SEM Investigation of the Variability of EnamelSurfaces After Simulated Clinical Acid Etchingfor Pit and Fissure Sealants

G. W. MARSHALL, L. M. OLSON, and C. V. LEEDepartment of Biological Materials, Northwestern University Dental School,Chicago, Illinois 60611, USA

Etching preferentially occu-s on cuspal in-clines and on premolars vs molars. Afterlonger times, etching occurs near the pitsand fissures. Enamel rods etch at cores andprism borders simultaneously in the samearea. Longer etching times produce moreenamel surface relief, permitting bettersealant adaption to the enamel.

Pit and fissure caries is a very destructiveand rapidly advancing disease of the teeth.Several methods have been tried in an at-tempt to stop or retard this process. Amongthe more well-known and possibly more ef-fective methods are fluoride treatments, goodoral hygiene, and the relatively new methodof pit and fissure sealants.The main objective of pit and fissure

sealants is to prevent bacterial colonizationand plaque formation which are implicatedin the cariogenesis of developmental pits,fissures, and grooves. A tight seal deniesplaque colonization and physically preventsaccumulation of food debris both of whichsupport the bacteria at these preferred sites.One of the major problems in pit and fissuretreatment is to effectively bond the sealantto the enamel surface.Buonocorel first used acidic conditioning

treatments of enamel surfaces to increasethe adhesion of acrylic to enamel. Acidetching introduces microirregularities intothe enamel structure that are penetratedby the fluid sealant and then mechanically

This article was awarded first place at the Annual Re-search Award Competition, Chicago Section, AADR,1974.

Received for publication June 24, 1974.Accepted for publication June 27, 1975.

1222

locked into place on polymerization of thesealant. Clinical reports2,3 have shown thatetching with 50% phosphoric acid solutionscan be used with BIS-GMA methyl metha-crylate polymer sealants to reduce pit andfissure caries in permanent and deciduousteeth. Ohsawa4 found that the improvementin adhesion after etching was not a functionof the amount of decalcification that tookplace but was dependent on the surfacecharacteristics created by the acid etching.Gwinnett and Buonocore5 reported that acidetching was limited to the inclined planesof the cusps and did not reach into the pitsand fissures of the tooth. This probably haslittle effect on the initial adhesion of thesealant, but with sealant attrition on thecusps because of masticatory wear, it couldresult in loss of sealant.

Organic and mineral acids reportedly dis-solve enamel prism necks and cores, whereasethylenediaminetetraacetic acid initially de-mineralizes the enamel rod periphery.6.'1Etching patterns parallel to the major axisof the enamel rods are usually described asfish scales or keyholes, but a variety of pat-erns have been reported.12(PP 187,188)

Gwinnett13 described the structure of aprismless layer on the surface of permanentteeth. This layer may be more resistant todissolution than the underlying prismaticenamel. The term "prismless enamel" isused here to describe portions of the enamelthat lack the prismatic pattern.

Lee, Phillips, and Schwartz14 found thatetching enamel with 50% phosphoric acidproduced a mechanical strength with thepolymerized sealant that exceeded thestrength of the sealing material. Martinezl5found that several commercial sealants

ACID ETCHING FOR PIT & FISSURE SEALANT 1223

slhowed good in vitro sealability after tooth-brushing and temperature cycling.The present investigation attempted to

clharacterize the variety of acid-etched pat-terns producedl during simulated clinicaletchiing witlh 50% plhosphoric acid contain-ing 7% dissolved zinc oxidea as a functionof etchiing time on the enamel surfaces ofpremolars and molars. The resulting pat-terrns were related to the adaption of a com-mercial pit anid fissure sealant when appliedto the treated enamel suLrface.

