- Email: [email protected]
Using Extracted Teeth for Research
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Using extracted teeth for research The effect of storage medium and sterilization on dentin bond strengths Jason Jonghyuk Lee, DDS; Anthony Nettey-Marbell, DDS; Archie Cook Jr, DDS; Luiz A.F. Pimenta, DDS, MS, PhD; Ralph Leonard, DDS, MPH; André V. Ritter, DDS, MS
ne of the ways in which clinicians select products for their practices is to compare the products’ performances in in vivo and in vitro studies. One method used often to assess the performance of dental adhesives is to test how well they bond to dentin by measuring their composite-todentin bond strengths. When dental specimens are used for in vitro adhesive studies, one of the factors that can affect the study outcome is how the specimens are stored and sterilized. The Centers for Disease Control and Prevention (CDC) has adopted guidelines for infection control of extracted teeth used for research and teaching.1 These guidelines require that teeth not containing amalgam be heat-sterilized by an autoclave cycle for 40 minutes before use. Teeth that contain amalgam should be stored in 10 percent formalin for two weeks before use. Although autoclaving does not seem to alter the tooth’s physical properties sufficiently for research purposes, it is unknown whether autoclaving affects the chemical and micromechanical relationship between dentin and dental
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ABSTRACT Background. The Centers for Disease Control and Prevention has adopted guidelines for infection control of extracted teeth used for research and teaching, requiring that teeth be sterilized before use. The authors conducted a study to test the null hypothesis that the storage medium and sterilization method have no effect on composite-to-dentin bond strengths. Materials and Methods. The authors collected 170 bovine incisors, cleaned them and placed them randomly into one of six storage media at 37°C for 60 days: distilled water (dH2O), 0.9 percent sodium chloride, 0.5 percent chloramine-T, 5.25 percent sodium hypochlorite (NaClO), 2 percent glutaraldehyde and 10 percent formalin. For sterilization, they autoclaved a subset of 10 specimens from every sample, while they stored another subset of 10 specimens from every sample (except for the 10 percent formalin sample) in 10 percent formalin for 14 days. The authors then embedded the specimens in epoxy and ground flat the facial surface to expose middledepth dentin, which they polished to 600 grit. They used a dental adhesive to apply composite to the exposed dentin. The authors tested the compositeto-dentin shear bond strength 24 hours after bonding. They analyzed the data using global analysis of variance and, when appropriate, multiple post hoc tests (P = .05). Results. Storage in NaClO resulted in significantly lower bond strength than that of the other treatment specimens. Sterilization with the autoclave negatively affected the bond strength of specimens stored initially in dH2O or 10 percent formalin, while sterilization with formalin alone had no significant effect on bond strengths. Conclusions. Storing bovine teeth in 5.25 percent NaClO may negatively affect composite-to-dentin bond strengths. Immersion in 10 percent formalin might be the best option for storage and sterilization of bovine teeth that are to be used in dental bonding studies in vitro. Key Words. Storage medium; extracted bovine teeth; sterilization of teeth; dentin bonding. JADA 2007;138(12):1599-1603.
At the time this study was conducted, Dr. Lee was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is a staff dentist and clinical supervisor, Tri-County Family Dental Center, Dunn, N.C. At the time this study was conducted, Dr. Nettey-Marbell was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is in private practice in Raleigh, N.C. At the time this study was conducted, Dr. Cook was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is a captain in the U.S. Air Force, assigned to the 375th Dental Squadron, Scott Air Force Base, Ill. Dr. Pimenta is a clinical professor, Department of Dental Ecology, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, N.C. Dr. Leonard is a clinical professor, Department of Diagnostic Sciences and General Dentistry, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, N.C. Dr. Ritter is an associate professor, Department of Operative Dentistry, University of North Carolina at Chapel Hill School of Dentistry, 441 Brauer Hall, CB#7450, Chapel Hill, N.C. 27599-7450, e-mail “[email protected]”. Address reprint requests to Dr. Ritter.
