Abstract
Objectives
The occurrence of recurrent caries at the interface of dental materials and the enamel surface is an important performance issue. The objective of this study was to investigate the most effective way to control the release rate of bioavailable phosphate ions contained in aqueous solutions within ion permeable microcapsules formulated in to rosin based varnishes and resin based sealants, in order to promote remineralization.
Methods
Microcapsules that contained aqueous solutions of K 2 HPO 4 with concentrations from 0.8 to 7.4 M were prepared. 3–50 w/w% of microcapsules were loaded into both rosin and resin based dental formulations.
Results
The effect of initial salt solution concentration inside the microcapsules and weight percent loading of the microcapsules on release rate were contrasted. The effect of microcapsule loading was found to be highly dependent on the continuous phase. In rosin, 3–15 w/w% loading resulted in rapid release of ions. Higher weight percent loadings were initially slower but resulted in sustained release of ions. In resin, 3–15 w/w% formulations slowly released ions for at least 300 days, while higher loading formulations released an initial burst of ions. Initial salt solution concentration contained inside the microcapsule affected ion release rate. Initial rate of ion release was greatest at a concentration that was less than the maximum concentration studied in both continuous phases.
Significance
Phosphate ion release can be controlled from resin or rosin based dental material by adjusting initial salt solution concentration in microcapsules or percent loading of microcapsules. The potential for burst release from a varnish or slow, sustained release from a sealant has been demonstrated.
1
Introduction
From a historical perspective, the field of dentistry is beginning to broaden its direction of patient care from surgical procedures (e.g. restorations) to preventative approaches that work to interrupt the caries process . Tooth decay continues to affect adults and children, and can be influenced by factors such as consumption, hygiene, and fluoride exposure . As this epidemic lingers among society the methods of prevention have been forced to evolve. This is partly because when successful, preventative methods can provide a cost-effective treatment plan to those susceptible to decay and to those with limited options for treatment. Oral hygiene is the easiest means of preventing the initiation and spread of bacterial decay. Current products such as varnish, toothpaste and sealants have all targeted this process. One approach to treat this disease is through a course known as tooth remineralization. Remineralization is accomplished when ions such as calcium, fluoride, and phosphate contribute to the rebuilding of enamel. Although there are plenty of products which claim to inhibit demineralization and rebuild tooth structure, the dental field continues to search for new methods that challenge the progression of caries.
The cycle of remineralization and demineralization is a continuous one that takes place naturally in the oral environment, with and without dental therapies. Typically, it starts at the enamel surface. Dental enamel is composed primarily of inorganic mineral (approximately 95–98%), and is often described as hydroxyapatite (HAP) with a molecular formula of Ca 10 (PO 4 ) 6 (OH) 2 . However, actual biological apatite has other impurities (such as carbonate) which can influence the solubility of the tooth structure . It is more accurate to describe enamel as carbonated hydroxyapatite, which has slightly different properties from pure hydroxyapatite .
Caries is a disease that affects the tooth by disrupting its inorganic structure. It begins with the ingestion of sugars, often in the form of carbohydrates that are metabolized by bacteria ( Streptococcus mutans ) into organic acids. If the acidic environment is persistent, the tooth structure becomes highly susceptible to decay . S. mutans is often affiliated with the caries process because it is both acidogenic and aciduric. When bacterial metabolism occurs, there is a decrease in pH in the fluids surrounding the teeth and gums. HAP dissolves if the pH drops below the critical pH and if the saliva is undersaturated with respect to the tooth . The concentration of ions (e.g. calcium, phosphate) present in the saliva help to determine whether HAP will precipitate or dissociate in the oral environment. The longer the tooth is exposed to a lower pH, the faster the rate of dissolution. Often times tooth decay appears as a white spot, and lesions with this appearance are described as areas of demineralization below the surface of the tooth . The outer surface is believed to form as calcium and phosphate ions diffuse from the enamel and into the plaque fluid .
