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Ion release, fluoride charge of and adhesion of an orthodontic cement paste containing microcapsules
Journal of Dentistry 45 (2016) 32–38
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Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Ion release, fluoride charge of and adhesion of an orthodontic cement paste containing microcapsules Brant D. Burbanka , Michael Slatera , Alyssa Kavab , James Doyleb , William A. McHalec, Mark A. Lattaa , Stephen M. Grossa,b,* a b c
Department of Oral Biology, School of Dentistry, Creighton University, Omaha, NE 68178, United States Department of Chemistry, College of Arts and Sciences, Creighton University, Omaha, NE 68178, United States Premier Dental Products Company, Plymouth Meeting, PA 19462, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2015 Received in revised form 24 November 2015 Accepted 30 November 2015
Objectives: Dental materials capable of releasing calcium, phosphate and fluoride are of great interest for remineralization. Microencapsulated aqueous solutions of these ions in orthodontic cement demonstrate slow, sustained release by passive diffusion through a permeable membrane without the need for dissolution or etching of fillers. The potential to charge a dental material formulated with microencapsulated water with fluoride by toothbrushing with over the counter toothpaste and the effect of microcapsules on cement adhesion to enamel was determined. Methods: Orthodontic cements that contained microcapsules with water and controls without microcapsules were brushed with over-the-counter toothpaste and fluoride release was measured. Adhesion measurements were performed loading orthodontic brackets to failure. Cements that contained microencapsulated solutions of 5.0 M Ca(NO3)2, 0.8 M NaF, 6.0 M K2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. Results: A greater fluoride charge and re-release from toothbrushing was demonstrated compared to a control with no microcapsules. Adhesion of an orthodontic cement that contained microencapsulated remineralizing agents was 8.5 2.5 MPa compared to the control without microcapsules which was of 8.3 1.7 MPa. Sustained release of fluoride, calcium and phosphate ions from cement formulated with microencapsulated remineralizing agents was demonstrated. Conclusions: Orthodontic cements with microcapsules show a release of bioavailable fluoride, calcium, and phosphate ions near the tooth surface while having the ability to charge with fluoride and not effect the adhesion of the material to enamel. Incorporation of microcapsules in dental materials is promising for promoting remineralization. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Bioactive Fluoride Calcium Phosphate Recharge Orthodontic cement Composites Bioactive Ion release
1. Introduction Orthodontic cements and other organic composites are commonly used in dental restoratives due to the ease of use and aesthetic quality the patient desires. These materials function to help straighten teeth and restore proper tooth function upon enamel loss. However, secondary caries continue to form. Undesired side effects of orthodontic cements are white spot lesions, a form of demineralization [1–3]. If demineralization of the tooth enamel continues around the orthodontic bracket then an opaque square shaped lesion develops called a white spot lesion.
* Corresponding author at: Stephen Gross, Creighton University, 2500 California Plaza, Omaha, NE 68178, United States. E-mail address: [email protected] (S.M. Gross). http://dx.doi.org/10.1016/j.jdent.2015.11.009 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
Reviews show that exposure to fluoride ions on the demineralized tooth structure will assist in the treatment of white spot lesions after an orthodontic procedure [4–6]. A recent review has extensively covered approaches to enamel remineralization [7]. Fluoride ions, in the presence of hydroxyapatite may promote the formation of fluorapatite (Ca10(PO4)6F2) [8– 10]. Fluorapatite has a lower solubility product (KSP) than hydroxyapatite and the critical pH for dissolution of the mineral is lower [11]. As a result, fluoride has been introduced into drinking water, mouth rinses and toothpastes. Brushing with fluoridated toothpaste may help with the remineralization process by removing bacteria from the immediate surface of the tooth and exposing the demineralized enamel to fluoride ions, possibly forming fluorapatite. A review has been published showing other dental materials besides toothpaste that release fluoride ions to promote the formation of fluorapatite [12]. Most materials contain
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an initial burst of fluoride ions when the material is first placed and a slow release over the course of the following day [13]. After fluoride ions are present in the oral environment the limiting factor in remineralization may be the concentration of calcium and phosphate ions [14,15]. Several approaches have attempted to address the release of calcium and phosphate ions in the oral environment as well as fluoride ions. Some studies have reported that increased levels of calcium in the plaque increase the ability of the plaque to retain fluoride, thereby extending the potential time of fluoride availability in the oral environment [16]. Several other approaches to promote remineralization of enamel are bioactive glass systems, unstabilized and stabilized amorphous calcium phosphate systems, and calcium orthophosphates [17– 29]. In many cases the source of a bioactivity is from some form of bioactive glass (BAG) [30,31]. One example is a material that utilizes casein phosphopeptide amorphous calcium phosphate (CPP-ACP), which releases calcium and phosphate ions near the enamel surface to promote mineral formation [32,33]. CPP-ACP uses a milk derived protein that can stabilize calcium, phosphate and fluoride ions into a more available amorphous complex while binding adjacent to the tooth to allow the ions to reach subsurface enamel lesions [34]. CPP-ACP transform from an amorphous phase to a crystalline phase of hydroxyapatite. Recently CPP-ACPs have been used with zirconia for a slower release of calcium and phosphate ions [35]. Despite the use of fluoride, calcium and phosphate ions, demineralization remains a challenge in oral healthcare. Regardless of the deficiencies of the quantification of fluoride uptake into the enamel, reviews of materials such as glass ionomers and resin based materials show the ability to release fluoride ions initially and recharge to release fluoride ions again to continue the promotion of fluorapatite formation on the enamel [31]. All of these materials, when incorporated directly near the tooth improve remineralization in some form [36–38]. The ion-releasing agents require the dissolution or etching of the mineral fillers in order to release remineralizing ions. As a result, it would be ideal to innovate a filler material that can release fluoride, calcium and phosphate ions in their bioavailable forms adjacent to the enamel without dissolution of the dental material for remineralization. The ability to recharge ion releasing dental materials with fluoride is also of great interest [39]. A novel approach to promote remineralization through bioactive fillers has been reported. This approach incorporated microcapsules that could be loaded into any type of dental material and are capable of releasing fluoride, calcium and phosphate in a bioavailable form. Davidson, et al. showed microcapsules composed out of different polymers were able to release bioavailable forms of calcium, phosphate, and fluoride ions through a semipermeable membrane. The membranes used showed a difference of permeability and the potential to control the rate of ion release [40]. Falbo, et al. extended this work by incorporating these microcapsules into rosin varnishes and resin glazes [41]. The study showed some of the key variables of controlling ion release rates over extended periods of time. In this study, several orthodontic cement paste formulations were prepared with and without microcapsules. Initially, the ability of a non-fluoridated composite orthodontic cement paste formulated with microcapsules that had only water contained within the shell was studied. This was done to determine if the microcapsules promoted fluoride charge in the oral environment from a simple, over the counter toothpaste. This sample was compared to a control cement with no microcapsules formulated within. This was accomplished by cycling forty brushing cycles with toothpaste on both of these samples and then checked for the subsequent rerelease of fluoride ions from these cements over time. From there, the release of fluoride, calcium and phosphate
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ions from orthodontic cement pastes formulated with microcapsules containing these remineralizing ions was established. Finally, incorporating microcapsules into orthodontic cement paste and how the adhesion was affected of the orthodontic bracket to the tooth was examined. This study demonstrates the potential for an orthodontic cement that can release calcium, phosphate and fluoride, be charged with greater fluoride levels by simple toothbrushing while maintaining the working standard for adhesion to the enamel surface. 2. Materials and methods 2.1. Prepolymer synthesis Microcapsules were prepared with a polyurethane shell via a prepolymer synthesis in an inert environment at 70 C. [40] A cyclohexanone solvent was used to react ethylene glycol (Fisher, New Jersey) with isophorone diisocyanate (Sigma–Aldrich, Steinheim) and left overnight. The prepolymer was then dried by vacuum. 2.2. Microcapsule synthesis The prepolymer, after being dried was added to an emulsifying agent and methyl benzoate (Acros Organics, New Jersey). Aqueous salt solutions of 6.0 M potassium phosphate dibasic (Fisher Scientific, New Jersey), 5.0 M calcium nitrate tetrahydrate (Alfa Aesar, Massachusetts) and 0.8 M sodium fluoride (MP Biomedicals, Ohio) were prepared. These were introduced to the prepolymer during synthesis after having prepared a reverse emulsion [40]. When the prepolymer oil solution is agitated and the aqueous salt solution is introduced a reverse emulsion forms. The oil solution was agitated in a custom made reactor at 70 C. while the aqueous salt solution was added slowly. The reaction was then quenched using ethylene glycol to drive the reaction to the desired product. These microcapsules were centrifuged in a Fisher Centrific 288 centrifuge using a diluent, rinsed and prepared for formulation into the orthodontic cements. 2.3. Orthodontic cement formulations This study included different formulations of orthodontic cements. An orthodontic cement formulation was formulated with microcapsules containing 7 w/w% nanopure water. Nanopure water was generated by a Thermo Scientific Barnstead filtration system. There was only water within the microcapsules of this formulation to unambiguously determine the source of fluoride for use in the charging experiment. Three custom manufactured formulations were obtained from BJM Laboratories (Or Yehuda). These formulations were acrylate-based composites that contained standard boroaluminosilicate glass fillers. The first of these were formulated to incorporate 5 w/w% of microcapsules that contained 0.8 M sodium fluoride aqueous salt solution. The second formulation consisted of microcapsules containing a mixture of 2 w/w% 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate tetrahydrate and 1 w/w% 6.0 M potassium phosphate dibasic aqueous salt solutions (2/2/1), all the ions needed for remineralization. The third formulation was prepared as a control in which no microcapsules were added. Two more formulations were prepared in lab for ion release studies by adding microcapsules into the formulation containing no microcapsules to complement the BJM manufactured formulations. The first formulation included microcapsules containing 3 w/w% 6.0 M potassium phosphate dibasic aqueous salt solution. The second formulation included microcapsules containing 3 w/w % 5.0 M calcium nitrate tetrahydrate aqueous salt solution.
