Caries Inhibiting Effectiveness of the Origin SmartCrown An in-vitro Comparative Study

Caries Inhibiting Effectiveness of the Origin SmartCrown An in-vitro Comparative Study B&D Dental Corporation Key Words Resin modified glass ionomer; Interproximal caries; Adjacent surfaces; Fluoride releasing; Caries inhibition; Tooth Demineralization; SmartCrown ; Abstract Objective: Caries are difficult to detect and treat in the proximal area of teeth. Fluoride releasing restorative dental materials have been effective in inhibiting or reducing caries development. The Origin SmartCrown incorporates fluoride releasing materials, specifically resin-modified glass ionomer (RMGI), in the mesiodistal portion of the crown with the potential for long term prevention of caries. This study evaluated the effectiveness of different varieties of dental restorative materials, consisting of resin-modified glass ionomers, fluoride releasing composites, and non-fluoride releasing composites, on the adjacent tooth surfaces in inhibiting interproximal caries. Method: Teeth blocks were cut and placed in contact with various dental restorative materials using tooth models and an assembly base. The assembled samples were immersed in an artificial caries lesion solution to induce caries. The enamel samples were inspected for structure as well as morphology, tomography, density and surface roughness at different intervals before treatment and after three weeks of treatment. Conclusion: The SmartCrown with RMGI was the most effective in caries inhibition. 1. Introduction Caries formation in proximal tooth surfaces are difficult to detect in early stages and is often already progressed to advanced stages by the time detection occurs. Even with early detection caries are difficult to treat [1]. Caries develop from demineralization of Fig. 1 - Origin SmartCrown RMGI teeth due to acids secreted by oral bacteria after digestion of carbohydrates such as sugar. Restorative dental materials are used to inhibit or reduce caries. A primary method by which restorative materials perform this function is by releasing fluoride ions which contain caries inhibiting properties and promote remineralization of dental dentin and enamel [2,3,4]. It has been recognized that the initial carious lesion should be exposed to fluoride in the aqueous phase to achieve the cariostatic effect [5,6]. Fluoride has been incorporated in many dental materials such as silicates, glass ionomer, amalgam, liners, pit and fissure sealants, and resins [7]. Glass ionomer materials can be considered as a potential caries preventive material because of their high fluoride release capacity and ability to recharge fluoride for long term fluoride release. The Origin SmartCrown is a crown based dental prosthetic that is filled with a fluoride source material in the 1 B&D Dental Technologies

