Australian Dental Journal

Australian Dental Journal The official journal of the Australian Dental Association SCIENTIFIC ARTICLE Australian Dental Journal 2012; 57: 151 156 doi: 10.1111/j.1834-7819.2012.01677.x Enamel roughness and depth profile after phosphoric acid etching of healthy and fluorotic enamel I Torres-Gallegos,* V Zavala-Alonso,* N Patiño-Marín,* GA Martinez-Castañon,* K Anusavice, JP Loyola-Rodríguez* *The Master s Degree in Dental Science with Specialization in Advanced Education General Dentistry Program, Faculty of Dentistry, San Luis Potosi University, México. Center for Dental Biomaterials, College of Dentistry, University of Florida, Gainsville, Florida, USA. ABSTRACT Background: Dental fluorosis requires aesthetic treatment to improve appearance and etching of enamel surfaces with phosphoric acid is a key step for adhesive restorations. The aim of this study was to evaluate surface roughness and a depth profile in healthy and fluorotic enamel before and after phosphoric acid etching at 15, 30 and 60 seconds. Methods: One hundred and sixty enamel samples from third molars with no fluorosis to severe fluorosis were evaluated by atomic force microscopy. Results: Healthy enamel showed a statistically significant difference (p < 0.05) between mean surface roughness at 15 seconds (180.3 nm), 30 seconds (260.9 nm) and 60 seconds (346.5 nm); depth profiles revealed a significant difference for the 60 second treatment (4240.2 nm). For mild fluorosis, there was a statistically significant difference (p < 0.05) between mean surface roughness for 30 second (307.8 nm) and 60 second (346.6 nm) treatments; differences in depth profiles were statistically significant at 15 seconds (2546.7 nm), 30 seconds (3884.2 nm) and 60 seconds (3612.1 nm). For moderate fluorosis, a statistically significant difference (p < 0.05) was observed for surface roughness for 30 second (324.5 nm) and 60 second (396.6 nm) treatments. Conclusions: Surface roughness and depth profile analyses revealed that the best etching results were obtained at 15 seconds for the no fluorosis and mild fluorosis groups, and at 30 seconds for the moderate fluorosis group. Increasing the etching time for severe fluorosis decreased surface roughness and the depth profile, which suggests less micromechanical enamel retention for adhesive bonding applications. Keywords: Healthy enamel, fluorosed enamel, phosphoric acid etching, dental fluorosis. Abbreviations and acronyms: AFM = atomic force microscopy; DF = dental fluorosis; F = fluoride; SEM = scanning electron microscopy; SiN = silicon nitride; TSIF = Tooth Surface Index of Fluorosis. (Accepted for publication 21 August 2011.) INTRODUCTION Healthy enamel formation occurs in four time periods: presecretory, secretory, transition and maturation stages. In the last stage, ameloblasts exhibit increased growth activity and lose their protein secretory properties; 1 any disturbance during this stage produces enamel pathologies such as enamel hypoplasia, amelogenesis imperfecta and dental fluorosis (DF). 2,3 The latter condition is caused by excessive intake of fluoride (F) in drinking water during enamel formation, which produces flattened hexagonal crystals with more protein content than healthy enamel. An interaction between F and Ca 10 (PO 4 ) 6 (OH) 2 occurs depending on the F concentrations: for 1 ppm F, fluorapatite (Ca 10 (PO 4 ) 6 F 2 ) is formed, which hypermineralizes the enamel surface, thereby increasing apatite crystallinity. 4 For 5 to 10 ppm F, fluorite (CaF 2 ) is formed and apatite is partly dissolved, which produces a contraction in the width of enamel crystals, increasing enamel surface roughness. 4 6 Eight degrees of freedom have been described, ranging from grade 0 (healthy enamel) to grade 7 grade (severely affected enamel). 7 Adhesive materials are used to restore bonding brackets to fluorosed enamel and several studies recommend cleaning the enamel for increasing micromechanical adhesion to resin-based materials. 8,9 Subsequently, phosphoric acid etching is used: (a) to transform the enamel surface from a lower to a higher surface energy; and (b) to dissolve and demineralize the inorganic Ca 10 (PO 4 ) 6 (OH) 2 matrix, creating micropores and microgrooves to improve mechanical retention. It has ª 2012 Australian Dental Association 151

