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Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
Journal of Dentistry (2004) 32, 75–81
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Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents ¨feb, S. Atalayb, B. Bottb M. Hanniga,*, A. Gra a
Department of Operative Dentistry and Periodontology, Saarland University, Building 73, D-66421 Homburg/Saar, Germany b Clinic of Operative Dentistry and Periodontology, Christian-Albrechts-University of Kiel, Arnold-Heller-Str. 16, D-24105 Kiel, Germany Received 1 June 2003; revised 24 June 2003; accepted 21 August 2003
KEYWORDS Dental materials; Fissure sealant; Self-etching primer; Enamel bonding; Microleakage; Airabrasion
Summary Objectives. This study evaluated the microleakage and internal seal of fissure sealants placed by use of self-etching priming agents in comparison to phosphoric acid etching of enamel. Methods. Seventy-two caries-free extracted human molars were divided into six groups with 12 teeth each. Occlusal surfaces were cleansed by either pumicing (Groups I, III, V) or by 15-s air-abrasion treatment with 25 mm aluminum oxide particles (Groups II, IV, VI). Fissures were sealed with the self-etching priming systems, Clearfil Liner Bond 2 (Groups I, II) or Resulcin AquaPrime (Groups III, IV). In Groups V and VI, sealants were placed after phosphoric acid etching. Half of the teeth in each group were thermocycled. After staining with 0.5% methylene blue, the teeth were sectioned for evaluation of microleakage. Internal adaptation of the fissure sealants was analyzed by SEM on replicas of cross sections. Results. Independent of the methods used for cleansing of the occlusal surfaces, fissure sealants in Groups I and II showed significantly more microleakage and less sufficient internal seal as compared to sealants placed in Groups III to VI. Sealants placed by Resulcin AquaPrime (Groups III, IV) leaked significantly more than sealants applied after phosphoric acid etching (Groups V, VI) of the enamel. However, statistical analysis (H-test) did not reveal significant differences concerning the internal adaptation of sealants placed in Groups III, IV, V and VI. Conclusions. Concerning the microleakage data, use of the self-etching bonding systems, Clearfil Liner Bond 2 and Resulcin AquaPrime, cannot be recommended for fissure sealing, since the sealing ability is less effective as compared to the conventional acid-etching technique. q 2003 Elsevier Ltd. All rights reserved.
Introduction Fissure sealants have been introduced as a way to prevent occlusal caries almost more than 30 *Corresponding author. Tel.: þ 49-6841-1624960; fax: þ 496841-1624954. E-mail address: [email protected]
years ago.1 Since then, usage of fissure sealants has increased steadily.2 Most resin materials used for fissure sealing are based on Bis-GMA. Usually, resin sealants are placed after cleansing and phosphoric acid etching of the fissure enamel. Phosphoric acid etching removes surface contaminants and creates an irregular, microporous enamel surface that is infiltrated by the resinous
0300-5712/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2003.08.005
76
sealing material. After polymerization of the sealing material, a durable bonding to the enamel surface is achieved by micromechanical retention via rheological and geometrical effects. Recently, so-called self-etching priming agents have been introduced, which enable composite-toenamel bonding without previous phosphoric acid etching of the enamel surface.3 – 6 These selfetching priming agents contain hydrophilic, acidic monomers capable of etching and penetrating the enamel surface simultaneously. The reactive molecules in self-etching primers are esters from bivalent alcohols with methacrylic acid and phosphoric acid or derivatives (Table 1). While the phosphate residue of the molecule is thought to etch the enamel, the methacrylate component is available for polymerization and co-polymerization with the bonding agent. Bond strength measurements and analysis of marginal adaptation offer evidence that selfetching adhesive systems can be used for composite-to-enamel bonding without phosphoric acid etching.3 – 5,7,8 However, data concerning the applicability and potential of self-etching priming agents in fissure sealing are rare.9 The purpose of this in vitro study was to evaluate the microleakage and internal seal of fissure sealants placed by use of two self-etching priming agents in comparison to conventional phosphoric acid etching of enamel.
Materials and methods A total of 72 caries-free extracted human molars stored in water at 4 8C were selected for this in vitro study. The teeth were randomly divided to six groups with 12 teeth each, and treated and sealed using the following methods and materials (Table 1): Group I The occlusal surfaces of the teeth were cleansed with a water slurry of fine flour of pumice using a brush in a slow-speed handpiece. Sealing of fissures took place with the Clearfil Liner Bond 2 system according to the mode of application described in detail in Table 1. Group II Fissures were cleansed and roughened by airabrasion treatment with 25 mm aluminum oxide particles (Air-Flow prep K1; EMS SA, Nyon, Switzerland) over a 15-s period. Subsequently, fissures were sealed with the Clearfil Liner Bond 2 system as described for Group I.
M. Hannig et al.
Group III Fissures were cleansed by use of a rotary brush and pumice (see Group I) and sealed with the Resulcin AquaPrime þ Monobond System (Table 1). Group IV Fissures were cleansed and roughened by shortterm air-abrasion (see Group II) and sealed with Resulcin AquaPrime þ Monobond (see Group III). Group V Fissures were cleansed by use of a rotary brush and pumice (see Group I). Sealing was performed with Resulcin Monobond after conventional phos¨ tzgel Minitip, Espe; Seefeld, phoric acid etching (A Germany) of the enamel surface. Group VI Fissures were cleansed and roughened by shortterm air-abrasion (see Group II), phosphoric acid etched and sealed with Resulcin Monobond. In all groups, light-curing of the sealing materials was performed with an Astralis 5 (Vivadent; Schaan, Liechtenstein) curing unit. The output of the light tip, as measured by a digital curing radiometer (Cure Rite, Caulk Dentsply, Milford, DE, USA), was 515 mW/cm2. After placement of the sealants, all teeth were stored in tap water at 4 8C for 48 h. Six teeth of each group were thermocycled for 2500 cycles between 5 and 55 8C with 60-s dwell times at the minimum and maximum temperatures. Subsequently, apices of all teeth were occluded with resin composite, and a layer of nail varnish was applied to all tooth surfaces except of a 1mm window around the restoration. Specimens were immersed in 0.5% methylene blue solution at room temperature for 24 h, thoroughly brushed to remove any excess dye and stored in tap water at 4 8C until sectioned. All sealed teeth were cut bucco-lingually into four sections using a slow-speed rotating diamond blade. Dye penetration was evaluated with a stereomicroscope at 40-fold magnification on the two outer sections and the opposite sites of the two inner sections. The extent of dye penetration was recorded by one person blinded to the treatment groups using the following ordinal scale: 0 1
2
no dye penetration at the interface between enamel and fissure sealant, dye penetration at the interface between enamel and fissure sealant up to 500 mm depth, dye penetration beyond 500 mm of the enamel – fissure sealant interface.
Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
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Table 1 Enamel surface pre-treatment and materials used for application of fissure sealants in experimental Groups I–VI. Group
Enamel pre-treatment (mode of fissure cleansing)
Materials (manufacturer) components
Principle ingredients
Mode of application
I
Pumicing (rotary brush)
Clearfil Liner Bond 2 (Kuraray Co, Osaka, Japan) LB PRIMER liquid A, Lot #41134
2-methacryloyloxyethylphenyl-hydrogen-phosphate, n-methacryloyl-5-aminosalicylic acid
Mix LB PRIMER liquid A and liquid B Apply to enamel for 60 s Air blow gently Apply LB bond Air blow gently Light cure for 20 s
II
Air-abrasion (Air-Flow prep K1)
LB PRIMER liquid B, Lot #41134
Hydrophilic dimethacrylate, 2-hydroxyethylmethacrylate, ethanol, water 10-methacryloyloxydecyldihydrogen phosphate, 2hydroxyethylmethacrylate, hydrophobic dimethacrylate, bis-phenol A diglycidyldimethacrylate, SiO2
LB Bond, Lot #41178
III
Pumicing (rotary brush)
Resulcin AquaPrime þ MonoBond (Merz Dental, Lu ¨tjenburg, Germany) AquaPrime, Lot #97200061
2-Methacryloyloxyethyldihydrogen-phosphate
Mix AquaPrime with water (1:1) Scrub into the enamel surface for 60 s Gently air dry Apply MonoBond Air blow gently
IV
Air-abrasion (Air-Flow prep K1)
MonoBond, Lot #97200061
Bis-phenol A diglycidyldimethacrylate, triethylenglycoldimethacrylate, polymethacryl-oligomaleic acid
Light cure for 20 s
V
Pumicing (rotary brush)
Resulcin MonoBond, (Merz Dental, Lu ¨tjenburg, Germany)
Bis-phenol A diglycidyldimethacrylate, triethylenglycoldimethacrylate, polymethacryl-oligomaleic acid
Etch the enamel with phosphoric acid for 60 s Rinse with water for 30 s Thoroughly air dry
VI
Air-abrasion (Air-Flow prep K1)
Lot #97200061
Subsequently, surfaces of the sections were polished by wet-grinding with 4.000-grit abrasive paper. Internal adaptation of the fissure sealants was analyzed by scanning electron microscopy performed on epoxy resin replica models (Stycast, Grace; Westerlo, Belgium) of the polished sections. Quality of the internal adaptation was evaluated at 320-fold magnification by one person blinded to the treatment groups. Internal adaptation was ranked according to the following ordinal scale:
Apply MonoBond Air blow gently Light cure for 20 s
0 1 2
perfect gap-free internal adaptation, gap formation between enamel and fissure sealant up to 500 mm depth, gap formation at the enamel –fissure sealant interface beyond 500 mm depth.
The Kruskal – Wallis H-test followed by the Mann – Withney U-test were used for statistical analyses (comparison of groups, comparison of thermocycled and non-thermocycled specimens). The level of significance was set at p , 0:05:
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M. Hannig et al.
Table 2 Results of dye penetration test: frequency distribution of microleakage scores (number of sections in each individual experimental group with scores 0, 1 and 2), and results of statistical analysis. Experimental group
Enamel pre-treatment
Thermocycling
Microleakage scores
h
0
1
2
7a,h 1b,i
12f,g,j 9
5 14
12c,d,h 5e,i
5j 13
7 6
18a 8b
4f 5
2 11
I
Pumicing þ Clearfil Liner Bond 2
No Yes
II
Air-abrasion þ Clearfil Liner Bond 2
No Yes
III
Pumicing þ Resulcin AquaPrime
No Yes
IV
Air-abrasion þ Resulcin AquaPrime
No Yes
19c 11e
4 6
1 7
V
Pumicing þ Phosphoric acid
No Yes
24a 17b
0g 4
0 3
VI
Air-abrasion þ Phosphoric acid
No Yes
23d 16e
1 5
0 3
h
h
Brackets indicate significant differences between thermocycled and non-thermocycled specimens for p , 0:05: Same superscript letters indicate statistically significant differences between groups for p , 0:05:
Results Microleakage scores observed in the six experimental groups are summarized in Table 2. Table 3 represents the results of the SEM evaluation. Fissure sealants placed by use of the self-etching primer Liner Bond 2 (Groups I, II) exhibited significantly more microleakage and less gap-free internal adaptation than did sealants applied by use of the self-etching priming system, Resulcin
AquaPrime þ Monobond or sealants placed after phosphoric acid treatment of the enamel (Tables 2 and 3). Sealants placed by Resulcin AquaPrime (Groups III, IV) leaked significantly more than sealants applied after phosphoric acid etching (Groups V, VI) of the enamel (Table 2). However, concerning the internal adaptation at the sealant – enamel interfaces, there did not exist significant differences between sealants placed by use of Resulcin AquaPrime (Groups III, IV) and those
Table 3 Results of SEM analysis: frequency distribution (number of sections in individual experimental groups) of evaluation criteria 0, 1 and 2 for the internal adaptation at the enamel– fissure sealant interface, and results of statistical analysis. Experimental group
Enamel pre-treatment
Thermocycling
SEM scores
h
0
1
11a,b 0c,d,m
6q 0q
8g,h 3i,j,m
9 2
23b 12d
1 7
22h 11i
2 3
2
h
I
Pumicing þ Clearfil Liner Bond 2
No Yes
II
Air-abrasion þ Clearfil Liner Bond 2
No Yes
III
Pumicing þ Resulcin AquaPrime
No Yes
IV
Air-abrasion þ Resulcin AquaPrime
No Yes
V
Pumicing þ Phosphoric acid
No Yes
21a 17c
3 4
0e 3
VI
Air-abrasion þ Posphoric acid
No Yes
22g 16j
2 5
0 3l
h h
h
7 24e,f 7 19k,l 0 5f
h
0 10k
Brackets indicate significant differences between thermocycled and non-thermocycled specimens for p , 0:05: Same superscript letters indicate statistically significant differences between groups for p , 0:05:
Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
applied after phosphoric acid treatment of the enamel surface (Groups V, VI), (Table 3). Statistically significant differences between thermocycled and non-thermocycled specimens were recorded for microleakage score 0 in Groups I, III and VI, as well as for SEM evaluation score 0 in Groups I, III, IV, score 1 in Group I, and score 2 in Groups I, II, IV (Tables 2 and 3). In case of fissure sealants applied by use of the Clearfil Liner Bond 2 system, a significant decrease in microleakage was observed in air-abraded fissures as compared to pumiced fissures. However, no significant differences were detected in microleakage of sealants placed by use of the Resulcin AquaPrime adhesive system or after phosphoric acid treatment depending on the fissure cleansing method used.