interproximal areas to prevent primary interproximal caries (Fig.1). Previous studies had been performed on in vivo and in vitro settings to observe the long term effects of fluoride releasing restorative materials such as glass ionomers on adjacent proximal surfaces to the restorations. The studies have concluded that fluoride releasing restorative materials inhibit or reduce caries in adjacent surfaces compared to nonfluoridated restorative materials [8,9]. This suggests the potential of glass ionomers, which release fluoride ions, in treating patients with high caries risk can inhibit further caries. The aim of this study was to evaluate the effects of various restorative materials utilized with the Origin SmartCrown on the adjacent tooth surfaces. This study demonstrates the Origin SmartCrown's effect of inhibiting interproximal caries and compares the effectiveness of caries inhibiting properties for each the restorative materials at different intervals of time. 2. Material and methods 2.1 Materials The dental filling materials evaluatedduring this study are described in Table 1. Resin modified Glass ionomers (RMGI) were purchased from 3M ESPE (Ketac nano), GC America (Equia). Ivoclar Vivadent (Heliomolar) and 3M ESPE (Filtek Supreme Ultra) were used as the fluoride releasing composite and non-fluoride releasing composite, respectively. Acetic acid, calcium chloride, sodium fluoride, potassium dihydrogen phosphate, potassium hydroxide (All from Sigma-Aldrich) were used directly without further purification for the artificial caries solution. 2.2 Tooth specimens Twenty-eight enamel tooth blocks with the dentinoenamel junction were prepared from 10 extracted human third molars. Experimental procedures were followed from previous studies as cited [10,11]. The molars were recently extracted by orthodontists during surgical procedures. The enamel of each tooth was inspected for defects and the teeth without defects were selected for the study. The teeth were stored in a 1% thymol solution for at least one week as a disinfectant procedure. Tooth specimans were cut into 28 square pieces with dimensions of 3mm in length, width and height. A high-speed dental hand piece and a diamond wheel with copious amounts of water was used (Fig. 2a). The specimens were ground with waterlubricated 600-1200 Si-C grit abrasive paper to obtain a flat enamel surface (Allied Multprep, USA). Subsequently, the specimens were polished with diamond films (3M, USA) with 5µm, 0.1µm and 50nm alumina suspension (Fig. 2b), then cleaned with an ethanol solution in an ultrasonic bath for 3 minutes, rinsed under de-ionized water to remove alumina powder and dried at room temperature. The tooth blocks were scanned with the Origin Intelligence 3D dental scanner (B&D Dental, USA) (Fig. 2c) and then Exocad software (Exocad, Germany) was used to create a tooth model with the mesiodistal area designed specifically to fit each tooth block and testing material(fig 2d). The Objet 3D Printer (Stratasys, USA) was used to produce the tooth models (Fig 2e). The tooth specimens were coated in acid-resistant nail varnish, except for the contact area where dental filling material is located (Fig 2f). The specimens were then embedded into the individual tooth models (Fig. 2g). The base holder was also produced using the 3D printer and as used for assembly of the specimen models and dental material models to provide tooth contact. 2.3 Dental materials Four different dental filling materials were prepared for the study. Exocad software was used to design tooth models with the mesiodistal opening for the dental materials (Fig. 2d). The dental materials were prepared according to manufacturer instructions and were filled into the tooth models (Fig. 2h). 2.4 Artificial carious lesions Artificial carious lesions were created by immersing the specimens in 100ml of demineralization solution. The demineralization solution contained 50mM acetic acid, 1.5mM CaCl 2, 0.9mM KH 2 PO 4 and 0.05ppm F. The ph was adjusted to 5.0 with 1M KOH [12]. The specimens were assembled with the base holder, specimen model and dental material model so that each tooth specimen was in contact with a dental material for the duration of the study (Fig. 2i). The specimens were then immersed in the demineralization solution at a temperature of 37 o C for 3 weeks (Fig. 2j). Characterization analysis was performed at the initial period and at 3 weeks. 2 B&D Dental Technologies

Table 1. Materials used in the study Material Product name Manufacturer Lot Number Composition a Resin modified glass ionomer (RMGI) Ketac Nano 3M ESPE N570444 FAS glass, nanoparticles and nanoclusters, blend including HEMA, methacrylatemodified polyalkenoic acid Resin modified glass ionomer (RMGI) Fluoride releasing composite Equia GC ameria 1305071 Heliomolar Ivoclar Vivadent T11921 FAS glass, polyacrylic acid bis-gma, UDMA, silicon dioxide, ytterbium trifluoride Non-fluoride releasing composite a Approximate composition as given by manufacturers. Filtek Supreme Ultra 3M ESPE N582052 Nanoparticles and nanoclusters, bis- GMA, UDMA, TEGDMA, bis- EMA Fig. 2 - Photos of caries inhibiting experimental procedure 3 B&D Dental Technologies

2.5 Characterization of the demineralized enamel surface 2.5.1 Tomographic images and density profile The tomographic images and density profile of the samples adjacent to the RMGI, fluoride releasing composite and non-fluoride composite materials were obtained by micro X-ray CT (Xradia Bio MicroCT, MicroXCT-400). The X-ray computed tomography (XCT) was taken of 994slices of data with 6.01 μm voxel resolution. Three-dimensional images were reconstructed from individual tomograghs and Drishti v2.4. MIPAV v7.1.1 software was used to calculate the density of the surface of specimens. Data from the study was processed with Statistical Package for Social Science (SPSS) Ver. 18. Analysis was conducted of the mean and standard deviation (SD) values for each set of data. A one-way ANOVA was performed to analyze the various surface roughness parameters. 2.5.2 The structure and morphology (SEM) The structure as well as the morphology of the enamel surface contacting the resin-modified glass ionomer (RMGI), fluoride releasing composite and non-fluoride composite at specific periods (before the study and 3 weeks after) were observed with the scanning electron microscope (SEM, FEI Quanta 600 FEG, USA). To reduce charging at higher magnifications, an Au/Pd coating of 6 nm thickness was applied on the enamel surface (precision etching coating system 682, Gatan, USA). The parameters used for the SEM image were 3.5 spot sizes, 25kV voltage. 2.5.3 Surface roughness The surface roughness of the enamel adjacent to the RMGI, fluoride releasing composite and non-fluoride releasing composite was measured using an optical profiler (Zygo NewView). The parameters used for the analysis included a 30X optical lens, FDA resolution at normal settings and a scan length of 30μm. Three scale parameters representing arithmetical peak-to-valley (PV), average roughness (R a ), and the root mean square roughness (R q ) were determined. 2.5.4 Elemental composition of the enamel The surface elemental composition was analyzed by X-ray photoelectron microscopy (XPS, Kratos axis ultra DLD). Each sample was placed inside the load lock of the instrument which was evacuated before transfer into the UHV system. X-ray photoelectron spectra were taken using the monochromatic Al Kα source (1486.7 ev) at a 300 x 700 µm spot size. Low resolution survey and high resolution region scans were taken for each sample. The low resolution scan was done at a dwell time of 200 µs to obtain the survey spectra. The high resolution scan was also Fig 3 - Effects of the SmartCrown on the proximal tooth 4 B&D Dental Technologies

