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Surface Analysis of Etched Molar Enamel by Gas Adsorption
1 Orofacial Sciences, Division of Orthodontics, School of Dentistry, University of California, San Francisco, 707 Parnassus Ave., D-1011, Box 0438, San Francisco, CA 94143-0438, USA; Correspondence: * corresponding author, maria.orellana{at}ucsf.edu
Much research has been devoted to the study of etched enamel, since it is critical to bonding. Currently, there are no precise data regarding the etched-enamel specific surface area. The aim of this study was to characterize, by two different methods, the surface of human dental enamel in vitro after being etched. It was hypothesized that differences would be observed between specimens in terms of specific surface area and grade of etching. Sixteen third molar enamel samples were etched for 30 sec with 37% phosphoric acid prior to being viewed by SEM. Etched enamel surfaces were graded according to the Galil and Wright classification. The total surface area of etched samples was determined by the BET gas absorption method. A substantial variability in total surface area was observed between and among samples. A Pearson’s Correlation Coefficient showed a lack of relationship between etch pattern and total surface area.
Key Words: enamel • SEM • etched • gas adsorption • BET
The introduction of the acid-etch bonding technique (Buonocore, 1955) represents one of the most important advances in dentistry, and bonding to enamel has become a routine practice in many areas of dentistry. Phosphoric acid treatment, as described by Buonocore (Buonocore, 1955), creates a porous enamel surface layer that, when penetrated by a low-viscosity resin-bonding agent, facilitates the interlocking between composite resin and enamel. The retentive ability displayed by etched enamel for composite resin has historically been assumed to be a function of the increase in surface area due to etching and in the wettability of the etched enamel (Gwinnett, 1971; Silverstone, 1974). Much research has been devoted to the characterization of the physical and chemical properties of dental enamel in general, and the surface topography of acid-etched enamel in particular. Scanning electron microscopy (SEM) has been widely used in dentistry to explore the surface and microstructure of enamel (Mahoney et al., 2004; Ceppi et al., 2006), as well as the effect of acid-etching on enamel surfaces (Oliver, 1987; Gardner and Hobson, 2001; Hobson et al., 2001, 2002; Hobson and McCabe, 2002). Through the application of the SEM technique, a variation in quality and quantity of etched enamel was observed by Poole and Johnson (1967), who are recognized as the first to classify etched enamel patterns. Other investigators (Marshall et al., 1975; Silverstone et al., 1975; Galil and Wright, 1979a,b) have further developed and/or modified the original Poole and Johnson classification. More recent studies have adopted one of these grading scales or have used their own modified versions thereof (Oliver, 1987; Johnston et al., 1996; Hobson and McCabe, 2002). The qualitative nature of the data, along with the lack of consensus on which enamel-etching grading scale should be universally adopted, makes comparisons among studies difficult. The adoption of a standardized classification is long overdue. Furthermore, these grading scales are subjective and, at best, require reproducibility testing.
The absorption of gases in multimolecular layers, as a means of determining surface area of materials, was first described by Brunauer and co-workers in 1938 (Brunauer et al., 1938). Since then, the Brunauer-Emmett-Teller (BET) gas adsorption method has become the most widely used standard procedure for the determination of the surface areas of finely divided and porous materials (Sing et al., 1985). The BET equation is used to determine the volume of gas needed to form a monolayer on the surface of a sample. A known volume of gas (adsorbate) is added to a solid material. At cryogenic temperatures, weak molecular attraction forces will cause the gas molecules to attach to the surface of the solid material. Gas (usually nitrogen) is added to the sample in controlled doses, and the pressure in the sample container is measured after each dosing. A direct relationship exists between the pressure and the volume of gas in the sample container. The volume of gas adsorbed by the sample can be determined by measurement of the reduced pressure due to the adsorption. This relationship is known as an adsorption isotherm (Gregg and Sing, 1982; Adamson and Gast, 1997). The actual surface area can be calculated from knowledge of the size and number of the adsorbed gas molecules (Fig. 1
The aim of this study was to characterize, in vitro, human dental enamel surface area after being etched. This was achieved by a quantitative method, the gas adsorption technique, and by SEM, a qualitative method. Our secondary aim was to determine if a correlation exists between the BET enamel surface area and different grades of etchings as determined by visual examination.