Materials and MethodsNoncarious, extracted human molars and

piemolais were selected for acid etclhling andsealant studies uIsing a scanning election mi-croscope (SE NI) 1, 'Ilhe teetlh were scr tbbedwithi watei app))lied witlh a cotton pledgetand were stored in commercial grade alcoholafter extraction. They were allowed tolench (Iry 24 liours before SET\I examinationl.No p)rophylaxis treatments were used. TiheuinetchiedI enamel surfaces were examined forlater comparison witlh the etclhed surfaces.This initial exposure to the SEMN atmosphieredid not produce any apparent structuiralmodification since the same etching patternswere observed even if this step was elim-inated.

After study of the uinetclhed surfaces, theteethl were etclhed Lusing the recommendedclinical application teclhniquies. The occlusalsuirfaces of the teeth were etched witlh buf-fered 50%) phiosplhoric acid applied with acotton swal) for time periods of 30, 60, 75,90, 120, or 240 seconds. The teeth werethen rinsed witlh tap water, air dried, andreexamined under tlhe SEM. Th-e occlusalsurfaces of representative teeth etched foreach of these time periods were coated with acommercial sealante and polymerized withtultraviolet liglht foi 60 seconds. Several ofthese teetlh were sectioned to yield mesio-distal or buccolingual sections through thepits or fisstrres. These sections were lateruLsed to study the adaptation of the sealantat the enamel-sealant interface.A thin layer (200 A) of gold was vacuum

evaporated onto the sealant suLrface afterthe sealing or sectioning procedures. Thegold laver was necessary to prevent charge

nNova-Seal con(litioning agent, L. D. Caulk Co., Mil-ford, I)el.

b Stereoscan S-4 scanninig electron microscope, KentCambridge Scientific Instruments, Ltd., Cambridge, Eng.

e Nuva-Seal, L. D. Caulk Co., Milford, Del.

FIC 1.-SEM photomicrograph of unetchedenamel. Upper portion shows relationship ofcusps and central groove of molar. Groove con-tains debris. Lower portion shows typical ap-pearanace of unetchedt enamel on cuspal inclinesat right on upper portion at highi magnification.Relatively smooth enamel is visible with num-erous minor depressions in enamel before etchi-inig. (. = pm)

buildup on the electrically insulating sealantdIuring electron bombardment. Each of thesealed or sealed-and-sectioned teeth was ex-amined withl the SEM for surface defects inthe sealant an(d for sealant bonding at theenamel-sealant interface. The SEM was op-erated at ain accelerating potential of 20 kvwitlh a 200-micrometer (btm) diameter finala-)erture for all samples in this investigation.

FIG 2.-Photomicrograph of enamel on cuspalincline midway between cusp tip and grooveafter 30-second etching treatment with 50%phosphoric acid. Enamel was lightly etched onsurfaces of premolars. Cores of enamel rodswere preferentially dissolved by etchant. (s=pm)

Vol 54 No. 6

1224 MARSHALL, OLSON, AND LEE

FIw 3,-Photormicrographs showv effect of 60-seconid etchinig trcatmenlt onl occlusal su-faceof priemolar. Enamel on cuspal inclines was fairly well etched (left), but only light, non-uLniform etchiing occurred near the fossa (right) (,u =-1m)

ResultsB3efore acidl etclinrg, the enamel surfaces

weie relatively smooth over the entire inneraspect of tlhe cusps. Frequently, debris couldhe seen in thie cential groove, pits, and fis-sures of the teeth. Figture I slows a typicalenamel surface from a molar before etclhing.The top portion of Figure I is a low-magni-ficattion, SENI photomicrograph sthowing thegeneral culsp aniatomy and debris in thegroove. The lower portion of this figureslhows the fairly smooth aspect of the enamelon tile cLuspal inclines of tlhe same tootlh. Anumher of minior depressions can be seenb)efore tde etclhing treatment. After 30-secondetcliing times withi phosphoric acid, the cus-pal inclinies of molairs and premolars showedsigns of liglht etclhing (Fig 2). The begin-

ning of etched prism patterns is clearlyseen in this figure and tlhe etcliing attackwas concentrated in the core region of theenamel irods. T1he etchlinig pattern was un-cxen and fui -thier dlown tlhe ctusps, little evi-dleuce of etcling could le found. Figure 3slhows the clharacteristic etching patterns ob-sei-ved on- prernolars after etclhing for 60 sec-ondcls. EI'lie acid dissolution of enamel higheron- the culsps in Figure 3, left appeared to hedeeper and more uniform than the etchingnear the developmental pit in Figure 3,i-ighlt. The 60-secondI etching patterns onmolars were less well developedl (Fig 4).Near tlle cu1sp ti p, Figure 4, left, etclhingeffects were evideent, but a prismless layer atthe lower right apparently interfered witlthe continuLouis development of enamel rod