I Torres-Gallegos et al. been reported that from 35% to 37% phosphoric acid (H 3 PO 4 ) applied for 15 to 20 seconds creates ideal enamel etching patterns for the retention of aesthetic restorative materials. 10 12 Self-etching systems are currently used for enamel and dentine bonding, but some studies have shown that adhesion to uncut enamel with these systems can be enhanced by supplementary phosphoric acid etching. 13,14 It has been suggested that fluorotic enamel needs longer acid etching times ranging from 75 seconds to 180 seconds. 15,16 These findings were supported by scanning electron microscopy (SEM) studies, but no quantitative parametric data were provided to support the observations. Information on enamel topography changes has been obtained from atomic force microscopy (AFM), which produces high atomic resolution images without the need for sample preparation. In addition, the resulting data are obtained at the nanometre (nm) scale in three dimensions (3-D). The aim of this study was to characterize by AFM the surface roughness and depth profiles of no fluorotic and fluorotic ground enamel that were etched at different times with 37% H 3 PO 4. The primary objective was to test the hypothesis that severely fluorosed enamel would not exhibit roughness and depth profiles comparable to those with milder fluorosis and no fluorosis enamel. MATERIALS AND METHODS Subjects and clinical samples preparation Patients undergoing third molar extractions at hospitals and private clinics were asked to donate their extracted teeth. Informed and voluntary written consent was obtained from the subjects prior to clinical examination according to the ethical principles of the Declaration of Helsinki (Version 2002) and approval was granted by the Institutional Review Board. A total of 20 recently erupted third molars were collected from Ciudad Valles (San Luis Potosí, State, México), which has a water fluoride level between 0.1 ppm and 0.6 ppm F; from San Luis Potosí City (México) with a natural fluoride level between 0.7 ppm and 2 ppm F; and from Salitral de Carrera (San Luis Potosí, México) with a natural fluoride level between 2 ppm and 5 ppm F. Selected teeth were stored in distilled water (Milli-Q, Millipore Co., Billerica, MA, USA) until they were cleaned and disinfected by sodium hypochlorite (10%) in an ultrasonic bath (Biosonic UC300-115B, Colténe Whaladent) with a Suprasson P5 Booster (Satelec, ZI du Phare 33700, Merignac, France). The selected teeth were then stored at room temperature in phosphate buffered saline containing 0.002% sodium azide for up to two weeks until experimental procedures were performed. Dental fluorosis severity was determined according to the Tooth Surface Index of Fluorosis (TSIF) [9]. The TSIF uses an 8-point scale, where zero represents the non-affected tooth and 7 the most severely affected tooth. Samples were divided into four categories: non-fluorotic enamel (N) [TSIF = 0]; mild fluorosis [TSIF = 1 2]; moderate fluorosis [TSIF = 3 5]; and severe fluorosis [TSIF = 6 7]. The study was blinded for the clinical diagnosis of dental fluorosis, and a second observer carried out AFM evaluations. Each molar was sectioned horizontally at the cemento-enamel junction by means of a water-cooled high-speed diamond wheel (#7910, fine-grain; Brasseler, Savannah, GA, USA). Vertical cuts were made perpendicular to the first cuts to produce mesial, middle and distal samples; they were placed in self-curing, 35 mm 35 mm acrylic cubes (Nic Tone Cross Linked #65, mdc dental and Nic Tone Cross Monomer Link self-curing, mdc dental, Guadalajara, México). All samples were cleaned with a rubber cup (Crescent, Dentsply, DF, México) and prophylaxis paste (Nupro, Dentsply, DF, México), followed by cleansing in an ultrasonic bath containing distilled water for three minutes at room temperature. Drying was performed with oil-free air. The samples were then submitted for AFM baseline measurements. Twenty ground samples of each DF grade were etched for 15 seconds with 37% H 3 PO 4 (Ultra-Etch, Ultradent, Utah, USA), washed with deionized water for 15 seconds, dried with oil-free air and analysed by AFM. These samples were etched for another 15 seconds, for a total etching time of 30 seconds, washed with deionized water for 15 seconds, dried with oil-free air and analysed by AFM. The samples were etched again for 30 seconds, or a total etching time of 60 seconds, washed with deionized water for 15 