimination of bacteria within 15 hours of incubation. The strongest effect was exhibited by ZUT 20, which only had 43 colonies while ZUT .2%,

ZUT 2%> and the two controls had greater than 1000 colonies at 10 hours. The non-ZUT materials exhibited no anti-microbial activity towards E. faecalis throughout the complete incubation period. A plot of the optical density versus incubation times revealed a gradual drop in turbidity over 30 hours for media that contained ZUT materials (Figure 4). This gradual drop in turbidity corresponds to the drop in colonies observed for the different materials in Figure 3. In contrast, media containing non-ZUT materials permitted growth of bacteria, which was exhibited by the high turbidity value.

A correlation coefficient (O calculated between optical density and colony growth was determined to be Y = 0.997. This correlation, obtained using a three-way analysis

(ANOVA) was highly significant (p<0.0001). A three way ANOVA was also used to assess the effects of media exposure time on the materials and their ability to sustain growth of bacteria as measured by optical density. The ZUT materials were shown to have a significant material/bacteria interaction (p<0.0001 ) for the 7 and 10 hour bacteria incubation periods (Figure 5), with all media exposure times (i.e. 14, 28.... and 84 days). Based on this analysis, it was also shown that there was no prominent anti-bacterial activity demonstrated by Groups 1 and 5 for any time interval.

There were no viable cells found on the ZUT materials, which demonstrated an absence of bacterial growth in the media (Table 1 ). Complete suppression of bacterial growth was observed at 15 hours for ZUT materials.

Table 1 : Viable E. faecalis cells removed from the surface of ZUT materials is indicated by '+' at 10 hours. Absence of cells is denoted by '-', indicating no cells were recovered after 15 hours.

Trace elemental analysis was conducted to study the amount of silver being released from ZUT materials over the 84 period. Data in Figure 6 illustrate that the amount of silver ions released is in the order of parts per million. Note that a ten fold increase in zeolite content only translated to approximately a two fold increase on silver ion release over 28 days and an approximate 5 fold increase over 84 days (Figure 6). While there were slight variations in hardness values for ZUT materials between the 28 and 84 day period, no significant difference was found between them (Table 2).

Table 2: The Knoop values in the above table are of ZUT materials after they were incubated for 28 and 84 days. A weight of 300 ponds was used for all three materials. The microhardness of each material is measured in kp/mm2.

Discussion

The purpose of this study was to assess the ability of ZUT materials to suppress the growth of E. faecalis. Materials were exposed to culture media for different intervals over a period of 84 days and then exposed to bacteria. Groups 2, 3, 4, which contained Zeomic® in ratios of .2%, 2%, 20% proved to be effective against E. faecalis. E. faecalis is one of the few facultative microorganisms and the most resistant to be found in the root canal. A common isolate found in infected root canals, it makes up a small percentage of the root canal flora, and may be favoured by ecological changes that establish infections, which are difficult to treat. E. faecalis has been used previously in studies of root canal dis-infection and is known to survive in environments where the pH is as high as 1 1.5.

An important requirement of endodontic sealers is their insolubility. In this context ZUT is not soluble and therefore its anti-microbial activity could not be measured using a routine agar diffusion test. An effective approach was to measure its ability to inhibit bacterial growth on its surface. A direct contact test (DCT) is taught by Weiss et al, Assessment of Antibacterial Activity of endodontic sealers by a direct contact test, Endodontics and Dental Traumatology 1996, 12 179-184 The modification of the direct contact test (DCT) used in the current investigation was less dependent on the diffusion properties of the materials and was therefore advantageous to this study

During the 84 day period it was clearly demonstrated (Figure 6) that silver ions were diffusing into the surrounding media The amount of silver ions detected in media varied from 319 to 3523 ppb (Figure 6) and was dependent on the percentage of Zeomic® present in the material This release of silver ions showed a continuous accumulation through out the 84-day period, which indicates that the ion exchange is permitted over an extended period of time Based on the quantity of zeolite added to each disc the highest possible amount of silver ions that could be released into 2 5 ml of incubation stock was calculated to be 7000 ppb for a single ZUT 2% disc Therefore there would still be the potential for more silver ions to diffuse into the media and provide a sustained bactericidal effect beyond 84 days since ZUT 2% only released about one tenth of this amount (Figure 6) The levels of silver ions diffusing through the matrix were sufficient enough to reduce and ultimately resist adhesion of E. faecalis (Figures 2 & 3) ZUT materials were able to suppress the adherence of bacteria repeatedly for samples retrieved throughout the 84-day period In a study conducted by Moroz et al (Susceptibility of organisms which cause intestinal infections to removal by Silver, Khim Technol (Kiebv), 1980' 2"275), it was stated that 50 to 200 parts per billion of silver was sufficient enough to kill Salmonella and E. colt, as well as bacteria highly resistant to antibiotics (21) As indicated in Figure 6, over a period of 84 days an average of more than 600 parts per billion of silver ions were released by ZUT 2% This is three times the amount required to kill some bacteria, as reported by Moroz et al.