Discussion Fissure sealing is an effective treatment to prevent caries formation in occlusal surfaces.8, 10 – 13 Sealant retention and integrity of the enamel– sealant interface determine, to a great extent, the caries reduction ability and effectiveness of a fissure sealant.13,14 Prior to sealant application, the enamel surface usually is etched by phosphoric acid, thoroughly water-rinsed and air-dried. Subsequently, the resin sealing agent is applied and light-cured. Fissure sealants placed by this technique reveal a tight seal after application, as shown in the present study. However, due to thermocycling, loss of sealing ability could be detected. These in vitro results are in good accordance with the fact that fissure sealants under clinical conditions reveal a failure rate of 5 – 10% per year, even under best circumstances during placement, as reported in a recent review.13 Adequate isolation is one of the most critical aspects during sealant application.2 Salivary contamination of the tooth surface after acid etching has a deleterious effect on the ultimate bonding between resin and enamel.2 In case that the fissure sealant is placed without rubberdam, swallowing or tongue movement during and after water-rinsing of the tooth surface might cause flooding of the etched enamel with saliva. The resulting salivary coating cannot be completely removed by rinsing.15 Thus, adhesive materials which enable resin-toenamel bonding without previous acid-etching and water-rinsing could facilitate the clinical use of sealants in cases, where placement of rubberdam is not possible.
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In the present study, two self-etching priming agents, which have the potential to provide sufficient composite-to-enamel as well as composite-to-dentin bonding4,16 were evaluated for enamel pre-treatment in fissure sealing without prior use of phosphoric acid etching. However, results of the dye penetration test indicate that both self-etching adhesive systems are less effective in fissure sealing than the bonding agent applied after phosphoric acid etching of the enamel. These findings can be explained by the fact that in the area of the occlusal fissures, the enamel surface shows a mainly a prismatic or prismless configuration.17 – 19 Due to phosphoric acid etching and subsequent water-rinsing of the etched enamel, the prismless enamel surface layer is removed. Thereby the prismatic structured enamel is exposed providing sufficient microretentive bonding of the fissure sealant. In contrast, treatment with self-etching priming agents does not remove a significant amount of the prismless enamel surface layer, since no rinsing takes place after application of the primer.6,7 Possibly, the prismless enamel surface layer prevents the permeation of self-etching primers, thus leaving some areas partially unetched.7 It has been shown recently that self-etching primers produce high bond strengths to ground enamel,4,7 but significantly lower bond strengths to intact enamel surfaces.7 Therefore, the insufficient etching and deficient resin penetration of the self-etching priming agents into the fissure enamel may have caused the high extent of microleakage observed in Groups I to IV. It has been reported previously from SEM investigations that the etching ability of different self-etching priming agents reveals distinct differences.4 Whereas the Clearfil Liner Bond 2 system produces a relatively shallow enamel etching pattern,4 – 7 use of the Resulcin AquaPrime systems causes a comparatively stronger etching effect at ground enamel surfaces.4,6 These differences in etching capability can be attributed to differences in the composition of the selfetching primers,4 and could be the reason for the significantly less microleakage and better internal adaptation observed in specimens sealed by use of Resulcin AquaPrime as compared to Clearfil Liner Bond 2. The LB Primer of the Clearfil Liner Bond 2 system contains phenyl-hydrogen-phosphate along with carbonic acid, while the Resulcin AquaPrime contains the more acidic 2methacryloyloxyethyl-dihydrogen-phosphate as reactive components (Table 1).4 Prior to acid etching and sealant application, it is important to make sure that the occlusal tooth surface and the fissure areas are free from
80
plaque and any debris that might interfere with the etching and sealing procedure. Air-polishing has been suggested to improve the removal of debris,20,21 and to increase sealant penetration22 and micro-mechanical retention of the sealant.23 – 25 However, the present study has shown that fissure sealants placed after short-term air-abrasion or pumicing of the occlusal surface did not differ significantly with regard to microleakage and internal seal. Only when Clearfil Liner Bond 2 was used for placement of fissure sealants, the 15-s air-abrasion treatment caused a significant improvement of the sealing effect compared to cleansing of the occlusal surface with pumice. The increase in enamel surface roughness due to air-abrasion treatment possibly has improved the etching and sealing ability of the Clearfil Liner Bond 2 system. In Groups V and VI (fissure sealants placed after conventional phosphoric acid etching), results of the dye penetration test were confirmed by the results of the SEM evaluation indicating that the infiltration of dye at the enamel – sealant interface was caused by gap formation. However, in Groups I, II, III and IV, certain differences between corresponding dye penetration scores and SEM data were observed. In Groups III and IV (sealants placed by use of Resulcin AquaPrime), these differences were caused by the fact that the priming agent was stained by methylene blue without the existence of gaps. This observation might be related to the phenomenon of nanoleakage, describing leakage in sub-micron wide spaces and channels that could not be detected by SEM analysis performed at 320-fold magnification. However, in Groups I and II (Clearfil Liner Bond 2), more interfacial gaps were detected by SEM analysis as indicated by dye penetration scores in corresponding tooth sections. These findings are difficult to explain. Possibly, problems in wettability of the bonding resin that contains hydrophobic dimethacrylates by the watery methylene blue solution might have hindered complete infiltration of the dye in marginal gaps. Thus, the present data indicate that both dye penetration test as well as SEM analysis should be combined in order to get reliable data on the sealing ability obtainable with self-etching priming agents. Without additional SEM evaluation, the dye penetration test might reveal both, false negative as well as false positive results, regarding microleakage caused by gap formation at the tooth– sealant interface. Analysis of marginal and internal seal based solely on SEM investigation might lead to nondetection of nanoleakage phenomena at the enamel –resin interface.