seconds, dried with oil-free air, and analysed by AFM. Measurements were performed in three distinct regions of the same area of each sample. Atomic force microscopy Enamel surface roughness was quantified using surface roughness and depth profile parameters. Surface roughness represents the arithmetic mean of the absolute values of the scanned surface roughness profile and the depth profile (peak-to-valley height) represents the mean of the absolute heights of the five highest profile peaks to the five deepest valleys within the sampling length on the scanned surface. AFM was used in the contact mode as for measurement of surface roughness of the enamel. All samples were evaluated at the same scan size (49.5 49.5 lm 2 ) by triplicate in different areas, all of which were selected at random and the mean roughness and peak-to-valley depth (profile) obtained for each sample. The evaluations of enamel surface roughness and absolute depth profile in enamel were carried out at a scanning rate of 49.5 lm s by using an AFM 152 ª 2012 Australian Dental Association

Etching in healthy and fluorotic enamel (Nanosurf Easy Scan 2, SPM Electronics, Liestal, Switzerland) in the contact mode with a silicon nitride (SiN), probe having a tip height of 14 lm and a tip radius of <10 nm. The conditions used for the short cantilever contact mode were as follows: spring constant, 0.1 N m; resonant frequency, 28 khz; length, 225 lm; mean width, 28 lm; thickness, 1 lm; tip height, 14 lm; radius, <10 nm. The Nanosurf Easy Scan 2 software (Version 1.6) was used to measure the AFM parameters. The feedback gains with a set point of 20 nn were as follows: P-Gain: 10 000; I-Gain: 1000; and D-Gain: 0. A calibration grid of silicon oxide on silicon material (Nanosurf AG, CH-4410, SPM Electronics, Liestal, Switzerland) with an XY periodicity of 10 lm and a Z height of 119 nm was used for calibration prior to each evaluation session. Statistical analysis Examiners were calibrated by an expert in DF using the intra-class correlation coefficient, which is an assessment of consistency or reproducibility of quantitative measurements made by different observers. All data were expressed as a mean ± standard deviation. Shapiro-Wilks and Brown Forsythe methods were used to test the normality of the data distribution. The Student t test was used to analyse the differences between means prior to and after etching, and ANOVA was used to compare difference in mean roughness and peak-to-valley height as a function of acid etching time and fluorosis severity. JMP program version 9.0 and Stata version 11.0 (Stata Corp LP, College Station, TX, USA) were used for statistical analysis. Statistical significance was set at a = 0.05. RESULTS The inter-observer reliability analysis of DF achieved by the examiner and the expert (i.e. the intra-class correlation coefficient), was 0.99. The mean, standard deviation, the range of surface roughness and the depth profile were calculated for each group. Table 1 shows the surface roughness of healthy and fluorotic ground enamel at baseline and after etching. Comparisons between groups were made by simultaneously considering the baseline measure and phosphoric acid etching time. Depth profiles in healthy ground enamel showed gradual increases after the three etching times, and the differences between means were statistically significant (p < 0.05). Mild fluorosis did not exhibit significant increases in roughness and depth profile at 15 seconds (p > 0.05), but it did at 30 and 60 seconds (p < 0.05). For moderate fluorosis, surface roughness decreased after 15 seconds of etching, but for 30 and 60 seconds treatments, the roughness profile increased significantly (p < 0.05). For severe fluorosis, surface roughness decreased for 15 and 60 second treatments, but at 30 seconds it increased, although the difference was not statistically significant (p > 0.05). Table 2 lists the mean depth profiles for healthy and fluorotic ground enamel at baseline and after etching. In healthy ground enamel, there was a gradual increase in depth profile for the three etching times, but the change was statistically significant (p < 0.05) only after 60 seconds of etching. For mild fluorosis, there were statistically significant increases in depth profile after 15, 30 and 60 seconds of etching (p < 0.05). Moderate fluorosis showed a gradual increase in depth profile for