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Oral fluid contamination of etched enamel surfaces: an SEM study
A r t ic l e s
In as little time as 1 second, salivary exposure will compromise bonding of resin to enamel.
Oral fluid contamination of etched enamel surfaces: an SEM study Leon M. Silverstone, DDSc, PhD, BChD, LDS, RCS(Eng) M. John Hicks, DDS, M S, PhD M ary J. Featherstone
T
.1 . he acid-etch technique is used exten sively in aspects of preventive and re storative dentistry as well as in orthodon tic treatment. Whether a sealant or a com posite material is being placed or ortho dontic brackets are being attached, the durability of the bond is paramount to clinical success. One of the major reasons for a poor or failed bond is salivary con tamination of the etched enamel surface before resin placement.1 This factor was highlighted as a probable reason for fail ure in early clinical trials more than a decade ago.2,3 Relatively few studies, either in vivo4,5 or in vitro,5,9 have at tempted to evaluate the effect of salivary contamination on the acid-etch tech nique. Although bond strength and sur face topography for saliva-contaminated etched enamel have been shown to be al tered, no investigation has studied the ef fect of the length of time that etched enamel is exposed to saliva. The purpose of this in vitro study was to determine the effect of differing salivary contamination periods on the surface topography of acid-etched human enamel. In addition, the appearance of the contaminated sur face after washing with an air/water syringe was assessed. A scanning elec tron microscope (SEM) was used to eval uate the enamel surfaces. Materials and methods Permanent premolars and molars with macro-
Fig 1 ■ Microtome provides serial longitudinal undemineralized and unembedded sections of teeth.
scopically sound buccal and lingual enamel surfaces were chosen for this in vitro study. Each tooth was sectioned into three portions buccolingually using the Silverstone-Taylor Hard T issu e M icrotom e (Fig 1). An acidresistant varnish was painted on the specimens so that windows of sound enamel in the middle thirds of both the buccal and lingual surfaces of the crowns were left exposed. This allowed three treatment comparisons to be made on a single tooth from a sim ilar region of sound enamel. Before the varnish was applied, the e n am el w as c le a n e d th o ro u g h ly w ith a fluoride-free prophylaxis paste. The buccal and lingual windows of sound enamel on each specimen were etched for 60 seconds with a
freshly prepared solution of 30% wt/wt phos phoric acid. The experimental design is outlined in Fig ure 2. For each tooth, one portion was left as a control after etching, whereas the other two were exposed to saliva in vitro. Whole saliva was collected freshly from one of the inves tigators. (As the whole salivary fluid obtained on expectoration con tain s m ore than ju st saliva, it is referred to as oral fluid.) The re maining two portions of the tooth were ex posed to oral fluid for a single exposure period. The exposure periods used in these experi ments were 60, 30, 10, 5, 1, or 0.5 seconds. With the 1-second and 0.5-second expo sures, the specimens were dipped into oral JADA, Vol. 110, M arch 1985 ■ 329
ARTICLES
with warm compressed air. The specimens were then prepared for SEM exam ination (JEOL JSM 35C microscope) to determine the effect of the treatment regimen on the surface topography of the enamel and to compare it with the etched control.
i
ï (A)
(B)
(C)
Acid-etch only
Acid-etch and oral fluid
Acid-etch, oral fluid, and wash
(B)
(C)
(A)
Scanning electron m icroscopy (SEM)
Fig 2 ■ Diagram of experimental design.
fluid and then rapidly removed, according to a stop clock. After exposure to oral fluid, one portion was left to air dry, and the remaining portion was vigorously washed for 30 seconds with an air/water spray and then dried for 30 seconds
The experimental design resulted in a direct comparison of three different treatment effects on the surface appearance of enamel: acidetching of sound enamel, salivary contamina tion of etched sound enamel, and washing of saliva-contaminated etched sound enamel. In addition, the effect of six different periods of oral fluid exposure could be compared. A total of 20 teeth were used in this study, resulting in 120 windows of buccal and lingual enamel available for study, as each tooth was divided into three portions. Forty windows were avail able for each of the three treatment groups and six windows were examined for each of the different periods of oral fluid exposure.
