Effect of Endodontic Irrigants on Microtensile Bond Strength to Dentin After Thermocycling and Long-Term Water Storage

Headnote

Abstract

Objective: The bond strength of adhesives in irrigated dentin behaves differently over time. The aim of this study was to evaluate the influence of long-term water storage and thermocycling on the microtensile bond strength of adhesive systems to dentin irrigated with endodontic solutions.

Materials and Methods: Sixty human molars were used after removal of the oc- clusal portion and exposure of the dentin by grinding. The specimens were irri- gated with 2.5% NaOCl for 30 minutes and then 17% EDTA for 5 minutes and assigned to six groups according to the adhesive system (n=10): Gl and G2- Clearfil SE Bond; G3 and G4-Single Bond 2; and G5 and G6-XP Bond. The teeth were restored with composite and were subjected to water storage for different time periods. Gl, G3 and G5 were stored for 24 h; G2, G4 and G6 were stored for 6 months and were subjected to thermocycling (12,000 cycles, 5°C to 55°C, 500 cycles per week for 6 months). After storage, the tooth/restoration assembly was sectioned to obtain four sticks of approximately 1 mm2, for microtensile bond strength testing. The results were analyzed by two-way ANOVA and Tukey's test.

Results: Significant differences were observed among the adhesives (p<0.01). No significant differences were observed in the microtensile bond strength between samples after 24 hours of storage without thermocycling and after 6-month sto- rage with 12,000 cycles (p<0.05).

Conclusion: The bond strengths of G5 and G6 after irrigation with 2.5% NaOCl and 17% EDTA were significantly different from those of other groups. Long- term water storage/thermocycling had no effect on bond strength to dentin.

Key Words: Microtensile Bond Strength; Self-etch; Total-etch; NaOCl, EDTA

Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2013; VoL 10, No. 5)

INTRODUCTION

Root canal irrigation during endodontic treat- ment is necessary for successful chemical- mechanical preparation [1,2], The function of endodontic irrigants is to facilitate preparation and disinfect the root canal [2],

These irrigants should not affect the dentin surface or interfere with restorative materials [3-6], The deleterious effects of certain irriga- tors, such as sodium hypochlorite, have been confirmed by previous studies, which have shown that bond strength is decreased [3,5-7],

Sodium hypochlorite is an alkaline solution [8] with a wide antibacterial spectrum and sporicidal and virucidal potential [9],

The concentration ranges between 0.5 and 5.25%, and its use is conditioned by factors such as time and the type of instruments used [10].

Another substance used as an irrigant is EDTA, which is a chelating agent capable of decalcifying dentin after 5 minutes of use, at 20 to 30 micrometers of depth [8],

Regardless of the category of the adhesive system, current adhesion depends on the inte- raction between dental adhesives and the smear layer [11], Etch-and-rinse adhesives remove the smear layer and superficial hy- droxyapatite through etching with a separate acid gel. Self-etch adhesives render the smear layer permeable without removing it com- pletely [11],

Adhesion durability is a parameter of the suc- cess of restorations. Degradation of the adhe- sive interface was demonstrated by Shono et al. [12], who found decreased bond strength as a function of storage time.

A commonly used in vitro aging technique is thermocycling that simulates the ingestion of hot and cold foods [13],

Given the importance of dentin sealing for a better prognosis with endodontic treatment, the evidence from previous studies that irri- gant substances directly affect adhesion [2,3,4] and the lack of information about the effects of endodontic irrigants on dentin after adhe- sive interface degradation, the aim of this in vitro study was to evaluate the influence of long-term water storage and thermocycling on the microtensile bond strength of adhesive systems to dentin irrigated with endodontic solutions.

MATHERIALS AND METHODS

Sixty caries-free, extracted human third mo- lars were selected for this study (obtained from the bank of teeth FORP-USP). The fol- lowing protocol was prescribed, revised and approved by the Committee of Ethics in Hu- man Research (2008.1.157.58.8). The teeth were stored in 0.2% thymol solution at 4°C for up to 6 months after extraction.

The teeth were embedded in polyester resin (Milflex ind. Química LTDA., Säo Bernardo do Campo, SP, Brazil) using plastic cylinders (15 mm in height and 20 mm in diameter), 1 mm apical to the limit between the crown and the root. Next, the teeth were mounted in a cutting machine (Isomet 1000, Buehler Ltd. Lake Bluff, IL, USA) and were sectioned to remove the occlusal surface at the middle third. The dentin surfaces were planed using #400 grit silicon carbide sandpaper (Buehler Ltd., Lake Bluff, IL, USA) under water cool- ing. The polishing procedure was performed until the deep dentin was exposed, as identi- fied by observation of the pulp horn by trans- parency. To produce standardized smear lay- ers, the teeth were subjected to polishing using #600 grit silicon carbide sandpaper (Buehler Ltd., Lake Bluff, IL, USA) under water cool- ing for 15 seconds.

