1 CAROLINA PAES TORRES, 2 JACIARA MIRANDA GOMES-SILVA, 2 MICHELLE ALEXANDRA CHINELATTI, 3 FERNANDO CARLOS HUEB DE MENEZES, 4 REGINA GUENKA PALMA-DIBB, 5 AND MARIA CRISTINA BORSATTO 6 * 1 Department of Pediatric Dentistry, Dental School of Uberaba, University of Uberaba, Uberaba, Minas Gerais, Brazil 2 Department of Pediatric Clinics, Preventive and Community Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil 3 Department of Restorative Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil 4 Department of Restorative Dentistry, Dental School of Uberaba, Uberaba, Minas Gerais, Brazil 5 Department of Restorative Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil 6 Department of Pediatric Clinics, Preventive and Community Dentistry, Dental School of Ribeira o Preto, University of Sa o Paulo, Ribeira o Preto, Sa o Paulo, Brazil KEY WORDS dental enamel; scanning electron microscopy; X-ray diffraction; energy dispersive X-ray spectrometer ABSTRACT Purpose: This study evaluated and compared in vitro the microstructure and min- eral composition of permanent and deciduous teeths dental enamel. Methods: Sound third molars (n 5 12) and second primary molars (n 5 12) were selected and randomly assigned to the following groups, according to the analysis method performed (n 5 4): Scanning electron microscopy (SEM), X-Ray diffraction (XRD) and Energy dispersive X-ray spectrometer (EDS). Qualitative and quanti- tative comparisons of the dental enamel were done. The microscopic ndings were analyzed statis- tically by a nonparametric test (Kruskal-Wallis). The measurements of the prisms number and thickness were done in SEM photomicrographs. The relative amounts of calcium (Ca) and phospho- rus (P) were determined by EDS investigation. Chemical phases present in both types of teeth were observed by the XRD analysis. Results: The mean thickness measurements observed in the deciduous teeth enamel was 1.14 mm and in the permanent teeth enamel was 2.58 mm. The mean rod head diameter in deciduous teeth was statistically similar to that of permanent teeth enamel, and a slightly decrease from the outer enamel surface to the region next to the enamel-dentine junction was assessed. The numerical density of enamel rods was higher in the deciduous teeth, mainly near EDJ, that showed statistically signicant difference. The percentage of Ca and P was higher in the permanent teeth enamel. Conclusions: The primary enamel structure showed a lower level of Ca and P, thinner thickness and higher numerical density of rods. Microsc. Res. Tech. 73:572577, 2010. V VC 2009 Wiley-Liss, Inc. INTRODUCTION The crowns of teeth are covered by dental enamel, which is the hardest tissue in the human body, and is composed of 9296% inorganic matter or mineral phase, and 4% of organic material and plasma by weight. The mineral phase consists primarily of cal- cium phosphate in the form of hydroxyapatite crystals, which are carbonated or uoridated (Gwinnett, 1992; Ten Cate, 1989). The primary crown average growth is from 6 to 14 months, whereas the permanent teeth average growth is from 3 to 4 years. So, primary teeth present lesser thickness of enamel than its successor teeth (Arau jo et al., 1995; Mortimer, 1970). The enamel microstructure consists of crystals arranged in prisms or rods, which run approximately perpendicular from the dentine-enamel junction towards the tooth surface (Fava et al., 1999; Ten Cate, 1989). The interfacial area between prisms is protein- rich, and termed interprismatic enamel (Fejerskov et al., 1984). In the prismless enamel layer, the hydroxyapatite crystals are disposed parallel to each other and perpendicular to the enamel surface (Fava et al., 1997). Studies have described the presence of a prismless enamel layer that is thicker and uniform in deciduous teeth compared to permanent teeth (Fava et al., 1997). According to Mortimer (1970), the thinness of the enamel in deciduous teeth and its lower level of miner- alization (80.6% in primary teeth enamel and 89.7% in permanent teeth enamel) are the main differences between the primary teeth enamel when compared to the permanent teeth. As the chemical, morphological, and physiological aspects vary between permanent teeth and deciduous *Correspondence to: Profa Dra. Dr. Maria Cristina Borsatto, Faculdade de Odontologia de Ribeirao Preto/USP, Departamento de Cl nica Infantil, Odontolo- gia Preventiva e Social, Avenida