With the help of saliva, remineralization is a process that can naturally take place in the oral environment. Remineralization is stimulated by the supersaturation of ions in the saliva, especially when the pH is greater than the critical pH and the mineral can precipitate . When the pH drops below the critical pH, the saliva is no longer saturated and the tooth will demineralize . The cycle of remineralization and demineralization in the mouth is a continuous one, but if ion loss continues unchecked, a permanent carious lesion will result . Mineral loss and repair is determined by the degree of saturation of the fluid; typically, ions needed for tooth repair are provided by saliva in the oral environment . Thermodynamic supersaturation of enamel is determined by the ions which contribute to the structure and the solubility product of the mineral . The progression of mineral loss has been replicated in numerous experiments as an attempt to understand how the process occurs. While enamel loss in vitro can occur rapidly under certain conditions, caries development in vivo is commonly understood to occur over a longer period of time .
The role of fluoride in the process of remineralization has recently been investigated. Aside from its incorporation into drinking water, fluoride has been utilized in products such as cavity varnish, toothpaste, and mouthwash. The greatest challenge facing products aimed at rebuilding tooth structure is the bioavailability of ions able to perform this task. In terms of fluoride (as provided in a clinical setting), bioavailability has been described by White as, “the amount of fluoride taken up by enamel/dentin, the retention of fluoride within enamel and/or dentin, or the ability of the treated surfaces to provide low levels of fluoride in saliva.” It has been claimed that fluoride may be capable of replacing hydroxyl ions in the enamel structure, creating fluoridated hydroxyapatite (FAP) . However, Ten Cate et al. also claimed that the precipitation of fluoridated hydroxyapatite onto the enamel (or rather, unincorporated in the tooth) may also be beneficial . The use of fluoride provides a unique benefit to the rebuilding of tooth structure, because when FAP is formed, it provides better protection against acidic environments. This could be explained by the lower critical pH and a lower solubility product of FAP relative to tooth structure .
In addition to the importance of fluoride ions for remineralization, calcium and phosphate ions must be present for the process of remineralization to occur . This justifies the incentive to create new products which utilize calcium and phosphate for enamel repair. For instance, formulations have included casein phosphopeptide to create calcium phosphate derivatives such casein phosphopeptide amorphous calcium phosphate (CPP-ACP) . This was devised as an effort to stabilize calcium and phosphate and localize ion release near the tooth surface . The role of CPP as a binding agent has been claimed to be successful because of its ability to localize calcium and phosphate for enamel repair, and has been utilized in products such as Recaldent™ . Amorphous calcium phosphate (ACP) products have also been marketed without the CPP component. Even as an independent structure, ACP is believed to precipitate as HAP . ACP has been utilized in toothpaste and dental composites in an effort to remineralize . Another approach uses a calcium sodium phosphate silicate bioactive glass that releases ions when combined with saliva . Ideally, when placed in the oral cavity, calcium and phosphate dissociate from the structure and precipitate near the surface of enamel .
Despite current efforts, the challenge still remains to create a ubiquitous platform that can deliver bioavailable ions from a variety of dental materials that are capable of promoting remineralization and preventing demineralization. Our research group has recently reported the synthesis of ion permeable microcapsules containing aqueous salt solutions useful for remineralization . Salts that contained either a calcium, phosphate, or fluoride ion were solvated in an aqueous solution within the microcapsule. Key variables that affected the rate of ion release from the microcapsule into nanopure water were identified in the previous study. This approach confirmed the potential of solvated salts to exogenously release bioavailable calcium, phosphate, and fluoride ions into the saliva for a targeted period of time. The presence of bioavailable ions is critical because remineralization cannot occur unless ions are capable of releasing into the plaque fluid and supersaturate near the enamel surface.
The purpose of this study was to determine the most effective method of delivering phosphate ions from a dental material. Ion permeable microcapsules containing solvated phosphate ions were incorporated into two different types of continuous phases. Phosphate ion release profiles were measured as a function of the initial concentration of the solvated salt within the microcapsule and by the percent loading of the microcapsules. This study explores the possibility of controlling the rate and longevity of phosphate ion release and whether it can be controlled to conform to a variety of dental therapies through the release of either an immediate abundance of ions (e.g. useful in a rosin varnish) or long term sustained release (e.g. useful in a glaze or sealant).
