Caries Res. 9: 373-387 (1975) Variation in the Pattern of Acid Etching of Human Dental Enamel Examined by Scanning Electron Microscopy LL. M. SiLverstone, C. A. Saxon, I. L, DoGon and O. ERSKOV The London Hospital Medical College, Dental School, London, ibbs Dental Research, Unilever Lid., Isleworth Laboratory, Isleworth, England Harvard School of Dental Medicine, Boston, Mass., and The Royal Dental College, Aarhus Key Words. Acid etching * Enamel ° Etching patterns * Prisms Absiract. This study has shown that the exposure of human dental enamel to solutions in vitro produces three basic etching patterns. In the most common, ng pattern, prism core material was preferentially removed leav- ing the prism peripheres relatively intact. In the second, type 2 etching pattern, the reverse pattern was observed. The peripheral regions of prisms were removed preferentially, leaving prism cores remaining relatively unaffected. In the type 3 etching pattern, there was a more random pattern, areas of which corresponded to types 1 and 2 damage together with regions in which the pattern of etching could not be related to prism morphology. These findings differ from previous studies in which the type I patiern was ascribed to acid action and type 2 etching pattern to ck by chelators. The results therefore suggest that there is no one specific etching pattern produced in human dental enamel by the action of acid solutions. uch differences produced by acids are difficult to explain on the basis of varia- tion in chemical composition, and crystallite orientation. This further highlights the variation in structure that can occur in enamel not only from tooth to tooth, or surface to surface, but also from site to site on a single tooth surface In a previous publication Poote and JoHNSON [1967] described dif- ferences in etching patterns produced by the action of dilute mineral ac ids, dilute organic acids at pH 4.0, and ethylene diamine tetra-acetic acid (EDTA) at pH 7.0, on the surface of human dental enamel seen with the scanning electron microscope. When the natural surface of a tooth, or a surface prepared transverse to prism direction, was exposed to acid, a characteristic honeycomb pattern was produced. This was in~ terpreted as resulting from preferential loss of prism centres. Mineral374 SU.VERSTONE/SAXTON/DOGON/FEIERSKOV and organic acids apparently had similar effects. The EDTA solution al- ways produced what appeared to be the opposite effect. Cylinders of material were left projecting towards the original surface, separated by gaps produced by an apparent preferential dissolution of the peripheral parts of the prism, These findings were first recorded by MULLER and Scuarr [1957] using replica techniques and have subsequently been con- firmed by Horrman et al. [1968, 1969a, b] using the scanning electron microscope. In a further study, JoHNSON et al. [1971] found, once again, that acid solutions preferentially removed prism centres whilst EDTA, at neutral pH, preferentially removed prism peripheries. However, short exposures to dilute acids produced narrow clefts at prism peripheries as well as shallow concavities in the body of the prism. Gwinverr [1971] examined enamel after exposure to acid solutions and found different surface characteristics in experimental regions, none of which were ex- clusive to any one particular acid. The most common finding was that in which prism core material was preferentially removed. The reverse of this pattern was observed in some cases after acid action. GWINNETT et al. [1972] and SHEYKHOLESLAM and BuoNocorE [1972] also showed etching patierns in which there was either a preferential loss of prism core material or prism peripheries, both patterns being produced by the cone acid solution, Thus, the pattern of etching in which prism cores are left relatively intact with a preferential removal of the peripheral’ materi- al does not necessarily appear to be confined to attack on enamel by chelating agents. This study was carried out to see if there is one specific etching pat- tern produced by exposure of surfaces of human dental enamel to acid solutions, Materials and Methods Sixty sound human premolar teeth, extracted for orthodontic purposes, were employed in this study. Small window regions of enamel were prepared on cither smooth surfaces or cuspal slopes by painting the rest of the tooth with varnish and then they were exposed to one particular acid solution. The acid solutions employed were unbuffered phosphoric acid in the concentration range 20-70%, and buffered solutions of lactic acid at 1, O.1 and 0.001 at pH 4.5. Exposure times varied from 1 to 10min when employing phosphoric acid solutions and 1-5 days with lactic acid solutions. None of the solutions was stirred during an expo- sure, In this manner, damage to the superficial layers of enamel was observed in the group of experiments employing phosphoric acid whilst with lactic acid, etchVariation in the Pattern of Acid Etching 18 patterns were observed in the inner regions of enamel. After exposure, speci- mens were thoroughly washed with distilled water and dried with compressed air. Specimens were then glued to aluminium stubs with the exposure window upper- most and vacuum-coated with a layer of carbon followed by silver or gold, for ex- amination in the scanning electron microscope. The total thickness of the coating was estimated