Results Acid-etching of the sound enamel control sur faces produced characteristic etching patterns (Fig 3). Types 1 ,2 , and 3 etching patterns were observed with no one etching pattern pre dominating. Regions of prisms that had lost their cores (type 1 etching pattern) were found adjacent to other regions with either prisms that had lost their peripheries (type 2 etching pattern) or areas w ith generalized surface roughening and porosity, but no distinct prism morphology (type 3 etching pattern). Figure 4 shows a high-power view of part of the enamel surface in Figure 3. This region is referred to as a type 1 etching pattern as the periphery of the prism is relatively intact with the core showing greatest destruction. However, this photomi crograph demonstrates clearly the high poros ity of the entire surface after acid etching. In all instances, the control surfaces were suffi ciently etched to provide porosities for pene tration of resin material. When the etched surfaces were contamin ated with oral fluid, the surface topography was altered dramatically. An adherent surface
Fig 3 ■ Etched enamel showing examples of three etching patterns. Areas show ing preferential loss of prism cores where prism peripheries are prominent (type 1) can be seen in addition to regions in which prism cores have remained (type 2). There is also band of porous enamel without evidence of prism structure (type 3) (orig mag x 1,000). Space bar represents 10 //III. 330 a JADA, Vol. 110, M arch 1985
coating that obscured the underlying etchec enamel to a substantial extent was observed Figure 5 shows an etched enamel surface thal was exposed to oral fluid for 60 seconds. There is no evidence of the etched surface as it hat been completely obscured by an adherent layei of organic material. Figure 6 shows the adja cent region of etched enamel that was also ex posed to oral fluid for 60 seconds. In this specimen, the surface was thoroughly washec for 30 seconds and then air dried. However, the washing regimen had no apparent effect as the adherent layer is still present. Figure 7 shows an etched enamel surface that was exposed to oral fluid for 5 seconds The etched surface was completely masked b j the organic precipitate in spite of the relatively short contamination period. Figure 8 shows the adjacent etched surface, also exposed tc oral fluid for 5 seconds, but followed by a 30 second wash. The surface appears similar tc the unwashed specimen in that the etching pattern is still completely masked. Thus, the washing regimen did not remove the salivary contaminants and, therefore, a resin would no bond successfully to such a surface. Figure 9 is a high-power micrograph focusec on a group of enamel prisms. This region hac been etched in the conventional manner for 6( seconds, exposed to oral fluid for just 1 second and then thoroughly washed. In spite of the short 1-Second contamination period and the 30-second washing regimen, a layer of adhe rent organic material can still be seen over the prism structure. For comparison, Figure 1C shows the comparable region on the etchec control. There is a significant difference in ap pearance as the normal porosity of etchec enamel can be seen in the control specimen. These experiments all show a sim ilar trenc in that surface contamination occurred aftei exposure to oral fluid, using the entire series o: exposure periods. With respect to the washing regimen, surface contaminants were not re moved with exposure periods of 1 second oi greater. Only when specimens were contamin ated for less than 1 second could the organic layer be removed by the washing regimen.
Fig 4 ■ High-power view of part of Figure 3 showing high degree of porosity c etched enamel. This porosity is necessary so that fluid resin can penetrate am form a strong mechanical bond with tissue (orig mag x 5,400). Space bar repre sents 1 fj.m.
ARTICLES
Fig 5 ■ Etched enamel surface after 60-second contamination period with oral fluid. Etched region is completely obscured by organic deposit (orig mag x 1,000). Space bar represents 10 ¿im.
Fig 6 ■ Region adjacent to that shown in Figure 5, also having 60-second con tamination period with oral fluid, but followed by 30-second vigorous wash. Sur face still shows presence of adherent layer that masks etched region (orig mag x 1,800). Space bar represents 10 /xm.
Fig 7 ■ Etched enamel surface was exposed to oral fluid for 5 seconds. Organic layer has completely masked etched region (orig mag x 400). Space bar repre sents 10 /xm.
Fig 8 ■ Region of etched enamel adjacent to that seen in previous figure. After 5-second contamination period, surface w as washed for 30 seconds and then air dried. However, washing regimen has not been successful in removing organic coating (orig mag x 1,500). Space bar represents 10 /xm.
