By JOSHUA FULTON A THESIS

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3 THE EFFECT OF CONTINUOUS PHASE AND GLASS CONTENT ON FRACTURE TOUGHNESS AND CALCIUM AND PHOSPHATE ION RELEASE FROM ION PERMEABLE MICROCAPSULES IN GLAZE FORMULATIONS By JOSHUA FULTON A THESIS Submitted to the faculty of the Graduate School of the Creighton University in Partial Fulfillment of the Requirements for the degree of Master of Science in the Department of Oral Biology Omaha, NE April 3, 2013

4 ABSTRACT This study expands on the work of developing dental resin based restorations capable of releasing phosphate, calcium, and fluoride ions. This approach has the capability of combating recurrent caries. In an attempt to develop a glaze formulation, the role of monomer composition and glass filler incorporation on ion release was studied. Ion permeable microcapsules containing bioavailable calcium and phosphate salts integrated into various resin formulations that differ in monomer types and ratios of the continuous phase along with how the presence of silanated glass filler effects the release of remineralizing ions is measured. The objective of this research is to develop a glaze formulation that can sustain a controlled release of bioavailable calcium and phosphate ions for extended periods, ideally between prophylactic examinations. Second, mechanical testing will be conducted to see how the inclusion of ion permeable microcapsules affects the fracture toughness of the glaze, with the aim to understand how to develop a bioactive dental composite that can withstand occlusal stresses of large restorations in posterior dentition. iii

5 ACKNOWLEDGMENTS I would like to thank Creighton University and the School of Dentistry, specifically Dr. Stephen Gross, Dr. Mark Latta, Dr. Neil Norton, Dr. Wayne Barkmeier, and Dr. Margaret Jergenson for allowing me the opportunity to contribute to the field of dentistry through my investigative efforts. I would also like to thank my colleagues, Ryan Cooper, Michelle Falbo, and Matthew Schmidt for their aid in completion of this project. Furthermore, I would like to thank Creighton University and the Premier Dental Products Co for supporting this research. iv

6 Table of Contents ABSTRACT iii ACKNOWLEDGMENTS.. iv LIST OF FIGURES.. vi SECTION 1: INTRODUCTION Prevalence of caries Tooth structure Demineralization of enamel Direct restorative dental materials Recurrent caries Ion permeable microcapsules 12 SECTION 2: MATERIALS AND METHODS Materials Pre- polymer synthesis Oil solution preparation Salt solution preparation Microcapsule synthesis Glaze formulations Ion release measurement preparation Phosphate ion detection Calcium ion detection Fracture toughness specimen preparation Fracture toughness testing SECTION 3: RESULTS Effect of glass content on phosphate ion release from two different continuous phases Effect of glass content on calcium ion release from two different continuous phases Effect of MMA concentration on the initial release of phosphate ions and calcium ions from a glaze formulation Comparison of the release rate of phosphate ions and calcium ions as a function of the diluent monomer Effect of glass content on fracture toughness Effect of microcapsules on fracture toughness Effect of glass and microcapsules on fracture toughness of a continuous phase with MMA Comparison of fracture toughness based upon the diluent monomer.. 51 SECTION 4: DISCUSSION REFERENCES v

7 LIST OF FIGURES Figure 1: Image of formulation films used for ion release measurements Figure 2: Single notch specimen for fracture toughness measurements. 32 Figure 3A: Effect of varying glass content on release of phosphate ions. 36 Figure 3B: Release of phosphate ions as a function of glass content at 0 w/w%, 10 w/w%, 20 w/w%, and 30 w/w% Figure 4A: Effect of glass content on phosphate ion release Figure 4B: Effect of glass content on calcium ion release.. 39 Figure 5: Comparison of the release of phosphate and calcium ions from two different continuous phases Figure 6A: Effect of MMA concentration on the initial release rate of phosphate ions Figure 6B: Release of phosphate ions as a function of MMA content. 42 Figure 6C: Effect of MMA concentration on the initial release rate of phosphate ions Figure 6D: Release of calcium ions as a function of MMA content Figure 7:Effect of diluent monomer type on calcium and phosphate ion release.. 46 Figure 8: Fracture toughness as a function of glass content 47 Figure 9: Fracture toughness as a function of microcapsules content.. 48 Figure10: Fracture toughness as a function of glass and microcapsule content. 49 Figure11: Fracture toughness as a function of glass and microcapsule content. 50 Figure 12: Fracture toughness as a function of diluent monomer LIST OF TABLES Table 1: Glaze formulations for ion release measurements Table 2: Glaze formulations for fracture toughness measurements vi

8 Section 1 Introduction 1

9 1.1. Prevalence of Caries Dental caries is one of the most prevalent chronic diseases worldwide despite the fact that it is almost entirely preventable. 1 A report issued by the United States Surgeon General in 2000 stated that dental caries is the most common disease in children ages 5-17 years old, surpassing asthma and hay fever. 2 In the United States over half of the children ages 5-9 have at least one cavity or restoration and that number increases to 78% among 17 year- olds. 2 Consequences of school- aged children acquiring caries are a decrease in quality of life (e.g. difficulty eating, speaking, lowering self- esteem) and may lead to further health problems. Additionally, it is estimated that more than 51 million school hours are lost to dental related illness each year in the United States. 3 Some of the more prevalent factors that predispose an individual to caries include socioeconomic status, education level, diet, oral hygiene habits and access to dental care Tooth structure Teeth are comprised of four components, with varying physical and biological properties. These components are enamel, dentin, pulp and cementum. Enamel is an acellular tissue comprised of a highly mineralized crystalline structure wt% inorganic hydroxyapatite (HAP), 1-2 wt% organic (carbonate), 2-3wt% water and trace elements (e.g. sodium, magnesium, potassium, chloride and zinc). 4 Enamel is the hardest substance in the human body and provides a protective cap for the vital tissues of the tooth. HAP crystals in enamel form the shape of millions of interlocking rods that extend from the dentin to the enamel surface. Dentin 2

10 makes up the largest portion of the tooth and is characterized as flexible and softer than enamel thus acting as a cushion so that enamel is not prone to fracture during the forces of mastication. The composition of dentin is 75 wt% inorganic HAP, 20 wt% organic (primarily collagen) and 5 wt% water. 5 Cementum is the outer most lining of the root, and is composed of wt% HAP and wt% organic matter and water. Pulp occupies the pulp cavity within the tooth. Pulp functions to supply the tooth with nutrients, provide sensory perception, stimulate repair of organic components of dentin and cementum, along with containing lymphatic tissues Demineralization of Enamel Dental caries is a multifactorial disease that includes the participation of cariogenic and noncariogenic bacteria, salivary components and dietary sources of fermentable carbohydrates. 4 The caries process begins with the dental pellicle, which is an acellular base layer of protective proteins that has a strong affinity for HAP. The function of the pellicle is to protect the tooth surface from abrasion and grinding forces during mastication. 6 The dental pellicle forms immediately on newly erupted teeth and on tooth surfaces exposed to saliva after being cleaned. A drawback of the dental pellicle is that it serves as a substrate for bacterial adhesion to initiate bioflim formation, allowing colonization of a vast array of bacterial species. Dental biofilms harbor cariogenic bacteria that are acidogenic (produce acid) and aciduric (tolerate acidic environments). 5 Cariogenic bacteria include Streptococcus mutans and Lactobaccilli. The colonization of S. mutans and 3