at approximately 50. nm. After exposure to phosphoric acid, a number of specimens were coated with a fissure sealant. Two materials were used, both of which were polymerized by ul- traviolet light (Nuva-Seal® and TP 226) [Suvexsrone, 1974]. After application of the sealant and its subsequent polymerization, specimens were demineralized in A2-percent hydrochloric acid for 5h. In this manner, all of the dental tissues were removed, leaving just the plastic-type sealant remaining. The fitting surface of the sealant thus acted as a replica of the previously etched enamel surface. These specimens were also vacuum-coated for examination in the scanning electron mi- eroscope. Results Phosphoric Acid When using phosphoric acid solutions within the short exposure times employed in this study, three patterns of enamel surface etching were seen. In the first, called type I etching pattern, there was a generalized roughening of the enamel surface, but with a distinct pattern showing hollowing of prism centres with relatively intact peripheral regions (fig. 1). The average diameter of the hollowed regions was 3 xm. This was found to be the most common of the three patterns observed. In the second, or type 2 etching pattern, prism peripheries appeared to be removed, or heavily damaged. Therefore, the prism cores were left projecting towards the original enamel surface (fig. 2). This apparent damage of the peripheral regions of the prisms was seen to extend along the length of the prism, thus aiding in delineating individual prisms (fig. 2). When viewed from the original surface (fig. 3), separate bundles cor columns of material are seen, the gaps separating them corresponding to the peripheral regions of the prisms. Thus, this type 2 etching pattern was the reverse of the honeycomb pattern of type 1 damage, and both patterns were produced by exposure to a similar solution of phosphoric acid for an identical exposure time. Some etched regions showed neither type 1 nor type 2 etching pat- terns exclusively. These areas appeared as a generalized surface rough- ening with regions resembling hollowed prism centres adjacent to areas in which the pattern was more consistent with the loss of prism peri- pheries. In addition, within these areas were regions in which the etching376 SU.VERSTONE/SAXTON/DOGON/FESERSKOV ME Sonieat « Fig. 1. Scanning electron micrograph of an enamel surface that was exposed to 37-percent phosphoric acid for 1 min, The surface shows a pattern in which there is a distinct hollowing of prism centres with relatively intact prism peripheries. This type of damage, called type 1 etching pattern, was the most common seen in this study. Fig. 2. Scanning electron micrograph of an enamel surface also exposed to 37- percent phosphoric acid for 1 min. However, in this specimen, the prism peripher- ies have been removed preferentially, leaving the prism cores projecting towards the original enamel surface. This type 2 etching pattern can be seen to extend along the length of the rods, thus aiding in delineating individual prisms Fig. 3. Scanning electron micrograph of part of the surface seen in the pre- vious figure, now at a higher magnification and viewed from the original surface, Separate prism cores are seen, the gaps separating them corresponding to. prism peripheries. This is a type 2 etching pattern.SILVERSTONE/SAxTON/DOGON/FEIERSKOV Fig. 4. Scanning electron micrograph of an enamel surface exposed to 30-per- cent phosphoric acid for I min. In this, areas can be seen showing a type 1 etching pattern in which there is a preferential removal of prism cores. However, adjacent regions show a type 2 etching pattern in which the reverse pattern can be seen. In addition, in many areas the pattern of surface damage is difficult to relate to prism structure. Such a field is referred to in this study as a type 3 etching pattern. patterns did not apparently conform to prism morphology. These regions were referred to as a type3 etching pattern (fig. 4). Sometimes, the whole region in the type 3 etching pattern was one in which the entire surface topography could not be related to a prism pattern (fig. 5). Fig.5, Scanning electron micrograph of the inner fitting surface of a fissure sealant (TP 226) [SitveRsroNe, 1974] after removal of the enamel by deminerali al etched enamel surface, which had previously been exposed to 50-percent phosphoric acid for 2 min, In ad- as suggesting types 1 and 2 etching patterns, the topography of the major part of the sealant cannot be related directly to prism structure. Thus, this whole region represents a type 3 etching pattern, zation, This surface thus acts as a replica of the origi dition to some a Fig.6. Scanning electron micrograph of an enamel surface exposed to 0.18 lactic acid for 48h. The pattern of destruction is one in which there has been a preferential loss of prism core material. Thus, this represents a type 1 etching pat tern and, due to the greater degree of tissue damage, is more clearly defined than the similar pattern seen in figure 1. This was also found to be the most common pattern of damage in the experiments with lactic acid.