Fig 9 ■ High-power view showing group of prisms in area that was etched, ex posed to oral fluid for 1 second, and then washed for 30 seconds. Surface film of organic material is seen as contaminant on etched prisms. This would adversely affect bonding of resin to surface (orig mag x 6,000). Space bar represents 1 /xm.
Fig 10 ■ Region on etched control specimen comparable to that seen in previous figure. Prism structure appears as porous region that would be ideal for bonding (orig mag x 6,000). Space bar represents 1 fim.
Silverstone-Hicks-Featherstone : CONTAMINATION OF ENAMEL SURFACES ■ 331
A R T IC LES
Discussion The ability of salivary contamination to affect surface topography was shown to be independent of the length of exposure of etched enamel to oral fluid when the exposure periods defined in this study were used. When the exposure period was 1 second or greater, a tenacious sur face coating was present, regardless of whether a 30-second air/water wash was used. Only with an exposure time of less than 1 second was the organic surface coating removed by a vigorous 30-second washing. These results show that should the etched enamel surface be contamin ated by saliva for 1 second or longer, the surface will still possess an adherent sur face coating after an air/water wash. These findings are in agreement with previous work that has shown that an or ganic pellicle forms quickly over etched enamel after exposure to the oral environment.4,5,10'12 In fact, acquisition of cal cium and phosphate phases (remineralization) by the etched enamel surfaces had occurred as shown by SEM,5'10 microprobe,11 and acid solubility12,13 tech niques. However, the length of exposure to oral fluid for the etched enamel sur faces ranged from 1 hour to 4 days in these studies. In the present study, the longest con tamination period was 60 seconds. The surface coating formed during such a short contamination period would most likely represent the formation of an or ganic pellicle on the etched surface. The tenacity of this newly formed pellicle may be caused by the highly reactive na ture of the etched enamel surface as well as its porosity. The etched enamel surface would provide reactive sites of exposed calcium and phosphate ions to which the proteins present in the organic pellicle could attach by electrostatic interac tions.14 The effect of salivary contamination on bond strength has also been deter mined.6'8 The bond strength of resin applied to etched surfaces contaminated by saliva was reduced by up to 50% when compared with uncontaminated etched enamel controls. It was also noted that even if the contaminated surface was washed and re-etched, the bond strength was still less than that for etched enamel that had not been contaminated. The most frequent type of fracture found with specimens contaminated with saliva for 60 seconds was an adhesive fracture rather than a cohesive type of fracture. Cohesive fractures were found most fre quently with uncontaminated speci mens.8 In other words, the contaminated specimens were fractured at the enamelresin interface, whereas in the control specimens, fractures occurred within the resin itself. When a resin material was 332 ■ JADA, Vol. 110, M arch 1985
placed directly on saliva-contaminated surfaces, all fracture sites were at the enamel-resin interface. Thus, the effect of contaminating the etched surface was to weaken the bond between the two mate rials. More than 65% of the specimens that had been contaminated, washed, dried, and then sealed had fractures at the enamel-resin interface. These findings are explained by the presence of a surface coating partially obscuring the underly ing etched enamel prisms, as observed in the present study. The findings in the present study em phasize the importance of maintaining a dry field while placing acid-etch preven tive and restorative materials. Should salivary contamination of an etched enamel surface occur, the procedure should be stopped, the area dried thoroughly, and acid-etching repeated before any attempt to bond a resin mate rial. To avoid salivary contamination, rubber dam isolation is preferred. In cases in which a rubber dam cannot be placed because of access limitations, a newly erupting tooth, an uncooperative patient, or the unwillingness of the operator, cot ton roll isolation with saliva suction is likely to be adequate to avoid contamina tion.14,15 Recent research has shown that sealant placement with cotton roll isola tion resulted in 88% of the sealants being fully retained after 2 years, whereas rub ber dam isolation resulted in 96% full re tention. However, there was no statisti cally significant difference between these two results, although the authors stated that it was less comfortable for the patient and a more difficult technique for the operator when cotton rolls were used for isolation.15 Therefore, this study has demonstrated that the effect of contamination of etched enamel with oral fluid, even for a single second, results in an adherent organic coating that is not removed by conven tional washing techniques. This in turn will compromise the bond between the enamel and the resin material. As any contamination occurring clinically will inevitably exist for at least a 1-second period, the operator should not proceed with the bonding technique until the sur face has been dried and re-etched.