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2.4. Fluoride charge and release Specimens were prepared by gluing (Amazing Goop1, Eclectic Products Inc., Pineville) plastic 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 mm2, total volume: 57.9 mm3) onto microscopes slides (Fisherfinest1 premium microscope slides, 3” 1” 1 mm). The specimens were weighed before being filled with an orthodontic cement paste sample. Samples (n = 60) were formulated with the microcapsules filled with nanopure water. The microcapsules were filled with nanopure water to unambiguously determine the source for the fluoride charging experiment. Control samples (n = 60) that contained orthodontic cement with no microcapsules were also placed in separate washers on microscope sides. All samples (n = 120) were cured for 40 s using a Spectrum 800 curing light at 600 mW/cm2 on each side before being placed in a DENTSPLY Triad1 Visible Light Cure System for five minutes. The specimens were polished using a 320 grit polish wheel to obtain even surface areas and weighed again before overnight storage in 100% humidity. The samples were placed on a custom toothbrushing instrument as shown in Fig. 1 and exposed to a toothpaste slurry. The slurry was prepared by stirring a 50 w/w% Arm and Hammer Advance White Stain Defense Extreme Whitening regular fluoridated toothpaste containing 0.243% sodium fluoride with 50 w/w% nanopure water. A 0.4 mL dollop of the slurry of 1:1 toothpaste to nanopure water was placed on each sample and brushed for the recommended time of two minutes. After each two-minute brushing period the samples were rinsed with nanopure water and wiped with a kimwipe for the brushing cycles to continue. Each sample was brushed for forty separate 2 min cycles. The brushing instrument was set at 1 Hz, therefore, there were 120 strokes of the toothbrush for each cycle. The control samples were treated identically for comparison. After brushing, the slides were cleaned and dried. The microscope slides were loaded back to back into disinfected slide dishes with 200 mL of nanopure water. Each slide dish consisted of 20 slides, for a total of 60 washers with films. 1.0 mL aliquots were taken to measure fluoride release at time zero, 1 h, 1 day, 4 days, 1 week, and 2 weeks. Nanopure water was restored for the aliquot taken to maintain total volume in the slide dish. Potentiometry was used to measure the fluoride ion concentration. Each aliquot was measured three times. The average ion release was divided by the total mass of orthodontic cement within the slide container for normalization. The term normalized ion release on the y-axis of Fig. 2 refers to the fact that the measured ppm of ion release is
Fig. 2. Normalized concentration of fluoride ions released from microcapsules containing nanopure water loaded in orthodontic cement and the normalized concentration of fluoride ions released from orthodontic cement paste formulated without microcapsules. The plot is the concentration of fluoride ions in ppm released from the orthodontic cement as a function of time in days.
reported per gram of orthodontic cement submerged in nanopure water. 2.5. Static ion release set-up The orthodontic cements were placed into nylon washers which were fixed to glass microscope slides using a standard water resistant adhesive as done in Section 2.4 but with each cement formulated with an individual ion. Static ion release profiles were set up using the orthodontic cements that were formulated with microcapsules containing potassium phosphate dibasic or calcium nitrate tetrahydrate aqueous salt solutions added to the BJM manufactured cement in the lab, or the BJM manufactured fluoride or 2/2/1 aqueous salt solutions. The orthodontic cements were cured by using a Spectrum 800 curing light at 600 mW/cm2 for 1 min on the top and one minute on the bottom of the washer. Specimens were cured for an additional 5 min using a DENTSPLY Triad1 Visible Light Cure System. The microscope slides were loaded back to back into disinfected slide dishes with 200 mL of nanopure water. Each slide dish consisted of 20 slides, for a total of 60 washers with films. 1.0 mL aliquots were taken immediately the first day at time zero and at 1 h and then at days 1, 4, 7, 30, 60 and at three month intervals thereafter. The volume taken from the slide dish during each aliquot was consistently refreshed with the same volume of nanopure water. The term normalized ion release on the y-axis of Figs. 3–6 refers to the fact that the measured ppm of ion
Fig. 1. (a) A picture of a custom built toothbrushing instrument where four orthodontic cement films could be simultaneously brushed by four toothbrushes. (b) Four slides that each contained three washers were brushed for two minutes each for 40 cycles. The slide position was easily adjusted underneath the toothbrush in order to brush each film on the slide separately.
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Fig. 3. Normalized concentration of fluoride ions released from microcapsules containing 0.8 M sodium fluoride loaded in orthodontic cement paste. The plot is the concentration of fluoride ions in ppm released from microcapsules loaded at 5 w/w% in an orthodontic cement paste formulation as a function of time in days.
