Journal of Dental Research

Journal of Dental Research http://jdr.sagepub.com/

Internal Morphology of Surface Zones from Acid-etched Caries-like Lesions: A Scanning Electron Microscopic Study M.J. Hicks and L.M. Silverstone J DENT RES 1985 64: 1296 DOI: 10.1177/00220345850640110901 The online version of this article can be found at: http://jdr.sagepub.com/content/64/11/1296

Published by: http://www.sagepublications.com

On behalf of: International and American Associations for Dental Research

Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/64/11/1296.refs.html

Downloaded from jdr.sagepub.com by guest on July 11, 2011 For personal use only. No other uses without permission.

CLINICAL SCIENCE Internal Morphology of Surface Zones from Acid-etched Caries-like Lesions: A Scanning Electron Microscopic Study M. J. HICKS1'2 and L. M. SILVERSTONE' Dental Research Unit' and Department of Growth and Denver, Colorado 80262

Development2,

Using scanning electron microscopic techniques, we compared the surface topography and internal surface morphology of acid-etched caries-like lesions of enamel with those of sound enamel and carieslike lesions of enamel. The results indicated that acid-etching of caries-like lesions of enamel yielded etching patterns similar to those previously described for sound enamel. The internal morphology of the acid-etched lesion indicated that porosities were created which may allow access to the subsurface demineralized area of the lesion and could prove beneficial in facilitating remineralization. J Dent Res 64(11):1296-1301, November, 1985

Introduction. Sound enamel is composed of approximately 96% inorganic material (primarily hydroxyapatite), 1% organic material, and 3% water by weight (Berkovitz et al., 1978; Yaegar, 1980). When a thin section of sound enamel is viewed with a polarized light microscope after imbibition with water, striae of Retzius, prism boundaries, and "wedge-shaped" defects in the enamel surface may be seen. Qualitatively, sound enamel appears to be negatively birefringent with respect to prism direction, in contrast to the positive birefringence of the underlying sound dentin. By using quantitative polarized light techniques, researchers have shown the pore volume of sound enamel to be 0.1% spaces (Darling et al., 1961; Silverstone, 1973). During lesion formation, four distinct zones of enamel caries the translucent zone and the may be observed. Two zones body of the lesion- represent areas of demineralization; while the dark zone and the surface zone represent areas of remineralization within the lesion of enamel (Silverstone, 1973, 1983). This study concerns only the surface zone, the most superficial zone of enamel caries. Both the surface zone and the body of the lesion may be observed when an enamel lesion is viewed with the polarized light microscope after imbibition with water. The surface zone appears to be relatively unaffected when compared with adjacent sound enamel. However, it typically has a pore volume of between 1 and 5% and is negatively birefringent. Thus, although the surface zone appears relatively unaffected, it is between 10 and 50 times more porous than sound enamel. In contrast, the underlying body of the lesion appears positively birefringent and has a minimum pore volume of 5% of spaces at its periphery, with a pore volume in excess of 25% in its central portion. Acid-etching of enamel caries has been proposed as a means to enhance the degree and rate of remineralization of incipient lesions by rendering the intact, relatively unaffected surface zone slightly porous (Hicks and Silverstone, 1984 a,b). The histopathologic and topographic appearances of surface zones

School of Dentistry, University of Colorado Health Sciences Center,

from acid-etched caries-like lesions have been described. Although acid-etching for 60 sec with an unbuffered phosphoric acid solution resulted in a 12-rxm loss of surface enamel, the surface zone was found to be intact but somewhat porous. The etched surface zone was composed of two layers: a superficial pseudo-isotropic layer and a negatively birefringent layer adjacent to the body of the lesion. The pseudo-isotropic layer represents an area with a pore volume of 5% spaces; whereas the underlying negatively birefringent layer has a pore volume of less than 5% spaces. The purpose of this study was to determine whether acidetching of caries-like lesions of enamel created porosities in surface zones. This was performed by investigating and comparing the appearances of the internal morphologies of surface zones from caries-like lesions and acid-etched caries-like lesions using a microdissection technique (Featherstone and Silverstone, 1982) and scanning electron microscopy.