2
Materials and methods
2.1
Prepolymer synthesis
A polyurethane based prepolymer was prepared as the material for the microcapsule shell. Ethylene glycol (Fisher, New Jersey) was used as the diol source, and was combined with toluene-2,4-diisocyanate (Sigma–Aldrich, Steinheim). A 1:2 stoichiometrically limited ratio of diol to diisocyanate functional groups reacted in cyclohexanone at 70 °C in an inert atmosphere. The solvent was removed by vacuum.
2.2
Microcapsule synthesis
The oil phase consisted of the ethylene glycol polyurethane prepolymer, an emulsifying agent, and methyl benzoate (Acros Organics, New Jersey). Potassium phosphate dibasic (Fisher Scientific, New Jersey) aqueous solutions were made using ionic concentrations of 0.8 M, 2.4 M, 4.0 M, and 7.4 M with respect to the phosphate ion. Microcapsules were synthesized using a reverse emulsion which had an excess of oil to water by volume . The oil solution was heated to 70 °C in a custom-made stainless steel reactor that was submerged in a Büchi BS-490 heating bath. To create a reverse emulsion, the aqueous phosphate solution was added to the reactor slowly. The reactor mixed at 4000 rpm by a Caframo BDC 6015 stirrer. A stoichiometric excess of the associated diol-containing monomers was subsequently added to the reaction to quench the isocyanate functionalities remaining in the prepolymer. After the synthesis, microcapsules were isolated via centrifugation using a Fisher Centrific 288 centrifuge.
2.3
Dental formulation
2.3.1
Rosin based formulation
Rosin varnish formulations were created using Rosin CH (partially hydrogenated rosin, Staybelite ® -type Resin, Wilmington) combined with ethanol. For our purposes, rosin/ethanol was combined at a ratio of 75/25. The rosin was manually ground using a mortar and pestle and added to ethanol. The rosin and ethanol mixture were mixed at 3540 rpm for 2-min intervals using a Speed Mixer™ DAC150FVZ four to five times, or until the mixture was homogenous. Microcapsules were added next and the formulation mixed for another 2 min. The varnishes were transferred into washers (Nylon standard flat washers obtained from Washers USA. Outer dimension: 15.875 mm, inner dimension: 9.525 mm, thickness: 0.8128 mm, surface area: 71.3 mm 2 , total volume: 57.9 mm 3 ) which were fixed to glass microscope slides (Fisherfinest ® premium microscope slides, 3″ × 1″ × 1 mm) using a standard water resistant adhesive (Amazing Goop ® , Eclectic Products Inc, Pineville). Each slide consisted of three washers. The slide and varnish specimens dried overnight.
2.3.2
Resin based formulation
Resin formulations were made by first mixing the monomers TEGMA (Sigma–Aldrich, St. Louis):MMA (Acros Organics, New Jersey):UDMA (Esstech, Pennsylvania) at a ratio of 45:45:10 for 2 min at 3540 rpm (resulting in a relative centrifugal force of approximately 680 × g ) using a Speed Mixer™ DAC150FVZ (Flacktek Inc., South Carolina). Microcapsules were then added (at a targeted weight percent) to the combined monomers and the formulation mixed for 2 min at 3540 rpm. The quantity of microcapsules in the formulations varied, however, the ratio of TEGMA:MMA:UDMA remained 45:45:10 in all formulations. The photoinitiators, camphorquinone (CQ) and ethyl-4-dimethylaminobenzoate (E4DMAB) (Sigma–Aldrich, St. Louis), were added next and the formulation mixed for another 2 min at 3540 rpm. The formulations were transferred into washers (Nylon standard flat washers obtained from Washers USA. Outer dimension: 15.875 mm, inner dimension: 9.525 mm, thickness: 0.8128 mm, surface area: 71.3 mm 2 , total volume: 57.9 mm 3 ) which were fixed to glass microscope slides (Fisherfinest ® premium microscope slides, 3″ × 1″ × 1 mm) using a standard water resistant adhesive (Amazing Goop ® ). Each slide consisted of three washers. The resin varnish was irradiated by using a Spectrum 800 curing light at 600 mW/cm 2 for 2 min (per washer). Specimens cured for an additional 5 min using a DENTSPLY Triad ® Visible Light Cure System.