release is reported per gram of orthodontic cement submerged in nanopure water. 2.6. Phosphate ion detection In order to determine the concentration of phosphate ion in a given aliquot, the classic molybdenum blue method was used [42]. The concentration of the dibasic form of the anion is reported in Figs. 5 and 6. A Tecan Infinite M200 spectrophotometer was used to measure absorbance values of the molybdenum complex at 882 nm. Each measurement was performed in triplicate. 2.7. Calcium and fluoride ion detection Potentiometry was used to determine the concentration of calcium and fluoride ions [43]. An ELIT 1265 calcium specific electrode was used for the calcium ion; An ELIT 8221 fluoride specific electrode was used for the fluoride ion. Both of the aforementioned electrodes were used in conjunction with an ELIT 001N silver chloride reference electrode. 2.8. Adhesion measurement procedure Bovine teeth were sectioned buccolingually and placed in an acrylic mold with the enamel exposed. The teeth were acid conditioned using 35% phosphoric acid (Ultradent, Utah) for 20 s, rinsed and air-dried. Primer was applied to the tooth and visible light cured by a Spectrum 800 curing light at 600 mW/cm2 for 20 s. Orthodontic cement was applied to 3 M Victory Roth orthodontic brackets (n = 10) containing the BJM manufactured 2/2/1 formulation with microcapsules. A control (n = 10) paste was prepared using the formulation without microcapsules. These orthodontic cement pastes were pressed to exude excess cement, cleaned and light cured for 20 s per each of the four sides. The light was held at a 45 angle for each of the 4 exposures. The specimens were stored in water at 37 C for 7 days before being loaded to failure at 1 mm per minute using a crosshead-shearing fixture rounded to fit the bracket on an Instron (model) test frame. The surface area (determined by using the outline dimension) of each bracket was measured and calculated to be 13.86 mm2. Shear bond strength was measured and reported in Megapascals (MPa). Each debonded surface was examined visually at a magnification of 20X to assess the qualitative characteristics of the failure interface. 3. Results Charging of a dental material with fluoride is a potential strategy for the promotion of remineralization in the oral environment. Introduction of water filled microcapsules into an
orthodontic cement to determine if the presence of these microcapsules affects the ability of the material to charge and release fluoride ions was investigated. One orthodontic cement was formulated with microcapsules containing only nanopure water to unambiguously determine the source of the fluoride ion while a control cement was prepared without microcapsules. These two separate orthodontic cements were brushed for 40 cycles with an over the counter toothpaste. These formulations were then soaked in nanopure water and the release of fluoride was measured over time. In addition to charging an orthodontic cement by brushing, it would potentially be advantageous to have an orthodontic cement capable of slow, sustained release of ions useful for remineralization. Orthodontic cements were also prepared containing encapsulated aqueous solutions of calcium, phosphate or fluoride ions or mixtures of all three. Finally, the effect of microcapsules on the adhesion of the orthodontic cement with the bracket in place to the enamel was determined. 3.1. Fluoride charge and release The effect of loading microcapsules into an orthodontic cement and the subsequent release of fluoride from the cement following toothbrushing was determined. Fig. 2 depicts the part per million of fluoride being released per gram of orthodontic cement as a function of time for two orthodontic cement formulations. One formulation was formulated with microcapsules that contained nanopure water. The other formulation was an orthodontic cement with no microcapsules. Both formulations, after being exposed to 40 cycles of toothbrushing released fluoride ions over time. As seen in Fig. 2, after 14 days, the control released 0.010 ppm of fluoride/g of cement. The formulation with microcapsules released 0.069 ppm of fluoride/g of cement. 3.2. Static ion release The ion release profile of fluoride ions from an orthodontic cement manufactured to contain 5 w/w% microcapsules that contained an aqueous solution of 0.8 M sodium fluoride is shown in Fig. 3. The graph depicts the ppm of fluoride ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of calcium ions from an orthodontic cement formulated with 3 w/w% microcapsules that contained an aqueous solution of 5.0 M calcium nitrate tetrahydrate is shown in Fig. 4. The graph depicts the ppm of calcium ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of phosphate ions from an orthodontic cement formulated with 3 w/w% microcapsules that contained an aqueous solution of 6.0 M potassium phosphate dibasic is shown in Fig. 5.
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the ppm of each individual ion released per gram of orthodontic cement formulation as a function of time. 3.3. Adhesion
Fig. 4. Normalized concentration of calcium ions released from microcapsules containing 5.0 M calcium nitrate tetrahydrate loaded in orthodontic cement paste. The plot is the concentration of calcium ions in ppm released from microcapsules loaded at 3 w/w% in an orthodontic cement paste formulation as a function of time in days.
Fig. 5. Normalized concentration of phosphate ions released from microcapsules containing 6.0 M potassium phosphate dibasic loaded in orthodontic cement paste. The plot is the concentration of phosphate ions in ppm released from microcapsules loaded at 3 w/w% in an orthodontic cement formulation as a function of time in days.
The graph depicts the ppm of phosphate ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of fluoride, calcium and phosphate ions from an orthodontic cement manufactured to contain 2 w/w% microcapsules that contained an aqueous solution of 0.8 M sodium fluoride, 2 w/w% microcapsules that contained an aqueous solution of 5.0 M calcium nitrate tetrahydrate, and 1 w/w% microcapsules that contained an aqueous solution of 6.0 M potassium phosphate dibasic is shown in Fig. 6. The graph depicts
Fig. 6. Normalized concentration of fluoride, calcium and phosphate ions released from 2 w/w% microcapsules containing 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate and 1 w/w% 6.0 M potassium phosphate dibasic loaded in orthodontic cement paste. The plot is the concentration of fluoride, calcium and phosphate ions in ppm released from microcapsules loaded at 5 total w/w% in an orthodontic cement formulation as a function of time in days.
The adhesion of the orthodontic cement to the enamel with the bracket in place was determined. Fig. 7 depicts the pressure the orthodontic bracket underwent before failure and is compared side by side after quartiles are obtained. The whiskers demonstrate the maximums and minimums. The average for the control formulation without microcapsules was 8.32 1.71 MPa. The formulation with the microcapsules had an average of 8.56 2.52 MPa. 4. Discussion Considering the delicate balance of demineralization and remineralization of the enamel in the oral environment, the purpose of this study was to develop a new approach to promote remineralization using polyurethane based microcapsules as a filler in orthodontic cement. To promote remineralization, the orthodontic cement would ideally release remineralizing ions (e.g., calcium, phosphate, fluoride) contained within the microcapsules. Furthermore, if inclusion of microcapsules enhanced the uptake and release of additional fluoride ions due to toothbrushing while not adversely effecting adhesion, an improvement in orthodontic cement could potentially be achieved. In this study, the question of whether the presence of the semipermeable membrane in the form of the microcapsule shell could possibly assist in the absorption or adsorption of fluoride ions for release after realistic daily exposure from toothbrushing was explored. In orthodontic cement formulated with microcapsules that contained only water, the microcapsule could potentially absorb or adsorb fluoride and release it into the environment. In this study, fluoride ions were introduced to an orthodontic cement that contained microcapsules through toothbrushing with an over the counter toothpaste. Additionally, the effect of microcapsules on adhesion of a material to enamel was explored for the first time. Finally, in Falbo et al., only the release of phosphate ions from varnish and glaze formulations was reported. In this paper, the release of calcium and fluoride ions, in addition to phosphate ions was studied from orthodontic cement formulated with microencapsulated aqueous solutions of sodium fluoride, calcium nitrate and potassium phosphate dibasic. Previous work demonstrated the passive diffusion of ions from dental materials formulated with the microcapsules that is primarily driven by a concentration gradient. In this study, the possibility of charging a dental material with microcapsules that contained nanopure water with fluoride was examined. In order to
Fig. 7. Box plot of pressure loaded before failure of orthodontic cement with microcapsules containing 2 w/w% microcapsules containing 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate and 1 w/w% 6.0 M potassium phosphate dibasic and orthodontic cement formulated without.