11 Lactobaccilli metabolize fermentable carbohydrates, such as sucrose, to produce organic acids in the oral environment. The resulting decrease in the ph at the biofilm- enamel interface initiates demineralization of HAP. 7 HAP is composed of millions of enamel rods that are made up of many hexagonal prisms that hold the calcium, phosphate, hydroxyl and other impurities. The hydroxyl group is centrally located with a triangle of calcium surrounding it. A triangle of phosphate surrounds the calcium triangle. The whole structure is then surrounded by a hexagon of calcium. 8 These structures are then stacked on top of one another with each rotated 60 degrees to its neighboring hexagon. The chemical process of demineralization of enamel begins with understanding critical ph and solubility product (Ksp) of enamel. First, the critical ph describes the ph at which a solution is just saturated with respect to a mineral, such as enamel at the biofilm interface. If the ph is greater than the critical ph the solution is supersaturated with respect to the mineral and more of that mineral will tend to precipitate. On the other hand, if the ph is less than the critical ph, the solution is unsaturated and more of the mineral will dissolve into the solution. The chemical formulation of HAP is Ca10(PO4)6(OH)2 and the individual ions of HAP undergo constant precipitation and dissolution in the oral environment until an equilibrium is achieved between the two. Ksp is defined as the product of the concentrations (mol/l) of the component ions raised to the appropriate power, in a saturated solution. The Ksp for HAP is [Ca] 10 [PO4] 6 [OH] 2 which is approximately The Ksp for a mineral is always 4

12 constant, however the concentrations of the three constituents can change and, in the case of HAP, impurities within the crystals have an effect on Ksp. HAP is not homogeneous and impurities such as carbonate (which can occupy the hydroxyl or phosphate positions in the lattice) create a higher solubility product of HAP making it more acid- soluble. 8,10 Featherstone et al. created an equation to illustrate how these impurities change the constituents of HAP: {Ca10- a(na)a(po4)6- b(co3)c(oh)2- d(f)d}. 7 These forms of carbonated apatite are the most acid soluble form of apatite found in enamel and are the most common form of apatite in enamel. Concentrations of carbonate and sodium are dependent on the partial pressure of CO2 during crystal development. 8 After the consumption of fermentable carbohydrates, the ph in the oral environment is lowered due to the metabolic activity of acidogenic bacteria and the carbonate and hydroxyl groups of HAP are most susceptible to dissolution. Carbonate readily dissolves due to having a poorer fit in the HAP crystal and is therefore less stable. 8 The elevated level of H3O + ions readily dissolve the calcium ions and phosphate ions into solution. 7 As dental plaque accumulates and the ph of the plaque decreases, the aciduric bacteria S. mutans and lactobacilli bacteria begin to proliferate and become the prominent microflora of the dental plaque and further exasperate the caries process. 11 The oral environment can be viewed as a dynamic environment experiencing an oscillating ph range which results in periods of progression and remission of dental caries with the outcome being decided on which factor (pathological or protective) outweighs the other. These phases of demineralization and remineralization can vary between locations within the same lesion. 8 5

13 In order to repair the damage done by demineralization, prevention, intervention, and remineralization can be achieved by either reducing pathological factors (bacteria and carbohydrates) or enhancing protective factors (saliva, calcium, phosphate, fluoride). 9 Reduction in pathological factors may include antimicrobial mouthrinses that contain chlorhexidine gluconate 12 and decreasing carbohydrate consumption. Fluoride has been widely used as an ion that can promote remineralization of HAP through the ability to occupy the vacancy left from the dissolution of the hydroxyl ion of HAP to form fluorapatite (Ca10(PO4)6F2). 12 A desirable outcome from the binding of fluoride into the HAP lattice is a more stabilized lattice that has a lower Ksp and therefore is more resistant to acid attack. 7 Fluoride is available through many sources (e.g. fluoridated tap water, toothpaste, mouthwash, and other dentifrices). However, fluoride alone has not put an end to the demineralization of HAP. Studies have shown that fluoride preferentially binds to the external surface of HAP and can have an inhibitory effect on the penetration of other fluoride ions to deeper areas of the carious lesion. Current dental products that promote remineralization of enamel are bioactive glass systems, unstabilized amorphous calcium phosphate systems, and stabilized amorphous calcium phosphate systems. Bioactive glass systems are based on a calcium sodium phosphosilicate glass that theoretically should release calcium and phosphate ions into the oral environment to stimulate remineralization. NovaMin is an example of this type of system, which incorporates a calcium sodium phosphosilicate glass in a dentifrice. 13 6

14 An example of unstabilized amorphous calcium phosphate was Enamelon toothpaste. Calcium ions and phosphate ions were introduced as a dentifrice separately in a dual chamber device claiming to form amorphous calcium phosphate in situ. It is claimed that formation of the amorphous complex promotes remineralization. 13 Recaldent is a product that uses casein phosphopepetide- stabilized amorphous calcium phosphate (CCP- ACP). CCPs are a milk derived protein that have the ability to stabilize calcium, phosphate and fluoride ions as bioavailable amorphous complexes. It is hypothesized that CCPs can facilitate the stabilization of high concentrations of ionically available calcium and phosphate even in the presence of fluoride. 14 This is proposed to happen by the dinding of CCP to the pellicle and plaque. The CCP would then prevent the formation of dental calculus as the ions diffuse down the concentration gradient to subsurface enamel lesions facilitating remineralization. Recaldent can be applied to teeth by sugar- free gum (Trident XtraCare with Recaldent) or professional tooth crème (MI Paste, MI Paste Plus, GC s Tooth Mousse) Direct Restorative Dental Materials Purpose When demineralization progresses far beyond preventative treatment, the carious tooth structure needs to be surgically removed and replaced with a prosthetic device that replaces the lost tooth structure and retains the mechanical properties of the tooth. Restorations are classified based on what surface of a 7

15 particular tooth the restoration covers. These are classified as Class I Class VI. A Class I restoration is one that involves pits and fissures of all teeth. Class II describes a carious lesion on a proximal surface of a posterior tooth (i.e. molars and premolars). Class III restorations involve proximal surfaces of anterior teeth (i.e. incisors and canines). Class IV restorations involve a proximal surface of an anterior tooth in which an incisal angle is also involved. Class V describes a carious lesion in the gingival 1/3 of any tooth. Class VI denotes a carious lesion on cusp tips of posterior teeth or incisal edges of anterior teeth. 5 Dental Amalgam An amalgam is referred to an alloy of mercury with another metal or metals. In dentistry the alloy is comprised of silver, tin, copper and zinc with each metal contributing to specific mechanical properties of the amalgam. Silver increases strength and expansion while decreasing flow. Tin increases contraction and reduces strength. Copper increases both strength and hardness while decreasing brittleness. Zinc decreases brittleness, increases plasticity and minimizes oxide formation. Most dental alloys are categorized as being high copper, with the copper content being wt%, this decreases corrosion and increases life expectancy of the restoration material. 5 Advantages of dental amalgam are that they have been proven to be clinically successful, it is cheap, has a compressive strength of MPa which is similar to enamel and dentin (384 MPa, 297 MPa respectively), easy to use, simplicity of procedure, high tensile strength, less time consuming, excellent wear resistance. 8

16 Disadvantages are that the tooth preparation is more complex and requires removal of healthy enamel and dentin. Furthermore, amalgam is non- insulating, not esthetic, has an initial marginal leakage, common marginal breakdown and contains mercury which has handling, disposal and health concerns. Health and environmental concerns relating to amalgam fillings arise from the high mercury content in the alloy. During mastication mercury vapor is released from the restoration and absorbed into the blood stream and can target the nervous system and kidney. A study of cadavers found a positive correlation between the number of occlusal amalgam surfaces and concentration of mercury in a number of tissues (pituitary gland, cerebral cortex, thyroid, renal cortex) (Gianpaolo). Glass Ionomer Cements Glass ionomer cements fall into two categories: conventional glass ionomer cements (GIC) and resin- modified glass ionomer cements (RMGIC). GICs are composed of a polyacrylic acid liquid and glass powder containing aluminum, fluoride, calcium, sodium, and silica. The setting reaction is an acid base reaction in which the fluoroaluminosilicate glass is attacked by H3O + ions from the polyacrylic acid, which releases aluminum, calcium, sodium and fluoride ions. Crosslinking occurs when the free aluminum and calcium ions crosslink with two or three iononized carboxyl groups of the polyacid to form a gel. This polycarboxylate salt begins to precipitate until the cement is hard. Advantages of GICs include cariostatic potential due to fluoride release, good adhesion to tooth structure, low setting shrinkage, and a coefficient of thermal expansion approximately equal to tooth structure (Summit 2006). Disadvantages of GICs are that they are very sensitive to 9