do Cafe, s/n, Monte Alegre. CEP: 14040-904 Ribeira o Preto, SP, Brasil. E-mail: borsatto@forp.usp.br Received 13 August 2009; accepted in revised form 4 October 2009 DOI 10.1002/jemt.20796 Published online 23 November 2009 in Wiley InterScience (www.interscience. wiley.com). V VC 2009 WILEY-LISS, INC. MICROSCOPY RESEARCH AND TECHNIQUE 73:572577 (2010) teeth (Mortimer, 1970; Sonju-Clasen and Ruyter, 1997), the behavior of primary teeth seems to be differ- ent under conditions such as caries, erosion process, and bond strength (Hunter et al., 2000; Marquezan et al., 2008; Wang et al., 2006). The majority of studies are performed solely in per- manent or in deciduous teeth, and frequently the results obtained with permanent teeth are extrapo- lated to the primary teeth. Presently, the literature is still scarce in data comparing the microstructure and composition between the permanent and deciduous teeth and the available studies shows contradictory results. The analysis of the primary and permanent teeth enamel in the same research is important in order to provide the methodology standardization and, there- fore, a reliable comparison between these substrates. New and improved research techniques have enable investigators to improve understanding of the dental structures. In this way, it is of a great scientic relevance the accomplishment of a comparison between these sub- strates in order to establish specic preventive and restorative protocols. Therefore, the purpose of this study was to evaluate and compare in vitro the micro- structure and the mineral composition of permanent and deciduous teeths dental enamel. MATERIALS AND METHODS Sample Selection This study was approved by the Ethics in Research Committee of the School of Dentistry of Ribeira o Preto, University of Sa o Paulo. Freshly extracted sound third molars (n 5 12) with complete root formation and second primary molars (n 5 12) with at least 2/3 of the dental root length (from the Human Tooth Bank of the School of Dentistry of Ribeira o Preto, University of Sa o Paulo, Brazil), were hand scaled and cleaned with water/pumice slurry in rotating bristle brushes to remove calculus and surface-adhered debris, and were examined under a 320 magnier to discard those with structural defects. The selected teeth were randomly assigned to the following groups, according to the analysis method performed to analyze the substrates (n 5 4): Scanning Electron Microscopy (SEM), X-Ray diffraction (XRD) and Energy dispersive X-ray spectrometer (EDS). SEMAnalysis The roots were sectioned 3 mm below the cement- enamel junction. Crowns were xed with wax in plex- glass 1 plates and bisected longitudinally in a mesiodis- tal direction using a double-faced diamond disk (KG Sorensen, 7015, Barueri-SP, Brasil) mounted at a low- speed handpiece. The cut surfaces were then attened and polished with #600 and #1200-grit silicon carbide paper (Norton/Saint-Gobain Abrasivos, Guarulhos, SP, Brazil) and 0.5-lm alumina paste (Struers A/S, Copen- hagen, Denmark). Next, the enamel thickness was measured in a surface-junction direction using a digital electronic paquimeter (Digimatic Caliper/code number 071-467B, Mitutoyo, Suzano, Brazil), with 0.01 mm resolution. Then, the specimens were immersed in 3% glutaral- dehyde (Sigma-Aldrich, St. Louis, MO) in 0.05 M sodium cacodylate buffer at pH 7.4 (Merck KGaA, Darmstadt, Germany) for 24 h at 48C. After xation, the samples were rinsed with distilled water several times for 1 h. The enamel surfaces were etched with a 35% phos- phoric acid gel (Scotchbond etchant, 3M/ESPE) for 15 s and rinsed thoroughly for 15 s. Then, the specimens were immersed for 10 min in a ultrasonic cleaner (T- 1449-D, Odontobra s Ind. e Com, Ribeira o Preto, SP, Brazil) containing distilled water and sequentially dehydrated in an ascending ethanol (Labsynth Produ- tos para Laboratorio, Diadema-SP, Brasil) series (25% for 20 min; 50% for 20 min; 75% for 20 min; 90% for 30 min; 100% for 60 min). Next, the specimens were immersed in hexamethyldisizilane (HMDS; Merck KGaA, Darmstadt, Germany), for 10 min, placed on absorbing paper inside glass plates and left drying in an exhaust system. Specimens were mounted on stubs, sputter-coated with gold and examined with a scanning electron microscope (Philips-FEG SEM, Philips Electron Optics, Netherlands) belonging to the Laboratory of Structural