the three etching times, which was statistically significant only for the 60 second treatment (p < 0.05). For severe fluorosis, there was a slight increase observed at 30 seconds, but all changes were not statistically significant (p > 0.05). There were no statistically significant differences (p > 0.05) when comparing the depth profile among different degrees of dental fluorosis and different etching times; however, there was statistical significant difference (p < 0.05) in surface roughness when comparing the healthy enamel group with the moderate and severe fluorosis groups that were etched for 15 and 30 seconds. Figure 1 depicts a representative 3-D image of mild fluorosis, in which the peaks rise and the valleys deepen with increased etching time. Figure 2 shows a representative 3-D image of moderate fluorosis, for which the maximum roughness profile was observed after 60 seconds of etching time. Figure 3 depicts a representative 3-D image of severe fluorosis. Since the baseline measurements revealed increasing surface roughness as etching time increased, the roughness profile was lost. There is only a slight increase visible for the 30 seconds treatment group. Table 1. Roughness profile values of healthy and fluorotic ground enamel before and after etching Healthy Mild fluorosis Moderate fluorosis Severe fluorosis X ±DE p X±DE p X±DE p X±DE p Baseline 127.9 ± 55.1 203.9 ± 85.9 260.6 ± 115.2 321.9 ± 209.9 15 s *180.3 ± 61.1 <0.05 204.3 ± 69.0 >0.05 256.2 ± 69.0 >0.05 264.0 ± 103.0 >0.05 30 s *260.9 ± 76.9 <0.05 *307.8 ± 102.5 <0.05 *324.5 ± 102.5 <0.05 352.6 ± 132.0 >0.05 60 s *346.5 ± 179.3 <0.05 *346.6 ± 122.5 <0.05 *396.6 ± 122.5 <0.05 293.3 ± 119.6 >0.05 X: median. SD: standard deviation. p values are derived from Student test analyses. Baseline: values before etching. 15 s, 30 s and 60 s: roughness profile after etching time in seconds. Student t test. Values in nanometres. n = 20 samples. Values are expressed as nanometres. *Statistically different. ª 2012 Australian Dental Association 153

I Torres-Gallegos et al. Table 2. Depth profile values for healthy and fluorotic ground enamel before and after etching Healthy Mild fluorosis Moderate fluorosis Severe fluorosis X ±DE p X±DE p X±DE p X±DE p Baseline 2221.7 ± 3231.5 2016.7 ± 755.6 2884.7 ± 2548.6 3908.2 ± 3480.8 15 s 3295.4 ± 2237.5 >0.05 *2546.7 ± 727.6 <0.05 3164.2 ± 866.7 >0.05 2869.7 ± 865.6 >0.05 30 s 3322.6 ± 708.9 >0.05 *3884.2 ± 1510.1 <0.05 3586.4 ± 2353.4 >0.05 4302.2 ± 1935.9 >0.05 60 s *4240.2 ± 2797.1 <0.05 *3612.1 ± 1016.1 <0.05 *4327.7 ± 1939.2 <0.05 3233.1 ± 1316.7 >0.05 X: median. SD: standard deviation. p values are from Student t test. Baseline: values before etching. 15 s, 30 s and 60 s: depth profile after etching time in seconds. Student t test. Values in nanometres. n = 20 samples. Values are expressed as nanometres. *Statistically different. (a) (b) 622n 1,08µ Line fit 1,7µm 467n 1,1µ Line fit 1,56µm (c) (d) 882n 1,15µ Line fit 2,04µm 1,11µ 1,2µ Line fit 2,31µm Fig. 1 Representative 3-D images show the surface roughness of mild fluorotic ground enamel at baseline and after acid etching at the different times used: (a) baseline sample; (b) sample etched for 15 seconds; (c) sample etched for 30 seconds; and (d) sample etched for 60 seconds. (a) (b) 1,39µ 2,12µ Line fit 3,51µm 492n 760n Line fit 1,25µm (c) (d) 955n 971n Line fit 1,93µm 585n 642n Line fit 1,23µm Fig. 2 Representative 3-D images show the surface roughness of moderate fluorotic ground enamel at baseline and after acid etching at the different times used: (a) baseline sample; (b) sample etched 15 seconds; (c) sample etched 30 seconds; and (d) sample etched 60 seconds. 154 ª 2012 Australian Dental Association

Etching in healthy and fluorotic enamel (a) (b) 1,21µ 1,6µ Line fit 2,81µm 2,37µ 2,27µ Line fit 4,63µm (c) (d) 1,69µ 2,17µ Line fit 3,86µm 2,08µ 2,46µ Line fit 4,54µm X* 49,9µm Y* 49,9µm Fig. 3 Representative 3-D images show the surface roughness of severe fluorotic ground enamel at baseline and after acid etching at the different times used: (a) baseline sample; (b) sample etched for 15 seconds; (c) sample etched for 30 seconds; and (d) sample etched for 60 seconds. DISCUSSION Despite improved dental adhesive materials and techniques, some treatments fail, either because of material misuse or because of the inability of the etched enamel structure to form adequate retentive features. 