17 moisture and dessication, short life span, have a low initial ph and may have a post cementation sensitivity. 5 Additionally a study found that one half of GIC restorations had to be removed due to secondary caries. 15 Resin modified glass ionomer cements (RMGICs) combine the chemistry of GICs with a small amount of light cured resin to help overcome the challenges of GICs while still maintaining desirable physical properties. 16 These cements follow up the same acid- base reaction as GICs but then add a light- activated polymerization of the resin. Advantages are that the RMGIC has increased physical properties over the GICs while maintaining fluoride release. Disadvantages are that although the physical properties are superior to GICs, they are still not sufficient to be used as a restorative material and their use has been relegated mainly to baseliners. Resin based Composites Resin based composites7were introduced in the 1960 s as an esthetic alternative to dental amalgam. 18 Because of the esthetic, physical and mechanical properties, resin composites have become one of the most common and widely used materials for the direct restoration of both anterior and posterior teeth. 18,19 Currently used commercial restorative composites contain a mixture of various cross linking dimethacrylates, inorganic fillers, and a photoinitiator system. 20 Common base monomers used in resins are bisphenol A glycidyl dimethacrylate (Bis- GMA) and urethane dimethacrylate (UDMA). These monomers are hydrophobic, have high molecular weights, high viscosities and multiple functional groups that require the use of a low viscosity diluent monomer such as triethylene 10

18 glycol dimethacrylate (TEGDMA) or methyl methacrylate (MMA)to make the resin flowable. 21,22 Diluent monomers tend to decrease viscosity, composite curing and mechanical properties and increase volumetric shrinkage and conversion. The filler has several roles, including enhancing modulus, radiopacity, altering thermal expansion behavior, and reducing polymerization shrinkage. 22 Glass fillers, such as barium borosilicate glass (BBS) are able to fill all the roles needed of a filler. Silanated groups on the glass surface are able to chemically bind to the continuous phase of the resin matrix; it has a high coefficient of thermal expansion; and the high barium content increases radiopacity. The curing reaction in composite restoration materials involves visible light initiated photopolymerization in the presence of a photoinitiator and coinitiator. In most instances, camphorquinone (CQ) serves as the photoinitiator and a tertiary amine as the coinitiator. The setting reaction begins with a blue light producing wavelengths of nm to fit the absorbance spectra of CQ, which has a peak absorbance of 470 nm. 23 The blue light causes CQ to generate a short lived excited state, ultimately produces an amine radical. This amine radical attacks the carbon- carbon π bond of the monomers, propagating to form new radicals in the monomers. 22,23 A result of the splitting of the carbon- carbon π bonds of the monomers in order to form the polymer network is a closer packing of the molecules resulting in volumetric contraction. 24 One way to lessen this contraction is the incorporation of fillers such as quartz, silica, silicates and silica glass containing barium, strontium, and zirconium. 18 The volumetric contraction of resin based composites is of special clinical significance because it can result in stresses at 11

19 the resin- dentin/enamel interface that could lead to fracture of the tooth and microleakage at the cavosurface- enamel junction and is a major cause of recurrent caries Recurrent Caries Recurrent caries are the most frequent reason for the replacement of all types of restorations and make up about 50 percent of restorations. 25 Costs associated with replacement of restorations were estimated to be in the billions of dollars annually. Recurrent caries, also referred to as secondary caries, is defined as caries of the tooth at the margin of a restoration. 25 During polymerization of resin based composites, there is a volumetric reduction of the composite which creates a gap at the restoration cavosurface interface. This gap may result in microleakage of cariogenic bacteria or acids which can create a carious lesion at the restoration cavosurface interface. Also, stresses created by polymerization shrinkage pulling on tooth structures can create micofractures that are also susceptible to caries or failure of the restoration. Recurrent caries are more common to occur with Class II V restorations due to the fact that everyday cleaning in the gingival third is more difficult than occlusial surfaces. Further, during placement, the preparation could be contaminated by gingival fluid. Finally, polymerization shrinkage of resin based composites can cause margins at the gingival- cavosurface interface Ion Permeable Microcapsules 12

20 Davidson et al. demonstrated the potential of diffusion of calcium, phosphate and fluoride ions from various polyurethane- based microcapsules submerged in nanopure water. 26 This initial study reported that the chemical structure of the microcapsule shell and initial salt concentration in the microcapsule could control the rate of ion release of phosphate, calcium, and fluoride ions. Experiments expanding on this work (Elassal and Falbo) demonstrated a continuous release of phosphate, calcium and fluoride ions from polyurethane based microcapsules embedded in resin based composite and rosin based varnish formulations. 27,28 This study expands on the work of developing dental resin based restorations capable of releasing phosphate, calcium, and fluoride ions. This approach has the capability of combating recurrent caries. In an attempt to develop a glaze formulation, the role of monomer composition and glass filler incorporation on ion release was studied. Ion permeable microcapsules containing bioavailable calcium and phosphate salts integrated into various resin formulations that differ in monomer types and ratios of the continuous phase along with how the presence of silanated glass filler effects the release of remineralizing ions is measured. The objective of this research is to develop a glaze formulation that can sustain a controlled release of bioavailable calcium and phosphate ions for extended periods, ideally between prophylactic examinations. Second, mechanical testing will be conducted to see how the inclusion of ion permeable microcapsules affects the fracture toughness of the glaze, with the aim to understand how to develop a bioactive dental composite that can withstand occlusal stresses of large restorations in posterior dentition 13

21 Section 2 Materials and Methods 14

22 The aim of this study was to understand a few of the variables that effect the ion release rate from microcapsules in a continuous phase, in order to lead to the development of a bioactive glaze formulation. The role that both glass fillers and MMA content in the continuous phase of the formulation has on the initial release rate of calcium and phosphate ions from ion permeable microcapsules was studied. In this section the synthesis of microcapsules will be described first. This was accomplished by synthesizing a polymer that ultimately becomes the shell of a microcapsule that forms in the presence of a reverse emulsion of an aqueous salt solution of potassium phosphate dibasic or calcium nitrate and an oil phase. The preparation of these solutions is described followed by the synthesis of the microcapsules. The formulation of the dental glaze is described next followed by the description of how the release rate of phosphate ions and calcium ions were measured. Additionally, the description of the fracture toughness measurements of the formulation is described Materials The following chemicals were obtained from Acros Organics: cyclohexanone, ethyl- 4- dimethylaminobenzoate, calcium nitrate tetrahydrate, and methyl benzoate. Chemicals from Sigma Aldrich included: diurethane dimethacrylate, tri(ethylene glycol)dimethacrylate, methyl methacrylate, ammonium heptamoylbdate tetrahydrate, L- ascorbic acid, and potassium antimonyl tartrate. Chemicals obtained from Fischer Sceintific were: ethylene glycol and potassium phosphate dibasic. DL- camphorquinone was purchased from MP Biomedicals. Barium 15

23 borosilicate glass was purchased from Esstech. Toluene- 2,4- diisocyanate was obtained from Alfa Aesar. Nanopure water was produced through a NANOpure Diamond filtration system manufactured by Barnstead International Pre-polymer synthesis The microcapusles consisted of a polyurethane shell. This polyurethane is synthesized from a diol and diisocyanate, two components that, together, create the repeating urethane linkages (- NH- CO- O- ) of a polyurethane. In this experiment, a solution polymerization was used to synthesize the polyurethane. A solution polymerization uses a non- reactive solvent, in this case cyclohexanone, in which the monomers are soluble or miscible. For this experiment, a polyurethane was synthesized from ethylene glycol and tolulene- 2,4- diisocyanate. To perform this synthesis, a clean 500mL, 3- neck round bottom flask (RBF) was used. The RBF was equipped with a stir bar and two 24/40 septa (one on each of the outer necks) that were secured with copper wire. Vacuum grease was then applied to a 24/40 flow adapter that was inserted into the middle neck of the 3- neck RBF and secured with a Keck clip. A vacuum hose from a manifold attached to a vacuum pump was then attached to the flow adapter and the flask was evacuated using the vacuum pump. The process to evacuate the RBF was performed first. This entailed turning on the vacuum, then opening the vacuum port on the manifold, and finally opening the valve on the flow adapter. The RBF was evacuated for 5 minutes. Next, residual moisture in the RBF needed to be removed. To accomplish this, a Bunsen burner was used to heat the RBF while still being evacuated. This flame 16