performed for each element of interest, and was run at a dwell time of 400 µsec. To minimize charging, samples were flooded with low-energy electrons and ions from the instrument s built-in charge neutralizer. The surface of the samples were also sputtered with high energy Ar + ions for 5 minutes to remove surface contamination. The pressure of the Ar gas during the sputtering was about 5x10-8 torr, producing a current of 2.3 ma. Data was analyzed using CASA XPS software. Binding energy corrections on high resolution region scans were done by referencing the Ar 2p3/2 peak to 241.9 ev. 3. Results and discussion Enamel demineralization or remineralization is a dynamic physicochemical process which occurs when oral bacteria form on the enamel surface and is exposed to fermentable dietary carbohydrates, with sucrose being the most cariogenic of them [13]. Enamel is dissolved by the lower ph in dental plaque due to acid production [14]. However, with the presence of fluoride, fluorapatite (FA) is formed (remineralization) at the same time that hydroxyapatite (HA) is dissolved (demineralization) at lower ph (> ph 4.5) [15]. Fluoride ions aid in enhanced enamel remineralization which prevents mineral loss from oral bacteria. Fig. 3 illustrates the effect of the SmartCrown on the proximal tooth. The SmartCrown contains Fluoride releasing dental filling materials in the mesiodistal side that can increase tooth remineralization in the interproximal area with continuous fluoride release. Fig. 4 is the photo of the tooth blocks treated with various dental filling materials after being immersed in the artificial caries solution for 3 weeks. The top surface of each specimen in the picture was in contact with the various restorative materials. (a) (b) (c) (d) Fig. 4 - Images of the tooth blocks that were immersed in the artificial caries solution with various dental restorative materials (Ketac nano, Equia, Helliomolar, Filtek supreme ultra) for 3 weeks. 5 B&D Dental Technologies

The enamel surface adjacent to the SmartCrowns containing RMGI filling materials (Ketac nano, Equia) did not show any demineralization, whereas the fluoride releasing composite (Helliomolar) and nonfluoride releasing composite (Filtek supreme ultra) did show demineralization when inspected with the naked eye. This result shows evidence of the prevention of caries on the enamel surface due to the presence of fluoride ions. The enamel surface contacting the fluoride releasing composite (Helliomolar)(Fig. 4(c)) demonstrated demineralization on the surface edge which indicates that the fluoride releasing composite does have some anti caries effect on the enamel but is not sufficient enough to protect the entire tooth block due to their low level fluoride releasing capability. Based on existing literature, the resin modified glass ionmer is observed to release over 21 times more fluoride ions than fluoride releasing composites [16]. The permeability is a major factor in the mechanism of fluoride release. Fluoride releasing composites are. known to have a relatively low water permeability that lead to a lower fluoride release than RMGI materials [17]. RMGI materials, conversely, consists of an acid-base reaction that is physically inhibited by the presence of a polymer network, making the materials more permeable for longer time intervals [18].The enamel surface contacting the non-fluoride releasing composite(filtek supreme ultra) (Fig. 4(d)) had more of an extensive demineralization area than all other samples which indicates that non-fluoride releasing composite does not have any anti caries effect on the enamel surface. 3.1 Tomographic images and density profile Fig. 5 shows a 3D reconstructed image of the tooth blocks treated with the various dentals filling materials. The enamel surfaces contacting the RMGI materials (Ketac nano, equia) did not show any difference from before the artificial caries solution was introduced and after 3 weeks of immersion. Fig. 5-3D reconstructed images of tooth blocks treated with RMGI materials (Ketac nano, equia), fluoride releasing composite (Helliomollar) and non-fluoride releasing composite (Filtek supreme ultra) from the x-ray CT analysis data before and 3 weeks after treatment 6 B&D Dental Technologies