Materials and methods. Ten caries-free human teeth were cleaned thoroughly with fluoride-free prophylaxis paste. Prior to exposure to the artificial caries system (Silverstone, 1967), an acid-resistant varnish was applied to the teeth, leaving windows of sound enamel exposed on the buccal and lingual surfaces. Four window areas of 0.5 mm occlusocervically and 4 mm mesiodistally were available for lesion formation. The teeth were then suspended in a 15% acidified acid gel, brought to pH 4.0 with the addition a

Received for publication April 30, 1985 Accepted for publication July 16, 1985 This investigation was supported by NIH New Investigator Research Award 5 R23 DE06829. Presented at the General Session of the International Association for Dental Research, March, 1984 1296

Fig.

1

Experimental design for microdissection of caries-like lesions.


Vol. 64 No. II

INTERNAL MORPHOLOGY OF ACID-ETCHED SURFACE ZONES: AN SEM STUDY

1297

Fig. 2 Lesion segments positioned for SEM examination. The fractured surfaces of the lesions (arrow) may be viewed

directly.

Fig. 3 Occasional surface depressions (arrow) may be observed on this representative sound enamel surface.

of lactic acid. After a 10-to-12-week exposure period to the imbibition with water. Two zones of enamel caries the acidified gel, the teeth were removed and sectioned into mesial negatively birefringent surface zone and positively birefringent and distal tooth halves. With each tooth, one tooth half was body of the lesion- can be identified when a caries-like lesion is viewed in water with a polarized light microscope. Contour randomly assigned to the control group, while the other half was assigned to the acid-etch group. The tooth halves in the maps of the lesions were constructed to determine the positions acid-etch group were exposed to 30% phosphoric acid for 60 occupied by the zones relative to the enamel surface. Each seconds. Using a painting motion and a fine sable hairbrush, lesion was carefully fractured with a sharp scalpel blade. Each we applied the acid to the surface of the lesion during the entire portion of the lesion was photographed in water with polarized exposure period. In order to ensure that only the lesion surface light, to ensure that proper identification of the region to be was exposed to the acid solution, we applied an acid-resistant examined with the scanning electron microscope (SEM) could varnish to the tooth halves, including the cut faces, leaving the be made. The lesion segments were then mounted on SEM window areas with enamel lesions exposed. Two longitudinal stubs such that the fractured surfaces of the lesion could be sections were then taken from each of the control and etched observed directly. Because the areas occupied by the surface tooth halves (Fig. 1). The control and acid-etched lesions for zone and body of the lesion were determined previously by each tooth were examined withDownloaded a polarizing after it was possible to characterize the microscope polarized light microscopy, from jdr.sagepub.com by guest on July 11, 2011 For personal use only. No other uses without permission.

1298

HICKS & SILVERSTONE

J Dent Res Novemberu 19855

Fig. 4 An increased number of surface depressions (arrow) are present on this representative surface of a caries-like lesion of enamel when compared with sound enamel surfaces.

Fig. 5 Acid-etching of a representative caries-like lesion of enamel has resulted in preferential loss of enamel prism cores, characteristic of a type I etching pattern.

internal morphology of the surface zone at different depths from the enamel surface for both control and acid-etched lesions (Fig. 2). In addition, lesion segments from control and acid-etched lesions and sound enamel segments were mounted on SEM stubs such that the enamel surfaces could be examined for comparative purposes. The specimens were then examined uncoated at 5 kV using the high-resolution upper stage of the ISI DS-130 SEMa. The internal morphologies of 30 control and 30 etched surface zones were examined from the fractured specimens. The topographies of the enamel surfaces from ten sound enamel segments, ten control lesions, and ten etched lesions were also examined. Photomicrographs were taken of the representive findings from the specimens.

Results. Surface morphology. The surface of sound enamel appeared to be relatively smooth with occasional surface depressions, representing prism ends terminating at the surface (Fig. 3). These structures were usually found at the base of the perikymata. The surface of the caries-like lesion was quite similar to that seen for sound enamel, with the exception of an increased number of surface depressions (Fig. 4). Despite these surface defects, the surface overlying the lesion was considered to be relatively smooth and intact. Following acid-etching with 30% phosphoric acid for 60 seconds, the surfaces of the lesions exhibited both type 1 (Fig. 5) and type 2 (Fig. 6) etching patterns. The etched surfaces of lesions with a type I etching pattern had preferentially lost the

"International Scientific Instruments Company, Santa Clara, CA 95051 Downloaded from jdr.sagepub.com by guest on July 11, 2011 For personal use only. No other uses without permission.