24 drying was conducted for two additional minutes. The RBF was then allowed to cool to room temperature and the flow adapter was closed, followed by closing of the vacuum manifold port and then turning the vacuum pump off. The RBF was filled with an inert gas (argon or nitrogen). The procedure for this was to open the valve on the inert gas tank, in this experiment nitrogen was used. The inert gas valve on the manifold was opened. This was followed by the gradual opening of the flow valve on the RBF. It was important to do this by sequentially rotating the valve to allow a gradual increase in pressure to prevent ejection of the septa or adapter. While continuing to flow nitrogen into the RBF, one septum was removed and the solvent, cyclohexanone, was added using a graduated cylinder. The septum was replaced and secured with copper wire. Toluene- 2,4- diisocyanate was then added using a syringe and inserting the needle of the syringe through the septum. The solution was allowed to stir for 30 minutes. After this, mol of ethylene glycol was added using a syringe by inserting the needle through the septum. The RBF was then lowered into a high temperature silicone oil bath. A degassing needle, which is connected to the inert gas port of the manifold, was inserted through the one septum. It is imperative that the degassing needle rest at the bottom of the flask. The manifold port for the degassing needle was then opened followed by closing of the flow valve and the inert gas port on the manifold connected to the flow valve. A pressure relief needle was then inserted into the opposite septa of the degassing needle. The solution was then allowed to degas for fifteen minutes at room temperature. After fifteen minutes, the pressure relief needle was removed. 17

25 Next, the manifold port for the degassing needle was closed followed by turning the inert gas tank off. The degassing needle was then removed and the reaction mixture was set to 80 C using a temperature controller which rested in the silicone oil bath. Once the temperature of the solution had reached 80 C the reaction was allowed to proceed overnight. The pre- polymer was isolated next by removing the solvent to obtain the pure polymer. The reaction solution was transferred to a single neck RBF and a lubricated 24/40 flow adapter was attached and secured with a keck clip. A vacuum line from the cold trap was then secured to the flow adapter. The cold trap dewar was filled approximately one third full with isopropanol and small pieces of dry ice were slowly added. Silicone grease was applied to the joint of the cold trap to prevent the two components from seizing together. The RBF was then lowered into the oil bath and the temperature controller was set to 100 C. Glass wool wrapped in aluminum foil was used to cover and insulate the RBF, flow adapter, and the top of the cold trap. After all components were covered and the temperature had reached 100 C, the vacuum pump was turned on and the flow adapter was opened. Total time for pulling the solvent took approximately one hour and the cold trap was emptied around every fifteen minutes. To empty the cold trap, the flow adapter was closed and then the vacuum pump was turned off. The hose connecting the top of the cold trap to the vacuum was then removed from the cold trap to break the vacuum seal. The cold trap was then separated and the solvent was poured into a beaker. In the event that the solvent had frozen, a heat gun was used to melt the solvent. Silicone grease was then reapplied to the joint of the cold trap and the 18

26 vacuum hose was reattached. The vacuum pump was turned on followed by opening the flow adapter. This process was repeated until the collection beaker reached approximately 150 ml (the original amount of solvent used). The polymer was collected from the RBF. The flow adapter was removed from the RBF and the residual silicone grease in the neck of the RBF was removed to prevent contamination of the polymer. To accomplish this, the RBF was held inverted and a Kimwipe saturated with hexanes was wiped on the inner surface of the neck. After the silicone grease was cleaned, the RBF was secured on a ring stand at a 45 degree angle with a 20 ml screw top vial beneath the opening of the RBF. A heat gun was used to allow the pre- polymer to flow from the RBF into the vial. Care was taken not to thermally decompose the polyurethane with the heat gun. A clean spatula was then used to scrape any remaining pre- polymer and the vials were labeled and stored at room temperature until needed for microcapsule synthesis Oil Solution preparation In order to synthesize the microcapsules by a reverse emulsion, an oil phase was prepared. Oil solutions were prepared at least 24 hours prior to microcapsule synthesis. The oil solution consisted of methyl benzoate, an emulsifying agent, and the polyurethane pre- polymer. The three components were mixed with a stirbar in an Erlenmeyer flask covered with parafilm Salt solution preparation 19

27 Aqueous salt solutions were ultimately encapsulated in the microcapsule. The salt solutions were made at targeted molarities for ion release studies. For this experiment, 4.0 M calcium nitrate and 4.0 M potassium phosphate dibasic were prepared. Salts were weighed and added sequentially to nanopure water (at a lower volume than the desired amount, initially) in an Erlenmeyer flask. Once the salt was transferred completely, the solution was stirred until completely dissolved. Once complete, each solution was transferred to a volumetric flask, where it was diluted to its proper volume. Solutions were then stored in Nalgene bottles at room temperature Microcapsule synthesis The purpose of this step was to create a polyurethane microcapsule that would encapsulate the aqueous salt solutions. Encapsulation requires an oil solution and an aqueous salt solution in a process known as a reverse emulsion. The reaction was carried out in a custom made stainless steel reactor, consisting of a canister, lid and propeller. The reactor was placed in a Büchi B- 495 heating bath filled with deionized water and set to 70 C. It was important to keep the reactor off the bottom of the heating bath, approximately one inch of clearance was used in this experiment. Clamps were then used to secure the lid to the canister with the propeller inside and an extra ring stand was used to level and support the reactor. A Caframo stirrer (Model BDC 6015) was mounted over the water bath and the propeller rod from the reactor was fitted into the chuck of the stirrer and tightened. The stirrer was positioned approximately two inches above the lid of the reactor. 20

28 Next, the stirrer was tested to make sure all components were leveled and secured. Testing was conducted at 100 rpm, 500 rpm, 1,000 rpm, and 4,000 rpm. If all speeds produced minimal vibration the device was ready to use. First, the reactor was heated for approximately 30 minutes to equilibrate the temperature. The oil solution was added through a funnel placed in the opening on the top of the canister. The motor was set to 1,500 rpm and stirred for 1-2 minutes. Next, 110 ml of either 4.0 M calcium nitrate tetrahydrate or 4.0 M potassium phosphate dibasic were added using a Pasteur pipette. Once complete, a rubber stopper was placed on the opening of the lid and the stirrer was set to 4,000 rpm. After 30 minutes of mixing, the rubber stopper was removed and 0.05 mol of ethylene glycol was added to the solution via syringe and needle. This was done to quench any remaining isocyanate groups of the pre- polymer. The rubber stopper was placed back on the opening and the emulsion mixed for three and a half more hours. Once complete, the stirrer was turned off and the canister was taken out of the water bath and allowed to cool. The contents of the canister were then poured into 15 ml centrifuge tubes and centrifuged for several minutes to pelletize the microcapsules. Microcapsules were stored in the capped 15 ml centrifuge tubes at room temperature Glaze formulations In these experiments, a potential bioactive glaze was being developed. As a result, the monomers used in the continuous phase, the ratio of these monomers used and the glass filler concentrations were varied. Regardless of the formulation, 21