8 Adhesive dentistry relies on the surface irregularities provided by phosphoric acid conditioning; however, an enamel developmental defect such as dental fluorosis inhibits the ability to obtain a suitable etched enamel surface. 16 It has been reported that the optimal etching time for dental fluorosis grade 4 ranges from 75 to 180 seconds, and the greater the extent of severe fluorosis, the longer the etching time that is needed. It has also been reported that the enamel surface roughness values of enamel is affected by different degrees of fluorosis, and fluorotic crests are higher in proportion to the severity of fluorosis. 16 Our results agree with this finding, but the surface roughness of fluorosed enamel decreased after phosphoric acid etching, which can be explained by a hypomineralization condition. Although there are reports on conditioning fluorotic enamel with phosphoric acid, none specify the topographic structural changes that occur in teeth that are affected by different degrees of dental fluorosis and treated for variable etching times. 17 In this study, surface roughness and depth profile were determined by AFM at different etching times for different dental fluorosis grades. These evaluations provide different findings compared with those from studies in which only SEM observations were made. 18 Only healthy enamel gradually increases surface roughness and depth profiles for increasing etching times, which is not observed in dental fluorosis groups. Several phosphoric acid manufacturers recommend etching for 15 seconds of healthy ground enamel, but there are no data on surface roughness and depth profiles; in this study, controlled etching yielded a mean surface roughness of 52.4 nm and a mean depth profile of 1074 nm, which suggest increased micromechanical retention enamel surface for adhesive dentistry. Clinically, mild fluorosis does not represent severely damaged tissue. Nanoscale images showed enamel alterations with deeper surface profiles with a gradual increase in surface roughness, but decreases in depth profile occurred after acid etching for 60 seconds. These findings suggest that 15 seconds of etching by phosphoric acid may provide a suitable micromechanical retention surface for aesthetic restorations. For moderate fluorosis, baseline samples were twice as rough compared with healthy enamel; however, the best conditions were obtained after 30 seconds of etching time. For severe fluorosis, baseline samples were rougher for all groups after etching for 15 and 60 seconds. Surface roughness and depth profile values decreased, although there was a slight increase in surface roughness and depth profile at 30 seconds, which may not be practical for adhesive dentistry. Thus, phosphoric acid etching for the severe fluorosis group is questionable. Moderate and severe fluorosis of enamel exhibited decreased surface roughness and depth profiles after etching, probably because of demineralization of enamel structure, and loss of enamel. The findings from the present study differ from other studies which suggest that the greater the degree of dental fluorosis, the greater the etching time is ª 2012 Australian Dental Association 155

I Torres-Gallegos et al. required. Furthermore, this study showed by AFM that increasing the etching time for severe fluorosis can decrease surface roughness and depth profile, which may produce a less effective micromechanical enamel surface for effective adhesive bonding. It has been reported that surface roughness is a reliable measure of the external enamel structure produced by dental fluorosis, so it is important to perform a correct dental fluorosis analysis before using the phosphoric acid treatment. 19 The basic principles of acid etching are to transform enamel surface of low surface energy into a high surface energy, and to dissolve and demineralize the inorganic [Ca 10 (PO 4 ) 6 (OH) 2 ] matrix, creating micropores and microgrooves to improve mechanical retention. 