FIG 4.-Etched enamel patterns are shown after 60-second etching treatmelnt on molar. Nearcusp tip (left), enamel prism patterns can be seen in upper left, but much of surface wascovered with prismless layer (lower right) Near central groove (right) , there wvas littleapparent effect of etching attack on enamel. (,u=-um)

J Dent Res November-December 1975

ACID ETCHING FOR PIT & FISSURE SEALANT 1225

end patterns as seen at the upper left. Littleevidence of etching near the pit was seen inthe molars (Fig 4, righit).

Figure 5 sihows an unusual result of etch-ing on the uipper part of the cuspal inclineson a premolar etched for 75 seconds. A high(legree of surface roughness and patchies ofwell-developed enamel rod patterns wereproduced. In the center portion of Figure5, a region of exposecd and etched dentin isapparent. (Note the dark holes whlich arethe etchled dentinal tubules.) This regioncould hlave been exposed because of a local-ized legioni of thin enamel that was etchedatwa. 1 hle dentin appears dark because it ismulch less hiiglhly mineralized and tlheteforeemits fewer secondary electrons per u-nitarea.

Characteristic etching patterns on tlhe cui-spal inclines of a1premolar after 75 seconidsof etcliing are slhown in Figutre 6. XVell-developed enamel rods are seen interspersedwitl)poorly etclhed areas in Figniie 6, top.Figuire 6, hottom is a hiigler-magnificationview of Figure 6, top and slhows that etchingof the enamel rod periphery predominatedin this portioni of the sample. This gave atypical fishi scale appearance to the enamelprisms. Etcling of the occlusal su-rfaces ofmolars resuLlted in less well-developed rodpattterns (Fig 7) A band of well-etchedenamel rods are seeni in Figuire 7, top, sand-wiched between prismless enamel regionsthat resisted etching. Figutle 7, bottom slIows

FiG 5.-Cuspal enamel from premolar etched75 seconds. General roughening of surface ancdai-eas of enamel rodIs are apparent. In centerof micrograph, region of exposed andI etclhedIdlentin canl be seen. Note dentinal tubules atcenter of region. (u=gim)

EF.; 6.-Typical etching patterns on cuspal in-clines of premolar after 75-second etching treat-ment. Urxeven or mottled appearance of patternis apparent (top). At higher magnification,tendency for acid attack on enamel prism peri-phery is shown (bottom). (L -gim)

only the faint beginnings of etched enamelpatterns throughl the prismless layer near thecentral fossa of the same tooth. It shouldalso be noted that in Figure 7, bottom etch-ing appeais to occur first at the prism cores.

Etching time periods of 90 seconds pro-duced well-developed prism patterns on thecuspal inclines as shown in Figure 8, left,witlh patclhes of a prismless layer still evident.Etching patterns extended downward alongthe cusps almost to the periphery of the de-velopmental pits as seen in Figure 8, right.