29 the contents were always added in the same order. Using a translucent Max 40 Cup (FlackTek Inc.), monomers were added first based on order of increasing viscosity [i.e. methyl methacrylate was always added first followed by tri(ethylene glycol)dimethacrylate and finally diurethane dimethacrylate]. The monomers were mixed in a Speed Mixer DAC150FVZ set at 3,540 rpm for a duration of two minutes after the addition of each monomer. Next, microcapsules were added by first pouring off the methyl benzoate and then discarding the top 1mm using a spatula. Microcapsules were added to the cup in increments of one third at a time with mixing at 3,540 rpm for two minutes after each addition. Next, barium borosilicate glass was added in increments of one third at a time with mixing at 3,540 rpm for two minutes after each addition. Finally, photoinitiators DL- camphorquinone and ethyl- 4- dimethylaminobenzoate (EDMAB) were added at the same time and then the glaze formulation was mixed at 3,540 rpm for two minutes. It is imperative that the photoinitiators be added under light that filters out blue light. Formulations tested for the phosphate ion release portion of this experiment were as follows: UDMA:TEGDMA (80:20 w/w%), 15 w/w% 4.0 M potassium phosphate dibasic microcapsules, and with varying glass content (0 w/w%, 10 w/w%, 20 w/w%, and 30 w/w%). MMA:TEGDMA:UDMA (45:45:10 w/w/w%), 15 w/w% 4.0 M potassium phosphate dibasic microcapsules, 20 w/w% glass content. MMA:TEGDMA:UDMA (30:45:25 w/w/w%), 15 w/w% 4.0 M potassium phosphate dibasic microcapsules, 20 w/w% glass content. MMA:TEGDMA:UDMA (15:45:40 w/w/w%), 15 w/w% 4.0 M potassium phosphate dibasic microcapsules, 20 w/w% glass content. UDMA:MMA (80:20 w/w%), 15 w/w% 4.0 M potassium phosphate 22

30 dibasic microcapsules, 20 w/w% glass content. DL- camphorquinone and EDMAB were added in a 1:1 w/w% ratio as 0.05 w/w% in all formulations and their masses were not included in the formulation mass (Table 1). Table 1: Glaze formulations for ion release experiments. U indicates UDMA; T indicates TEGDMA; M indicates MMA. Formulations tested for the calcium ion release were as follows: UDMA:TEGDMA (80:20 w/w%), 15 w/w% 4.0 M calcium nitrate microcapsules, and with varying glass content (0 w/w% and 20 w/w%). MMA:TEGDMA:UDMA (45:45:10 w/w/w%), 15 w/w% 4.0 M calcium nitrate microcapsules, and with varying glass content (0 w/w% and 20 w/w%). MMA:TEGDMA:UDMA (30:45:25 23

31 w/w/w%), 15 w/w% 4.0 M calcium nitrate microcapsules, and 20 w/w% glass content. MMA:TEGDMA:UDMA (15:45:40 w/w/w%), 15 w/w% 4.0M calcium nitrate microcapsules, and 20 w/w% glass content. UDMA:MMA (80:20 w/w%), 15 w/w% 4.0 M calcium nitrate microcapsules, and 20 w/w% glass content (Table 1). Table 2: Glaze formulations tested for fracture toughness measurements. U indicates UDMA; T indicates TEGDMA; M indicates MMA. 24

32 Formulations prepared for fracture toughness measurements are seen in Table 2. The formulations tested for fracture toughness are the same as those listed in Table 1, except for additional control samples that contained no microcapsules Ion release measurement preparation In order to determine the effect of specific variables on ion release, films of the formulations that contained microcapsules were prepared with a constant surface area and thickness. This was accomplished by pouring formulations into washers that were adhered to slides. To prepare slides to be used in static release experiments, premium glass microscope slides from Fisherfinest, measuring 3 inches x 1 inch x 1 mm were used. Nylon standard flat washers from Washers USA, measuring (outer dimension), : (inner dimension), thick, were adhered to the glass slides using a water resistant adhesive (Amazing Goop ). Three washers were placed on each slide (Figure 1). The slides with the attached washers were then numbered and weighed to determine the mass without glaze. Glaze formulations for the ion release profiles were transferred from the Max 40 Cups to the washers using either a spatula or Pasteur pipette (depending on viscosity). Each formulation in each individual washer was then photocured for two minutes using a Spectrum 800 curing light (Dentsply ) at 600 mw/cm 2 and then each slide was placed in a Triad 200 (Dentsply ) curing oven for five minutes. After curing in the oven, the oxygen inhibited layer of the glaze was removed by gentle circular wiping with a Kimwipe. The slides were then massed to determine the total mass of glaze on each slide. 25

33 20- slide staining dish units (Electron Microscopy Sciences ) were sterilized using a 5 vol% bleach solution followed by 70 vol% ethanol and nanopure water rinsing. Sterilization was conducted in close proximity to an ignited Bunsen burner. The dish units consist of a dish, Figure 1: Image of formulation films used for ion release measurement. Three superfluous washers were adhered to a glass slide and filled with a formulation and subsequently polymerized. These slides were then soaked in nanopure water for ion release measurements. cover, and rack. All components were allowed to dry and then the slides containing glaze were rinsed with nanopure water, blotted dry with a Kimwipe and placed on the holders. Slides were placed back to back in the slot holders so the washers faced outward into the surrounding water. Once all of the twenty slides were loaded, the rack was placed in the dish and 200 ml of nanopure water was added to the dish. 1 ml aliquots were taken immediately the first day at time zero and one hour. After, aliquots were taken at day 1, day 4, day 7, day 14, one month and then monthly. The volume extracted from the bath during each aliquot was refreshed afterwards with the same volume of nanopure water. Samples were stored in microcentrifuge test tubes (Fisherbrand ) until ion concentration measurements were performed Phosphate ion detection 26

34 In order to measure the phosphate ion release, the molybdenum blue method was used. 29 The molybdenum blue method utilizes a mixed reagent composing of sulfuric acid, L- ascorbic acid, ammonium heptamoylbdate tetrahydrate, and potassium antimonyl tartrate. It is important to note that the ascorbic acid rapidly oxidizes, therefore the ascorbic acid solution needs to be prepared fresh each day. The mixed reagent containers remain covered with aluminum foil to prevent photodegradation. Phosphate ion detection was accomplished using an Infinite M200 (Tecan ) plate reader with Magellan software. The measurements commenced by turning on the Infinite M200 followed by opening the Magellan software. Once in the Magellan software program, a plate ID and plate method is prepared. For plate ID, a 48 well template was used and the layout was labeled on the software as follows: Wells 1-6 were filled with a nanopure blank, ppm, ppm, ppm, 1.50 ppm, and 3.00 ppm standards, respectively. Standards and samples were added in triplicate (i.e. nanopure H2O was added to wells A, B, and C, and unknown samples would be added to wells D, E, and F). Each well was then labeled in Magellan. Next, a method was created. The parameters of the method used in this experiment were a duration of twenty minutes; a shaking duration of 60 seconds in orbital mode at an amplitude of 6 mm; absorbance was set to 882 nm (the wavelength at which molybdenum blue complex has a maximum absorption). Dilution values must then be entered for each well. To do this, the conc. dil, ref, values tab was selected. Nanopure and standards are left at 1 and values were then entered based on the 27

35 sample dilution (i.e. a pure sample would be entered as 1, where as a 1 in 150 dilution would be entered as 150 ). The method was then saved. Cellstar 48 Well cell culture plates (Greiner bio- one) were used in this experiment. Standards and pure samples were added as 750 µl followed by 150 µl of the molybdenum blue mixed reagent. A 1 in 10 dilution sample would contain 75 µl of the sample, 675 µl nanopure H2O, 150 µl molybdenum blue mixed reagent. Other dilutions followed the same format as diluting the phosphate sample to 750 µl with nanopure water followed by 150 µl molybdenum blue mixed reagent. The plate cover was then placed over the plate and the plate out icon was selected on the software. The plate was then loaded and the plate in icon was selected to bring the plate into the plate reader. The method and sample ID previously made were then selected and measurements were commenced. Following completion of measurements, the standard curve was observed and R needed to be between and If the R value was less than 0.999, a new plate with new samples and standards were prepared. If the R value was acceptable, the results were exported to Microsoft Excel for analysis Calcium ion detection Potentiometry was utilized to determine the concentration of calcium ions in aliquots. Ion specific electrodes were used for these measurements. An ELIT 1265 calcium specific electrode was used for calcium ion detection. An ELIT 001N silver chloride electrode served as the reference. The calcium ion specific electrode was 28