9,14 These criteria apply only to healthy, mild and moderately fluorotic enamel, but in the case of severe fluorosis the effect of the treatment is uncertain. However, we don t know if the final enamel etched surface can withstand the contraction forces of polymerization, or if these peaks and valleys can withstand chewing forces. CONCLUSIONS Healthy enamel exhibited gradual increases in surface roughening with increasing etching time, and statistically significant differences in surface roughness were found at different times. Surface roughness in healthy enamel increased to 180.3 nm and the depth profile reached 3295 nm, so we suggest that the optimum etching times are 15 seconds for healthy enamel and mild fluorotic enamel, and 30 seconds for moderately fluorotic enamel. For the severely fluorotic enamel, the etching did not provide optimal surface roughness and depth profile values, and the clinical success for this condition is uncertain. The use of a micro-abrasion to remove external layers of fluorosed enamel prior to application of phosphoric acid could help to improve the enamel surface, providing a better retention for adhesive bonding applications. ACKNOWLEDGEMENTS This investigation was supported by FMSLP-2008- C01-87090, SEP-UASLP-CA-84 and PIFI-2009-24MSU0011E. REFERENCES 1. Fincham AG, Belcourt AB, Termine JD. Changing patterns of enamel matrix proteins in the developing bovine tooth. Caries Res 1982;16:64 71. 2. DenBesten PK, Giambro N. Treatment of fluorosed and whitespot human enamel with calcium sucrose phosphate in vitro. Pediatr Dent 1995;17:340 345. 3. Lench NJ, Winter GB. Characterization of molecular defects in X-linked amelogenesis imperfecta (AIH1). Hum Mutat 1995;5: 251 259. 4. Bronckers AL, Lyaruu DM, DenBesten PK. The impact of fluoride on ameloblast and the mechanisms of enamel fluorosis. J Dent Res 2009;88:877 1274. 5. Chen H, Czajka-Jakubowska A, Spencer NJ, Mansfield JF, Robinson C, Clarkson BH. Effects of systemic fluoride and in vitro fluoride treatment on enamel crystals. J Dent Res 2006; 85:1042 1045. 6. Vieira A, Hancock R, Limeback H, Schwartz M, Grynpas M. How does fluoride concentration in the tooth affect apatite crystal size? J Dent Res 2003;82:909 913. 7. Horowitz HS, Driscoll WS, Meyers RJ, Heifetz SB, Kingman A. A new method for assessing the prevalence of dental fluorosis the Tooth Surface Index of Fluorosis. J Am Dent Assoc 1984;109: 37 41. 8. Toman M, Cal E, Türkün M, Ertugrul F. Bond strength of glassceramics on the fluorosed enamel surfaces. J Dent 2008;36: 281 286. 9. Noble J, Karaiskos N, Wiltshire W. In vivo bonding of orthodontic brackets to fluorosed enamel using an adhesion promotor. Angle Orthodontist 2008;78:357 360. 10. Armstrong S, Geraldeli S, Maia R, Raposo LH, Soares CJ, Yamagawa J. Adhesion to tooth structure: a critical review of micro bond strength test methods. Dent Mater 2010;26:50 62. 11. Gwinnett AJ. Structure and composition of enamel. Oper Dent 1992;5:10 17. 12. Silverstone LM, Saxton CA, Dogon IL, Fejerskov O. Variation in the pattern of acid etching of human dental enamel examined by scanning electron microscopy. Caries Res 1975;9:373 387. 13. Torii Y, Itou K, Hikasa R, Iwata S, Nishitani Y. Enamel tensile bond strength and morphology of resin-enamel interface created by acid etching system with or without moisture and self-etching priming system. J Oral Rehabil 2002;29:528 533. 14. Weerasinghe DS, Nikaido T, Wettasinghe KA, Abayakoon JB, Tagami J. Micro-shear bond strength and morphological analysis of a self-etching primer adhesive system to fluorosed enamel. J Dent 2005;33:419 426. 15. Al-Sugair MH, Akpata ES. Effect of fluorosis on etching of human enamel. J Oral Rehabil 1999;26:521 528. 16. Opinya GN, Pameiher CH. Tensile bond strength of fluorosed Kenyan teeth using the acid etch technique. Int Dent J 1986;36: 225 229. 17. Loyola-Rodriguez JP, Zavala-Alonso V, Reyes-Vela E, Patiño- Marin N, Ruiz F, Anusavice KJ. Atomic force microscopy observation of the enamel roughness and depth profile after phosphoric acid etching. J Electron Microsc 2010;59:119 125. 18. Weerasinghe DS, Nikaido T, Wettasinghe KA, Abayakoon JB, Tagami J. Micro-shear bond strength and morphological analysis of a self-etching primer adhesive system to fluorosed enamel. J Dent 2005;33:419 426. 19. Zavala-Alonso V, Martinez-Castañon GA, Patiño-Marin N, Terrones H, Anusavice K, Loyola-Rodriguez JP. Characterization of healthy and fluorotic enamel by atomic force microscopy. Microsc Microanal 2010;16:531 536. Address for correspondence: Dr Juan Pablo Loyola-Rodriguez Head and Chairman The Master s Degree in Dental Science with Specialization in Advanced General Dentistry Program Faculty of Dentistry San Luis Potosi University Mexico Email: jloyola@uaslp.mx 156 ª 2012 Australian Dental Association