M. Hannig et al.
Conclusions The present in vitro data indicate that use of the Clearfil Liner Bond 2 system for application of fissure sealants cannot be recommended. The Resulcin AquaPrime bonding system and the conventional acid-etching technique reveal a similar potential for placement of fissure sealants with regard to internal adaptation of the sealant. However, concerning microleakage, the fissure sealing ability of the self-etching primer Resulcin AquaPrime is less effective as compared to the conventional acid-etching technique.
References 1. Cueto EI, Buonocore MG. Sealing of pits and fissures with an adhesive resin. Its use in caries prevention. Journal of the American Dental Association 1967;75:121—8. 2. Waggoner WF, Siegal M. Pit and fissure sealant application: updating the technique. Journal of the American Dental Association 1996;127:351—61. 3. Barkmeier WW, Los SA, Triolo PT. Bond strengths and SEMevaluation of Clearfil Liner Bond 2. American Journal of Dentistry 1995;8:289—93. 4. Hannig M, Reinhardt KJ, Bott B. Self-etching primer vs phosphoric acid: an alternative concept for composite-to-enamel bonding. Operative Dentistry 1999;24: 172—80. 5. Perdiga ˜o J, Lopez L, Lambrechts P, Leitao J, Van Meerbeek B, Vanherle G. Effects of a self-etching primer enamel shear bond strengths and SEM morphology. American Journal of Dentistry 1997;10:141—6. 6. Hannig M, Bock H, Bott B, Hoth-Hannig W. Inter-crystallite nanoretention of self-etching adhesives at enamel imaged by transmission electron microscopy. European Journal of Oral Sciences 2002;110:464—70. 7. Kanemura N, Sano H, Tagami J. Tensile bond strength to and SEM evaluation of ground and intact enamel surfaces. Journal of Dentistry 1999;27:523—30. 8. Simonsen RJ. Retention and effectiveness of a single application of white sealant after 10 years. Journal of the American Dental Association 1987;115:31—6. 9. Gillet D, Nancy J, Dupuis V, Dorignac G. Microleakage and penetration depth of three types of materials in fissure sealant: self-etching primer vs etching: an in vitro study. Journal of Clinical Pediatric Dentistry 2002;26: 175—8. 10. Wendt LK, Koch G. Fissure sealant in permanent first molars after 10 years. Swedish Dental Journal 1988;12: 181—5. 11. Simonsen RJ. Retention and effectiveness of dental sealant after 15 years. Journal of the American Dental Association 1991;122:34—42. 12. Llodra JC, Bravo M, Delgado-Rodriguez M, Baca P, Galvez R. Factors influencing the effectiveness of sealants—a metaanalysis. Community Dentistry and Oral Epidemiology 1993; 21:261—8. 13. Feigal RJ. Sealants and preventive restorations: review of effectiveness and clinical changes for improvement. Pediatric Dentistry 1998;20:85—92.
Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
14. Ripa LW. Sealants revisited: an update of the effectiveness of pit-and-fissure sealants. Caries Research 1993;27(suppl 1):77—82. 15. Silverstone LM, Hicks MJ, Featherstone MJ. Oral fluid contamination of etched enamel surfaces: an SEM study. Journal of the American Dental Association 1985;110: 329—32. 16. Hannig M, Reinhardt KJ, Bott B. Composite-to-dentin bond strength, micromorphology of the bonded dentin interface, and marginal adaptation of Class II composite resin restorations using self-etching primers. Operative Dentistry 2001;26:157—65. 17. Gwinnet AJ. Human ‘prismless’ enamel and its influence on sealant penetration. Archives of Oral Biology 1973;18: 441—4. 18. Gwinnet AJ. The ultrastructure of the ‘prismless’ enamel of permanent human teeth. Archives of Oral Biology 1967;12: 381—8. 19. Kodaka T, Kuroiwa H, Higashi S. Structural and distribution patterns of surface prismless enamel in human permanent teeth. Caries Research 1991;25:7—20.
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20. Garcia-Godoy F, Medlock JW. An SEM study of the effects of air-polishing on fissure surfaces. Quintessence International 1988;19:465—7. 21. Vesterhus Strand G, Raada M. The efficiency of cleaning fissures with an air-polishing instrument. Acta Odontologica Scandinavia 1988;46:113—7. 22. Brocklehurst PR, Joshi RI, Northeast SE. The effect of airpolishing occlusal surfaces on the penetration of fissures by a sealant. International Journal of Pediatric Dentistry 1992;2: 157—62. 23. Brockmann SL, Scott RL, Eick JD. A scanning electron microscopic study of the effect of air-polishing on the enamel sealant surface. Quintessence International 1990; 21:201—6. 24. Brockmann SL, Scott RL, Eick JD. The effect of an airpolishing device on tensile bond strength of a dental sealant. Quintessence International 1989;20: 211—7. 25. Scott L, Greer D. The effect of an air-polishing device on sealant bond strength. Journal of Prosthetic Dentistry 1987; 58:384—7.