=. was #5 oe norte Sirs mee oe380 SILVERSTONE/SAXTON/DOGON/FEJERSKOV Fig. 7, Scanning electron micrograph of an enamel surface also exposed to OLN lactic acid for 48h. However, a re the previous figure, even though identic: pattern of damage is seen relative to ‘perimental conditions were employed. Approximately 100 prisms can be seen in this field of view and they all show a type 2 etching pattern in which the prism cores are left relatively intact tial Loss of prism peripheries.Variation in the Pattern of Acid Etching 381 Fig. 8. Scanning electron micrograph of part of the enamel surface seen in the previous figure, now at a higher magnification. In this type 2 etching pattern prism cores are seen as bundles, clearly separated from each other by gaps repre senting the loss of prism peripheries. Individual arrays of crystallites can be identi- fied in the prism core material, showing that the remaining tissue is relatively po- ‘The inner fitting surfaces of fissure sealants, when examined with the scanning electron microscope, acted as accurate replicas of the acid- etched enamel surfaces. All three types of etching pattern were seen in these specimens. However, since the damaged surfaces were viewed in replicas, a reverse pattern was observed (fig. 5). That is, with a type 1 etching pattern, the plastic replica appeared as a type 2 pattern. Similar- ly the type 2 etching pattern, in which the prism cores were left stand- ing, appeared as a type 1 pattern in the replica. ‘The rate of damage to the enamel surface, in terms of both depth of etch and extent of porosity of the tissue, was found to be inversely pro- portional to the concentration of phosphoric acid employed, as reported previously [SILVERSTONE, 1974]382 SILVERSTONE/SAXTON/DOGON/FEIERSKOVVariation in the Pattern of Acid Etching 383 Lactic Acid Exposure of enamel surfaces to lactic acid for periods of 1-5 days produced a much greater degree of damage to the tissue than that found with phosphoric acid. The enamel was etched to depths varying from one quarter to three quarters of its entire thickness, With these experi- ments, all three types of etching patterns were again seen, but this time, the patterns were even more pronounced than in the previous series of experiments, Figure 6 shows a type 1 etching pattern in which there has been a preferential loss of prism core material, The prism cores are much more hollowed out than in the previous experiments using phosphoric acid, thus demonstrating more extensive damage of the tissue. The average di- ameter of the central depressions was 3-5 ym. As with the experiments employing phosphoric acid, this pattern was found to be the most com- mon. Figure 7 shows a specimen, also etched with lactic acid for 48h, in which the loss appears heaviest in relation to prism peripheries, and therefore demonstrates a type 2 pattern of enamel etching. Approximate- iy 100 prisms are seen in this field of view (fig. 7) and they all show an identical pattern of attack. The prism cores are projecting 20-30 wm above the general level of the tissue clearly demonstrating a type 2 etch- ing pattern of the enamel. The average width of the prism cores is 3m. At a higher magnification (fig. 8) the crystallite bundles within the prism cores can be seen clearly, indicating the extent of porosity of the remaining tissue. In figure 9, prisms can be seen in a more longitudinal direction. This also demonstrates the type 2 etching pattern, the loss of prism peripher- ies aiding in the identification of individual prisms. The damage to the peripheral regions of the prisms is seen to extend for a significant tance along the prism structure, Both type 1 and type 2 etching patterns were produced over the en- tire concentration range of lactic acid used in these experiments, and were apparently independent of exposure time. Some regions were identified as showing a type 3 etching pattern, but these regions were found to occur less often than when using phosphoric acid. Figure 10 shows an area of inner enamel demonstrating a type 3 shows a type2 etching pattern and is seen to extend a significant distance along. the prisms.384 SILVERSTONE/SAXTON/DOGON/FEIERSKOV Fig. 10. Scanning electron micrograph of an enamel surface exposed to 0.1N lactic acid for 24h. The surface shows a type 3 etching pattern in which there ap- pears to be a random pattern of damage which is difficult to relate to prism struc- ture, etching pattern in which it is extremely difficult to relate surface topog- raphy to prism structure. The various etching patterns produced were not related to specific regions of enamel. That is, all three patterns were found to occur on smooth enamel surfaces as well as on the occlusal surface of the crown, In this, the results are in agreement with the find- ings for phosphoric acid. Discussion When enamel was exposed to either phosphoric acid for short expo- sure periods, or lactic acid with long exposures, patterns were seen in‘Variation in the Pattern of Acid Etching 385 which there was a preferential loss of prism core material, called a type 1 etching patiern in this study. This finding supports previous ob- servations [MULLER and Scart, 1957; Poote and JouNsoN, 1967; HOFFMAN ef al., 1968, 1969a, b; JOHNSON e# al., 1971]. However, in contrast to the above-reported findings, the reverse pattern of destruc- tion was found in specimens exposed to identical conditions. That is, prism core material appeared relatively intact with the preferential loss of prism peripheries. This type 2 etching pattern was found in both se- ries of experiments reported in this study. Although this second etching