Fig. 6 - Density profiles of the tooth blocks before treated with various dental filling materials from the X-ray CT (a) Ketac nano before treatment (b) 3 weeks after (c) Equia before treatment (d) 3 weeks after (e) Helliomolar before treatment (f) 3 weeks after (g) Filtek supreme ultra before treatment (h) 3 weeks after The fluoride releasing composite and non-fluoride releasing composite treated tooth blocks, however, did show demineralized on the enamel surface after 3 weeks. During formation of caries, mineral ions in the enamel such as calcium and phosphate are lost due to acidic ph and result in appearance of porosities between the crystallites [19].When the calcium and phosphate ions are supersaturated in oral fluids, re-deposited enamel mineral is formed due to regrowth of existing crystallites [20, 21]. This forms the basis of the mechanism of remineralization. The presence of Fluoride ions 7 B&D Dental Technologies

Fig. 7 - Scanning Electron Microscopy (SEM) of the tooth blocks before and after being treated with various dental filling materials. (a) Ketac nano before (b) 3 weeks after (c) Equia before (d) 3 weeks after (e) Helliomolar before (f) 3 weeks after (g) Filtek supreme ultra before (h) 3 weeks after 8 B&D Dental Technologies

Fig. 8- Oblique plot of tooth blocks treated with various dental filling materials from optical profiler data, (a) Ketac nano before treatment (b) 3 weeks after (c) Equia before treatment (d) 3 weeks after (e) Helliomolar before treatment (f) 3 weeks after and (g) Filtek supreme ultra before treatment (g) 3 weeks after 9 B&D Dental Technologies

Table 2. Roughness parameters of the enamel surface treated withvarious materials Samples PV (μm) a RMS (nm) b Ra (μm) c Ketac nano 0.911 6.409 0.004 Before Equia 2.589 29.750 0.021 Helliomolar 1.149 10.628 0.007 Filltek Supreme Ultra 2.443 25.721 0.017 Ketac nano 2.822 41.168 0.028 After Equia 4.500 85.821 0.046 Helliomolar 6.595 424.708 0.310 a Maximum peak-to-valley height. The absolute value is between the highest and lowest peaks. PV = Rp + R v b Root-mean-square (rms) roughness. The average of the measured height deviations taken within the evaluation length or area and measured from the mean linear surface. ( ) Filltek Supreme Ultra 9.816 425.033 0.312 c The arithmetic average of the absolute values of the roughness profile. ( ) in the media causes acceleration of remineralization of the enamel [22, 23, 24] which is demonstrated in the result of the RMGI treated enamel with the inhibition of mineral loss. However, the enamel surface contacting the fluoride releasing composite and non-fluoride releasing composite showed demineralization due to the acidic area in the solution. Fig. 6 shows the density profiles of each tooth block before and after being treated in the artificial caries solution. There was no observed demineralized from the 2D sliced image in the RMGI treated tooth blocks. Density variation was found only between the enamel and dentin. The demineralized surface lesions developed in the enamel surface is seen as a dark (radiolucent) area (marked with white arrows in Fig 6 (f), (h)) which was observed in the tooth blocks contacting fluoride releasing composite and non-fluoride releasing composite. The density of mineral loss area was ca. 20~35% lower than the original enamel, and the demineralization depth was ca. 100 μm from the surface enamel [25]. 3.2 The structure and morphology (SEM) Fig.7 b and d are SEM images of the enamel surface enamel that was contacting the RMGI materials after 3 weeks of immersion in the artificial caries solution. The enamel surfaces had some void crystals which were formed by dissolving HAp (hydroxyapatite) crystals due to the acid media, but the majority of the surface remained as original due to the fluorapatite deposited on the enamel surface. The fluoride releasing composite and non-fluoride releasing composite, in comparison, displayed rough and loose surfaces caused by demineralization of the HAp crystals due to the lack of fluoride or the low level of fluoride ions in the solution. 3.3 Surface roughness The surface roughness of the enamel was analyzed by an optical profiler. The optical profiler uses the wave properties of light to compare the optical path difference between a test surface and reference surface for interference fringe, contrast, optionally combined with interference phase, to determine surface height [26]. The optical profiler provides quantitative maps of the surface topography of a million image points. Fig. 8 is the oblique plot of the tooth surfaces from the optical profiler. The oblique plot shows the polished original enamel surfaces were observed to be smooth and flat. After treatment with the artificial caries solution, however, the enamel surface of all samples were rougher than the original samples. The enamel surfaces contacting the fluoride releasing composite and non-fluoride 10 B&D Dental Technologies