CharacterizationDepartment of Materials Engineer- ingFederal University of Sa o Carlos (UFSCar, Sa o Carlos, SP, Brazil) operating at 20.0 kV. The following enamel regions were analyzed (the mean rod head diameter and the numerical density of enamel rods): a region near the outer surface and a region near the inner surface, next to the dentinoenamel junction (EDJ). The most representative areas were recorded surfaces at magnications of 5003, 1,5003, and 5,0003and a single previously calibrated examiner an- alyzed the morphological ndings, blinded to the groups to which the specimens belonged, by performing a visual and qualitative comparison of the dental enamel. In a previous data analysis since a non-normal distribution was detected, the microscopic ndings were analyzed statistically by a nonparametric test (Kruskal-Wallis). The measurements of the prisms number and thick- ness were done in SEM photomicrographs by mean of the computer software AxioVision (Zeiss). EDS Analysis The same sample that had been used in the SEM investigation was used for the EDS analysis and in the same regions: near the outer surface and near the inner surface, next to the dentinoenamel junction. For the analysis of the percentage component composition of the enamel, the X-ray detector system attached to a scanning electron microscope (Philips-FEG SEM, Philips Electron Optics, Netherlands) belonging to the Laboratory of Structural CharacterizationDepart- ment of Materials EngineeringFederal University of Sa o Carlos (UFSCar, Sa o Carlos, SP, Brazil) operating at 20.0 kV, spot size of 5 nm, was used. This method allowed the relative amounts of calcium (Ca) and phos- phorus (P) by volume percent to be determined. XRDAnalysis X-Rays are useful in identifying crystal structures since X-ray radiation represents the spectrum of radiation around 0.1 nm, which is an acceptable approximation of the size of atoms and ions. For the X-rays analysis the roots were sectioned 3 mm below Microscopy Research and Technique 573 PERMANENT AND PRIMARY ENAMEL MICROSTRUCTURE the cemento-enamel junction. Crowns were xed with wax in plexglass 1 plates and bisected longitudinally in a mesio-distal direction using a double-faced diamond disk (KG Sorensen, 7015, Barueri -SP, Brasil) mounted at a low-speed handpiece. In the cut fragments, the dentin substrate was removed using high-speed dia- mond dental burs with water-cooling, resulting in slabs measuring at least 1-mm thick. To determine the crystal structure of enamel, XRD was carried out using a diffractometer powder system (Rigaku Geigerex, Woodland, TX) with Ni-ltered CuKa radiation and a source operating at 40 kVand 25 mA, belonging to the Laboratory of Structural Charac- terization, Department of Materials Engineering, Fed- eral University of Sa o Carlos (UFSCar, Sa o Carlos, SP, Brazil). RESULTS SEMAnalysis The mean thickness measurements observed in the deciduous teeth enamel was 1.14 mm and in the per- manent teeth enamel was 2.58 mm. The morphological analysis showed that the mean rod head diameter in deciduous teeth was statistically similar to that of permanent teeth enamel. It was observed a slightly decrease in the rod head diameter from the outer enamel surface to the region next to the dentinoenamel junction in both types of teeth, but no statistically signicant difference was assessed (P > 0.05; Table 1). The numerical density of enamel rods was higher in the deciduous teeth than that recorded in the perma- nent teeth, mainly near EDJ, that showed statistically signicant difference (P < 0.05). Additionally, It was observed a slightly increase in the number of rods from the outer enamel surface to the region next to the den- tinoenamel junction in both types of teeth, but no stat- istically signicant difference was assessed (P > 0.05; Table 2). SEM micrographs representative of the two regions analyzed are illustrated in Figure 1. EDS Analysis The results of EDS analysis of the two regions in pri- mary and permanent enamel are shown in Table 3. The percentage of calcium and phosphorus was higher in the two regions analyzed of the permanent teeth enamel when compared to the deciduous teeth. XRDAnalysis The XRD patterns obtained from primary and per- manent enamel are shown in Figure 2. Phase analysis of the XRD patterns indicated that the phases present in deciduous teeth enamel, evidenced by the intensity of peaks, were hydroxyapatite, calcium