T1he prism patternis were more uniformon tlhe premolars etchled for 120 seconds(Fig 9) than the piism patterns on molarsetclhed for 240 seconds. After the 240-second

Vol 54 No. 6

1226 MARSHALL, OLSON, AND LEE

FIG 7.-Etching patterns on molar occlusalsuirface after 75-second etching tieatment. Prism-less layer covered much of surface on cuspal in-cline and interfered with etchling of enamel rods(top) Etched rods are seen at center with etch-ing at rodI bordeis. Very light etchiing wvas seennear fossa (bottom) . Acid attack through prism-less layer occurred at rod cores. (,= ,um)

FIc 9.-Well-developed enamel rod patternon cuspal surface of premolar after 120-secondletchiing treatment. (, = ,um)etclinlg periods, the enamel prism patternson the inclines of premolars were well de-veloped, and a typical mosaic pattern wasevident as seen in Figure 10, left. Figure 10,right is a higher magnification of the samestructure and shows the common observationthat some patches of rods were preferentiallyattacked at rod peripheries wlhereas otlherpatches suffered preferential dissolution ofthe rod centers and tails. Figure 11 showstlhat etclhing patterns were developed at theeIge but not on the steeply inclined wallsof developmental grooves after the 240-sec-o(id etcliing treatment.

\pplication and polymerization of thesealant resulted in a smooth surface coveringthe desvelopmental pits and fissures. Thesealant surfaces did contain numerous pores

FIG 8.-Etched enamel on premolar after 90-second etching treatment. Left, well-etchedenamel rods interspersed with remnants of prismless layer on cuspal incline. Right, lower-magnification view shows approach of etching patterns to edge of pit. (gi = fm)

J Dent Res November-December 1975

ACID ETCHING FOR PIT Qr FISSURE SEALANT 1227

FIG 10.-Enamel etching patterns on cuspal incline of premolar after 240-second etching treat-ment. Ieft, lower-magnification view shows mosaic or patchy appearance of etched enamel.Rig/lt, at lhigher magnification, patches of rods can be seen that etched initially at prism bordersand other patches can be seen etched preferentially at prism cores. Region of unetched orprismless enamel appears to separate most similarly etched patches. (j = xm)

over a wide-size range. Figure 12 shows atypical occlusal surface view of the sealantin a developmental pit of a molar. Largepores of more than 50-1tm diameter are evi-dent in the surface. Smaller pores are alsoevident, as shown at the arrow.

Figures 13 to 15 show the adaptation ofthe sealant to the etclhed enamel in sectionsthrougli the fis.sure. The relatively pooradaptation of the sealant at the enamel wallsafter etcling for 30 seconds witlh phosphoricaicidl can be seen in Figure 13. Figtire 14 isa photomicrograplh of a small portion of theeniamel-sealant interface obtained from a sec-tion tlhrough a fissuire with use of a 60-secondetching treatment. The adaptation of thesealant is much better than after the 30-second etclhing treatment. A similar view ofthe excellent adaptation of the sealant tothe etched enamel after a 240-second etclhingtreatment is shown in Figure 15.

DiscussionThe acidic conditioning treatments using

50% phosphoric acid increased the enamel

FIG 11.-SEM photomicrographs of etching .4patterns near central groove after 240-second 8etch of premolar. Enamel rod patterns are seeninterspersed with clumps of remaining prism-less layer up to steep inclines of walls (top)Higher magnification view at groove wall showsborder between well-etched and poorly etched Ienamel at gx-oove (bottom). (g in)

Vol 54 No. 6

1228 MARSHALL, OLSON, AND LEE

FiG 12.-SEM view of sealant on occlusal sur-face of molar. Varying diameter bubbles wereobserved as defect of application. They rangedin size from relatively large ones shown at uspperleft to very small micropores (arrow)

surface area and produced a variety of etclh-ing patterns into whlichi the sealant could keyand form a mechanical bond. Slhorter etch-ing times resulted in etclhed enamel rodpatterns on the cuspal inclines but did notetclh the groove region significantly as shownin Figures 2 to 4. Generally, increased etchi-ing time resulted in the development ofetching patterns over a greater portion ofthe enamel surface. Longer etclhes producedprism patterns that reachedl the periphieryof the steep inclines of tlevelopmentalgrooves or pits and fissures. This effect can

FIG 14.-High-magnification photomicrographof enam-lel-sealant interface in section throtigihfissure after 60-second etching treatment. En-amel is at left and sealant at right. Tighterjiuinction between enamel and scalant is evidentas compared with Figure 13. (,A=im)

he seen by comparing Figure 8, right andFiguire 11. to1). The grooves, pits, and fis-sures were Inot etcled, possibly as a result ofa film of organic material an(d debris cover-inig these areas. This film apparently offersa bai-rier to the general etching attack onthe enamel. However, it is to he expectedthat localized areas of the pits and fissurescouldlibe attackedl in view of the prevalenceof pit and fissure caries. Suclh localized at-tack in the pits would not le beneficial foraclhieving sealant adhesion.