Conclusions Salivary contamination of etched enamel, even for a single second, resulted in an adherent coating that masked the under lying etched enamel. Only with salivary exposure times of less than 1 second could the contaminant be successfully removed by washing. Should salivary contamination occur, the operator should not proceed until the enamel surface has been dried and re-etched before applica tion of a resin.
Summary The purpose of this in vitro study was to determine the effects of differing salivary contamination periods on the surface to pography of etched enamel surfaces using SEM techniques. The results indi cated that salivary contamination of etched enamel for 1 second or longer re sulted in the formation of surface coatings that could not be removed by a water wash. It was concluded that should sali vary contamination of an etched enamel surface occur, it would be necessary to repeat the etching procedure to ensure adequate bonding of a resin material.
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This investigation was supported in part by NIDR research grant no. 2 R01 DE 06564. The authors have no financial interest in the microtome described in the text. The authors thank Ms. Wanda Valentine for assis tance with the preparation of this manuscript. Dr. Silverstone is associate dean for research, and director, dental research unit; Dr. Hicks is assistant professor, dental research unit and department of growth and development; and Ms. Featherstone is professional research assistant, dental research unit, School of Dentistry—C284, University of Colorado Health Sciences Center, Denver, 80262. Address re quests for reprints to Dr. Silverstone. 1. Silverstone, L.M. State-of-the-art on sealant re search and priorities for further research. In Proceed ings of dental sealants in the prevention of tooth de cay. J Dent Educ 48 (Suppl):107-118, 1984. 2. Rock, W.P. Fissure sealants: results obtained with two different sealants after one year. Br Dent J 133:146-151, 1972. 3. Burt, A.B., and others. Retention of a fissure sealant six m onths after app lication. B r Dent J 138:98-102, 1975. 4. Garberoglio, R., and Cozzani, G. In-vivo effect of oral environment on etched enamel: a scanning elec tron microscopic study. J Dent Res 58:1859-1865, 1979. 5. Kastendieck, M.J., and Silverstone, L.M. Re mineralization of acid-etched human enamel by ex posure to oral fluid in vivo and in vitro. J Dent Res 58:163, 1979. 6. Bates, D., and others. Effects of acid etch param eters on enamel topography and composite resinenamel bond strength. Pediatr Dent 4 :1 06-110,1982. 7. Wood, L.W., and Barkmeier, W.W. The effect of salivary contam ination on the retention of acidetched retained composite resin. J Neb Dent Assoc 1 4 -1 8 ,1 9 7 9 . 8. Hormati, A.A., and others. Effects of contamina tion and mechanical disturbance on the quality of acid-etched enamel. JADA 100(l):34-38, 1980. 9. Albert, M., and Grenoble, D.E. An in vivo study of enamel remineralization after acid-etching. J South Calif Dent Assoc 39:747-751, 1971. 10. W ei, S.H. Remineralization of enamel and d e n tin -^ review. J Dent Child 34:444-451, 1967. 11. Silverstone, L.M. Fissure sealants: the suscep tib ility to d issolution of acid -etch ed and sub sequently abraded enamel in vitro. Caries Res 11:465 1 ,1 9 7 7 . 12. Silverstone, L.M. The susceptibility to dissolu tion of fissure-sealed enamel surfaces artificially abraded in vitro. Helv Odont Acta 17:64, 1973. 13. Rolla, G. Effects of fluoride on initiation of plaque formation. Caries Res 11:243-261, 1977. 14. Poulsen, S., and Peltoniemi, A.L. Retention of fissure sealant in primary second molars after 6 months. Scand J Dent Res 87:328-330, 1979. 15. Eidelman, E., and others. The retention of fis sure sealants: rubber dam or cotton rolls in a private practice. J Dent Child 50:259-261, 1983.