Enamel Sample Preparation Sixteen unerupted human third molars extracted from individuals as part of their dental treatment were stored in a 0.1% thymol solution prior to use. All individuals gave informed consent. Confidentiality of the donors was protected, and all procedures were approved by the Health Research Ethics Board of the University of Alberta. Approximately 5 x 2 mm samples were obtained from the buccal aspects of the molars (from approximately the middle third of the surface). The dentinal layers were completely removed from the cut sections with a football-shaped diamond bur (Brasseler Dental Instrumentation, Savannah, GA, USA). A 5x surgical loupe and a light source were used to examine each specimen during removal. The intact enamel surfaces were etched for 30 sec with 37% phosphoric acid, rinsed with air and water for 5 sec, and air-dried. The treated samples were cut into 2 smaller samples of approximately 2 x 2 mm. One sample was analyzed by SEM and the other by gas absorption.
SEM
In our study, both observers had agreed a priori to grade each sample according to the enamel type that was present in the greatest proportion. This approach was taken because it was evident that many of the previous SEM studies did not use a systematic method to evaluate the etch patterns observed. After a three-week interval, the assessments were repeated by the two evaluators (MO three times and DB once).
Gas Adsorption Measurement
SEM Results SEM images of etched enamel surfaces were analyzed by two independent observers. The intraclass correlation coefficient (ICC) showed intra-operator reliability of 0.84 and inter-operator reliability of 0.86. The SEM micrograph (Fig. 2a  ) represents the most prevalent etching pattern. This type of enamel etching was graded as type 4 and was found in 10 (62.5%) of our samples. Three enamel samples (19%) were graded as type 5 (Fig. 2b), 2 (12.5%) as type 3 (Fig. 2c), and only 1 (6%) as type 1, or "ideal etch" (Fig. 2d).
Krypton Adsorption Results The estimation of specific surface areas, SBET, took place with use of the traditional BET plots from the relevant adsorption isotherms (Fig. 3a ). The BET surface area is the multipoint surface area, calculated with 3 or more pressure points (Fig. 3b).
The BET surface area for each of our samples is depicted below (Fig. 4 ). A great variation between and among samples was observed. Specimen specific surface area ranged from 0.160 m2/g to 0.528 m2/g, with a mean of 0.359 m2/g. We performed a Pearson correlation test to examine a relationship between BET surface area and types of etched enamel. A lack of linear relationship between these two variables was demonstrated by a Pearson correlation of 0.079.
SEM has been the traditional method for studying the microscopic "surface structure" of tooth tissues in general (Oliver, 1987; Johnston et al., 1996; Gardner and Hobson, 2001; Hobson et al., 2001, 2002; Hobson and McCabe, 2002; Mahoney et al., 2004; Ceppi et al., 2006), although it is not a surface-specific technique in the strictest sense. A significant difficulty with the use of SEM in examining etched enamel is the lack of consensus on grading systems. Several enamel-etch type classifications have arisen since Poole and Johnson’s original grading (Poole and Johnson, 1967). Silverstone’s (Silverstone et al., 1975) basic etching pattern types are still widely used (Tandon and Mathew, 1997; Seow and Amaratunge, 1998; Lopes et al., 2006). In type 1, the prism cores are preferentially removed, while the prism peripheries remain relatively intact. In type 2, the reverse pattern is observed. In type 3, areas of types 1 and 2 appear to co-exist in the same regions. Galil and Wright’s (Galil and Wright, 1979b) five-point classification appears to have been adopted by the largest percentage of investigators (Oliver, 1987; Johnston et al., 1996; Gardner and Hobson, 2001; Hobson and McCabe, 2002). Hobson et al. reported difficulty distinguishing between type 1 and type 2 enamel, according to Galil and Wright, and converted their five-point scale to a four-point scale (Hobson et al., 2002). In the present study, the Galil and Wright classification was utilized. The grading system from 1 to 5 is comprehensive, and it seems to be the most widely accepted. Several micrographs presented more than one type of etching pattern; thus, giving each micrograph a "global score" presented some challenges. Gardner and Hobson (2001) used histometric point-sampling to address this issue and reported different etch patterns on the same sample. Our SEM studies showed only one sample as type 1 or an "ideal etch". These findings are in agreement with Mattick and Hobson’s report that only a small percentage of enamel surface area is etched ideally upon application of 37% phosphoric acid for 30 sec (Mattick and Hobson, 2000). Ten of our enamel samples were graded as type 4, meaning that 62.5% were poorly etched, or, in other words, displayed a "suboptimal etched pattern". Perdigão et al.