The specimens previously obtained were di- vided randomly into six experimental groups. Subsequently, the teeth were immersed in 2.5% NaOCl (Farmácia da Terra, Ribeiräo Preto, SP, Brazil) for 30 minutes [14] and then immersed in 17% EDTA (Merck, Darmstadt, Germany) [15] for 5 minutes and rinsed in deionized water for 10 seconds in order to si- mulate the amount of time that dentin is in contact with endodontic irrigants.

The adhesive systems were applied to the den- tin surfaces following endodontic irrigation, according to the manufacturers' instructions (Table 1). The specimens in groups Gl and G2 were bonded with Clearfil SE Bond - CF (Ku- raray Co. Ltd., Umeda, Osaka, Japan) that was light cured for 10 seconds.

The specimens in groups G3, G4, G5 and G6 were conditioned with 37% phosphoric acid (Dentsply, York, PA, USA) for 15 seconds and rinsed for 30 seconds. Then, the excess water was removed with absorbent paper, keeping the dentin moist. In groups G3 and G4, Single Bond 2 - SB (3M Dental Products, St. Paul, MN, USA) was applied, and in G5 and G6, XP Bond -XP (Dentsply, York, PA, USA) was applied and light cured (Deme- tron/Kerr, Danbury, USA) for 10 seconds.

The teeth were restored using a microhybrid composite resin (Z100, 3M ESPE, shade A3, St. Paul, MN, USA).

Composite resin blocks (4 mm in height) were built with 4 increments of 1 mm thickness each. Each increment was light cured for 20 seconds, in a continuous mode as recommend- ed by the manufacturer.

Then, the specimens were kept in deionized water at 37oC for the respective storage times. After 24 hours, the teeth in groups Gl, G2 and G3 were attached to a cutting machine and were sectioned using a low-speed diamond saw; then, they were rotated 90o to produce sticks (1.0±0.2 mm2).

Approximately 8 sticks were obtained per tooth. No premature failures were observed during sectioning. Four sticks were selected from the central portion of each tooth. There- fore, 40 sticks were obtained for each group (Fig 1). For statistical purposes, the mean bond strength of all four sticks was consi- dered.

The specimens from groups G2, G4 and G6 were subjected to thermocycling (12,000 cycles, 5°C to 55°C, 30 second dwell time, 500 cycles per week for 6 months) (Etica Equipamentos Científicos S.A., Säo Paulo, SP, Brazil). The specimens were kept in deionized water at 37oC between the cycles that was re- placed weekly. At the end of water storage and thermocycling, the samples were cut into sticks according to the aforementioned tech- nique. The sticks were attached to a Universal Testing Machine (EMIC Equipamentos e Sis- temas de Ensaio LTD A, Säo José dos Pinhais, PR, Brazil), using a cyanoacrylate-based adhe- sive (Super Bonder, Henkel Ltda., Säo Paulo, SP, Brazil). The test was performed at a speed of 0.5 mm/min, using a 500 N load cell.

The bond strengths were recorded in MPa, and the cross-section areas of the sticks were measured using digital calipers (Mitutoyo, Tokyo, Japan).

Statistical analysis was performed using two- way ANOVA and Tukey's test at a 5% signi- ficance level.

Fracture types were determined using a ste- reomicroscope (40x magnification) (Leica S6 D Stereozoom, Leica Microsystems AG, Heerbrugg, Switzerland).

Failures were classified as adhesive (fracture of the dentin-adhesive interface), cohesive in dentin (fracture of dentin structure), cohesive in resin (resin fracture), and mixed (combina- tion of the previous types).

One tooth from each group was used. The specimens were then sectioned into two slabs of ~2 mm. The slabs were embedded in po- lyester resin, sanded with ascending grains (#600, #1200, #2400) and polished with 3 pm alumina paste (Struers A/S, Copenhagen, Denmark).

The specimens were placed in an ultrasonic cleaner (1440D, Odontobras, Ribeiräo Preto, Brazil) and cleaned with EDTA for 5 minutes, fixed in glutaraldehyde for 24 h, dehydrated in a battery of increasing concentrations of alco- hol, and gold sputter coated (Fig 2).

RESULTS

Two-way ANOVA revealed statistically sig- nificant differences among adhesives (p=0.007). There were no statistically signifi- cant differences with regard to aging (p=0,088) or the interaction (p=0.226) be- tween the adhesives and aging (Table 2).