In as little time as 1 second, salivary exposure will compromise bonding of resin to enamel.
Oral fluid contamination of etched enamel surfaces: an SEM study Leon M. Silverstone, DDSc, PhD, BChD, LDS, RCS(Eng) M. John Hicks, DDS, M S, PhD M ary J. Featherstone
T
.1 . he acid-etch technique is used exten sively in aspects of preventive and re storative dentistry as well as in orthodon tic treatment. Whether a sealant or a com posite material is being placed or ortho dontic brackets are being attached, the durability of the bond is paramount to clinical success. One of the major reasons for a poor or failed bond is salivary con tamination of the etched enamel surface before resin placement.1 This factor was highlighted as a probable reason for fail ure in early clinical trials more than a decade ago.2,3 Relatively few studies, either in vivo4,5 or in vitro,5,9 have at tempted to evaluate the effect of salivary contamination on the acid-etch tech nique. Although bond strength and sur face topography for saliva-contaminated etched enamel have been shown to be al tered, no investigation has studied the ef fect of the length of time that etched enamel is exposed to saliva. The purpose of this in vitro study was to determine the effect of differing salivary contamination periods on the surface topography of acid-etched human enamel. In addition, the appearance of the contaminated sur face after washing with an air/water syringe was assessed. A scanning elec tron microscope (SEM) was used to eval uate the enamel surfaces. Materials and methods Permanent premolars and molars with macro-
Fig 1 ■ Microtome provides serial longitudinal undemineralized and unembedded sections of teeth.
scopically sound buccal and lingual enamel surfaces were chosen for this in vitro study. Each tooth was sectioned into three portions buccolingually using the Silverstone-Taylor Hard T issu e M icrotom e (Fig 1). An acidresistant varnish was painted on the specimens so that windows of sound enamel in the middle thirds of both the buccal and lingual surfaces of the crowns were left exposed. This allowed three treatment comparisons to be made on a single tooth from a sim ilar region of sound enamel. Before the varnish was applied, the e n am el w as c le a n e d th o ro u g h ly w ith a fluoride-free prophylaxis paste. The buccal and lingual windows of sound enamel on each specimen were etched for 60 seconds with a
freshly prepared solution of 30% wt/wt phos phoric acid. The experimental design is outlined in Fig ure 2. For each tooth, one portion was left as a control after etching, whereas the other two were exposed to saliva in vitro. Whole saliva was collected freshly from one of the inves tigators. (As the whole salivary fluid obtained on expectoration con tain s m ore than ju st saliva, it is referred to as oral fluid.) The re maining two portions of the tooth were ex posed to oral fluid for a single exposure period. The exposure periods used in these experi ments were 60, 30, 10, 5, 1, or 0.5 seconds. With the 1-second and 0.5-second expo sures, the specimens were dipped into oral JADA, Vol. 110, M arch 1985 ■ 329
ARTICLES
with warm compressed air. The specimens were then prepared for SEM exam ination (JEOL JSM 35C microscope) to determine the effect of the treatment regimen on the surface topography of the enamel and to compare it with the etched control.
i
ï (A)
(B)
(C)
Acid-etch only
Acid-etch and oral fluid
Acid-etch, oral fluid, and wash
(B)
(C)
(A)
Scanning electron m icroscopy (SEM)
Fig 2 ■ Diagram of experimental design.
fluid and then rapidly removed, according to a stop clock. After exposure to oral fluid, one portion was left to air dry, and the remaining portion was vigorously washed for 30 seconds with an air/water spray and then dried for 30 seconds
The experimental design resulted in a direct comparison of three different treatment effects on the surface appearance of enamel: acidetching of sound enamel, salivary contamina tion of etched sound enamel, and washing of saliva-contaminated etched sound enamel. In addition, the effect of six different periods of oral fluid exposure could be compared. A total of 20 teeth were used in this study, resulting in 120 windows of buccal and lingual enamel available for study, as each tooth was divided into three portions. Forty windows were avail able for each of the three treatment groups and six windows were examined for each of the different periods of oral fluid exposure.