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Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents ¨feb, S. Atalayb, B. Bottb M. Hanniga,*, A. Gra a
Department of Operative Dentistry and Periodontology, Saarland University, Building 73, D-66421 Homburg/Saar, Germany b Clinic of Operative Dentistry and Periodontology, Christian-Albrechts-University of Kiel, Arnold-Heller-Str. 16, D-24105 Kiel, Germany Received 1 June 2003; revised 24 June 2003; accepted 21 August 2003
KEYWORDS Dental materials; Fissure sealant; Self-etching primer; Enamel bonding; Microleakage; Airabrasion
Summary Objectives. This study evaluated the microleakage and internal seal of fissure sealants placed by use of self-etching priming agents in comparison to phosphoric acid etching of enamel. Methods. Seventy-two caries-free extracted human molars were divided into six groups with 12 teeth each. Occlusal surfaces were cleansed by either pumicing (Groups I, III, V) or by 15-s air-abrasion treatment with 25 mm aluminum oxide particles (Groups II, IV, VI). Fissures were sealed with the self-etching priming systems, Clearfil Liner Bond 2 (Groups I, II) or Resulcin AquaPrime (Groups III, IV). In Groups V and VI, sealants were placed after phosphoric acid etching. Half of the teeth in each group were thermocycled. After staining with 0.5% methylene blue, the teeth were sectioned for evaluation of microleakage. Internal adaptation of the fissure sealants was analyzed by SEM on replicas of cross sections. Results. Independent of the methods used for cleansing of the occlusal surfaces, fissure sealants in Groups I and II showed significantly more microleakage and less sufficient internal seal as compared to sealants placed in Groups III to VI. Sealants placed by Resulcin AquaPrime (Groups III, IV) leaked significantly more than sealants applied after phosphoric acid etching (Groups V, VI) of the enamel. However, statistical analysis (H-test) did not reveal significant differences concerning the internal adaptation of sealants placed in Groups III, IV, V and VI. Conclusions. Concerning the microleakage data, use of the self-etching bonding systems, Clearfil Liner Bond 2 and Resulcin AquaPrime, cannot be recommended for fissure sealing, since the sealing ability is less effective as compared to the conventional acid-etching technique. q 2003 Elsevier Ltd. All rights reserved.
Introduction Fissure sealants have been introduced as a way to prevent occlusal caries almost more than 30 *Corresponding author. Tel.: þ 49-6841-1624960; fax: þ 496841-1624954. E-mail address: [email protected]
years ago.1 Since then, usage of fissure sealants has increased steadily.2 Most resin materials used for fissure sealing are based on Bis-GMA. Usually, resin sealants are placed after cleansing and phosphoric acid etching of the fissure enamel. Phosphoric acid etching removes surface contaminants and creates an irregular, microporous enamel surface that is infiltrated by the resinous
0300-5712/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2003.08.005
76
sealing material. After polymerization of the sealing material, a durable bonding to the enamel surface is achieved by micromechanical retention via rheological and geometrical effects. Recently, so-called self-etching priming agents have been introduced, which enable composite-toenamel bonding without previous phosphoric acid etching of the enamel surface.3 – 6 These selfetching priming agents contain hydrophilic, acidic monomers capable of etching and penetrating the enamel surface simultaneously. The reactive molecules in self-etching primers are esters from bivalent alcohols with methacrylic acid and phosphoric acid or derivatives (Table 1). While the phosphate residue of the molecule is thought to etch the enamel, the methacrylate component is available for polymerization and co-polymerization with the bonding agent. Bond strength measurements and analysis of marginal adaptation offer evidence that selfetching adhesive systems can be used for composite-to-enamel bonding without phosphoric acid etching.3 – 5,7,8 However, data concerning the applicability and potential of self-etching priming agents in fissure sealing are rare.9 The purpose of this in vitro study was to evaluate the microleakage and internal seal of fissure sealants placed by use of two self-etching priming agents in comparison to conventional phosphoric acid etching of enamel.
Materials and methods A total of 72 caries-free extracted human molars stored in water at 4 8C were selected for this in vitro study. The teeth were randomly divided to six groups with 12 teeth each, and treated and sealed using the following methods and materials (Table 1): Group I The occlusal surfaces of the teeth were cleansed with a water slurry of fine flour of pumice using a brush in a slow-speed handpiece. Sealing of fissures took place with the Clearfil Liner Bond 2 system according to the mode of application described in detail in Table 1. Group II Fissures were cleansed and roughened by airabrasion treatment with 25 mm aluminum oxide particles (Air-Flow prep K1; EMS SA, Nyon, Switzerland) over a 15-s period. Subsequently, fissures were sealed with the Clearfil Liner Bond 2 system as described for Group I.
M. Hannig et al.
Group III Fissures were cleansed by use of a rotary brush and pumice (see Group I) and sealed with the Resulcin AquaPrime þ Monobond System (Table 1). Group IV Fissures were cleansed and roughened by shortterm air-abrasion (see Group II) and sealed with Resulcin AquaPrime þ Monobond (see Group III). Group V Fissures were cleansed by use of a rotary brush and pumice (see Group I). Sealing was performed with Resulcin Monobond after conventional phos¨ tzgel Minitip, Espe; Seefeld, phoric acid etching (A Germany) of the enamel surface. Group VI Fissures were cleansed and roughened by shortterm air-abrasion (see Group II), phosphoric acid etched and sealed with Resulcin Monobond. In all groups, light-curing of the sealing materials was performed with an Astralis 5 (Vivadent; Schaan, Liechtenstein) curing unit. The output of the light tip, as measured by a digital curing radiometer (Cure Rite, Caulk Dentsply, Milford, DE, USA), was 515 mW/cm2. After placement of the sealants, all teeth were stored in tap water at 4 8C for 48 h. Six teeth of each group were thermocycled for 2500 cycles between 5 and 55 8C with 60-s dwell times at the minimum and maximum temperatures. Subsequently, apices of all teeth were occluded with resin composite, and a layer of nail varnish was applied to all tooth surfaces except of a 1mm window around the restoration. Specimens were immersed in 0.5% methylene blue solution at room temperature for 24 h, thoroughly brushed to remove any excess dye and stored in tap water at 4 8C until sectioned. All sealed teeth were cut bucco-lingually into four sections using a slow-speed rotating diamond blade. Dye penetration was evaluated with a stereomicroscope at 40-fold magnification on the two outer sections and the opposite sites of the two inner sections. The extent of dye penetration was recorded by one person blinded to the treatment groups using the following ordinal scale: 0 1
2
no dye penetration at the interface between enamel and fissure sealant, dye penetration at the interface between enamel and fissure sealant up to 500 mm depth, dye penetration beyond 500 mm of the enamel – fissure sealant interface.
Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
77
Table 1 Enamel surface pre-treatment and materials used for application of fissure sealants in experimental Groups I–VI. Group
Enamel pre-treatment (mode of fissure cleansing)
Materials (manufacturer) components
Principle ingredients
Mode of application
I
Pumicing (rotary brush)
Clearfil Liner Bond 2 (Kuraray Co, Osaka, Japan) LB PRIMER liquid A, Lot #41134
2-methacryloyloxyethylphenyl-hydrogen-phosphate, n-methacryloyl-5-aminosalicylic acid
Mix LB PRIMER liquid A and liquid B Apply to enamel for 60 s Air blow gently Apply LB bond Air blow gently Light cure for 20 s
II
Air-abrasion (Air-Flow prep K1)
LB PRIMER liquid B, Lot #41134
Hydrophilic dimethacrylate, 2-hydroxyethylmethacrylate, ethanol, water 10-methacryloyloxydecyldihydrogen phosphate, 2hydroxyethylmethacrylate, hydrophobic dimethacrylate, bis-phenol A diglycidyldimethacrylate, SiO2
LB Bond, Lot #41178
III
Pumicing (rotary brush)
Resulcin AquaPrime þ MonoBond (Merz Dental, Lu ¨tjenburg, Germany) AquaPrime, Lot #97200061
2-Methacryloyloxyethyldihydrogen-phosphate
Mix AquaPrime with water (1:1) Scrub into the enamel surface for 60 s Gently air dry Apply MonoBond Air blow gently
IV
Air-abrasion (Air-Flow prep K1)
MonoBond, Lot #97200061
Bis-phenol A diglycidyldimethacrylate, triethylenglycoldimethacrylate, polymethacryl-oligomaleic acid
Light cure for 20 s
V
Pumicing (rotary brush)
Resulcin MonoBond, (Merz Dental, Lu ¨tjenburg, Germany)
Bis-phenol A diglycidyldimethacrylate, triethylenglycoldimethacrylate, polymethacryl-oligomaleic acid
Etch the enamel with phosphoric acid for 60 s Rinse with water for 30 s Thoroughly air dry
VI
Air-abrasion (Air-Flow prep K1)
Lot #97200061
Subsequently, surfaces of the sections were polished by wet-grinding with 4.000-grit abrasive paper. Internal adaptation of the fissure sealants was analyzed by scanning electron microscopy performed on epoxy resin replica models (Stycast, Grace; Westerlo, Belgium) of the polished sections. Quality of the internal adaptation was evaluated at 320-fold magnification by one person blinded to the treatment groups. Internal adaptation was ranked according to the following ordinal scale:
Apply MonoBond Air blow gently Light cure for 20 s
0 1 2
perfect gap-free internal adaptation, gap formation between enamel and fissure sealant up to 500 mm depth, gap formation at the enamel –fissure sealant interface beyond 500 mm depth.
The Kruskal – Wallis H-test followed by the Mann – Withney U-test were used for statistical analyses (comparison of groups, comparison of thermocycled and non-thermocycled specimens). The level of significance was set at p , 0:05:
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M. Hannig et al.
Table 2 Results of dye penetration test: frequency distribution of microleakage scores (number of sections in each individual experimental group with scores 0, 1 and 2), and results of statistical analysis. Experimental group
Enamel pre-treatment
Thermocycling
Microleakage scores
h
0
1
2
7a,h 1b,i
12f,g,j 9
5 14
12c,d,h 5e,i
5j 13
7 6
18a 8b
4f 5
2 11
I
Pumicing þ Clearfil Liner Bond 2
No Yes
II
Air-abrasion þ Clearfil Liner Bond 2
No Yes
III
Pumicing þ Resulcin AquaPrime
No Yes
IV
Air-abrasion þ Resulcin AquaPrime
No Yes
19c 11e
4 6
1 7
V
Pumicing þ Phosphoric acid
No Yes
24a 17b
0g 4
0 3
VI
Air-abrasion þ Phosphoric acid
No Yes
23d 16e
1 5
0 3
h
h
Brackets indicate significant differences between thermocycled and non-thermocycled specimens for p , 0:05: Same superscript letters indicate statistically significant differences between groups for p , 0:05:
Results Microleakage scores observed in the six experimental groups are summarized in Table 2. Table 3 represents the results of the SEM evaluation. Fissure sealants placed by use of the self-etching primer Liner Bond 2 (Groups I, II) exhibited significantly more microleakage and less gap-free internal adaptation than did sealants applied by use of the self-etching priming system, Resulcin
AquaPrime þ Monobond or sealants placed after phosphoric acid treatment of the enamel (Tables 2 and 3). Sealants placed by Resulcin AquaPrime (Groups III, IV) leaked significantly more than sealants applied after phosphoric acid etching (Groups V, VI) of the enamel (Table 2). However, concerning the internal adaptation at the sealant – enamel interfaces, there did not exist significant differences between sealants placed by use of Resulcin AquaPrime (Groups III, IV) and those
Table 3 Results of SEM analysis: frequency distribution (number of sections in individual experimental groups) of evaluation criteria 0, 1 and 2 for the internal adaptation at the enamel– fissure sealant interface, and results of statistical analysis. Experimental group
Enamel pre-treatment
Thermocycling
SEM scores
h
0
1
11a,b 0c,d,m
6q 0q
8g,h 3i,j,m
9 2
23b 12d
1 7
22h 11i
2 3
2
h
I
Pumicing þ Clearfil Liner Bond 2
No Yes
II
Air-abrasion þ Clearfil Liner Bond 2
No Yes
III
Pumicing þ Resulcin AquaPrime
No Yes
IV
Air-abrasion þ Resulcin AquaPrime
No Yes
V
Pumicing þ Phosphoric acid
No Yes
21a 17c
3 4
0e 3
VI
Air-abrasion þ Posphoric acid
No Yes
22g 16j
2 5
0 3l
h h
h
7 24e,f 7 19k,l 0 5f
h
0 10k
Brackets indicate significant differences between thermocycled and non-thermocycled specimens for p , 0:05: Same superscript letters indicate statistically significant differences between groups for p , 0:05:
Microleakage and SEM evaluation of fissure sealants placed by use of self-etching priming agents
applied after phosphoric acid treatment of the enamel surface (Groups V, VI), (Table 3). Statistically significant differences between thermocycled and non-thermocycled specimens were recorded for microleakage score 0 in Groups I, III and VI, as well as for SEM evaluation score 0 in Groups I, III, IV, score 1 in Group I, and score 2 in Groups I, II, IV (Tables 2 and 3). In case of fissure sealants applied by use of the Clearfil Liner Bond 2 system, a significant decrease in microleakage was observed in air-abraded fissures as compared to pumiced fissures. However, no significant differences were detected in microleakage of sealants placed by use of the Resulcin AquaPrime adhesive system or after phosphoric acid treatment depending on the fissure cleansing method used.