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unambiguously determine the source of fluoride ions measured, the orthodontic cements were formulated to contain microcapsules with nanopure water before being exposed to fluoride ions through simple brushing. As a control, an orthodontic cement with no microcapsules was also prepared. When the orthodontic cement paste formulations were brushed, both formulations either absorbed or adsorbed and subsequently released fluoride. As seen in Fig. 2, the formulation containing microcapsules released 68% more fluoride per gram of formulation than the control. After the similar initial burst of fluoride ions from both formulations, the control formulation stopped releasing fluoride between 1 and 7 days, whereas the formulation containing microcapsules still continued to release fluoride ions after one week. The formulation with water containing microcapsules could have released more fluoride after toothbrushing for a number of potential reasons. The fluoride ions could have absorbed into the more polar semipermeable polyurethane membrane found at the surface of the material and absorb into the interior for rerelease. Another explanation could be that the addition of the polar microcapsules into the nonpolar, hydrophobic dental material has changed the overall polarity of the material where ions may be more attracted to the material than one formulated without any microcapsules. Therefore the more polar orthodontic cement could enhance charging relative to a less polar, hydrophobic control. After charging water filled microcapsules within the orthodontic cement with fluoride through simple toothbrushing, a study was set up to show if an orthodontic cement paste formulated with microencapsulated remineralizing agents can release ions (calcium, fluoride and phosphate) at a slow, sustained rate over an extended period of time. Separate orthodontic cement pastes were formulated to contain microcapsules with aqueous solutions of calcium, fluoride or phosphate ions preloaded inside of the polyurethane shell. These orthodontic cements were prepared with each individual ion to give a sense of ion release potential without precipitation. Orthodontic cement paste was formulated with these microcapsules and the release was reported as a function of time in days that are shown a release of these ions in days in Figs. 3–5. Ion release is demonstrated over nine months. Based on these results and previous studies of phosphate ion release from glazes, the potential to release ions throughout the projected lifetime of an orthodontic cement in the oral environment should be achievable [41]. Fig. 6 shows a release profile of fluoride, calcium and phosphate ions from an orthodontic cement paste containing 2 w/ w% of 0.8 M sodium fluoride, 2 w/w% of 5.0 M calcium nitrate and 1 w/w% of 6.0 M potassium phosphate dibasic formulation or a mixture of all three ions. Potential for precipitation of insoluble calcium phosphates and calcium fluoride could effectively explain the difference between individual ion release profiles with that of mixed microcapsule formulations. Although mixed microcapsule formulations appear to have a lower ion release rate, it may be the precipitation of insoluble calcium phosphates or calcium fluoride. If this occurs in the presence of the enamel surface, remineralization would be promoted. A final aspect of this current study examined whether the adhesion properties of an orthodontic cement that contained microcapsules was different from a control formulation that was manufactured without microcapsules. An orthodontic cement paste that contained 2 w/w% of 0.8 M sodium fluoride, 2 w/w% of 5.0 M calcium nitrate and 1 w/w% of potassium phosphate dibasic formulation and a control with the same composition without microcapsules was prepared. Fig. 7 shows the adhesion data of these two different formulations. This experiment demonstrated little effect on the adhesion to conditioned enamel based on the presence of the microcapsules in the cement formula. This suggests that whatever viscosity variation occurs with the
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microcapsule formula, the prime influence on adhesion is derived from the acid conditioning and the application of the primer. There was no visual evidence of any difference in the adaptation of the cement to the bracket mesh between the two formulas. 5. Conclusion The development of an orthodontic cement paste that releases fluoride (in addition to calcium and phosphate ions) with acceptable adhesion properties could result from the mechanism of fluoride release being from simple diffusion of fluoride through a semipermeable membrane triggered by concentration gradients as opposed to etching and dissolution mechanisms. The addition of polyurethane based semipermeable microcapsules as a filler within orthodontic cement paste could promote remineralization of the tooth. Acknowledgements The authors would like to acknowledge the Premier Dental Products Company and the NSF/EPSCoR program under grant EPS1004094 for support of this research. The authors would like to acknowledge BJM Laboratories for manufacturing the orthodontic cements used in this study. ML and SG have a financial interest in the Premier Dental Products Company. References [1] L. Gorelick, A.M. Geiger, A.J. Gwinnett, Incidence of white spot formation after bonding and banding, Am. J. Orthod. 81 (1982) 93–98. [2] M.M. O'Reilly, J.D.B. Featherstone, Demineralization and remineralization around orthodontic appliances: an in vivo study, Am. J. Orthod. Dentofacial Orthop. 92 (1987) 33–40. [3] J.A. Chapman, W.E. Roberts, G.J. Eckert, K.S. Kula, C. González-Cabezas, Risk factors for incidence and severity of white spot lesions during treatment with fixed orthodontic appliances, Am. J. Orthod. Dentofacial Orthop. 138 (2010) 188–194. [4] F. Bergstrand, S. Twetman, Evidence for the efficacy of various methods of treating white-spot lesions after debonding of fixed orthodontic appliances, J. Clin. Orthodont. 37 (2003) 19–21. [5] P.E. Benson, N. Parkin, D.T. Millett, F.E. Dyer, S. Vine, A. Shah, Fluorides for the prevention of white spots on teeth during fixed brace treatment, Database System. Rev. 3 (2004) CD003809. [6] B.L. Chadwick, J. Roy, J. Knox, E.T. Treasure, The effect of topical fluorides on decalcification in patients with fixed orthodontic appliances: a systematic review, Am. J. Orthod. Dentofacial Orthop. 128 (2005) 601–606. [7] R.J. Lynch, R. Navada, R. Walia, Low-levels of fluoride in plaque and saliva and their effects on the demineralization and remineralization of enamel; role of fluoride toothpastes, Int. Dent. J. 54 (2004) 304–309. [8] C. ten, J.M. ate, Current concepts on the theories of the mechanism of action of fluoride, Acta Odontol. Scand. 57 (1999) 325–329. [9] J. ten Cate, J.D. Featherstone, Mechanistic aspects of the interactions between fluoride and dental enamel, Crit. Rev. Oral Biol. Med. 2 (1991) 283–296. [10] E.C. Reynolds, Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review, Spec. Care Dentist. 8 (1998) 8– 16. [11] A. Wiegand, W. Buchalla, T. Attin, Review on fluoride-releasing restorative materials-fluoride release and uptake characteristics, antibacterial activity and influence on caries formation, Dent. Mater. 23 (2007) 343–362. [12] D. Wood, R. Hill, Structure-property relationships in ionomer glasses, Clin. Mater. 7 (1991) 301–312. [13] E.C. Reynolds, Calcium phosphate-based remineralization systems: scientific evidence? Aust. Dent. J. 53 (2008) 268–273. [14] Li Xiaoke, The remineralization of enamel: a review of the literature, J. Dentist. (2014) S12–S20. [15] RM Duckworth, E. Huntington, On the relationship between calculus and caries. In: The Teeth and their Environment Duckworth RM, editor. Monographs in Oral Science 2006; 19: 1–28. [16] E.C. Reynolds, C.J. Cain, F.L. Webber, C.L. Black, P.F. Riley, I.H. Johnson, Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat, J. Dent. Res. 74 (1995) 1272–1279. [17] E.C. Reynolds, Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review, Spec. Care Dentist. 8 (1998) 8– 16. [18] E.C. Reynolds, Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions, J. Dent. Res. 76 (1997) 1587–1595.
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[19] E.C. Reynolds, C.L. Black, F. Cai, K.J. Cross, D. Eakins, N.L. Huq, Anticariogenic casein phosphopeptide-amorphous calcium phosphate, J. Clin. Dentist. 10 (1999) 86–88. [20] P. Shen, F. Cai, A. Nowiciki, J. Vincent, E.C. Reynolds, Remineralization of enamel sub surface lesions by sugar free chewing gum containing casein phosphopeptide amorphous calcium phosphate, J. Dent. Res. 80 (2001) 2066– 2070. [21] L.L. Hench, H.A. Paschall, Histo-chemical responses at a biomaterials interface, J. Biomed. Mater. Res. 5 (1974) 49–64. [22] A. Merrollli, L.P. Tranquilli, P.L. Guidi, Preparation of porous apatite granules from calcium phosphate cement, J. Mater. Sci. Mater. Med. 11 (2000) 219–222. [23] C. Ohtsuki, T. Kokubo, Compositional dependence of bioactivity of glasses in the system CaO-SiO2-Al2O3—its in vitro evaluation, J. Mater. Sci. Mater. Med. 3 (1992) 119–125. [24] L.L. Hench, Bioceramics: from concept to clinic, J. Am. Ceram. Soc. 74 (1991) 1487–1510. [25] F. Zhang, A.J. Allen, L.E. Levine, M.D. Vaudin, D. Skrtic, J.M. Antonucci, K.M. Hoffman, A.A. Giuseppetti, J. Ilavsky, Structural and dynamical studies of acidmediated conversion in amorphous-calcium-phosphate based dental composites, Dent. Mater. 30 (2014) 1113–1125. [26] C. Combes, C. Rey, Amorphous calcium phosphates: synthesis, properties and uses in biomaterials, Acta Biomater. 6 (2010) 3362–3378. [27] S.V. Dorozhkin, Calcium orthophosphates, J. Mater. Sci. 42 (2007) 1061–1095. [28] S.V. Dorozhkin, Amorphous calcium (ortho) phosphates, Acta Biomater. 6 (2010) 4457–4475. [29] S.V. Dorozhkin, Biphasic, triphasic and multiphasic calcium orthophosphates, Acta Biomater. 8 (2012) 963–977. [30] Li Xiaoke, The remineralization of enamel: a review of the literature, J. Dentist. (2014) S12–S20. [31] A. Wiegand, W. Buchalla, T. Attin, Review on fluoride-releasing restorateive materials-fluoride release and uptake characteristics, antibacterial activity and influence on caries formation, Dent. Mater. 23 (2007) 343–362. [32] W.D. Chan, L. Yang, W. Wankel, A.S. Rizkalla, Fluoride release from dental cements and composites: a mechanistic study, Dent. Mater. 22 (2006) 366–373.