Results Acid-etching of the sound enamel control sur faces produced characteristic etching patterns (Fig 3). Types 1 ,2 , and 3 etching patterns were observed with no one etching pattern pre dominating. Regions of prisms that had lost their cores (type 1 etching pattern) were found adjacent to other regions with either prisms that had lost their peripheries (type 2 etching pattern) or areas w ith generalized surface roughening and porosity, but no distinct prism morphology (type 3 etching pattern). Figure 4 shows a high-power view of part of the enamel surface in Figure 3. This region is referred to as a type 1 etching pattern as the periphery of the prism is relatively intact with the core showing greatest destruction. However, this photomi crograph demonstrates clearly the high poros ity of the entire surface after acid etching. In all instances, the control surfaces were suffi ciently etched to provide porosities for pene tration of resin material. When the etched surfaces were contamin ated with oral fluid, the surface topography was altered dramatically. An adherent surface
Fig 3 ■ Etched enamel showing examples of three etching patterns. Areas show ing preferential loss of prism cores where prism peripheries are prominent (type 1) can be seen in addition to regions in which prism cores have remained (type 2). There is also band of porous enamel without evidence of prism structure (type 3) (orig mag x 1,000). Space bar represents 10 //III. 330 a JADA, Vol. 110, M arch 1985
coating that obscured the underlying etchec enamel to a substantial extent was observed Figure 5 shows an etched enamel surface thal was exposed to oral fluid for 60 seconds. There is no evidence of the etched surface as it hat been completely obscured by an adherent layei of organic material. Figure 6 shows the adja cent region of etched enamel that was also ex posed to oral fluid for 60 seconds. In this specimen, the surface was thoroughly washec for 30 seconds and then air dried. However, the washing regimen had no apparent effect as the adherent layer is still present. Figure 7 shows an etched enamel surface that was exposed to oral fluid for 5 seconds The etched surface was completely masked b j the organic precipitate in spite of the relatively short contamination period. Figure 8 shows the adjacent etched surface, also exposed tc oral fluid for 5 seconds, but followed by a 30 second wash. The surface appears similar tc the unwashed specimen in that the etching pattern is still completely masked. Thus, the washing regimen did not remove the salivary contaminants and, therefore, a resin would no bond successfully to such a surface. Figure 9 is a high-power micrograph focusec on a group of enamel prisms. This region hac been etched in the conventional manner for 6( seconds, exposed to oral fluid for just 1 second and then thoroughly washed. In spite of the short 1-Second contamination period and the 30-second washing regimen, a layer of adhe rent organic material can still be seen over the prism structure. For comparison, Figure 1C shows the comparable region on the etchec control. There is a significant difference in ap pearance as the normal porosity of etchec enamel can be seen in the control specimen. These experiments all show a sim ilar trenc in that surface contamination occurred aftei exposure to oral fluid, using the entire series o: exposure periods. With respect to the washing regimen, surface contaminants were not re moved with exposure periods of 1 second oi greater. Only when specimens were contamin ated for less than 1 second could the organic layer be removed by the washing regimen.
Fig 4 ■ High-power view of part of Figure 3 showing high degree of porosity c etched enamel. This porosity is necessary so that fluid resin can penetrate am form a strong mechanical bond with tissue (orig mag x 5,400). Space bar repre sents 1 fj.m.
ARTICLES
Fig 5 ■ Etched enamel surface after 60-second contamination period with oral fluid. Etched region is completely obscured by organic deposit (orig mag x 1,000). Space bar represents 10 ¿im.
Fig 6 ■ Region adjacent to that shown in Figure 5, also having 60-second con tamination period with oral fluid, but followed by 30-second vigorous wash. Sur face still shows presence of adherent layer that masks etched region (orig mag x 1,800). Space bar represents 10 /xm.
Fig 7 ■ Etched enamel surface was exposed to oral fluid for 5 seconds. Organic layer has completely masked etched region (orig mag x 400). Space bar repre sents 10 /xm.
Fig 8 ■ Region of etched enamel adjacent to that seen in previous figure. After 5-second contamination period, surface w as washed for 30 seconds and then air dried. However, washing regimen has not been successful in removing organic coating (orig mag x 1,500). Space bar represents 10 /xm.