36 primed by soaking in a 1000 ppm calcium nitrate solution for a duration of at least 30 minutes prior to use. Standard stock solutions of known calcium concentrations were prepared. While the electrode was soaking, several 50 ml beakers, 10 ml volumetric flasks, volumetric flask caps, and stir bars were cleaned with soap, acetone, and rinsed three times with nanopure water. All glassware was thoroughly dried with Kimwipes, however, residual water droplets could be left in the volumetric flasks as they would contribute to the dilution of the samples. Clean stir bars were placed in the 50 ml beakers and the standard solutions to fit the desired calibration range were poured into the beakers, about 15 ml in each. The range of the standards varied for each run, depending on the predicted ppm of the samples being tested. Generally a broad range of 0.5 ppm to 5.0 ppm was initially used and then narrowed to improve accuracy (i.e. if the calculated ppm is 0.8 the standards used would be 0.5 ppm to 1.0 ppm). Once the calcium ion specific electrode was ready for use, it was rinsed with a stream of nanopure water and blotted dry with a Kimwipe. Next, the reference electrode was rinsed with nanopure water and blotted dry with a Kimwipe. Both the reference and calcium ion electrodes were inserted into the 201 dual head BNC connector. A beaker containing a standard solution was placed on a Thermix Model 210- T (Fischer Scientific) stir plate under the electrodes and the electrodes were lowered into the standard solution. The stir plate was set at level 2 on the stir control knob. It was important to inspect the submerged electrodes for air bubbles around the membranes. If air bubbles were present, they were removed by flicking the electrode with an index finger. 29

37 The electrodes were then calibrated. This was accomplished using the software. The potentiometry icon was selected on the computer and Ca was selected as the ion being measured. The calibrate selection was chosen and the corresponding ppm of the standard solution on the stir plate was entered. Millivolts were measured after two minutes, and the remaining standards were measured in the same fashion. To check the slope of the standard curve, the finish option was selected and the slope measurement was recorded. The slope needed to be in the range of 26 ± 3 mv/decade. If the slope was out of range, the calcium electrode was soaked in the 1000 ppm calcium solution for another hour. Once an acceptable slope is obtained, the samples were diluted to fit a projected standard curve. Samples were diluted in a 10 ml volumetric flask using nanopure water, covered with a stopper and inverted at least ten times. Contents of the volumetric flask were then transferred to a 50 ml beaker with a stir bar and placed on the stir plate. The electrodes were then lowered in the sample beaker, the measure icon was then selected on the computer and a timer was set for two minutes. After two minutes, the measured millivolts and ppm were recorded. After each measurement, a new calibration had to be conducted before taking a new measurement. At least three measurements were taken for each sample. In order to determine the actual ppm of the original sample, the Nernst equation was used. The Nernst equation can be used to determine the equilibrium reduction potential of a half- cell in an electrochemical cell and is written as: Ered=E Θ red - (RT/nF)lnQ. Ered is the half- cell reduction potential; E Θ red is the standard half- cell reduction potential; R is the universal gas constant; T is the absolute 30

38 temperature; n is the number of moles of electrons transferred; F is Faradays constant; Q is the reaction quotient. The Nernst equation can be rewritten to fit the slope- intercept from the straight- line equation y = mx + b. Where y is Ered; m is (RT/nF), also for the slope generated by the computer; x is lnq; and b is E Θ red which is also the y- intercept. Rearranging these terms leads to: mv = y- intercept + slope (log [Ca 2+ ]). From this equation it is possible to take the computer calculated millivolts of a sample (mv) minus the y- intercept then divide that total by the slope. The natural log of this number gives a ppm value that needs to be multiplied by the dilution factor originally used to dilute the sample (i.e. if 500 µl of the sample was diluted to 10 ml, the dilution factor would be 10/0.5 or 20) Fracture toughness specimen preparation Fracture toughness molds used were stainless steel and had an inner measurement of 24 mm x 5 mm x 2.5 mm. The single- edge notched method was used which required a razorblade to be placed in the notch and positioned perpendicular to the long axis of the mold. The glaze was transferred into the mold using a spatula or if the glaze had a very low viscosity, a Pasteur pipette was used. Samples were then irradiated by using a Spectrum 800 curing light at 600 mw/cm 2. The curing scheme was center, left, right for 30 seconds at each position. The specimen was then flipped and the curing scheme was repeated. Next, the specimen was placed in a curing oven for five minutes. After removal of the specimen from the mold, the specimen was shaped using 600 grit wet sand paper and then placed in a Max 40 Cup. Specimens were stored in a model 200A Bacteriological Incubator 31

39 (Blue M Electric) set at 37 C for eight days to allow for sufficient secondary cure before being tested Fracture toughness testing Before commencing the fracture toughness (KIc) measurements, the dimensions of the samples were measured. This was accomplished using a micrometer and taking three measurements at the three different dimensions (length (L), width (b), height (w)) of the specimen (Figure 2). Next, the fracture toughness machine is prepared for measurement by attaching the 250 N load cell to the MTS Insight 1SL (Material Testing Systems) universal testing machine and screwing the fracture toughness head into the load cell. The machine is then turned on and the MTS icon is opened on the computer. The method was selected for Figure 2: Single notch specimen for fracture toughness measurements. Dimensions used were: w=5mm, b= 2.5mm, L=24mm, and a=2.5mm. K1c = [3PLa 1/2 /2bw 2 ] f (a/w) a 250 N load cell with a movement rate of 0.25 mm/minute. The motor reset icon is selected to activate the motor. The specimen is then placed on the specimen holder on the stage. It is essential to make sure that the specimen holder and specimen are centered under the fracture toughness head. This was accomplished by pressing the unlock button on the hand- held unit and selecting the down arrow 32

40 to move the head close to the specimen. The dial on the hand- held controller was used to make fine height adjustments until the head is almost touching the specimen. After the specimen and holder are centered, the lock button on the hand- held unit was selected. In the load cell window, zero channel was selected. Next, the run button was selected. After the specimen fractures, the peak load data is selected and the units are changed from kilograms/force to Newtons. The peak load was recorded. The half of the specimen that has a ledge on the notch was located and used to measure the distance in two spots and record each (these measurements are used to calculate a in Figure 2). In order to calculate the KIc value for a composite, the following equation: KIc = [3PLa 1/2 /2bw 2 ] f (a/w) is used (refer to Figure 2). f(a/w) = (a/w) (a/w) (a/w) (a/w) 4. 33

41 Section 3 Results 34

42 Phosphate and calcium ions contribute to the process of remineralization, therefore, release profiles for these ions from various potential glaze formulations were studied. Aqueous salt solutions of potassium phosphate dibasic and calcium nitrate were encapsulated and loaded into composite resin formulations at fifteen weight percent. The figures in this section demonstrate release profiles and release rates of various monomer formulations and different glass filler contents. Furthermore, the effects of incorporating two different types of fillers (ethylene glycol based polyurethane microcapsules filled with an aqueous salt solution and glass) on the fracture toughness of the glaze formulations were examined. Release profiles are reported as normalized ion concentrations. This refers to the total phosphate ion or calcium ion concentration (in ppm) divided by the total mass of the formulation (in grams) that was submerged in a constant volume of nanopure water Effect of glass content on phosphate ion release from two different continuous phases The relationship between glass filler content and the release rate of phosphate ions from a resin glaze formulation was studied. Figure 3A shows the normalized ion release as a function of time from a glaze formulated with 15 w/w% ethylene glycol based polyurethane microcapsules containing an aqueous solution of 4.0 M potassium phosphate dibasic. The continuous phase consisted of UDMA and TEGDMA in a monomer ratio of 80:20 (w/w%). As seen in Figure 3A, the glass filler content and initial phosphate ion release rate is directly related for this 35