silicate, hydrated calcium phosphate, calcium phosphate sili- cate, and potassium calcium phosphate. Phase analysis of the XRD patterns indicated the phases present in permanent teeth enamel, evidenced by the intensity of peaks, were hydroxyapatite, calcium silicate, hydrated calcium phosphate, and hydrated calcium silicate. DISCUSSION Concerning the morphological aspect of the dental enamel, it was observed that the mean rod head diame- ter in deciduous teeth was statistically similar to that of permanent teeth enamel, with a slightly decrease from the outer enamel surface to the region next to the dentinoenamel junction. The mean rod head diameter varies from 3.22 lm (60.45) to 3.47 lm (60.48) for the primary teeth and from 3.84 lm (60.73) to 4.34 lm (60.95) lm for the permanent teeth. Different results were reported by Fosse (1968) and Mortimer (1970) that noted higher enamel prism diameter values in canines permanent teeth (6 lm) and deciduous teeth (47 lm). In the current investigation, the mean numerical density of rods in the permanent teeth enamel was 13.582/mm 2 (6600) in the outer region and 14.010/ mm 2 (6391) in the region nearest the dentinoenamel junction. In contrast, Fosse (1964) calculated a mean number of 21,904 prisms per mm 2 on the outer surface and a mean number of 47,089 prisms per mm 2 on the inner enamel surface. Moreover, the numerical density of enamel rods was higher in the deciduous teeth than that recorded in the permanent teeth and increased from the supercial surface to the deep layer nearest the junction. Consist- ent with the present study, Fosse (1964) found that the total number of prism on the inner surface of the enamel exceeded the number on the outer surface. This characteristic may cause more interprismatic region in the permanent teeth enamel, which forms diffusion pathways. The diffusion rate of substances, ions, and molecules through the pores in the enamel plays a cru- cial role in the dynamics of the caries process (Linden et al., 1986). As regards the amount of calcium and phosphorus, their percentage was higher in the permanent teeth enamel when compared to the deciduous teeth. In this context, Derise et al. (1974) reported that the mean concentration of Ca and P were 37.1 and 18.1% in the permanent teeth enamel, while Lakoma and Rytomaa (1977) did not observed differences in the amount of theses ions between primary and permanent teeth enamel. The different values of Ca and P reported in the current study may be due to factors such as the region where the teeth were collected, variations between individual teeth, the type, age and ethnicity and even the methodology performed at the researches. As the mineralization level is related to the crystals density (Gwinnett, 1992), it might be speculated that TABLE 1. Mean head diameter of the enamel rods (lm) to permanent and deciduous teeth in the different regions Outer surface Means (SD) Near EDJ Means (SD) Deciduous teeth 3.47 (60.48) 3.22 (60.45) Permanent teeth 4.34 (60.95) 3.84 (60.73) TABLE 2. Mean number of rods (per square mm) to permanent and deciduous teeth in the different regions Outer surface Means (SD) Near EDJ Means (SD) Deciduous teeth 14.149 (61.009) 15.244 (6648) Permanent teeth 13.582 (6600) 14.010 (6391) Microscopy Research and Technique 574 M.A.H. DE MENEZES OLIVEIRA ET AL. the overall crystals density should be lower in primary enamel when compared to the permanent teeth. It has been disclosed that overall mineral density was lower in the outermost layers, but showed no signicant dif- ferences closer the dentinoenamel junction (Wilson and Beynon, 1989). The thinness of the enamel in deciduous teeth and its lower level of mineralization could be responsible to its whiter appearance compared to their permanent counterparts. Additionally, it could play some roles to the faster and higher rate of dental caries and erosion progression in primary teeth enamel reported in previ- ous investigations (Amaechi et al., 1999; Featherstone and Mellberg, 1981; Johansson et al., 2001; Wang et al., 2006). It has been suggested that the mechanical properties of a calcied tissue are generally linked to its mineral content (Kodaka et al., 1992).Therefore, the mineral content and the crystals arrangement differences between primary and permanent enamel might account to the lower level of mechanical properties observed in the deciduous teeth (Low