Discussion Fissure sealing is an effective treatment to prevent caries formation in occlusal surfaces.8, 10 – 13 Sealant retention and integrity of the enamel– sealant interface determine, to a great extent, the caries reduction ability and effectiveness of a fissure sealant.13,14 Prior to sealant application, the enamel surface usually is etched by phosphoric acid, thoroughly water-rinsed and air-dried. Subsequently, the resin sealing agent is applied and light-cured. Fissure sealants placed by this technique reveal a tight seal after application, as shown in the present study. However, due to thermocycling, loss of sealing ability could be detected. These in vitro results are in good accordance with the fact that fissure sealants under clinical conditions reveal a failure rate of 5 – 10% per year, even under best circumstances during placement, as reported in a recent review.13 Adequate isolation is one of the most critical aspects during sealant application.2 Salivary contamination of the tooth surface after acid etching has a deleterious effect on the ultimate bonding between resin and enamel.2 In case that the fissure sealant is placed without rubberdam, swallowing or tongue movement during and after water-rinsing of the tooth surface might cause flooding of the etched enamel with saliva. The resulting salivary coating cannot be completely removed by rinsing.15 Thus, adhesive materials which enable resin-toenamel bonding without previous acid-etching and water-rinsing could facilitate the clinical use of sealants in cases, where placement of rubberdam is not possible.
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In the present study, two self-etching priming agents, which have the potential to provide sufficient composite-to-enamel as well as composite-to-dentin bonding4,16 were evaluated for enamel pre-treatment in fissure sealing without prior use of phosphoric acid etching. However, results of the dye penetration test indicate that both self-etching adhesive systems are less effective in fissure sealing than the bonding agent applied after phosphoric acid etching of the enamel. These findings can be explained by the fact that in the area of the occlusal fissures, the enamel surface shows a mainly a prismatic or prismless configuration.17 – 19 Due to phosphoric acid etching and subsequent water-rinsing of the etched enamel, the prismless enamel surface layer is removed. Thereby the prismatic structured enamel is exposed providing sufficient microretentive bonding of the fissure sealant. In contrast, treatment with self-etching priming agents does not remove a significant amount of the prismless enamel surface layer, since no rinsing takes place after application of the primer.6,7 Possibly, the prismless enamel surface layer prevents the permeation of self-etching primers, thus leaving some areas partially unetched.7 It has been shown recently that self-etching primers produce high bond strengths to ground enamel,4,7 but significantly lower bond strengths to intact enamel surfaces.7 Therefore, the insufficient etching and deficient resin penetration of the self-etching priming agents into the fissure enamel may have caused the high extent of microleakage observed in Groups I to IV. It has been reported previously from SEM investigations that the etching ability of different self-etching priming agents reveals distinct differences.4 Whereas the Clearfil Liner Bond 2 system produces a relatively shallow enamel etching pattern,4 – 7 use of the Resulcin AquaPrime systems causes a comparatively stronger etching effect at ground enamel surfaces.4,6 These differences in etching capability can be attributed to differences in the composition of the selfetching primers,4 and could be the reason for the significantly less microleakage and better internal adaptation observed in specimens sealed by use of Resulcin AquaPrime as compared to Clearfil Liner Bond 2. The LB Primer of the Clearfil Liner Bond 2 system contains phenyl-hydrogen-phosphate along with carbonic acid, while the Resulcin AquaPrime contains the more acidic 2methacryloyloxyethyl-dihydrogen-phosphate as reactive components (Table 1).4 Prior to acid etching and sealant application, it is important to make sure that the occlusal tooth surface and the fissure areas are free from
80
plaque and any debris that might interfere with the etching and sealing procedure. Air-polishing has been suggested to improve the removal of debris,20,21 and to increase sealant penetration22 and micro-mechanical retention of the sealant.23 – 25 However, the present study has shown that fissure sealants placed after short-term air-abrasion or pumicing of the occlusal surface did not differ significantly with regard to microleakage and internal seal. Only when Clearfil Liner Bond 2 was used for placement of fissure sealants, the 15-s air-abrasion treatment caused a significant improvement of the sealing effect compared to cleansing of the occlusal surface with pumice. The increase in enamel surface roughness due to air-abrasion treatment possibly has improved the etching and sealing ability of the Clearfil Liner Bond 2 system. In Groups V and VI (fissure sealants placed after conventional phosphoric acid etching), results of the dye penetration test were confirmed by the results of the SEM evaluation indicating that the infiltration of dye at the enamel – sealant interface was caused by gap formation. However, in Groups I, II, III and IV, certain differences between corresponding dye penetration scores and SEM data were observed. In Groups III and IV (sealants placed by use of Resulcin AquaPrime), these differences were caused by the fact that the priming agent was stained by methylene blue without the existence of gaps. This observation might be related to the phenomenon of nanoleakage, describing leakage in sub-micron wide spaces and channels that could not be detected by SEM analysis performed at 320-fold magnification. However, in Groups I and II (Clearfil Liner Bond 2), more interfacial gaps were detected by SEM analysis as indicated by dye penetration scores in corresponding tooth sections. These findings are difficult to explain. Possibly, problems in wettability of the bonding resin that contains hydrophobic dimethacrylates by the watery methylene blue solution might have hindered complete infiltration of the dye in marginal gaps. Thus, the present data indicate that both dye penetration test as well as SEM analysis should be combined in order to get reliable data on the sealing ability obtainable with self-etching priming agents. Without additional SEM evaluation, the dye penetration test might reveal both, false negative as well as false positive results, regarding microleakage caused by gap formation at the tooth– sealant interface. Analysis of marginal and internal seal based solely on SEM investigation might lead to nondetection of nanoleakage phenomena at the enamel –resin interface.