FiG. 13.-Enamel sealant interface in sectionthrough fissure after 30-second etch and poly- FIc. 15.-SEM view of very tight seal ob-merization of sealant. Bubble in sealant pene- tained in section after etching of enamel fortrated halfway through sealant at upper middle. 240 seconds. Sealant-enamel interface was soSealant was poorly adapted at interface. (,u= well adapted that it is difficult to locate bound-gm) ary. Sealant at left, enamel at right. ( =- inm)

J Dent Res November-December 1975

ACID ETCHING FOR PIT & FISSURE SEALANT 1229

A high degree of variation was noted inthe etching patterns from tooth to tooth andin different parts of the same tooth given thesame "clinical" etching procedure. Less well-developed etching patterns were found onmolars than on premolars given the sametreatment, for example, compare Figures 7and 8. Local anatomy would be expected tohave an effect on the etching patterns' char-acteristics. Teeth with less steeply inclineddevelopmental grooves gave etching pat-terns at inferior positions on the walls ascompared with teeth with steeper inclines.Etching patterns did not develop within thegrooves or fissures for any of the samplesstudied. This is apparent in Figures 4, right,7., bottom, and 11, and is in agreement withthe observations of Gwinnett and Buono-core.5The nature of the acid etching attack on

the enamel surfaces studied can be charac-terized as patchy. This phenomenon hadtwo major manifestations. First, certain por-tions of the enamel surface appeared to havea thicker, denser, or more tightly adheringprismless layer that impeded dissolution ofthe underlying enamel prisms. This layerwas more evident near the pits than on thecuspal inclines as shown in Figure 2 and wasalso more evident in molars than premolarsas seen by comparison of Figures 7 and 8.Occasional patches of this layer could befound on various surfaces of all teeth, evenafter the longest etching times used. Theetched surface of Figure 11, top is coveredwith clumps of this layer that are surroundedby well-etched enamel rods. Increased etch-ing times tended to promote a more evenlyroughened surface with fewer patches ofthe prismless layer. These more uniformlyetched and cleaned surfaces are required forgood adhesion of the sealant.The second form of the patchy nature of

the etched surfaces is characterized in Figure10, although it was a commonly observedphenomenon. Portions of the enamel rodswere preferentially etched at prism cores andnecks in some areas, whereas in other adja-cent areas the periphery of the enamel rodswere etched away. Both forms of etchingdissolution were seen throughout this in-vestigation. Generally, a small portion ofclosely packed and similarly etched rodswere bounded by a narrow, prismless region.This gives the impression of a structure sim-ilar to grains in metallographic specimens in

which regions of high order etch differentlythan their boundaries. These observationssuggest that patches of enamel rods tend toalign and are separated by regions of mis-match to accommodate the curved surface ofthe tooth. This structure would be in con-trast to a structure composed of enamel rodswith more or less uniform mismatch to ac-commodate the variations in the surface to-pography of the teeth. Transmission elec-tron micrographs suggest that some orienta-tional mismatches do occur between adjacentenamel rods.12 (p182) Continued investigationwould be necessary to determine how therelative rod orientations change with smallradii of curvature at the tooth surfaces.A second explanation of the mosaic ap-

pearance of the etched enamel could dependon the adhesion of the prismless layer dis-cussed previously. Each patch of similarlyetched enamel rods could be a result of be-ing covered with a prismless layer that wasdissolved away at one time. Thus, regionssuch as that at the center of Figure 7, topwould be etched similarly and the areas oneither side that are still covered with theprotective layer might etch differently. Itshould be noted that many of the boundariessurrounding similarly etched rods in Figure10 appear similar to the layer covering themajor portion of the surface in Figure 7, top.