(1997) showed no correlation between the less-well-defined enamel-etching patterns and shear bond strength, and Hobson and McCabe (2002) inferred that high bond strength does not depend on an ideal etch pattern. Several studies have concluded that there is no relationship between bond strength to acid-etched enamel and etching conditions (Barkmeier et al., 1987; Legler et al., 1989; Hotta and Nakabayashi, 1992; Shinchi et al., 2000). Moreover, two studies by Legler et al.(1989, 1990) described a "poor" relationship between resin penetration depth and resin-enamel bond strength. Nakabayashi and Pashley (1998) have hypothesized that resin-enamel bond strength depends on the cumulative cross-sectional area of the resin tags that infiltrate the etched enamel. The length of the tags has no effect on the cross-sectional area. The work of Nakabayashi and Pashley (1998) demonstrated that exposure of enamel crystallites is more important than the display of "ideal" etch patterns, and that penetration of adhesive resins into porous enamel creates a new structure. This structure is part enamel and part resin, and it is considered to be a hybrid layer (Nakabayashi and Pashley, 1998; Tay and Pashley, 2001). Hybridization is used routinely to achieve bonding to dentin. The findings from these studies support the suggestion that enamel porosity is more important than a defined etch pattern. When phosphoric acid is applied to the dental enamel surface, it creates microscopic pores by selective dissolution of the enamel (Beech and Jalaly, 1980). It is assumed that an increase in porosity will result in an increase in surface area, and that a large exposed area of enamel is optimal for hybrid layer formation. The need for methods that will measure etched enamel surface area as well as pore diameter and distribution is obvious. It is not possible to determine the surface areas of etched enamel by optical or electron microscopy, because of the size and complexity of the pores. Gas adsorption has been studied theoretically for most of the 20th century, and the simplest of the resulting theories has provided the insight needed for most applications. Still, a literature search identified only two studies in dental enamel that utilized the gas absorption method (Misra et al., 1978; Fridell et al., 1988). Only one was performed on human dental enamel (Misra et al., 1978). From the specific surface area of each of our enamel samples, it is evident that 3rd molars from different persons behave differently as a result of 30 sec of exposure to 37% phosphoric acid. Our unpublished observations confirmed a diverse surface composition of third molar enamel from different individuals. That is, the atomic surface concentration percentages, as well as the ratio between components, were different in all tested 3rd lower molars. This could explain the different behavior after etching. A Pearson’s correlation test did not show a relationship between BET surface area and enamel etch pattern. In view of the above discussion, these results are not surprising. We could speculate on different reasons for this lack of relationship, the most obvious being that an "ideal" etch pattern does not imply a larger surface area and vice versa. Nonetheless, in our opinion, it is of pivotal importance that etched enamel surfaces be analyzed in terms of specific surface area and porosity. We have displayed the techniques that are currently available to accomplish this type of analysis, which is in concert with the aim of this study—to illustrate the possibilities that gas adsorption analysis offers to researchers in different dental disciplines.
Funding for this research was provided by the McIntyre Memorial Research Fund and by the University of Alberta Fund for Dentistry Grant #2005–06. The authors are grateful to Mrs. Joanne LaFrance for administrative support. Received for publication June 15, 2007. Revision received February 15, 2008. Accepted for publication February 28, 2008.
Journal of Dental Research, Vol. 87, No. 6,
532-536 (2008)
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