M. Hannig et al.
Conclusions The present in vitro data indicate that use of the Clearfil Liner Bond 2 system for application of fissure sealants cannot be recommended. The Resulcin AquaPrime bonding system and the conventional acid-etching technique reveal a similar potential for placement of fissure sealants with regard to internal adaptation of the sealant. However, concerning microleakage, the fissure sealing ability of the self-etching primer Resulcin AquaPrime is less effective as compared to the conventional acid-etching technique.
References 1. Cueto EI, Buonocore MG. Sealing of pits and fissures with an adhesive resin. Its use in caries prevention. Journal of the American Dental Association 1967;75:121—8. 2. Waggoner WF, Siegal M. Pit and fissure sealant application: updating the technique. Journal of the American Dental Association 1996;127:351—61. 3. Barkmeier WW, Los SA, Triolo PT. Bond strengths and SEMevaluation of Clearfil Liner Bond 2. American Journal of Dentistry 1995;8:289—93. 4. Hannig M, Reinhardt KJ, Bott B. Self-etching primer vs phosphoric acid: an alternative concept for composite-to-enamel bonding. Operative Dentistry 1999;24: 172—80. 5. Perdiga ˜o J, Lopez L, Lambrechts P, Leitao J, Van Meerbeek B, Vanherle G. Effects of a self-etching primer enamel shear bond strengths and SEM morphology. American Journal of Dentistry 1997;10:141—6. 6. Hannig M, Bock H, Bott B, Hoth-Hannig W. Inter-crystallite nanoretention of self-etching adhesives at enamel imaged by transmission electron microscopy. European Journal of Oral Sciences 2002;110:464—70. 7. Kanemura N, Sano H, Tagami J. Tensile bond strength to and SEM evaluation of ground and intact enamel surfaces. Journal of Dentistry 1999;27:523—30. 8. Simonsen RJ. Retention and effectiveness of a single application of white sealant after 10 years. Journal of the American Dental Association 1987;115:31—6. 9. Gillet D, Nancy J, Dupuis V, Dorignac G. Microleakage and penetration depth of three types of materials in fissure sealant: self-etching primer vs etching: an in vitro study. Journal of Clinical Pediatric Dentistry 2002;26: 175—8. 10. Wendt LK, Koch G. Fissure sealant in permanent first molars after 10 years. Swedish Dental Journal 1988;12: 181—5. 11. Simonsen RJ. Retention and effectiveness of dental sealant after 15 years. Journal of the American Dental Association 1991;122:34—42. 12. Llodra JC, Bravo M, Delgado-Rodriguez M, Baca P, Galvez R. Factors influencing the effectiveness of sealants—a metaanalysis. Community Dentistry and Oral Epidemiology 1993; 21:261—8. 13. Feigal RJ. Sealants and preventive restorations: review of effectiveness and clinical changes for improvement. Pediatric Dentistry 1998;20:85—92.
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14. Ripa LW. Sealants revisited: an update of the effectiveness of pit-and-fissure sealants. Caries Research 1993;27(suppl 1):77—82. 15. Silverstone LM, Hicks MJ, Featherstone MJ. Oral fluid contamination of etched enamel surfaces: an SEM study. Journal of the American Dental Association 1985;110: 329—32. 16. Hannig M, Reinhardt KJ, Bott B. Composite-to-dentin bond strength, micromorphology of the bonded dentin interface, and marginal adaptation of Class II composite resin restorations using self-etching primers. Operative Dentistry 2001;26:157—65. 17. Gwinnet AJ. Human ‘prismless’ enamel and its influence on sealant penetration. Archives of Oral Biology 1973;18: 441—4. 18. Gwinnet AJ. The ultrastructure of the ‘prismless’ enamel of permanent human teeth. Archives of Oral Biology 1967;12: 381—8. 19. Kodaka T, Kuroiwa H, Higashi S. Structural and distribution patterns of surface prismless enamel in human permanent teeth. Caries Research 1991;25:7—20.
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20. Garcia-Godoy F, Medlock JW. An SEM study of the effects of air-polishing on fissure surfaces. Quintessence International 1988;19:465—7. 21. Vesterhus Strand G, Raada M. The efficiency of cleaning fissures with an air-polishing instrument. Acta Odontologica Scandinavia 1988;46:113—7. 22. Brocklehurst PR, Joshi RI, Northeast SE. The effect of airpolishing occlusal surfaces on the penetration of fissures by a sealant. International Journal of Pediatric Dentistry 1992;2: 157—62. 23. Brockmann SL, Scott RL, Eick JD. A scanning electron microscopic study of the effect of air-polishing on the enamel sealant surface. Quintessence International 1990; 21:201—6. 24. Brockmann SL, Scott RL, Eick JD. The effect of an airpolishing device on tensile bond strength of a dental sealant. Quintessence International 1989;20: 211—7. 25. Scott L, Greer D. The effect of an air-polishing device on sealant bond strength. Journal of Prosthetic Dentistry 1987; 58:384—7.