For both the resin and rosin formulations, we studied the release of phosphate ions from microcapsules containing 2.4 M potassium phosphate dibasic when loaded into varnishes at 3 w/w%, 9 w/w%, 15 w/w%, 30 w/w% and 50 w/w%. We also studied the effect of the initial concentration of the salt within the microcapsule at 0.8 M, 2.4 M, 4.0 M and 7.4 M potassium phosphate dibasic when loaded into formulations at 15 w/w%.
2.4
Static release set-up
Microscope slides containing either resin or rosin formulations were loaded back to back into disinfected slide dishes and submerged in 200 mL of nanopure water. Each experiment consisted of 20 slides, for a total of 60 washers. One mL aliquots were taken immediately the first day at time zero, 15 min, 30 min, and 1 h. After, aliquots were taken at day 1, day 4, day 7, and at weekly intervals thereafter. The volume taken from the slide dish during each aliquot was consistently refreshed with the same volume of nanopure water.
2.5
Phosphate ion detection
In order to determine the concentration of phosphate ion in a given aliquot, the classic molybdenum blue method was used . A Tecan Infinite M200 spectrophotometer was used to measure absorbance values of the molybdenum complex at 882 nm. Each measurement was performed in triplicate.
2.6
Scanning electron microscopy
Scanning electron microscopy images were taken on microcapsules loaded in a resin and rosin formulation. The SEM was a Hitachi TM 3000 Tabletop Microscope.
2.7
Statistical analysis
Standard deviations were calculated for each sample and a Tukey’s comparison was performed on the data collected over the entire experiment. The figures report ‘normalized’ data, meaning that the concentrations refer to ion release per gram of rosin or resin formulation.
2
Materials and methods
2.1
Prepolymer synthesis
A polyurethane based prepolymer was prepared as the material for the microcapsule shell. Ethylene glycol (Fisher, New Jersey) was used as the diol source, and was combined with toluene-2,4-diisocyanate (Sigma–Aldrich, Steinheim). A 1:2 stoichiometrically limited ratio of diol to diisocyanate functional groups reacted in cyclohexanone at 70 °C in an inert atmosphere. The solvent was removed by vacuum.
2.2
Microcapsule synthesis
The oil phase consisted of the ethylene glycol polyurethane prepolymer, an emulsifying agent, and methyl benzoate (Acros Organics, New Jersey). Potassium phosphate dibasic (Fisher Scientific, New Jersey) aqueous solutions were made using ionic concentrations of 0.8 M, 2.4 M, 4.0 M, and 7.4 M with respect to the phosphate ion. Microcapsules were synthesized using a reverse emulsion which had an excess of oil to water by volume . The oil solution was heated to 70 °C in a custom-made stainless steel reactor that was submerged in a Büchi BS-490 heating bath. To create a reverse emulsion, the aqueous phosphate solution was added to the reactor slowly. The reactor mixed at 4000 rpm by a Caframo BDC 6015 stirrer. A stoichiometric excess of the associated diol-containing monomers was subsequently added to the reaction to quench the isocyanate functionalities remaining in the prepolymer. After the synthesis, microcapsules were isolated via centrifugation using a Fisher Centrific 288 centrifuge.