[33] E.C. Reynolds, Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions, J. Dent. Res. 76 (1997) 1587–1595. [34] S.V. Dorozhkin, Amorphous calcium (ortho) phosphates, Acta Biomater. 6 (2010) 4457–4475. [35] F. Zhang, A.J. Allen, L.E. Levine, M.D. Vaudin, D. Skrtic, J.M. Antonucci, K.M. Hoffman, A.A. Giuseppetti, J. Ilavsky, Structural and dynamical studies of acidmediated conversion in amorphous-calcium-phosphate based dental composites, Dent. Mater. 30 (2014) 1113–1125. [36] H. Forssh, L. Nase, L. Seppä, Fluoride concentration, mutans streptococci and lactobacilli in plaque from old glass-ionomer fillings, Caries Res. 29 (1995) 50– 53. [37] E.M. Benelli, M.C. Serra, A.L. Rodrigues Jr, J.A. Cury, In situ anticariogenic potential of glass ionomer cement, Caries Res. 27 (1993) 280–284. [38] T. Uysal, M. Amasyali, A.E. Koyuturk, D. Sagdic, Efficiency of amorphous calcium phosphate-containing orthodontic composite and resin modified glass ionomer on demineralization evaluated by a new laser fluorescence device, Eur. J. Dentist. 3 (2009) 127–134. [39] H.B. Davis, F. Gwinner, J.C. Mitchell, J.L. Ferracane, Ion release from, and fluoride recharge of a composite with a fluoride-containing bioactive glass, Dent. Mater. 30 (2014) 1187–1194. [40] M.T. Davidson, T.A. Greving, W.A. McHale, M.A. Latta, S.M. Gross, Ion Permeable Microcapsules for the release of biologically available ions for remineralization, J. Biomed. Res. Part A 100 (2012) 665–672. [41] M.M. Falbo, P. Elassal, T.A. Greving, W.A. McHale, M.A. Latta, S.M. Gross, The control of phosphate ion release from ion permeable microcapsules formulated into rosin varnish and resin glaze, Dental Mater. 29 (2013) 804– 813. [42] J. Murphy, J.P. Riley, A modified single solution method for the determination of phosphate in natural waters, Anal. Chim. Acta 27 (1962) 31–36. [43] J.E. Harwood, Use of an ion-selective electrode for routine fluoride analyses on water samples, Water Res. 3 (1969) 273–275.
Contents lists available at ScienceDirect
Journal of Dentistry journal homepage: www.intl.elsevierhealth.com/journals/jden
Ion release, fluoride charge of and adhesion of an orthodontic cement paste containing microcapsules Brant D. Burbanka , Michael Slatera , Alyssa Kavab , James Doyleb , William A. McHalec, Mark A. Lattaa , Stephen M. Grossa,b,* a b c
Department of Oral Biology, School of Dentistry, Creighton University, Omaha, NE 68178, United States Department of Chemistry, College of Arts and Sciences, Creighton University, Omaha, NE 68178, United States Premier Dental Products Company, Plymouth Meeting, PA 19462, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2015 Received in revised form 24 November 2015 Accepted 30 November 2015
Objectives: Dental materials capable of releasing calcium, phosphate and fluoride are of great interest for remineralization. Microencapsulated aqueous solutions of these ions in orthodontic cement demonstrate slow, sustained release by passive diffusion through a permeable membrane without the need for dissolution or etching of fillers. The potential to charge a dental material formulated with microencapsulated water with fluoride by toothbrushing with over the counter toothpaste and the effect of microcapsules on cement adhesion to enamel was determined. Methods: Orthodontic cements that contained microcapsules with water and controls without microcapsules were brushed with over-the-counter toothpaste and fluoride release was measured. Adhesion measurements were performed loading orthodontic brackets to failure. Cements that contained microencapsulated solutions of 5.0 M Ca(NO3)2, 0.8 M NaF, 6.0 M K2HPO4 or a mixture of all three were prepared. Ion release profiles were measured as a function of time. Results: A greater fluoride charge and re-release from toothbrushing was demonstrated compared to a control with no microcapsules. Adhesion of an orthodontic cement that contained microencapsulated remineralizing agents was 8.5 2.5 MPa compared to the control without microcapsules which was of 8.3 1.7 MPa. Sustained release of fluoride, calcium and phosphate ions from cement formulated with microencapsulated remineralizing agents was demonstrated. Conclusions: Orthodontic cements with microcapsules show a release of bioavailable fluoride, calcium, and phosphate ions near the tooth surface while having the ability to charge with fluoride and not effect the adhesion of the material to enamel. Incorporation of microcapsules in dental materials is promising for promoting remineralization. ã 2015 Elsevier Ltd. All rights reserved.
Keywords: Bioactive Fluoride Calcium Phosphate Recharge Orthodontic cement Composites Bioactive Ion release
1. Introduction Orthodontic cements and other organic composites are commonly used in dental restoratives due to the ease of use and aesthetic quality the patient desires. These materials function to help straighten teeth and restore proper tooth function upon enamel loss. However, secondary caries continue to form. Undesired side effects of orthodontic cements are white spot lesions, a form of demineralization [1–3]. If demineralization of the tooth enamel continues around the orthodontic bracket then an opaque square shaped lesion develops called a white spot lesion.
* Corresponding author at: Stephen Gross, Creighton University, 2500 California Plaza, Omaha, NE 68178, United States. E-mail address: [email protected] (S.M. Gross). http://dx.doi.org/10.1016/j.jdent.2015.11.009 0300-5712/ ã 2015 Elsevier Ltd. All rights reserved.
Reviews show that exposure to fluoride ions on the demineralized tooth structure will assist in the treatment of white spot lesions after an orthodontic procedure [4–6]. A recent review has extensively covered approaches to enamel remineralization [7]. Fluoride ions, in the presence of hydroxyapatite may promote the formation of fluorapatite (Ca10(PO4)6F2) [8– 10]. Fluorapatite has a lower solubility product (KSP) than hydroxyapatite and the critical pH for dissolution of the mineral is lower [11]. As a result, fluoride has been introduced into drinking water, mouth rinses and toothpastes. Brushing with fluoridated toothpaste may help with the remineralization process by removing bacteria from the immediate surface of the tooth and exposing the demineralized enamel to fluoride ions, possibly forming fluorapatite. A review has been published showing other dental materials besides toothpaste that release fluoride ions to promote the formation of fluorapatite [12]. Most materials contain
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an initial burst of fluoride ions when the material is first placed and a slow release over the course of the following day [13]. After fluoride ions are present in the oral environment the limiting factor in remineralization may be the concentration of calcium and phosphate ions [14,15]. Several approaches have attempted to address the release of calcium and phosphate ions in the oral environment as well as fluoride ions. Some studies have reported that increased levels of calcium in the plaque increase the ability of the plaque to retain fluoride, thereby extending the potential time of fluoride availability in the oral environment [16]. Several other approaches to promote remineralization of enamel are bioactive glass systems, unstabilized and stabilized amorphous calcium phosphate systems, and calcium orthophosphates [17– 29]. In many cases the source of a bioactivity is from some form of bioactive glass (BAG) [30,31]. One example is a material that utilizes casein phosphopeptide amorphous calcium phosphate (CPP-ACP), which releases calcium and phosphate ions near the enamel surface to promote mineral formation [32,33]. CPP-ACP uses a milk derived protein that can stabilize calcium, phosphate and fluoride ions into a more available amorphous complex while binding adjacent to the tooth to allow the ions to reach subsurface enamel lesions [34]. CPP-ACP transform from an amorphous phase to a crystalline phase of hydroxyapatite. Recently CPP-ACPs have been used with zirconia for a slower release of calcium and phosphate ions [35]. Despite the use of fluoride, calcium and phosphate ions, demineralization remains a challenge in oral healthcare. Regardless of the deficiencies of the quantification of fluoride uptake into the enamel, reviews of materials such as glass ionomers and resin based materials show the ability to release fluoride ions initially and recharge to release fluoride ions again to continue the promotion of fluorapatite formation on the enamel [31]. All of these materials, when incorporated directly near the tooth improve remineralization in some form [36–38]. The ion-releasing agents require the dissolution or etching of the mineral fillers in order to release remineralizing ions. As a result, it would be ideal to innovate a filler material that can release fluoride, calcium and phosphate ions in their bioavailable forms adjacent to the enamel without dissolution of the dental material for remineralization. The ability to recharge ion releasing dental materials with fluoride is also of great interest [39]. A novel approach to promote remineralization through bioactive fillers has been reported. This approach incorporated microcapsules that could be loaded into any type of dental material and are capable of releasing fluoride, calcium and phosphate in a bioavailable form. Davidson, et al. showed microcapsules composed out of different polymers were able to release bioavailable forms of calcium, phosphate, and fluoride ions through a semipermeable membrane. The membranes used showed a difference of permeability and the potential to control the rate of ion release [40]. Falbo, et al. extended this work by incorporating these microcapsules into rosin varnishes and resin glazes [41]. The study showed some of the key variables of controlling ion release rates over extended periods of time. In this study, several orthodontic cement paste formulations were prepared with and without microcapsules. Initially, the ability of a non-fluoridated composite orthodontic cement paste formulated with microcapsules that had only water contained within the shell was studied. This was done to determine if the microcapsules promoted fluoride charge in the oral environment from a simple, over the counter toothpaste. This sample was compared to a control cement with no microcapsules formulated within. This was accomplished by cycling forty brushing cycles with toothpaste on both of these samples and then checked for the subsequent rerelease of fluoride ions from these cements over time. From there, the release of fluoride, calcium and phosphate