releasing composite displayed significantly increased (P< 0.05) roughness compared to the RMGI treated samples. The results were shown in Table. 2. All roughness parameters (PV, RMS, Ra) were increased compared to before the treatment in the artificial caries solution. However, the RMGI treated samples were able to inhibit demineralization due to the fluoride ions released from the RMGI materials. Conversely, dental filling materials that release low levels of fluoride or do not release fluoride greatly increased their surface roughness in the acid media. This result was also demonstrated from previous analysis (naked eye, X-ray CT, SEM). The surface roughness of the teeth is a critical factor in oral health as it may affect the aesthetics of the enamel surface as well as the amount of plaque and bacteria formation [27].The inhibition of demineralization of the enamel surface by fluoride ions increases the resistance to bacteria adherence [28]. however, by fluoride ions that replaced the hydroxyl ions and formed into fluorapatite crystals (Ca 10 (PO 4 ) 6 F 2 ) which have greater resistance to elution of minerals in the acid media [29]. X-ray photoelectron spectroscopy was used to confirm the presence of fluoride in the enamel surface. Fig. 9 is the XPS spectrum of the original and RMGI treated enamel surface. The peaks at 344, 436eV corresponds to Ca 2p, Ca 2s, respectively. The binding energy at 130, 188eV can be ascribed to P 2p, P 2s respectively. Oxygen peaks appeared at 23(2s) and 523eV (1s). The peak at 682eV is contributed to fluoride 1s. Fluoride peaks only appeared in the SmartCrown treated samples as opposed to the original enamel. This result can be shown as evidence that fluorapatite in the enamel crystal structure was due to the accelerated remineralization by fluoride released from the SmartCrown with RMGI. 5.4 Elemental composition of the enamel Fig. 9- X-ray photoelectron spectroscopy (XPS) spectrum of the original and SmartCrown treated enamel. 4. Conclusion In this study, we have tested the caries preventing effect of the Origin SmartCrown on adjacent teeth. The SEM and optical profiler analysis demonstrated that the SmartCrown (with RMGI) was the most effective in preserving surface smoothness. Micro bio X-ray CT data showed that there was no density loss due to demineralization in the SmartCrown treated enamel. The XPS confirmed the presence of fluorapatite crystals(ca 10 (PO 4 ) 6 F 2 ) only in the SmartCrown treated samples which provides evidence of remineralization of enamel with fluoride ions. All these results show evidence that the SmartCrown with the resin-modified glass ionomer material is the only material to demonstrate a significant anti-caries effect on the enamel. The fluoride releasing composite (Ivoclar Heliomolar) does not have a significant anti-caries effect on tooth enamel due to insufficient fluoride release from the material. References X-ray photoelectron spectroscopy was used for analyzing the surface element composition of the [1] Long-term evaluation of the remineralization of original enamel and the enamel that had been interproximal caries-likelesions adjacent to glassionomer contacting the SmartCrown after 3 restorations: A micro-ct study, Am. J. weeks immersed in the acid media. The Dent. 129-132,2008 HAp(hydroxy-apatite) crystals mainly consisted of calcium ions (Ca 2+ ), phosphate ions (PO 3-4 ) [2] The chemistry of caries: remineralization and and Hydroxyl (OH - ),usually denoted as demineralization events with direct clinical Ca 10 (PO 4 ) 6 (OH) 2. As mentioned above, HAp relevance, Dent Clinic NA, 54(3) 469 478, 2010. crystals were dissolved by acid that lead to loss of minerals. The minerals were re-deposited, 11 B&D Dental Technologies