XP presented lower microtensile bond strength compared to SB (p<0.01), and it was similar to CF at 24 hours. At 6 months, the adhesive sys- tems were similar (Table 2).

There were no statistically significant differ- ences between the microtensile bond strengths of the specimens stored for 24 hours and those of the same group analyzed after 6 months of water storage and 12,000 thermal cycles (Ta- ble 2).

The analysis of the fracture patterns revealed that the predominant type of fracture was mixed: 72.5% in Gl; 60% in G2; 75% in G3; 82.5% in G4; 72.5% in G5; and 80% in G6.

DISCUSSION

Endodontic irrigant solutions are applied to dissolve organic tissue, act as antimicrobials and help to debride the canal system because they are non-toxic to periapical tissues [16], However, some studies have shown that these substances can influence the quality of adhe- sion to dentin [6,7], particularly when using endodontic solutions at higher concentrations [10].

Coronal sealing is essential for successful en- dodontic therapy [17,18], Adhesive materials have also contributed to improving coronal sealing and increasing the retention of restora- tive materials [19] to dentin walls, increasing the possibility of marginal sealing, mechanical resistance to mastication stress and the dura- bility of restorations [19,20], The adhesive systems used in this study were selected be- cause of their proven successful results, ob- tained in both in vitro [21,22] and in vivo stu- dies [21,23] Another factor for the use of these adhesives was their different solvent contents, as shown in Table 2.

Time can have a negative effect on the dura- bility of the coronal sealing of composite resin restorations [13,24], Thermocycling accele- rates the hydrolysis of the adhesive interface components and enhances the coefficient of thermal expansion of the restorative material, producing stress at the interface [25-28],

It has been estimated that 10,000 cycles cor- respond to 1 year of in vivo activity [17],

However, our results showed that 6 months and 12,000 thermal cycles did not decrease the bond strength of dentin irrigated with 2.5% NaOCl and EDTA. Thus, the application of EDTA, which provided smear layer removal [10] and create demineralized dentin zones 2-4 [29], might have caused these results, thus al- lowing greater interaction of the adhesive sys- tems with dentin and enabling adhesive main- tenance independent of the adhesive system.

According to Myiasaki et al. [30], a combina- tion of thermocycling and storage in deionized water can simulate the tension generated in the oral cavity. In addition, it has been shown that water storage significantly reduces bond strength values after short periods of time [31], SEM micrographs (Fig 3) revealed sig- nificant interaction of the adhesive systems with demineralized dentin. The SB and CF adhesive systems presented similar hybrid lay- ers and evident resin tags, even after thermo- cycling; however, the presence of an interfa- cial gap was observed with the XP adhesive system. The gap observed under SEM might have been the result of the polishing of the samples, and the vacuuming could also have damaged the bond interface, resulting in its rupture [32],

Cohesive failures were found in only 15.41% of the samples. This low incidence of cohesive failures corroborates the findings of another study [23], The high number of mixed frac- tures (Graphic 1) could have resulted from strong interaction between the adhesives and the dentin substrate, as evidenced in the SEM analysis (Fig 3) that showed hybrid layer for- mation and the presence of tags for the adhe- sive systems.

Decrease bond strengths have been shown elsewhere after irrigation of dentin with 5.25% sodium hypochlorite for 30 minutes [6], Other in vitro studies have found that the bond strengths were reduced after the application of 5% sodium hypochlorite for 1 minute [4,33].

This finding indicates that as long as there is contact with the dentin, irrigant solution re- duces adhesive strength.

Studies have shown that when 5% NaOCl has been applied to dentin, self-etch adhesive sys- tems have better bond strength values, com- pared to etch-and-rinse adhesives [4,6,17,33], These findings differ from those of the present study that applied to 2.5% NaOCl and 17% EDTA and displayed the best values in teeth restored with Single Bond, suggesting that the adhesive system could be dependent on the irrigating solution's concentration.

XP exhibited the lowest bond strength values, with a significant difference from the SB ad- hesive system; however, both systems were similar to CF.

A possible explanation for this finding is the lower amount of calcium present in dentin af- ter the application of 2.5% hypochlorite and EDTA. According to Sayin et al. [34], the ap- plication of combined solutions promotes greater removal of calcium from dentin. The presence of calcium plays an important role in the binding of adhesives [34],

If an adhesive system has the monomer PENTA that has strong affinity for calcium in its composition, a lower quantity of calcium could decrease the bond strength of this adhe- sive system [35,36],

This behavior was also observed for the 10- MDP monomer present in Clearfil SE Bond that showed a statistical similarity to XP Bond at 24 hours. A possible explanation for the higher bond strength of Single Bond is the carboxylic group of polyalkenoic acid present in this adhesive system, as well as hydroxya- patite on the dentin surface or incompletely dissolved hydroxyapatite on exposed collagen fibrils forming ionic bonds that could have contributed to resin-dentin bonds [37, 38], Further studies, using mechanical degradation of the adhesive interface over time, are re- quired to evaluate the bond strength of dentin irrigated with endodontic substances.