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ions from orthodontic cement pastes formulated with microcapsules containing these remineralizing ions was established. Finally, incorporating microcapsules into orthodontic cement paste and how the adhesion was affected of the orthodontic bracket to the tooth was examined. This study demonstrates the potential for an orthodontic cement that can release calcium, phosphate and fluoride, be charged with greater fluoride levels by simple toothbrushing while maintaining the working standard for adhesion to the enamel surface. 2. Materials and methods 2.1. Prepolymer synthesis Microcapsules were prepared with a polyurethane shell via a prepolymer synthesis in an inert environment at 70 C. [40] A cyclohexanone solvent was used to react ethylene glycol (Fisher, New Jersey) with isophorone diisocyanate (Sigma–Aldrich, Steinheim) and left overnight. The prepolymer was then dried by vacuum. 2.2. Microcapsule synthesis The prepolymer, after being dried was added to an emulsifying agent and methyl benzoate (Acros Organics, New Jersey). Aqueous salt solutions of 6.0 M potassium phosphate dibasic (Fisher Scientific, New Jersey), 5.0 M calcium nitrate tetrahydrate (Alfa Aesar, Massachusetts) and 0.8 M sodium fluoride (MP Biomedicals, Ohio) were prepared. These were introduced to the prepolymer during synthesis after having prepared a reverse emulsion [40]. When the prepolymer oil solution is agitated and the aqueous salt solution is introduced a reverse emulsion forms. The oil solution was agitated in a custom made reactor at 70 C. while the aqueous salt solution was added slowly. The reaction was then quenched using ethylene glycol to drive the reaction to the desired product. These microcapsules were centrifuged in a Fisher Centrific 288 centrifuge using a diluent, rinsed and prepared for formulation into the orthodontic cements. 2.3. Orthodontic cement formulations This study included different formulations of orthodontic cements. An orthodontic cement formulation was formulated with microcapsules containing 7 w/w% nanopure water. Nanopure water was generated by a Thermo Scientific Barnstead filtration system. There was only water within the microcapsules of this formulation to unambiguously determine the source of fluoride for use in the charging experiment. Three custom manufactured formulations were obtained from BJM Laboratories (Or Yehuda). These formulations were acrylate-based composites that contained standard boroaluminosilicate glass fillers. The first of these were formulated to incorporate 5 w/w% of microcapsules that contained 0.8 M sodium fluoride aqueous salt solution. The second formulation consisted of microcapsules containing a mixture of 2 w/w% 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate tetrahydrate and 1 w/w% 6.0 M potassium phosphate dibasic aqueous salt solutions (2/2/1), all the ions needed for remineralization. The third formulation was prepared as a control in which no microcapsules were added. Two more formulations were prepared in lab for ion release studies by adding microcapsules into the formulation containing no microcapsules to complement the BJM manufactured formulations. The first formulation included microcapsules containing 3 w/w% 6.0 M potassium phosphate dibasic aqueous salt solution. The second formulation included microcapsules containing 3 w/w % 5.0 M calcium nitrate tetrahydrate aqueous salt solution.
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2.4. Fluoride charge and release Specimens were prepared by gluing (Amazing Goop1, Eclectic Products Inc., Pineville) plastic 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 mm2, total volume: 57.9 mm3) onto microscopes slides (Fisherfinest1 premium microscope slides, 3” 1” 1 mm). The specimens were weighed before being filled with an orthodontic cement paste sample. Samples (n = 60) were formulated with the microcapsules filled with nanopure water. The microcapsules were filled with nanopure water to unambiguously determine the source for the fluoride charging experiment. Control samples (n = 60) that contained orthodontic cement with no microcapsules were also placed in separate washers on microscope sides. All samples (n = 120) were cured for 40 s using a Spectrum 800 curing light at 600 mW/cm2 on each side before being placed in a DENTSPLY Triad1 Visible Light Cure System for five minutes. The specimens were polished using a 320 grit polish wheel to obtain even surface areas and weighed again before overnight storage in 100% humidity. The samples were placed on a custom toothbrushing instrument as shown in Fig. 1 and exposed to a toothpaste slurry. The slurry was prepared by stirring a 50 w/w% Arm and Hammer Advance White Stain Defense Extreme Whitening regular fluoridated toothpaste containing 0.243% sodium fluoride with 50 w/w% nanopure water. A 0.4 mL dollop of the slurry of 1:1 toothpaste to nanopure water was placed on each sample and brushed for the recommended time of two minutes. After each two-minute brushing period the samples were rinsed with nanopure water and wiped with a kimwipe for the brushing cycles to continue. Each sample was brushed for forty separate 2 min cycles. The brushing instrument was set at 1 Hz, therefore, there were 120 strokes of the toothbrush for each cycle. The control samples were treated identically for comparison. After brushing, the slides were cleaned and dried. The microscope slides were loaded back to back into disinfected slide dishes with 200 mL of nanopure water. Each slide dish consisted of 20 slides, for a total of 60 washers with films. 1.0 mL aliquots were taken to measure fluoride release at time zero, 1 h, 1 day, 4 days, 1 week, and 2 weeks. Nanopure water was restored for the aliquot taken to maintain total volume in the slide dish. Potentiometry was used to measure the fluoride ion concentration. Each aliquot was measured three times. The average ion release was divided by the total mass of orthodontic cement within the slide container for normalization. The term normalized ion release on the y-axis of Fig. 2 refers to the fact that the measured ppm of ion release is
Fig. 2. Normalized concentration of fluoride ions released from microcapsules containing nanopure water loaded in orthodontic cement and the normalized concentration of fluoride ions released from orthodontic cement paste formulated without microcapsules. The plot is the concentration of fluoride ions in ppm released from the orthodontic cement as a function of time in days.
reported per gram of orthodontic cement submerged in nanopure water. 2.5. Static ion release set-up The orthodontic cements were placed into nylon washers which were fixed to glass microscope slides using a standard water resistant adhesive as done in Section 2.4 but with each cement formulated with an individual ion. Static ion release profiles were set up using the orthodontic cements that were formulated with microcapsules containing potassium phosphate dibasic or calcium nitrate tetrahydrate aqueous salt solutions added to the BJM manufactured cement in the lab, or the BJM manufactured fluoride or 2/2/1 aqueous salt solutions. The orthodontic cements were cured by using a Spectrum 800 curing light at 600 mW/cm2 for 1 min on the top and one minute on the bottom of the washer. Specimens were cured for an additional 5 min using a DENTSPLY Triad1 Visible Light Cure System. The microscope slides were loaded back to back into disinfected slide dishes with 200 mL of nanopure water. Each slide dish consisted of 20 slides, for a total of 60 washers with films. 1.0 mL aliquots were taken immediately the first day at time zero and at 1 h and then at days 1, 4, 7, 30, 60 and at three month intervals thereafter. The volume taken from the slide dish during each aliquot was consistently refreshed with the same volume of nanopure water. The term normalized ion release on the y-axis of Figs. 3–6 refers to the fact that the measured ppm of ion
Fig. 1. (a) A picture of a custom built toothbrushing instrument where four orthodontic cement films could be simultaneously brushed by four toothbrushes. (b) Four slides that each contained three washers were brushed for two minutes each for 40 cycles. The slide position was easily adjusted underneath the toothbrush in order to brush each film on the slide separately.