pattern has been produced by the action of strong chelating agents on enamel [POOLE and JOHNSON, 1967; HOFFMAN ef al., 1968, 1969a, b] it is obviously not unique to a chelator attack on human dental enamel as has been inferred to date. ‘These results suggest therefore that there is no one specific etching pattern produced by acid action on human dental enamel, as far as the two acids used in this study are concerned. Poote and JOHNSON [1967] suggested that the different etching pat- terns produced with either an acid, or a chelating agent, were due to dif- ferences in orientation of crystallites relative to the direction of attack, together with differences in chemical composition between central and peripheral parts of enamel prisms. Little is known of differences in chemical composition between these areas, and the fact that in this study both etching patterns were produced by exposure to acid solutions, indi- cates that differential solubilities between prism core and prism peri- phery, if they exist, may be of little significance with respect to the pat- tern of acid etching. Such differences, however, could be of significanc in the carious process which is a more diffusion-controlled attack rela- tive to the relatively crude etching employed in these studies. In addi- tion, the caries process consists of a dynamic series of events, with phas- es of both demineralization and remineralization [SiLveRstone, 1973], rather than a more simple, continuing dissolution. Thus, since there is an abrupt change in crystallite orientation at prism junctions, more in- tercrystallite space exists, This space is probably filled with hydrated organic matrix and so the organic content of the region will be higher, even though no discrete organic prism sheath is thought to exist in sound mature enamel [MECKEL et al., 1965; JonNson, 1967; SuNDsTROM and ZELANDER, 1968]. The role of crystallite orientation may be significant in determining differential etching effects in enamel. However, little is known regarding386 SILVERSTONE//SAXTON/DOGON/FEIFRSKOV differences in the reactivity of different faces of individual enamel erys- tallites to demineralizing agents. Previous studies [JoHNSoN, 1966; SHARPE, 1967; BoyDe, 1971] suggest that enamel crystallites dissolve in acid more quickly along their c-axes than in a direction perpendicular to this axis. However, TYLER [1969] has shown that calcium is liberated at the same rate from areas on various faces of large fluorapatite crystals exposed to acid. JOHNSON ef al. [1971] suggested, as one further reason for the differ- ences in etching patterns produced by either acids or chelators, that the difference in size and electric charge exhibited by the different deminer- alizing radicals might play a role. However, since in this study both types of etching patterns were produced with acid solutions alone, this tends to decrease the importance of this parameter as a likely explana- tion for differences in action between the two types of agent. This study has shown that the exposure of human dental enamel to acid solutions i vitro produces three basic etching patterns. In the most common, called type 1 etching pattern, prism core material was prefer- entially removed leaving the prism peripheries relatively intact. In the second, type 2 etching pattern, the reverse pattern was observed. The peripheral regions of prisms were removed preferentially, leaving prism cores remaining relatively unaffected. In the type 3 etching pattern, there was a more random pattern, areas of which corresponded to types 1 and 2 damage together with regions in which the pattern of etch- ing could not be related to prism morphology. These findings differ from previous studies in which the type 1 pattern was ascribed to acid action and type 2 etching pattern to attack by chelators. The results therefore suggest that there is no one specific etching pattern produced in human dental enamel by the action of acid solutions. Such differences produced by acids are difficult to explain on the basis of variation in chemical composition and crystallite orientation, This further highlights the varia~ tion in structure that can occur in enamel not only from tooth to tooth, or surface to surface, but also from site to site on a single tooth surface. 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Areh, oral Biol. 12: 1621-1634 (1967). SnarPE, A. N.: Influence of crystal orientation in human enamel on its reactivity to acid as shown by high resolution microradiography. Arch. oral Biol. 12: 583-591 (1967) SHEYKHOLESLAM, Z. and BUONOCORE, M. G.: Bonding of resins to phosphoric acid- etched enamel surfaces of permanent and deciduous teeth. J. dent. Res. 57: 1572-1576 (1972) Snverstoxe, L, M.: Structure of carious enamel, including the early lesion; MELCHER and Zann Dental enamel. Oral sciences reviews, No.3, pp. 100-160 (Munksgaard, Copenhagen 1973). Siverstoxe, L, M.: Fissure sealants; laboratory studies. Caries Res. 8: 2-26 (1974) NDSTROM, B. and ZeLANDER, T.: Routine decalcification of thin ground sections ‘of adult human enamel by means of a basic chromium (111) sulphate solution, of stable pH. Odont, Revy 19: 3-233 (1968). ‘Tyner, J. E Comparative dissolution rates on human enamel and fluorapatite. Caries Res. 4: 23-30 (1969). Prof. L. M. SILVERSTONE, University of Minnesota, School of Dentistry, Minnea- polis, MN 55455 (USA)