Fig 9 ■ High-power view showing group of prisms in area that was etched, ex posed to oral fluid for 1 second, and then washed for 30 seconds. Surface film of organic material is seen as contaminant on etched prisms. This would adversely affect bonding of resin to surface (orig mag x 6,000). Space bar represents 1 /xm.
Fig 10 ■ Region on etched control specimen comparable to that seen in previous figure. Prism structure appears as porous region that would be ideal for bonding (orig mag x 6,000). Space bar represents 1 fim.
Silverstone-Hicks-Featherstone : CONTAMINATION OF ENAMEL SURFACES ■ 331
A R T IC LES
Discussion The ability of salivary contamination to affect surface topography was shown to be independent of the length of exposure of etched enamel to oral fluid when the exposure periods defined in this study were used. When the exposure period was 1 second or greater, a tenacious sur face coating was present, regardless of whether a 30-second air/water wash was used. Only with an exposure time of less than 1 second was the organic surface coating removed by a vigorous 30-second washing. These results show that should the etched enamel surface be contamin ated by saliva for 1 second or longer, the surface will still possess an adherent sur face coating after an air/water wash. These findings are in agreement with previous work that has shown that an or ganic pellicle forms quickly over etched enamel after exposure to the oral environment.4,5,10'12 In fact, acquisition of cal cium and phosphate phases (remineralization) by the etched enamel surfaces had occurred as shown by SEM,5'10 microprobe,11 and acid solubility12,13 tech niques. However, the length of exposure to oral fluid for the etched enamel sur faces ranged from 1 hour to 4 days in these studies. In the present study, the longest con tamination period was 60 seconds. The surface coating formed during such a short contamination period would most likely represent the formation of an or ganic pellicle on the etched surface. The tenacity of this newly formed pellicle may be caused by the highly reactive na ture of the etched enamel surface as well as its porosity. The etched enamel surface would provide reactive sites of exposed calcium and phosphate ions to which the proteins present in the organic pellicle could attach by electrostatic interac tions.14 The effect of salivary contamination on bond strength has also been deter mined.6'8 The bond strength of resin applied to etched surfaces contaminated by saliva was reduced by up to 50% when compared with uncontaminated etched enamel controls. It was also noted that even if the contaminated surface was washed and re-etched, the bond strength was still less than that for etched enamel that had not been contaminated. The most frequent type of fracture found with specimens contaminated with saliva for 60 seconds was an adhesive fracture rather than a cohesive type of fracture. Cohesive fractures were found most fre quently with uncontaminated speci mens.8 In other words, the contaminated specimens were fractured at the enamelresin interface, whereas in the control specimens, fractures occurred within the resin itself. When a resin material was 332 ■ JADA, Vol. 110, M arch 1985
placed directly on saliva-contaminated surfaces, all fracture sites were at the enamel-resin interface. Thus, the effect of contaminating the etched surface was to weaken the bond between the two mate rials. More than 65% of the specimens that had been contaminated, washed, dried, and then sealed had fractures at the enamel-resin interface. These findings are explained by the presence of a surface coating partially obscuring the underly ing etched enamel prisms, as observed in the present study. The findings in the present study em phasize the importance of maintaining a dry field while placing acid-etch preven tive and restorative materials. Should salivary contamination of an etched enamel surface occur, the procedure should be stopped, the area dried thoroughly, and acid-etching repeated before any attempt to bond a resin mate rial. To avoid salivary contamination, rubber dam isolation is preferred. In cases in which a rubber dam cannot be placed because of access limitations, a newly erupting tooth, an uncooperative patient, or the unwillingness of the operator, cot ton roll isolation with saliva suction is likely to be adequate to avoid contamina tion.14,15 Recent research has shown that sealant placement with cotton roll isola tion resulted in 88% of the sealants being fully retained after 2 years, whereas rub ber dam isolation resulted in 96% full re tention. However, there was no statisti cally significant difference between these two results, although the authors stated that it was less comfortable for the patient and a more difficult technique for the operator when cotton rolls were used for isolation.15 Therefore, this study has demonstrated that the effect of contamination of etched enamel with oral fluid, even for a single second, results in an adherent organic coating that is not removed by conven tional washing techniques. This in turn will compromise the bond between the enamel and the resin material. As any contamination occurring clinically will inevitably exist for at least a 1-second period, the operator should not proceed with the bonding technique until the sur face has been dried and re-etched.