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Fig. 3. Normalized concentration of fluoride ions released from microcapsules containing 0.8 M sodium fluoride loaded in orthodontic cement paste. The plot is the concentration of fluoride ions in ppm released from microcapsules loaded at 5 w/w% in an orthodontic cement paste formulation as a function of time in days.
release is reported per gram of orthodontic cement submerged in nanopure water. 2.6. Phosphate ion detection In order to determine the concentration of phosphate ion in a given aliquot, the classic molybdenum blue method was used [42]. The concentration of the dibasic form of the anion is reported in Figs. 5 and 6. A Tecan Infinite M200 spectrophotometer was used to measure absorbance values of the molybdenum complex at 882 nm. Each measurement was performed in triplicate. 2.7. Calcium and fluoride ion detection Potentiometry was used to determine the concentration of calcium and fluoride ions [43]. An ELIT 1265 calcium specific electrode was used for the calcium ion; An ELIT 8221 fluoride specific electrode was used for the fluoride ion. Both of the aforementioned electrodes were used in conjunction with an ELIT 001N silver chloride reference electrode. 2.8. Adhesion measurement procedure Bovine teeth were sectioned buccolingually and placed in an acrylic mold with the enamel exposed. The teeth were acid conditioned using 35% phosphoric acid (Ultradent, Utah) for 20 s, rinsed and air-dried. Primer was applied to the tooth and visible light cured by a Spectrum 800 curing light at 600 mW/cm2 for 20 s. Orthodontic cement was applied to 3 M Victory Roth orthodontic brackets (n = 10) containing the BJM manufactured 2/2/1 formulation with microcapsules. A control (n = 10) paste was prepared using the formulation without microcapsules. These orthodontic cement pastes were pressed to exude excess cement, cleaned and light cured for 20 s per each of the four sides. The light was held at a 45 angle for each of the 4 exposures. The specimens were stored in water at 37 C for 7 days before being loaded to failure at 1 mm per minute using a crosshead-shearing fixture rounded to fit the bracket on an Instron (model) test frame. The surface area (determined by using the outline dimension) of each bracket was measured and calculated to be 13.86 mm2. Shear bond strength was measured and reported in Megapascals (MPa). Each debonded surface was examined visually at a magnification of 20X to assess the qualitative characteristics of the failure interface. 3. Results Charging of a dental material with fluoride is a potential strategy for the promotion of remineralization in the oral environment. Introduction of water filled microcapsules into an
orthodontic cement to determine if the presence of these microcapsules affects the ability of the material to charge and release fluoride ions was investigated. One orthodontic cement was formulated with microcapsules containing only nanopure water to unambiguously determine the source of the fluoride ion while a control cement was prepared without microcapsules. These two separate orthodontic cements were brushed for 40 cycles with an over the counter toothpaste. These formulations were then soaked in nanopure water and the release of fluoride was measured over time. In addition to charging an orthodontic cement by brushing, it would potentially be advantageous to have an orthodontic cement capable of slow, sustained release of ions useful for remineralization. Orthodontic cements were also prepared containing encapsulated aqueous solutions of calcium, phosphate or fluoride ions or mixtures of all three. Finally, the effect of microcapsules on the adhesion of the orthodontic cement with the bracket in place to the enamel was determined. 3.1. Fluoride charge and release The effect of loading microcapsules into an orthodontic cement and the subsequent release of fluoride from the cement following toothbrushing was determined. Fig. 2 depicts the part per million of fluoride being released per gram of orthodontic cement as a function of time for two orthodontic cement formulations. One formulation was formulated with microcapsules that contained nanopure water. The other formulation was an orthodontic cement with no microcapsules. Both formulations, after being exposed to 40 cycles of toothbrushing released fluoride ions over time. As seen in Fig. 2, after 14 days, the control released 0.010 ppm of fluoride/g of cement. The formulation with microcapsules released 0.069 ppm of fluoride/g of cement. 3.2. Static ion release The ion release profile of fluoride ions from an orthodontic cement manufactured to contain 5 w/w% microcapsules that contained an aqueous solution of 0.8 M sodium fluoride is shown in Fig. 3. The graph depicts the ppm of fluoride ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of calcium ions from an orthodontic cement formulated with 3 w/w% microcapsules that contained an aqueous solution of 5.0 M calcium nitrate tetrahydrate is shown in Fig. 4. The graph depicts the ppm of calcium ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of phosphate ions from an orthodontic cement formulated with 3 w/w% microcapsules that contained an aqueous solution of 6.0 M potassium phosphate dibasic is shown in Fig. 5.
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the ppm of each individual ion released per gram of orthodontic cement formulation as a function of time. 3.3. Adhesion
Fig. 4. Normalized concentration of calcium ions released from microcapsules containing 5.0 M calcium nitrate tetrahydrate loaded in orthodontic cement paste. The plot is the concentration of calcium ions in ppm released from microcapsules loaded at 3 w/w% in an orthodontic cement paste formulation as a function of time in days.
Fig. 5. Normalized concentration of phosphate ions released from microcapsules containing 6.0 M potassium phosphate dibasic loaded in orthodontic cement paste. The plot is the concentration of phosphate ions in ppm released from microcapsules loaded at 3 w/w% in an orthodontic cement formulation as a function of time in days.
The graph depicts the ppm of phosphate ion released per gram of orthodontic cement formulation as a function of time. The ion release profile of fluoride, calcium and phosphate ions from an orthodontic cement manufactured to contain 2 w/w% microcapsules that contained an aqueous solution of 0.8 M sodium fluoride, 2 w/w% microcapsules that contained an aqueous solution of 5.0 M calcium nitrate tetrahydrate, and 1 w/w% microcapsules that contained an aqueous solution of 6.0 M potassium phosphate dibasic is shown in Fig. 6. The graph depicts
Fig. 6. Normalized concentration of fluoride, calcium and phosphate ions released from 2 w/w% microcapsules containing 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate and 1 w/w% 6.0 M potassium phosphate dibasic loaded in orthodontic cement paste. The plot is the concentration of fluoride, calcium and phosphate ions in ppm released from microcapsules loaded at 5 total w/w% in an orthodontic cement formulation as a function of time in days.
The adhesion of the orthodontic cement to the enamel with the bracket in place was determined. Fig. 7 depicts the pressure the orthodontic bracket underwent before failure and is compared side by side after quartiles are obtained. The whiskers demonstrate the maximums and minimums. The average for the control formulation without microcapsules was 8.32 1.71 MPa. The formulation with the microcapsules had an average of 8.56 2.52 MPa. 4. Discussion Considering the delicate balance of demineralization and remineralization of the enamel in the oral environment, the purpose of this study was to develop a new approach to promote remineralization using polyurethane based microcapsules as a filler in orthodontic cement. To promote remineralization, the orthodontic cement would ideally release remineralizing ions (e.g., calcium, phosphate, fluoride) contained within the microcapsules. Furthermore, if inclusion of microcapsules enhanced the uptake and release of additional fluoride ions due to toothbrushing while not adversely effecting adhesion, an improvement in orthodontic cement could potentially be achieved. In this study, the question of whether the presence of the semipermeable membrane in the form of the microcapsule shell could possibly assist in the absorption or adsorption of fluoride ions for release after realistic daily exposure from toothbrushing was explored. In orthodontic cement formulated with microcapsules that contained only water, the microcapsule could potentially absorb or adsorb fluoride and release it into the environment. In this study, fluoride ions were introduced to an orthodontic cement that contained microcapsules through toothbrushing with an over the counter toothpaste. Additionally, the effect of microcapsules on adhesion of a material to enamel was explored for the first time. Finally, in Falbo et al., only the release of phosphate ions from varnish and glaze formulations was reported. In this paper, the release of calcium and fluoride ions, in addition to phosphate ions was studied from orthodontic cement formulated with microencapsulated aqueous solutions of sodium fluoride, calcium nitrate and potassium phosphate dibasic. Previous work demonstrated the passive diffusion of ions from dental materials formulated with the microcapsules that is primarily driven by a concentration gradient. In this study, the possibility of charging a dental material with microcapsules that contained nanopure water with fluoride was examined. In order to
Fig. 7. Box plot of pressure loaded before failure of orthodontic cement with microcapsules containing 2 w/w% microcapsules containing 0.8 M sodium fluoride, 2 w/w% 5.0 M calcium nitrate and 1 w/w% 6.0 M potassium phosphate dibasic and orthodontic cement formulated without.