43 continuous phase. As the glass filler increased, the rate of phosphate ion release increased. The inclusion of 10 w/w% glass filler content resulted in a concentration of phosphate ions that was approximately three times the concentration of the sample that had no glass filler. When the amount of glass is increased to 30 w/w%, the concentration of phosphate ions released after 240 days is over six times greater than the sample that did not incorporate glass filler. Figure 3A: Effect of varying glass content on release of phosphate ions. Normalized concentration of phosphate ions released from UDMA:TEGDMA (80:20 w/w) glaze formulation with 0, 10, 20, and 30 w/w% glass content. The formulations were loaded with 15 w/w% ethylene glycol based polyurethane microcapsules that contained a 4.0 M potassium phosphate dibasic solution. The data is plotted as the concentration of ions in ppm per gram of formulation released from glaze formulations as a function of time in days. Figure 3B reports the phosphate ion release as a function of glass content. As can be seen in Figure 3B, there is a correlation between glass content and release of phosphate ions in glaze formulated from a continuous phase of UDMA:TEGDMA (80:20 w/w%). 36

44 Figure 3B: Release of phosphate ions as a function of glass content at 0 w/w%, 10 w/w%, 20 w/w%, and 30 w/w%. Normalized concentration of phosphate ions released from UDMA:TEGDMA (80:20 w/w) glaze formulation with 0, 10, 20, and 30 w/w% glass content. The formulations were loaded with 15 w/w% ethylene glycol based polyurethane microcapsules that contained a 4.0 M potassium phosphate dibasic solution. The data is plotted as the concentration of ions in ppm per gram of formulation released from glaze formulations as a function of glass content in weight percent. Figure 4A incorporates phosphate ion release data from two different continuous phase formulations. A second formulation that incorporated MMA with a monomer ratio of UDMA:TEGDMA:MMA (10:45:45 w/w/w%) was used. In this formulation, the effect of glass content was explored in formulations with 0 and 20 w/w% glass content. In formulations that incorporate MMA, initial ion release rate of phosphate ions is greater than formulations consisting solely of UDMA and TEGDMA with the same discontinuous phases 37

45 Figure 4A: Effect of glass content on phosphate ion release. Normalized concentration of phosphate ions released from 4.0 M phosphate dibasic solutions in ethylene glycol based polyurethane microcapsules loaded in a glaze at 15 w/w%. The continuous phases reported here are UDMA:TEGDMA at 80:20 w/w with 0 and 20 w/w% glass content and UDMA:TEGDMA:MMA at 10:45:45 w/w with 0 and 20 w/w% glass content. The plot is the concentration of phosphate ion release in ppm per gram of composite formulation as a function of time in days Effect of glass content on calcium ion release from two different continuous phases The interaction between glass content and the release rate of calcium ions from a glaze formulation was investigated. Figure 4B illustrates the ion release of 4.0 M calcium nitrate from ethylene glycol based polyurethane microcapsules loaded at 15 w/w% in a continuous phase consisting of UDMA and TEGDMA in a 80:20 (w/w%) ratio. 38

46 Figure 4B: Effect of glass content on calcium ion release. Normalized concentration of calcium ions released from 4.0 M calcium nitrate solutions in ethylene glycol based polyurethane microcapsules loaded in a glaze at 15 w/w%. The continuous phases reported here are UDMA:TEGDMA at 80:20 w/w with 0 and 20 w/w% glass content and UDMA:TEGDMA:MMA at 10:45:45 w/w with 0 and 20 w/w% glass content. The plot is the concentration of phosphate ion release in ppm per gram of composite formulation as a function of time in days. As can be seen in Figure 4B, a continuous phase consisting of UDMA:TEGDMA:MMA in a w/w/w% ratio of 10:45:45, that incorporates 20 w/w% glass filler resulted in a slower initial release rate of calcium ions from the formulation. The presence of 20 w/w% glass content in this formulation resulted in an increase of 16 ppm concentration of calcium ions released over the first 210 days. On the other hand, for a formulation with a monomer ratio of UDMA:TEGDMA (80:20 w/w%), the addition of 20 w/w% glass filler increased the initial rate of release of calcium ions. After 210 days, the formulation that included 20 w/w% 39

47 glass content had approximately twice the calcium ion concentration released when compared to the sample without any glass. When comparing the two continuous phases of these glaze formulations with 20 w/w% glass included, the UDMA:TEGDMA:MMA (10:45:45 w/w/w%) sample released four times the amount of calcium ions than the UDMA:TEGDMA (80:20 w/w%) formulation over the first 210 days. When comparing the two samples without glass, the UDMA:TEGDMA:MMA (10:45:45 w/w/w%) sample released seven times the amount of calcium ions compared to the UDMA:TEGDMA (80:20 w/w%) sample at day 210. Figure 5: Comparison of the release of phosphate and calcium ions from two different continuous phases. Normalized concentration of phosphate ions and calcium ions released from UDMA:TEGDMA (80:20 w/w%) and UDMA:TEGDMA:MMA (10:45:45 w/w/w%) glaze formulations with 0 and 20 w/w% glass content. The formulations were loaded with 15 w/w% ethylene glycol based polyurethane microcapsules that contained either 4.0 M potassium phosphate dibasic or 4.0 M calcium nitrate solutions. The data is plotted as the concentration of ions in ppm per gram of formulation released from glaze formulations as a function of time in days. 40

48 Figure 5 compares the release rates of calcium and phosphate ions from formulations in Figures 4A and 4B that have a discontinuous phase containing 20 w/w% glass content and 15 w/w% polyurethane microcapsules. This data displays that for both continuous phase formulations, phosphate ions have a greater initial release rate compared to calcium ions Effect of MMA concentration on the initial release of phosphate ions and calcium ions from a glaze formulation Figure 6A: Effect of MMA concentration on the initial release rate of phosphate ions. Normalized concentration of phosphate ions released from three different glaze formulations: UDMA:TEGDMA:MMA (10:45:45 w/w/w%), UDMA:TEGDMA:MMA (25:45:30 w/w/w%), and UDMA:TEGDMA:MMA (40:45:15 w/w/w%) with 20 w/w% glass content in each formulation. The glaze formulations contained 15 w/w% ethylene glycol based polyurethane microcapsules that contained 4.0 M potassium phosphate dibasic solutions. The plot is the concentration of phosphate ions released in ppm per gram of formulation as a function of time. The effect of MMA concentration on the initial rate of calcium and phosphate ions from a glaze formulation containing UDMA, TEGDMA, and MMA was 41

49 investigated. The change in MMA concentration was related to changing the UDMA concentration, with TEGDMA remaining constant for all samples. The glass filler content was 20 w/w% in all of these samples. As seen in Figure 6A, a monomer ratio of UDMA:TEGDMA:MMA of 25:45:30 w/w/w% yielded the highest initial rate of phosphate ion release, whereas a UDMA:TEGDMA:MMA ratio of 10:45:45 w/w/w% resulted in the slowest initial rate of release of phosphate ions. Figure 6B: Release of phosphate ions as a function of MMA content. Normalized concentration of phosphate ions released from 4.0 M potassium phosphate dibasic solutions in ethylene glycol based polyurethane microcapsules loaded at 15 w/w% into glaze formulations of UDMA:TEGDMA:MMA in ratios of 10:45:45, 25:45:30, and 40:45:15 w/w/w with 20 w/w% glass content in each formulation. The plot is the concentration of phosphate ions released in ppm per gram of formulation as a function of MMA content. 42

50 Figure 6C: Effect of MMA content on the initial release rate of phosphate ions. Normalized concentration of phosphate ions released from three different glaze formulations: UDMA:TEGDMA:MMA (10:45:45 w/w/w%), UDMA:TEGDMA:MMA (25:45:30 w/w/w%), and UDMA:TEGDMA:MMA (40:45:15 w/w/w%) with 20 w/w% glass content in each formulation. The glaze formulations contained 15 w/w% ethylene glycol based polyurethane microcapsules that contained 4.0 M potassium phosphate dibasic solutions. The plot is the concentration of phosphate ions released in ppm per gram of formulation as a function of time. Figure 6B reports the phosphate ion release data as a function of MMA concentration. The formulation using a monomer ratio of UDMA:TEGDMA:MMA of 25:45:30 w/w/w% resulted in a formulation with a maximum initial ion release rate for phosphate ions. This formulation released 1.5 times faster than the 45 w/w% MMA formulation and nearly 1.2 times faster than that of the 15 w/w% MMA formulation after the first 90 days. Figure 6C depicts the release profiles of calcium ions using the same compositions of glass loading in the glaze formulations as the phosphate experiments described in section 3.3. In this experiment, 15 w/w% ethylene glycol based polyurethane microcapsules containing a 4.0 M calcium nitrate solution were 43