Conclusions Salivary contamination of etched enamel, even for a single second, resulted in an adherent coating that masked the under lying etched enamel. Only with salivary exposure times of less than 1 second could the contaminant be successfully removed by washing. Should salivary contamination occur, the operator should not proceed until the enamel surface has been dried and re-etched before applica tion of a resin.
Summary The purpose of this in vitro study was to determine the effects of differing salivary contamination periods on the surface to pography of etched enamel surfaces using SEM techniques. The results indi cated that salivary contamination of etched enamel for 1 second or longer re sulted in the formation of surface coatings that could not be removed by a water wash. It was concluded that should sali vary contamination of an etched enamel surface occur, it would be necessary to repeat the etching procedure to ensure adequate bonding of a resin material.
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This investigation was supported in part by NIDR research grant no. 2 R01 DE 06564. The authors have no financial interest in the microtome described in the text. The authors thank Ms. Wanda Valentine for assis tance with the preparation of this manuscript. Dr. Silverstone is associate dean for research, and director, dental research unit; Dr. Hicks is assistant professor, dental research unit and department of growth and development; and Ms. Featherstone is professional research assistant, dental research unit, School of Dentistry—C284, University of Colorado Health Sciences Center, Denver, 80262. Address re quests for reprints to Dr. Silverstone. 1. Silverstone, L.M. State-of-the-art on sealant re search and priorities for further research. In Proceed ings of dental sealants in the prevention of tooth de cay. J Dent Educ 48 (Suppl):107-118, 1984. 2. Rock, W.P. Fissure sealants: results obtained with two different sealants after one year. Br Dent J 133:146-151, 1972. 3. Burt, A.B., and others. Retention of a fissure sealant six m onths after app lication. B r Dent J 138:98-102, 1975. 4. Garberoglio, R., and Cozzani, G. In-vivo effect of oral environment on etched enamel: a scanning elec tron microscopic study. J Dent Res 58:1859-1865, 1979. 5. Kastendieck, M.J., and Silverstone, L.M. Re mineralization of acid-etched human enamel by ex posure to oral fluid in vivo and in vitro. J Dent Res 58:163, 1979. 6. Bates, D., and others. Effects of acid etch param eters on enamel topography and composite resinenamel bond strength. Pediatr Dent 4 :1 06-110,1982. 7. Wood, L.W., and Barkmeier, W.W. The effect of salivary contam ination on the retention of acidetched retained composite resin. J Neb Dent Assoc 1 4 -1 8 ,1 9 7 9 . 8. Hormati, A.A., and others. Effects of contamina tion and mechanical disturbance on the quality of acid-etched enamel. JADA 100(l):34-38, 1980. 9. Albert, M., and Grenoble, D.E. An in vivo study of enamel remineralization after acid-etching. J South Calif Dent Assoc 39:747-751, 1971. 10. W ei, S.H. Remineralization of enamel and d e n tin -^ review. J Dent Child 34:444-451, 1967. 11. Silverstone, L.M. Fissure sealants: the suscep tib ility to d issolution of acid -etch ed and sub sequently abraded enamel in vitro. Caries Res 11:465 1 ,1 9 7 7 . 12. Silverstone, L.M. The susceptibility to dissolu tion of fissure-sealed enamel surfaces artificially abraded in vitro. Helv Odont Acta 17:64, 1973. 13. Rolla, G. Effects of fluoride on initiation of plaque formation. Caries Res 11:243-261, 1977. 14. Poulsen, S., and Peltoniemi, A.L. Retention of fissure sealant in primary second molars after 6 months. Scand J Dent Res 87:328-330, 1979. 15. Eidelman, E., and others. The retention of fis sure sealants: rubber dam or cotton rolls in a private practice. J Dent Child 50:259-261, 1983.