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unambiguously determine the source of fluoride ions measured, the orthodontic cements were formulated to contain microcapsules with nanopure water before being exposed to fluoride ions through simple brushing. As a control, an orthodontic cement with no microcapsules was also prepared. When the orthodontic cement paste formulations were brushed, both formulations either absorbed or adsorbed and subsequently released fluoride. As seen in Fig. 2, the formulation containing microcapsules released 68% more fluoride per gram of formulation than the control. After the similar initial burst of fluoride ions from both formulations, the control formulation stopped releasing fluoride between 1 and 7 days, whereas the formulation containing microcapsules still continued to release fluoride ions after one week. The formulation with water containing microcapsules could have released more fluoride after toothbrushing for a number of potential reasons. The fluoride ions could have absorbed into the more polar semipermeable polyurethane membrane found at the surface of the material and absorb into the interior for rerelease. Another explanation could be that the addition of the polar microcapsules into the nonpolar, hydrophobic dental material has changed the overall polarity of the material where ions may be more attracted to the material than one formulated without any microcapsules. Therefore the more polar orthodontic cement could enhance charging relative to a less polar, hydrophobic control. After charging water filled microcapsules within the orthodontic cement with fluoride through simple toothbrushing, a study was set up to show if an orthodontic cement paste formulated with microencapsulated remineralizing agents can release ions (calcium, fluoride and phosphate) at a slow, sustained rate over an extended period of time. Separate orthodontic cement pastes were formulated to contain microcapsules with aqueous solutions of calcium, fluoride or phosphate ions preloaded inside of the polyurethane shell. These orthodontic cements were prepared with each individual ion to give a sense of ion release potential without precipitation. Orthodontic cement paste was formulated with these microcapsules and the release was reported as a function of time in days that are shown a release of these ions in days in Figs. 3–5. Ion release is demonstrated over nine months. Based on these results and previous studies of phosphate ion release from glazes, the potential to release ions throughout the projected lifetime of an orthodontic cement in the oral environment should be achievable [41]. Fig. 6 shows a release profile of fluoride, calcium and phosphate ions from an orthodontic cement paste containing 2 w/ w% of 0.8 M sodium fluoride, 2 w/w% of 5.0 M calcium nitrate and 1 w/w% of 6.0 M potassium phosphate dibasic formulation or a mixture of all three ions. Potential for precipitation of insoluble calcium phosphates and calcium fluoride could effectively explain the difference between individual ion release profiles with that of mixed microcapsule formulations. Although mixed microcapsule formulations appear to have a lower ion release rate, it may be the precipitation of insoluble calcium phosphates or calcium fluoride. If this occurs in the presence of the enamel surface, remineralization would be promoted. A final aspect of this current study examined whether the adhesion properties of an orthodontic cement that contained microcapsules was different from a control formulation that was manufactured without microcapsules. An orthodontic cement paste that contained 2 w/w% of 0.8 M sodium fluoride, 2 w/w% of 5.0 M calcium nitrate and 1 w/w% of potassium phosphate dibasic formulation and a control with the same composition without microcapsules was prepared. Fig. 7 shows the adhesion data of these two different formulations. This experiment demonstrated little effect on the adhesion to conditioned enamel based on the presence of the microcapsules in the cement formula. This suggests that whatever viscosity variation occurs with the
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microcapsule formula, the prime influence on adhesion is derived from the acid conditioning and the application of the primer. There was no visual evidence of any difference in the adaptation of the cement to the bracket mesh between the two formulas. 5. Conclusion The development of an orthodontic cement paste that releases fluoride (in addition to calcium and phosphate ions) with acceptable adhesion properties could result from the mechanism of fluoride release being from simple diffusion of fluoride through a semipermeable membrane triggered by concentration gradients as opposed to etching and dissolution mechanisms. The addition of polyurethane based semipermeable microcapsules as a filler within orthodontic cement paste could promote remineralization of the tooth. Acknowledgements The authors would like to acknowledge the Premier Dental Products Company and the NSF/EPSCoR program under grant EPS1004094 for support of this research. The authors would like to acknowledge BJM Laboratories for manufacturing the orthodontic cements used in this study. ML and SG have a financial interest in the Premier Dental Products Company. References [1] L. Gorelick, A.M. Geiger, A.J. Gwinnett, Incidence of white spot formation after bonding and banding, Am. J. Orthod. 81 (1982) 93–98. [2] M.M. O'Reilly, J.D.B. Featherstone, Demineralization and remineralization around orthodontic appliances: an in vivo study, Am. J. Orthod. Dentofacial Orthop. 92 (1987) 33–40. [3] J.A. Chapman, W.E. Roberts, G.J. Eckert, K.S. Kula, C. González-Cabezas, Risk factors for incidence and severity of white spot lesions during treatment with fixed orthodontic appliances, Am. J. Orthod. Dentofacial Orthop. 138 (2010) 188–194. [4] F. Bergstrand, S. Twetman, Evidence for the efficacy of various methods of treating white-spot lesions after debonding of fixed orthodontic appliances, J. Clin. Orthodont. 37 (2003) 19–21. [5] P.E. Benson, N. Parkin, D.T. Millett, F.E. Dyer, S. Vine, A. Shah, Fluorides for the prevention of white spots on teeth during fixed brace treatment, Database System. Rev. 3 (2004) CD003809. [6] B.L. Chadwick, J. Roy, J. Knox, E.T. Treasure, The effect of topical fluorides on decalcification in patients with fixed orthodontic appliances: a systematic review, Am. J. Orthod. Dentofacial Orthop. 128 (2005) 601–606. [7] R.J. Lynch, R. Navada, R. Walia, Low-levels of fluoride in plaque and saliva and their effects on the demineralization and remineralization of enamel; role of fluoride toothpastes, Int. Dent. J. 54 (2004) 304–309. [8] C. ten, J.M. ate, Current concepts on the theories of the mechanism of action of fluoride, Acta Odontol. Scand. 57 (1999) 325–329. [9] J. ten Cate, J.D. Featherstone, Mechanistic aspects of the interactions between fluoride and dental enamel, Crit. Rev. Oral Biol. Med. 2 (1991) 283–296. [10] E.C. Reynolds, Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review, Spec. Care Dentist. 8 (1998) 8– 16. [11] A. Wiegand, W. Buchalla, T. Attin, Review on fluoride-releasing restorative materials-fluoride release and uptake characteristics, antibacterial activity and influence on caries formation, Dent. Mater. 23 (2007) 343–362. [12] D. Wood, R. Hill, Structure-property relationships in ionomer glasses, Clin. Mater. 7 (1991) 301–312. [13] E.C. Reynolds, Calcium phosphate-based remineralization systems: scientific evidence? Aust. Dent. J. 53 (2008) 268–273. [14] Li Xiaoke, The remineralization of enamel: a review of the literature, J. Dentist. (2014) S12–S20. [15] RM Duckworth, E. Huntington, On the relationship between calculus and caries. In: The Teeth and their Environment Duckworth RM, editor. Monographs in Oral Science 2006; 19: 1–28. [16] E.C. Reynolds, C.J. Cain, F.L. Webber, C.L. Black, P.F. Riley, I.H. Johnson, Anticariogenicity of calcium phosphate complexes of tryptic casein phosphopeptides in the rat, J. Dent. Res. 74 (1995) 1272–1279. [17] E.C. Reynolds, Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: a review, Spec. Care Dentist. 8 (1998) 8– 16. [18] E.C. Reynolds, Remineralization of enamel subsurface lesions by casein phosphopeptide-stabilized calcium phosphate solutions, J. Dent. Res. 76 (1997) 1587–1595.
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