2.3
Dental formulation
2.3.1
Rosin based formulation
Rosin varnish formulations were created using Rosin CH (partially hydrogenated rosin, Staybelite ® -type Resin, Wilmington) combined with ethanol. For our purposes, rosin/ethanol was combined at a ratio of 75/25. The rosin was manually ground using a mortar and pestle and added to ethanol. The rosin and ethanol mixture were mixed at 3540 rpm for 2-min intervals using a Speed Mixer™ DAC150FVZ four to five times, or until the mixture was homogenous. Microcapsules were added next and the formulation mixed for another 2 min. The varnishes were transferred into washers (Nylon standard flat washers obtained from Washers USA. Outer dimension: 15.875 mm, inner dimension: 9.525 mm, thickness: 0.8128 mm, surface area: 71.3 mm 2 , total volume: 57.9 mm 3 ) which were fixed to glass microscope slides (Fisherfinest ® premium microscope slides, 3″ × 1″ × 1 mm) using a standard water resistant adhesive (Amazing Goop ® , Eclectic Products Inc, Pineville). Each slide consisted of three washers. The slide and varnish specimens dried overnight.
2.3.2
Resin based formulation
Resin formulations were made by first mixing the monomers TEGMA (Sigma–Aldrich, St. Louis):MMA (Acros Organics, New Jersey):UDMA (Esstech, Pennsylvania) at a ratio of 45:45:10 for 2 min at 3540 rpm (resulting in a relative centrifugal force of approximately 680 × g ) using a Speed Mixer™ DAC150FVZ (Flacktek Inc., South Carolina). Microcapsules were then added (at a targeted weight percent) to the combined monomers and the formulation mixed for 2 min at 3540 rpm. The quantity of microcapsules in the formulations varied, however, the ratio of TEGMA:MMA:UDMA remained 45:45:10 in all formulations. The photoinitiators, camphorquinone (CQ) and ethyl-4-dimethylaminobenzoate (E4DMAB) (Sigma–Aldrich, St. Louis), were added next and the formulation mixed for another 2 min at 3540 rpm. The formulations were transferred into washers (Nylon standard flat washers obtained from Washers USA. Outer dimension: 15.875 mm, inner dimension: 9.525 mm, thickness: 0.8128 mm, surface area: 71.3 mm 2 , total volume: 57.9 mm 3 ) which were fixed to glass microscope slides (Fisherfinest ® premium microscope slides, 3″ × 1″ × 1 mm) using a standard water resistant adhesive (Amazing Goop ® ). Each slide consisted of three washers. The resin varnish was irradiated by using a Spectrum 800 curing light at 600 mW/cm 2 for 2 min (per washer). Specimens cured for an additional 5 min using a DENTSPLY Triad ® Visible Light Cure System.
For both the resin and rosin formulations, we studied the release of phosphate ions from microcapsules containing 2.4 M potassium phosphate dibasic when loaded into varnishes at 3 w/w%, 9 w/w%, 15 w/w%, 30 w/w% and 50 w/w%. We also studied the effect of the initial concentration of the salt within the microcapsule at 0.8 M, 2.4 M, 4.0 M and 7.4 M potassium phosphate dibasic when loaded into formulations at 15 w/w%.
2.4
Static release set-up
Microscope slides containing either resin or rosin formulations were loaded back to back into disinfected slide dishes and submerged in 200 mL of nanopure water. Each experiment consisted of 20 slides, for a total of 60 washers. One mL aliquots were taken immediately the first day at time zero, 15 min, 30 min, and 1 h. After, aliquots were taken at day 1, day 4, day 7, and at weekly intervals thereafter. The volume taken from the slide dish during each aliquot was consistently refreshed with the same volume of nanopure water.
2.5
Phosphate ion detection
In order to determine the concentration of phosphate ion in a given aliquot, the classic molybdenum blue method was used . A Tecan Infinite M200 spectrophotometer was used to measure absorbance values of the molybdenum complex at 882 nm. Each measurement was performed in triplicate.
2.6
Scanning electron microscopy
Scanning electron microscopy images were taken on microcapsules loaded in a resin and rosin formulation. The SEM was a Hitachi TM 3000 Tabletop Microscope.
2.7
Statistical analysis
Standard deviations were calculated for each sample and a Tukey’s comparison was performed on the data collected over the entire experiment. The figures report ‘normalized’ data, meaning that the concentrations refer to ion release per gram of rosin or resin formulation.
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