Vol. 64 No. 11

INTERNAL MORPHOLOGY OF ACID-ETCHED SURFACE ZONES. AN SEM STUDY

1299

Fig. 6 - Acid-etching of a representative caries-like lesion of enamel has resulted in preferential loss of enamel prism peripheries, characteristic of a type 2

etching pattern.

Fir. 7 Direct observation of the internal morphology of a representative surface zone from a caries-like lesion reveals only the boundaries present between enamel prisms.

prism cores, with the prism peripheries appearing to be relatively intact (Fig. 5). Acid-etching of the lesion surfaces also resulted in areas with type 2 etching patterns characterized by loss of prism peripheries and the presence of etched prism cores projecting toward the original enamel surface (Fig. 6). No preferential etching pattern was identified following exposure of the lesion surfaces to phosphoric acid. In tact, adjacent areas of types I and 2 etching patterns were routinely observed on the etched lesion surfaces. In addition to creating etching patterns, small porosities were created in the enamel

7). The prism boundaries could be identified readily; however, minimal areas of porosity were observed. The internal morphology for acid-etched surface zones (Fig. 8) was remarkably different from that for control surface zones. Funnel-shaped structures extending from the surfaces of the etched lesions

into the surface zones were apparent. These structures penetrated the etched surface zones to depths ranging from 5 to 8 kJm. The diameters of the funnel-shaped structures were approximately 3 km. Extending from the bases of the funnelshaped structures deep into the surface zones were etch pits. The etch pits were usually from 0.5 to 0.75 gLm in diameter. surfaces. Both the funnel-shaped structures and etch pits were found at Internal morphology of surface zones. SEM examination similar locations on the corresponding fractured portions of the of the fractured lesion segments from control and acid-etched lesions and did not appear to represent artifacts due to the lesions indicated certain differences in the surface zones. With inicrodissection technique. lesion segments from caries-like lesions, fracturing of the lethe 15 to 20 vLm from the outer surface, the etch sion occurred along the long axes of enamel (Fig. Approximately prisms Downloaded from jdr.sagepub.com by guest on July 11, 2011 For personal use only. No other uses without permission.

1300

HICKS & SILVERSTONE

J Dent Res November 1985

Fig. 8

The internal morphology of from an acidetched caries-like lesion demonstrates both the superficially positioned funnel-shaped structures (f) and etch pits (ep) present at the bases of the funnel-shaped struca representative surface zone

tures.

,,.., . a now-i:'.. M 'p. ;: ...

l v

&

A

.@| AtS~\

The presence of etch pits Fig. 9 (ep) and small porosities (p) can be ob-

served in this SEM photomicrograph of the internal morphology of a representative etched surface zone. The junction between the etch pits and small porosities is located 20 pAm from the surface of the lesion.

pits were found to terminate, and smaller porosities were observed extending deep into the etched surface zones (Fig. 9). These porosities were typically from 100 to 300 nm in size and were observed to be present at the peripheries of the enamel prisms, as well as extending further into the surface zones along the direction of the etch pits. These porosities were also found at similar locations on the corresponding fractured portions of the lesion segments, indicating that the porosities were not artifacts due to the microdissection technique.

lesions was not surprising. The lesions were created in enamel exposed to a constant, diffusion-controlled, attack with lactic acid. In addition, it is well known that the surface zone typically has a pore volume of between I and 5% spaces and represents a zone of remineralization (Silverstone, 1968, 1973). When the pore volume for the surface zone is compared with that for sound enamel, a difference in surface morphology would be expected. Sound enamel has a pore volume of approximately 0.1%, being one-tenth to one-fiftieth of that expected for the surface zone. Even though a great disparity exists between the pore volumes for sound enamel and surface Discussion. zones, the surface zone has been described as intact and relatively unaffected based upon scanning electron and polarized Although certain differences were noted between the surface light appearances (Silverstone, 1967, 1973). appearances of sound enamel and the caries-like lesion of enamel, the surfaces were considered to be intact. The inAcid-etching of a caries-like lesion of enamel resulted in the creased number of surface depressions of Noa other with bycaries-like present relatively porous but intact surface zone. The Downloaded from jdr.sagepub.com guest on July 11, 2011 Forcreation personal use only. uses without permission.