Neither of these hypotheses for the mo-saic appearance of the etched enamel ac-counts for the differences in etching attackof enamel rods observed in adjacent areasof the samples. Previous work reportedthat acids preferentially dissolve the prismcores.5,12 (p182) The present investigationdemonstrates that after the 60-second etch-ing time, attack at prism borders is as likelyas attack at the core. It appears that thefirst layers etched may be characterized bypreferential attack at prism cores as shownin Figures 2, 3, right, and 7, bottom. Afteretching through the prismless layer and aninitial layer of prismatic enamel further dis-solution can occur at prism borders or coreswith apparent equal probability. This ob-servation is in conflict with most publishedwork, although etching at peripheries andcores has been reported.16 This suggests thatin deeper enamel, adjacent groups of prismsexist in different states of mineralization, orthat particular groups of prisms have ahigher affinity for the prismless layer. A de-finitive explanation for this difference in sol-

Vol 54 No. 6

1230 MARSHALL, OLSON, AND LEE

ubility will depend on a more complete un-derstanding of the dissolution mechanism.

Acid etching of enamel is the most pop-ular and presently most effective way to ob-tain adhesion of pit and fissure sealants toenamel. It is axiomatic that such treatmentsshould produce a sealed surface for as longas possible. Figure 12 shows the range ofsizes of bubble defects that developed dur-ing polymerization of the sealant. The mi-cropores are of doubtful clinical significance,but the larger defects represent severelyweakened areas that would break down un-der occlusal forces. Such sealant loss un-doubtedly would shorten the protective life-time of the sealant since much less sealantwould remain to be abraded.

Figures 13, 14, and 15 show the increasedadaption of the sealer to the etched enamelwith increased etching time. Closer adap-tion of the sealant to the enamel is inter-preted as both increased marginal sealabilityand adhesion. These results suggest thatthere is a significant advantage in having aclean, debrisfree, and uniformly roughenedsurface for good sealability and that this con-dition can be partially achieved by longeretching times, for example, up to four min-utes for molars. Furthermore, the tight adap-tion approached the steep inclines of thepits, fissures, and grooves because of themore uniformly etched enamel in this regionafter extended etching times. This shouldextend the effective lifetime of the sealantbecause the fissure would continue to besealed even after the sealant on the cuspalinclines was partially abraded.The objectives of using longer etching

times included enhanced removal of theprismless layer and increased etching closeto the steep walls of the pits and fissures.These factors could outweigh the importanceof longer etching to achieve increased depthin the microirregularities associated with theprisms, although such changes have beenreported by Silverstone.17 If other methodscan be developed to efficiently remove theprismless layer, then shorter etching timesmight be used to obtain more uniform etch-ing patterns and good adaption of sealant tothe etched enamel surface.

ConclusionsThe present investigation of etching pat-

terns on enamel obtained during simulatedclinical etching experiments in vitro was

undertaken to determine differences in theetching patterns that would be encounteredin the clinical situation and might affect theadhesion of pit and fissure sealants.

In agreement with previous studies, etch-ing with 50% phosphloric acid increased theenamel surface area and produced etchedenamel prisms. Deep pits fissures, andgrooves are not etched, and preferentialetclhing occurred on the cuspal inclines.

In contrast with other work, etching oc-curred both at prism borders as well asprism cores in subsurface enamel. Neitheiform appears to be dominant. The initiallayer of enamel appeared to be preferen-tially attacked at the core of the enamelprisms.

Significant differences in the etching pat-terns occurred on various portions of theocclusal surface of individual teeth and dif-ferences also occurred between molars andpremolars. Portions of a single tooth ap-peared to have a more adherent, prismlesslayer near the pits, fissures, and devel-opmental grooves that was resistant to etch-ing. All molars were more resistant to etch-ing as compared to premolars.To obtain a debrisfree, well-etched,

enamel structure, extended etching timeswere required. Better adaption of sealant toetched enamel walls occurred with longeretching times and etching was more uniformnear the pits and grooves. All the evidencegained in this investigation suggests thatlonger than normal etching times wouldproduce a more adherent and longer lastingsealant, especially in preventive treatmentof molars with pit and fissure sealants.