The findings of this /// vitro study suggest that ZUT materials that were in contact with the biological media sustain activity against adherence of E. faecalis for at least 84 days It had been reported in U S Patent 5,556,699 to Niira et al, issued September 17, 1996 that the silver can bind within the zeolitic structures and this most likely reflects the gradual and long lasting release of the ions, as well as the continued anti-microbial character of ZUT materials (Figure 2) Ag+, Zn2+, are a few of the cations that can be localized within the zeolitic structures, which are also potent anti-microbial agents Cations, such as silver are electrostatically attracted to the negatively charged microorganism and may then undergo reactions on the surface. Once the cations are in the cell, they can act at sites of RNA, DNA, or enzymes, eventually disrupting the entire mechanism of the cell. The released silver is believed to exert a bactericidal effect through its' reducing action.

Since present day glass ionomers are thought to loose their structural integrity through a complex process of adsorption, disintegration and outward transportation of ions, there was interest in assessing changes in surface hardness as a preliminary determination of material stability. There were no significant changes in the hardness values of the ZUT materials between 1 to 3 months (Table 2) which indicates that the diffusion of silver ions on the structural nature of the ZUT materials appears to minimal, although further work will be required to assess compressive and tensile strength properties.

In summary this study indicates that ZUT materials have the potential to suppress microbial growth and metabolism in the local micro-environment of the root canal. It offers the clinician some information regarding the quality and properties of ZUT-based GICs. The ZUT materials were shown to inhibit and suppress growth of E. faecalis. The anti-bacterial effects were provided with low concentrations of Zeomic® and therefore the handling properties of the materials still reflect those of traditional GIC materials that are close to an ideal filler, as required in endodontic practice.

Although the invention has been described in connection with certain preferred embodiments, it is not intended to be limited thereto. Rather, it is intended that the invention cover all alternate embodiments as may be within the scope of the following claims. The invention also includes all embodiments which are functional equivalents of the specific embodiments and features which have been described herein.

It will be further understood that, although various features of the invention have been described with respect to one or another of the embodiments of the invention, the various features and embodiments of the invention may be combined or used in conjunction with other fetures and embodiments of the invention as described herein.

Claims

Claims:

1. An antimicrobial ionomer cement composition, comprising

(a) a polyelectrolyte;

(b) glass ionomer particles; and

(c) antimicrobial zeolite particles, wherein, when the composition is cured to form a hardened cement, the glass particles and zeolite particles form ionic bonds with the polyelectrolyte, and wherein the zeolite particles are present in the composition in an amount sufficient to prevent growth of bacteria in the composition and the hardened cement formed by curing the composition.

2. The cement composition of claim 1, wherein the antimicrobial zeolite particles are comprised of a zeolite containing antimicrobial metal ions.

3. The cement composition of claim 2, wherein the antimicrobial metal ions are contained in the zeolite in an amount of from about 0.1 to about 15 percent weight of the zeolite.

4. The cement composition of claim 2, wherein the antimicrobial metal ions are selected from one or more members of the group comprising ions of silver, copper, zinc, mercury, tin, lead, bismuth, cadmium, chromium and thallium.

5. The cement composition of claim 4, wherein the antimicrobial metal ions comprise silver ions.

6. The cement composition of claim 4, wherein the zeolite additionally comprises ammonium ion in an amount not greater than about 20 percent by weight of the zeolite.

7. The cement composition of claim 1, wherein the cement composition is one or more of a dental cement, a dental restorative material, a dental sealer, a dental adhesive, a bone cement, an implant component and a bone substitute.

8. An antimicrobial cement composition, comprising:

(a) an acid selected from the group comprising phosphoric acid, polycarboxylic acids, and salicylic acid derivatives;

(b) filler particles selected from one or more members of the group comprising metal oxides, metal hydroxides, calcium hydroxyapatite bioglasses, silicate- cement glazes, and glass ionomer particles; and

(c) antimicrobial zeolite particles, wherein, when the composition is cured to form a hardened cement, the filler particles and zeolite particles form ionic bonds with the acid, and wherein the zeolite particles are present in the composition in an amount sufficient to prevent growth of bacteria in the composition and the hardened cement formed by curing the composition.

9. The cement composition of claim 8, wherein the antimicrobial zeolite particles are comprised of a zeolite containing antimicrobial metal ions.

10. The cement composition of claim 9, wherein the antimicrobial metal ions are contained in the zeolite in an amount of from about 0.1 to about 15 percent weight of the zeolite.

1 1. The cement composition of claim 9, wherein the antimicrobial metal ions are selected from one or more members of the group comprising ions of silver, copper, zinc, mercury, tin, lead, bismuth, cadmium, chromium and thallium.

12. The cement composition of claim 9, wherein the antimicrobial metal ions are The cement composition of claim 9, wherein the antimicrobial metal ions are comprise silver ions.

13. The cement composition of claim 1 1, wherein the zeolite additionally comprises ammonium ion in an amount not greater than about 20 percent by weight of the zeolite.

14. The cement composition of claim 8, wherein the cement composition is one or more of a dental cement, a dental restorative material, a dental sealer, a dental adhesive and a bone cement.

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SUBST1TUTE SHEET (RULE 26)