[3] Long-Term Fluoride Exchanges at Restoration Surfaces and Effects on Surface Mechanical Properties, ISRN Dentistry, 1-8, 2013 [4] Short- and long-term fluoride release from glass ionomers and other fluoride-containing filling materials in vitro. Scand J Dent Res 98: 179-85, 1990. [5] Glass ionomer materials as a rechargeable fluoride-release system, Int. J. Ped. Dent. 65, 1997 [6] Flouride: its role in dentistry, Braz Oral Res 24: 9-17 2010 [7] Fluoride-containing restorative materials, Int. Dent. J 56, 33-43, 2006 [8] Fluorides leaching from restorative materials and the effect on adjacent teeth, Int. Dent. J. 60(3), 156-16, 2010 [9] Long-term evaluation of the remineralization of interproximal caries-like lesions adjacent to glassionomer restorations: A micro-ct study, Am. J. Dent. 129-132,2008 [10] Fluoride release and dentin caries inhibition adjacent to resin-modified glass-ionomer luting cements., Clinical Dent. J., 24(3) 127-133, 2005 [11] In vitro remineralization of human dental enamel by bioactive glasses, j. mater. Sci., 46, 1591-1596, 2011 [12] Surface Modulation of Dental Hard Tissues, D. Tantbirojn, Ph.D. University of Minnesota, 1998 [13] What is the critical ph and why does a tooth dissolve in acid?, J. can. dent. assoc., 69:722-4, 2003. [14] Enamel remineralization: controlling the caries disease or treating early caries lesion?, Braz. Oral. Res., 1:23-30, 2009. [15] Chemical interactions between the tooth and oral fluids. dental caries - the disease and its clinical management. 2nd ed. Oxford: Blackwell Munksgaard; chap. 12, 2008. [16]Fluoride release from tooth-colored restorative materials: A 12-month report., Journal of the Canadian Dental Association 64 (8): 561 4, 568, 1998. [17] Water sorption characteristics of dental microfine composite filling materials, Biomaterials, 5:369-72, 1984. [18] The recharge of esthetic dental restorative materials with fluoride in vitro-two year's results., dental materials, 19:32-37, 2003. [19] Kinetic and thermodynamics aspect of enamel demineralization., Caries Res., 19:22-35, 1985. [20] Remineralization of artificial enamel lesions in vitro: III/ A study of the deposition mechanism. Caries Res. 14:351-8, 1980. [21] Remineralization of natural and artificial lesions in human dental enamel in vitro: Effect of calcium concentration of the calcifying fluid., Caries Res. 15:138-57, 1981. [22] Anticariogenic potential of fluoride releasing dental restorative material., J. Dent. Res., 67(A): 145, 1988 [23] Fluoride-releasing core build-up materials and artificial caries., Am. J. Dent. 4:207-10, 1991. [24]In vitro demineralization of enamel caries at restoration margins utilizing fluoride-releasing composite resin., Quintessence int. 25:355-8, 1994. [25] Chemical interactions between the tooth and oral fluids. dental caries - the disease and its clinical management. 2nd ed. Oxford: Blackwell Munksgaard; chap. 12, 2008. [26] Optical Measurement of Surface Topography, R. Leach, ed., 1st Edition. Springer Verlag Berlin, 187, 2011. [27] effect of different splint removal techniques on the surface roughness of human enamel: a three dimensional optical profilometry analysis, J. dent. 22:s13-6, 1994. [28] Decreased oral colonization of streptococcus mutans during aging of spraque-dawley rats., Infec. Immun., 16(1): 203-212, 1977. [29] Chemical interactions between the tooth and oral fluids. dental caries - the disease and its clinical management. 2nd ed. Oxford: Blackwell Munksgaard; chap. 12, 2008. 12 B&D Dental Technologies

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Supporting information The following testing equipment from the Univ. of Utah was used for the characterization analysis of the demineralized enamel surface of this test. 2.5.1 Tomographic images and density profile Micro X-ray CT (Xradia BioMicroCT, MicroXCT-400) (University of Utah, Dept of Metallurgical Engineering) Tooth Specimen RMGI (Tooth specimen, 3mmX3mmX3mm) 14 B&D Dental Technologies

2.5.2 The structure and morphology (SEM) Scanning Electron Microscope (SEM, FEI Quanta 600 FEG, USA). 2.5.3 Surface roughness Optical Profiler (Zygo NewView) 15 B&D Dental Technologies

2.5.4 Elemental composition of the enamel X-ray photoelectron microscopy (XPS, Kratos axis ultra DLD) ORIGIN SmartCrown Intellectual Properties -Patents (Pending) 61/892,345 61/993,894 62/055,915 62/033,490 62/095,269 PCT/US14/40554 61/909,812 14/292,601 14/452,199 29/503,255 62/105,586 www.smartcrown.com B&D Dental Technologies Updated 04/22/15