References1. BUONOCORE, M.G.: A Simple Method of In-

creasing the Adhesion of Acrylic Filling Ma-terials to Enamel Surfaces, J Dent Res 34:849-853, 1955.

2. BUONOCORE, M.G.: Adhesive Sealing of Pitsand Fissures for Caries Prevention with Useof Ultraviolet Light, JADA 80: 324-328, 1970.

3. BUONOCORE, M.G.: Caries Prevention in Pitsand Fissures Sealed with an Adhesive ResinPolymerized by Ultraviolet Light; A TwoYear Study of a Single Adhesive Application,JADA 82: 1090-1093, 1971.

4. OHSAWA, I.: Studies on the Solubility andAdhesion of the Enamel in Pre-Treatmentfor Caries Preventive Sealing, Bull TokyoDental Coll 13: 65-82, 1972.

J Dent Res November-December 1975

ACID ETCHING FOR PIT & FISSURE SEALANT 1231

5. GwINNErr, A.J., and BUONOCORE, M.G.: AScanning Electron Microscope Study of Pitand Fissure Surfaces Conditioned for Ad-hesive Sealing, Arch Oral Biol 17:415-423,1972.

6. POOLE, D.F.G., and JOHNSON, N.W.: TheEffects of Different Remineralizing Agentson Human Enamel Surfaces Studied byScanning Electron Microscopy, Arch OralBiol 12:1621-1634, 1967.

7. HOFFMAN, S.; McEwAN, W.S.; and DREW,C.M.: Scanning Electron Microscope Studiesof EDTA-Treated Enamel, J Dent Res 48:1234-1242, 1969.

8. ScoTT, D.B.; SIMMELINK, J.W.; and NYGAARD,V.: Structural Aspects of Dental Caries, JDent Res 53 (suppl): 165-178, 1974.

9. SHARPE, A.N.: Influence of the CrystalOrientation on Human Enamel and Its Re-activity to Acid as Shown by High Resolu-tion Micro-Radiography, Arch Oral Biol 12:583-591, 1967.

10. SWANCAR, J.R.; Sco rr, D.B.; and NJEMIRov-SKIJ, Z.: Studies of the Structure of HumanEnamel by the Replica Method, J Dent Res49: 1025-1033, 1970.

11. NICHOL, T.; JUDD, G.; and ANSELL, G.W.: A

Two Stage Model for Human Enamel De-mineralization as Determined by ScanningElectron Microscope Analysis, J Dent Res 52:487-493, 1973.

12. Scorr, J.J., and SYMONs, N.B.B.: Introductionto Dental Anatomy, 6th ed, Baltimore:Williams & Wilkins Co., 1971, pp 182, 187-188.

13. GWINNETr, A.J.: The Ultrastructure of the"Prismless" Enamel of Permanent HumanTeeth, Arch Oral Biol 12: 381-387, 1967.

14. LEE, B.D.; PHILLIPs, R.W.; and SWARTZ, M.L.:The Influence of Phosphoric Acid Etchingson the Retention of Acrylic Resin to BovineEnamel, JADA 82:1381-1386, 1971.

15. MARTINEZ, C.R.: The Effect of Three Vari-ables on the Seal of Three Commercial Pitand Fissure Sealants, MS thesis, North-western University, 1973.

16. GwINNETT, A.J.; BUONOCORE, M.G.; andSHEYKHOLESLAM, A.: Effect of Fluoride onEtched Human and Bovine Tooth EnamelSurfaces as Demonstrated by ScanningElectron Microscopy, Arch Oral Biol 17:271-278, 1972.

17. SILVERSTONE, L.M.: Fissure Sealants/Labora-tory Studies, Caries Res 8: 2-26, 1974.

Vol 54 No. 6