CONCLUSION

Under the limitations of this in vitro study, it can be concluded that the bond strength of dentin irrigated with 2.5% NaOCl and 17% EDTA is influenced by the adhesive system used. Aging by thermocycling and water sto- rage did not interfere with the bond strength of the tested adhesive systems.

References

REFERENCES

1- Marending M, Luder HU, Brunner TJ, Knecht S, Stark WJ, Zehnder M. Effect of so- dium hypochlorite on human root dentine - mechanical, chemical and structural evalua- tion. Int Endod J. 2007 Oct;40( 10):786-93.

2- Zehnder M. Root canal irrigants. J Endod. 2006 May;32(5):389-98.

3- Fawzi EM, Elkassas DW, Ghoneim AG. Bonding strategies to pulp chamber dentin treated with different endodontic irrigants: mi- croshear bond strength testing and SEM anal- ysis. J Adhes Dent. 2010 Feb;12(l):63-70.

4- Ozturk B, Özer F. Effect of NaOCl on bond strengths of bonding agents to pulp chamber lateral walls. J Endod. 2004 May;30(5):362-5.

5- Pelegrine RA, De Martin AS, Cunha RS, Pelegrine AA, da Silveira Bueno CE. In- fluence of chemical irrigants on the tensile bond strength of an adhesive system used to cement glass fiber posts to root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol En- dod. 2010 Nov; 110(5):e73-6.

6- Santos JN, Carrilho MR, De Goes MF, Zaia AA, Gomes BP, Souza-Filho FJ et al. Effect of chemical irrigants on the bond strength of a self-etching adhesive to pulp chamber dentin. J Endod. 2006 Nov;32(l 1): 1088-90.

7- Nikaido T, Takano Y, Sasafuchi Y, Burrow MF, Tagami J. Bond strengths to endodonti- cally-treated teeth. Am J Dent. 1999 Aug; 12(4): 177-80.

8- Silva LA, Leonardo MR, Assed S, Tanoma- ru Filho M. Histological study of the effect of some irrigating solutions on bacterial endotox- in in dogs. Braz Dental J. 2004;15(2):109-14.

9- McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999 Jan; 12(1): 147-79.

10- Spanó JC, Barbin EL, Santos TC, Gui- maräes LF, Pécora JD. Solvent action of so- dium hypochlorite on bovine pulp and physi- co-chemical properties of resulting liquid. BrazDent J. 2001;12(3):154-7.

11- Perdigäo J. New developments in dental adhesion. Dent Clin North Am. 2007 Apr;51(2):333-57.

12- Shono Y, Terashita M, Shimada J, Kozo- no Y, Carvalho RM, Russell CM et al. Dura- bility of resin-dentin bonds. J Adhes Dent. 1999 Autumn; 1(3):211-8.

13- Gale MS, Darvell BW. Thermal cycling procedures for laboratory testing of dental res- torations. J Dent. 1999 Feb;27(2):89-99.

14- Tirali RE, Turan Y, Akal N, Karahan ZC. In vitro antimicrobial activity of several con- centrations of NaOCl and Octanisept in elimi- nation of endodontic pathogens. Oral Sur Oral Med Oral Pathol Oral Radiol Endod. 2009 Nov; 108(5):el 17-20.

15- Cunha RS, De Martin AS, Barros PP, da Silva FM, Jacinto Rde C, Bueno CE. In vitro evaluation of the cleansing working time and analysis of the amount of gutta-percha or Resi- lon remnants in the root canal walls after in- strumentation for endodontic retreatment. J Endod. 2007 Dec;33(12): 1426-8.

16- Cheung GS, Stock CJ. In vitro cleaning ability of root canal irrigants with and without endosonics. Int Endod J. 1993 Nov;26(6):334- 43.

17- Belli S, Zhang Y, Pereira PN, Pashley DH. Adhesive sealing of the pulp chamber. J Endod. 2001 Aug;27(8):521-6.

18- Ozturk B, Özer F, Belli S. An in vitro comparison of adhesive systems to seal pulp chamber walls. Int Endod J. 2004 May;37(5):297-306.