et al., 2007). In this context, Kerebel et al. (1979) described that the enamel crystallites thickness are 26.3 nm in perma- nent teeth and 16.0 nm in primary teeth, while Low et al. (2006, 2007) observed larger and coarser crystals in primary teeth (185 nm) when compared to the per- manent teeth (94 nm). In this study, the deciduous teeth showed a thinner enamel layer when compared to the permanent one. Accordingly, Mortimer (1970) observed that the thick- ness of deciduous enamel, in general, was about half of that found on permanent teeth. Furthermore, Low Fig. 1. Qualitative analysis of the enamel regions by scanning electron microscopy. A: Deciduous teeth enamel near the outer surface. B: Permanent teeth enamel near the outer surface. C: Deciduous teeth enamel near the enamel-dentine junction. D: Permanent teeth enamel near the enamel-dentine junction. TABLE 3. Results of EDS analysis Permanent teeth Deciduous teeth Outer surface (%) Near EDJ (%) Outer surface (%) Near EDJ (%) P 21.19 21.11 17.23 17.36 Ca 52.50 56.62 35.11 35.80 Microscopy Research and Technique 575 PERMANENT AND PRIMARY ENAMEL MICROSTRUCTURE et al. (2007) reported that the deciduous teeth enamel was thinner, softer and more prone to fracture when compared to the permanent teeth. Several studies (Fava et al., 1999, 1997) have docu- mented the prismless enamel layer, in which the hy- droxyapatite crystals are disposed parallel to each other and perpendicular to the enamel surface on teeth. They also demonstrated that this layer shows a greater and uniform thickness in deciduous teeth. The presence of aprismatic enamel may interfere with the dental enamel acidic demineralization process (Kuhar et al., 1997). Therefore, some authors have sug- gested the prolonging of the primary enamel phos- phoric acid-etching time in restorative procedures in order to obtain the dissolution of the prismless layer (Bozalis et al., 1979; Hosoya, 1991). Others demon- strated that the acid etching during 15 s seems suf- cient to cross the prismless layer, resulting in adequate Fig. 2. XRD patterns of primary and permanent enamel. Microscopy Research and Technique 576 M.A.H. DE MENEZES OLIVEIRA ET AL. retention of the resin materials to the enamel surface (Garcia-Godoy and Gwinnett, 1991; Gwinnett et al., 1992). In the present research, the prismless layer could not be observed since the teeth selected probably did not present prismless layer due to physiological dental wear as described by Fava et al. (1999). The conicting data reported in studies may be due to factors such as the variations between individual teeth, the type, age, and ethnicity, the region where the teeth were collected, the number of teeth analyzed and even the methodology performed at these researches. Moreover, the literature is scarce in data comparing the deciduous to the permanent teeth enamel. There- fore, the lack of studies testing the same methodology tested in this study was a hindrance to stating a reli- able comparison with outcomes of previous investiga- tions. Although the ndings obtained from permanent teeth have been assumed to apply to primary teeth, the existence of remarkable differences between primary and permanent enamel substrates must be taken into account, reinforcing the need of specic and biological- based preventive and restorative protocols to be deter- mined in order to provide effective treatments to these different tissues. CONCLUSIONS Within the limitations of this study, the following conclusions can be drawn: The numerical density of enamel rods was higher in deciduous teeth when compared to permanent teeth, mainly near the amelo-dentinal junction; the percentage of calcium and phosphorus was higher in permanent teeth enamel when compared to deciduous teeth enamel; deciduous teeth showed a thinner enamel layer when compared to the permanent one. REFERENCES Amaechi BT, Higham SM, Edgar WM. 1999. Factors inuencing the development of dental erosion in vitro: enamel type, temperature and exposure time. 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(Reviews in Mineralogy and Geochemistry'', 54) Patricia M. Dove, James J. de Yoreo, Steve Weiner-Biomineralization-Mineralogical Society of America (2003)
Materials Science and Engineering - C Volume 39 Issue 2014 (Doi 10.1016/j.msec.2014.02.019) Nascimento, R.M. Faita, F.L. Agostini, D.L.S. Job, A.E. Guim - Production and Characterization of Natu