Vol. 64 No. 11

INTERNAL MORPHOLOGY OF ACID-ETCHED SURFACE ZONES: AN SEM STUDY

surface porosities, characterized by type 1 and 2 etching patterns, were found to be continuous, with funnel-shaped structures, etch pits, and small porosities in the subsurface area of

the etched surface zone. These porosities extended into the etched surface zone for some distance and approached the upper border of the underlying body of the lesion. The funnel-shaped structures represent extensions of hollowed-out prism cores and prism peripheries usually observed as type 1 and 2 etching patterns following acid-etching of sound enamel. When sound enamel is etched, the enamel has been shown to be affected at three different levels. Using polarized light techniques, we have identified three distinct histologic zones: (1) the etched zone, (2) the qualitative porous zone, and (3) the quantitative porous zone (Silverstone, 1974). With acid-etched caries-like lesions of enamel, both an etched zone and a qualitative porous zone similar to those present in etched sound enamel have been described (Hicks and Silverstone, 1984a). The qualitative porous zone reported for caries-like lesions etched with phosphoric acid is characterized as a pseudoisotropic layer comprising the superficial portion of the etched surface zone. From the results of this study, it appears that the polarized light appearance of the qualitative porous zone is most likely due to the superficially positioned funnel-shaped structures and more deeply positioned etch pits. The presence of the small porosities located in the etched surface zone superficial to the body of the lesion may correspond to the quantitative porous zone observed with etched sound enamel. The presence of this internal pore structure in the surface zone from an acid-etched caries-like lesion may provide a means for enhancement of the degree and rate of remineralization of the lesion. Remineralization of enamel caries is possible, but requires numerous exposures to calcifying fluids due to the intact surface zone. In this study, it has been shown that it is possible to render the surface zone porous, while maintaining a relatively intact surface zone, and thus perhaps a pathway for more rapid remineralization of the area of greatest mineral loss i.e., the body of the lesion may be created. This

1301

could result in a decrease in the length and number of exposures to calcifying fluids, facilitating remineralization of enamel caries.

Acknowledgments. The authors wish to acknowledge Ms. Denise Richardson and Ms. Wanda Valentine for typing the manuscript. REFERENCES

BERKOVITZ, B.K.B.; HOLLAND, G.R.; and MOXHAM, B.J. (1978): A Color Atlas and Textbook of Oral Anatomy, 2nd ed. London: Wolfe Medical Publishers, pp. 79, 153. DARLING, A.I.; MORTIMER, K.V.; POOLE, D.F.G.; and OLLIS, W.D. (1961): Molecular Sieve Behaviour of Normal and Carious Human Dental Enamel, Arch Oral Biol 5:251-273. FEATHERSTONE, M.J. and SILVERSTONE, L.M. (1982): Creation of Caries-like Lesions in Sections of Teeth Using Acid Gels, IADR Progr & Abst 61: No. 278. HICKS, M.J. and SILVERSTONE, L.M. (1984a): Acid-etching of Caries-like Lesions of Enamel: A Polarized Light Microscopic Study, Caries Res 18:315-326.

HICKS, M.J. and SILVERSTONE, L.M. (1984b): Acid-etching of Caries-like Lesions of Enamel: A Scanning Electron Microscopic Study, Caries Res 18:327-335. SILVERSTONE, L.M. (1967): The Histopathology of Enamel Caries Produced In Vitro and their Relation with Enamel Caries. PhD Thesis, University of Bristol. SILVERSTONE, L.M. (1968): The Surface Zone in Caries and Caries-like Lesions Produced In Vitro, Br Dent J 125:145-157. SILVERSTONE, L.M. (1973): The Structure of Carious Enamel, Including the Early Lesion, Oral Sci Rev 4:100-160. SILVERSTONE, L.M. (1974): Fissure Sealants: Laboratory Studies, Caries Res 8:2-26. SILVERSTONE, L.M. (1983): Remineralization and Enamel Caries: New Concepts, Dent Update 10:261-273. YAEGAR, J.A. (1980): Enamel. In: Orban's Oral Histology and Embryology, 9th ed., S.N. Bashkar, Ed., St. Louis: Mosby Publishing Co., p. 46.