19- Cecchin D, Farina AP, Galafassi D, Barbizam JV, Corona SA, Carlini-Júnior B. Influence of sodium hypochlorite and edta on the microtensile bond strength of a self- etching adhesive system. J Appl Oral Sei. 2010 Jul-Aug;18(4):385-9.

20- Fortin D, Swift EJ Jr, Denehy GE, Rein- hardt JW. Bond strength and microleakage of current dentin adhesives. Dent Mater. 1994 Jul;10(4):253-8.

21- Hebling J, Castro FL, Costa CA. Adhesive performance of dentin bonding agents applied in vivo and in vitro. Effect of intrapulpal pres- sure and dentin depth. J Biomed Mater Res B Appl Biomater. 2007 Nov;83(2):295-303.

22- Manhart J, Trumm C. Marginal adaptation of an etch-and-rinse adhesive with a new type of solvent in class II cavities after artificial aging. Clin Oral Investig. 2010 Dec;14(6):699-705.

23- Frankenberger R, Krämer N, Lohbauer U, Nikolaenko SA, Reich SM. Marginal integrity: is the clinical performance of bonded restora- tions predictable in vitro?. J Adhes Dent. 2007;9 Suppl 1:107-16.

24- Giannini M, Seixas CA, Reis AF, Pimenta LA. Six-month storage-time evaluation of one-bottle adhesive systems to dentin. J Esthet RestorDent. 2003; 15(1):43-8.

25- Bedran-de-Castro AK, Pereira PN, Pimen- ta LA, Thompson JY. Effect of thermal and mechanical load cycling on microtensile bond strength of a total-etch adhesive system. Oper Dent. 2004 Mar-Apr;29(2): 150-6.

26- De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M et al. A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res. 2005 Feb;84(2): 118-32.

27- Dos Santos PA, Garcia PP, Palma-Dibb RG. Shear bond strength of adhesive systems to enamel and dentin. Thermocycling influ- ence. J Mater Sei Mater Med. 2005 Aug;16(8):727-32.

28- Ebert J, Löffler C, Roggendorf MJ, Petschelt A, Frankenberger R. Clinical adhesive sealing of the pulp chamber follow- ing endodontic treatment: influence of ther- momechanical loading on microleakage. J Adhes Dent. 2009 Aug; 11(4):311-7.

29- García-Godoy F, Loushine RJ, Itthagarun A, Weller RN, Murray PE, Feilzer AJ et al. Application of biologically-oriented dentin bonding principles to the use of endodontic irrigants. Am J Dent. 2005 Aug; 18(4):281-90.

30- Miyazaki M, Sato M, Onose H, Moore BK. Influence of thermal cycling on dentin bond strength of two-step bonding systems. Am J Dent. 1998 Jun;l 1(3):118-22.

31- Okuda M, Pereira PN, Nakajima M, Ta- gami J, Pashley DH. Long-term durability of resin dentin interface: nanoleakage vs. micro- tensile bond strength. Oper Dent. 2002 May- Jun;27(3):289-96.

32- Van Meerbeek B, Vargas M, Inoue S, Yoshida Y, Perdigäo J, Lambrechts P et al. Microsco- py investigations. Techniques, results, limit ations. Am J Dent. 2000 Nov: 13 (Spec No):3D-18D.

33- Erdemir A, Ari H, Güngüneç H, Belli S. Effect of medications for root canal treatment on bonding to root canal dentin. J Endod. 2004 Feb;30(2):113-6.

34- Sayin TC, Serper A, Cehreli ZC, Kalayci S. Calcium loss from root canal dentin follow- ing EDTA, EGTA, EDTAC, and tetracycline- HC1 treatment with or without subsequent NaOCl irrigation. J Endod. 2007 May;33(5):581-4.

35- Inai N, Kanemura N, Tagami J, Watanabe LG, Marshall SJ, Marshall GW. Adhesion be- tween collagen depleted dentin and dentin ad- hesives. Am J Dent. 1998 Jun; 11(3): 123-7.

36- Toledano M, Perdigäo J, Osorio R, Osorio E. Effect of dentin deproteinization on micro- leakage of Class V composite restorations. Oper Dent. 2000 Nov-Dec;25(6):497-504.

37- Kim DS, Park SH, Choi GW, Choi KK, Kim SY. Effect of EDTA treatment on the hybrid layer durability in total- etch dentin adhesives. Dent Mater J. 2011;30(5):717-22.

38- Fukuda R, Yoshida Y, Nakayama Y, Oka- zaki M, Inoue S, Sano H et al. Bonding effica- cy of polyalkenoic acids to hydroxyapatite, enamel and dentin. Biomaterials. 2003 May;24(ll): 1861-7.