51 formulated into the glaze. The relationship between MMA and release of calcium ion is correlated to the MMA concentration in the glaze formulation. Using a continuous phase in which MMA comprises 45 weight percent of the monomer formulation yielded the highest initial rate of calcium ion release, whereas a fifteen weight percent MMA monomer formulation resulted n the slowest initial rate of calcium ion release. After 90 days, the 45 w/w% MMA glaze had released a total of 61 ppm calcium ions per gram of formulation compared to 44 ppm per gram of formulation and 36 ppm per gram of formulation from the 30 w/w% and 15 w/w% MMA glazes, respectively. Figure 6D: Release of calcium ions as a function of MMA content. Normalized concentration of calcium ions released from 4.0 M calcium nitrate solutions in ethylene glycol based polyurethane microcapsules loaded at 15 w/w% into glaze formulations of UDMA:TEGDMA:MMA in ratios of 10:45:45, 25:45:30, and 40:45:15 w/w/w% with 20 w/w% glass content in each formulation. The plot is the concentration of calcium ions released in ppm per gram of formulation as a function of MMA content. 44

52 Figure 6D reports the calcium ion released from the glaze formulations as a function of MMA content. All three glaze formulations resulted in statistically similar calcium ion release rates for the first seven days. Between day seven and day fourteen, the formulation containing 45 w/w% ratio of MMA released a greater amount of calcium ions. At this point, as the experiment continued, the overall calcium ion release was greatest with the largest amount of MMA in the formulation Comparison of the release rate of phosphate ions and calcium ions as a function of the diluent monomer The continuous phase of a glaze formulation using UDMA and either MMA or TEGDMA as the diluent monomer on the effect of initial ion release rate of phosphate ions and calcium ions was investigated. These formulations were comprised of UDMA:TEGDMA or UDMA:MMA in a monomer ratio of 80:20 w/w% with 15 w/w/% ethylene glycol based polyurethane microcapsules that contained either 4.0 M calcium nitrate or 4.0 M potassium phosphate dibasic solutions and 20 w/w% glass content. As seen in Figure 7, both calcium and phosphate ions were released at a slower initial rate from continuous phases with TEGDMA as the diluent monomer. Initially, calcium ions were released at a slower rate than phosphate ions from both glaze formulations. When MMA was used as the diluent monomer, phosphate ion concentration at day 60 measured 15 ppm per gram of formulation whereas calcium ion concentration at the same time measured 10 ppm per gram of formulation. It 45

53 appears that at up to 60 days, the concentration of phosphate ions released by using MMA compared to TEGDMA as the diluent monomer was statistically the same, whereas calcium ions released by the MMA formulation released 10 ppm per gram of formulation compared to 6 ppm per gram of formulation released by the TEGDMA formulation. Figure 7: Effect of diluent monomer type on calcium and phosphate ion release. Normalized concentrations of phosphate ion and calcium ions released from UDMA:MMA (80:20 w/w%) and UDMA:TEGDMA (80:20 w/w%) glaze formulations with 20 w/w% glass content in all samples. The formulations were loaded with 15 w/w% ethylene glycol based polyurethane microcapsules that contained either 4.0 M potassium phosphate dibasic or 4.0 M calcium nitrate solutions. The data is plotted as the concentration of ions in ppm per gram of formulation as a function of time in days Effect of glass content on fracture toughness Fracture toughness experiments were carried out to determine how percent loading of glass would effect the strength of a glaze formulation with a continuous 46

54 phase consisting of UDMA:TEGDMA (80:20 w/w%). Figure 8 reports the fracture toughness of UDMA:TEGDMA formulations in the absence of ethylene glycol based polyurethane microcapsules. The average KIc for samples containing 30 w/w% glass content was 2.29 MPa m ½ with a standard deviation of 0.12 compared to 2.16 MPa m ½ and a standard deviation of 0.26 for the samples without glass. Figure 8: Fracture toughness as a function of glass content. Fracture toughness tested on a continuous phase consisting of UDMA:TEGDMA in a 80:20 w/w% ratio loaded at 0, 10, 20, and 30 w/w% glass content. All samples were without ethylene glycol based polyurethane microcapsules. Fracture toughness is reported as MPa x m ½ Effect of microcapsules on fracture toughness The effect of the addition of 15 w/w% of ethylene glycol based polyurethane microcapsules to a dental glaze formulation of UDMA:TEGDMA in a 80:20 w/w% 47

55 ratio with glass content of 0, 10, 20, and 30 w/w% on fracture toughness was tested. Data from this experiment is reported in Figure 9. Addition of 15 w/w% of microcapsules resulted in an average KIc value of 1.77 MPa m ½. The average KIc values are reported for samples as glass content increased in formulations containing 15 w/w% microcapsules. Figure 9: Fracture toughness as a function of microcapsule content. Fracture toughness tested on a continuous phase consisting of UDMA:TEGDMA in a 80:20 w/w% ratio loaded at 0, 10, 20, and 30 w/w% glass content. All samples were loaded with 15 w/w% ethylene glycol based polyurethane microcapsules with 4.0 M potassium phosphate dibasic solution. Fracture toughness is reported as MPa x m ½. 48

56 3.7. Effect of glass and microcapsules on fracture toughness of a continuous phase with MMA Figure 10: Fracture toughness as a function of glass and microcapsules content. Fracture toughness tested on a continuous phase comprised of UDMA:TEGDMA:MMA (10:45:45 w/w/w) and having 0 and 20 w/w% glass content and 0 and 15 w/w% ethylene glycol based polyurethane microcapsules. Fracture toughness is reported as MPa x m ½. Using a continuous phase consisting of UDMA:TEGDMA:MMA in a 10:45:45 w/w/w% ratio, the effect of adding 15 w/w% ethylene glycol based polyurethane microcapsules and 20 w/w% glass content on KIc values was investigated. As seen in Figure 10, there is an increase in the average KIc when 20 w/w% glass is included in the formulation without the 15 w/w% microcapsules. The sample that incorporated both 15 w/w% microcapsules and 20 w/w% glass content had an 49

57 average KIc value of 1.30 MPa m ½. Whereas samples without microcapsules had an average KIc of 1.95 MPa m ½. Figure 11: Fracture toughness as a function of glass and microcapsules content. Fracture toughness tested on a continuous phase comprised of UDMA:TEGDMA:MMA (40:45:15 w/w/w) and having 0 and 20 w/w% glass content and 0 and 15 w/w% ethylene glycol based polyurethane microcapsules. Fracture toughness is reported as MPa x m ½. A second continuous phase formulated with UDMA:TEGDMA:MMA (40:45:15 w/w/w%) was also investigated to see the effect of loading microcapsules and glass content on fracture toughness. As seen in Figure 11, the sample without microcapsules had a fracture toughness of 1.98 MPa m ½ and the sample with both 50

58 microcapsules and glass content had the lowest fracture toughness of 1.44 MPa m ½ Comparison of fracture toughness based upon the diluent monomer Figure 12: Fracture toughness as a function of diluent monomer. Formulations had continuous phases of either UDMA:TEGDMA or UDMA:MMA in an 80:20 w/w% monomer ratio with 20 w/w% glass content and 15 w/w% ethylene glycol based polyurethane microcapsules. Fracture toughness is reported as MPa x m ½. The continuous phase of a glaze formulation using UDMA and either MMA or TEGDMA as the diluent monomer on the effect of fracture toughness was investigated (Figure 12). These formulations were UDMA:TEGDMA or UDMA:MMA 51