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materials.1 Teeth often are stored in a solution after they are collected and before sterilization, and it is not clear how storage solutions affect enamel and dentin. Storage solutions and sterilization methods might influence how these substrates respond to resin-based composite bonding. Investigators commonly use in vitro shear bond strength (SBS) tests to analyze quantitatively and rank the bonding performance of adhesive resins on enamel and dentin surfaces.2-4 Although SBS tests are used widely in vitro,5 they have been criticized for their lack of standardization and consistency, as well as for their potential to produce tensile failure rather than shear failure.6,7 However, when compared with tensile and microtensile bond tests, the SBS test has been shown to be appropriate and effective as a tool for the evaluation and comparison of different bonding systems in vitro.8,9 Nevertheless, it is unlikely that the results obtained in these mechanical tests can be extrapolated to predict the clinical performance of the materials tested,10 although they can be used as an effective screening tool. The purpose of our in vitro study was to evaluate the effect of storage medium and sterilization methods on composite-to-dentin SBS. The study tested the null hypothesis that storage medium and sterilization methods have no effect on composite-to-dentin bond strengths. MATERIALS AND METHODS
Storage medium. We used 170 bovine incisor crowns in this study (Randolph Packing, Asheboro, N.C.).11,12 We cleaned the specimens of debris and assigned them randomly to one of the following storage solutions: distilled water (dH2O) (n = 30), 0.9 percent sodium chloride (NaCl) (n = 30), 0.5 percent chloramine-T (n = 30), 5.25 percent sodium hypochlorite (NaClO) (n = 30), 2 percent glutaraldehyde (n = 30) and 10 percent neutral buffered formalin (n = 20). We stored the specimens at 37°C for 60 days. Sterilization. After the initial 60-day storage period, we selected 10 randomly chosen specimens from each sample and autoclaved them in dH2O for 40 minutes. Simultaneously, we randomly selected an additional 10 specimens from each sample (with the exception of the sample stored in formalin) and stored them in 10 percent formalin for 14 days. Bond strength testing. After completing the storage and sterilization treatments, we mounted 1600
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all specimens in phenolic rings (Buehler, Lake Bluff, Ill.) with epoxy resin (Buehler). We then ground flat the labial surface of each tooth with 120-grit silicon carbide abrasive paper (Buehler) under running water and using mild pressure to expose the middle-depth dentin. To create a standardized smear layer, we polished dentin surfaces with wet 240-, 400- and 600-grit silicon carbide abrasive paper (Buehler).13 We etched the exposed dentin surfaces with 35 percent phosphoric acid (Scotchbond Etchant, 3M ESPE, St. Paul, Minn.) for 15 seconds, rinsed the surfaces for 15 seconds and blot-dried them for two seconds. We applied to the dentin surfaces two consecutive coats of adhesive (Adper Single Bond Plus, 3M ESPE), dried them for two seconds and light-cured them for 10 seconds. We bonded resin-based composite (Filtek Z250 Universal Restorative, 3M ESPE) to the adhesivecoated dentin surface by using a split matrix. We inserted the resin-based composite into the matrix in two 2-millimeter thick increments and light-cured them for 20 seconds each. Immediately afterward, we opened the split matrix and light-cured the composite post for an additional 40 seconds. The matrix resulted in a uniform bonded area of 2.38 square millimeters. We chose these materials as representative examples of currently used adhesives and resin-based composites; however, they certainly do not represent every single category of dental adhesive and restorative material. For all light-curing procedures, we used a quartz-tungsten-halogen light-curing unit (XL 3000, 3M ESPE). We used a curing radiometer (Kerr, Orange, Calif.) to monitor the curing light intensity, which ranged from 450 to 500 milliwatts/square centimeter. After carefully examining each composite post for uniform diameter at the base, we immersed the specimens in distilled water at 37˚C immediately after bonding. After 24 hours, we tested the specimens using a universal testing machine (Instron, Canton, Mass.) at a crosshead speed of 0.5 mm/minutes. We placed the shear knife parallel to and approximately 0.2 mm from the dentin surface, perpendicular to the composite post.
ABBREVIATION KEY. CDC: Centers for Disease Control and Prevention. dH20: Distilled water. NaCl: Sodium chloride. NaClO: Sodium hypochlorite. SBS: Shear bond strength.
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Data analysis. We calculated the SBS by dividing the failure load by the bonded surface area and expressed the result in megapascals. We used software (TestWorks, MTS Systems, Eden Prairie, Minn.) to record the data. We carried out the statistical analysis by using the R software package (Free Software Foundation, Boston), subjecting the data to analysis of variance (ANOVA) at the .05 significance level. We performed a Fisher post hoc test when ANOVA revealed a statistically significant difference between the samples (P < .05). RESULTS
The mean and standard deviation SBS values are shown in the table. Raw mean SBS values varied greatly across all samples and ranged from 3.6 MPa (storage only, NaClO) to 18.9 MPa (storage only, dH2O). An ANOVA F test produced a P value of 2.755 × 10-12 (P < .05), indicating that significant differences existed between treatments. Bond strengths were influenced significantly by the storage medium. Autoclaving significantly affected the bond strengths of specimens stored in dH2O, NaClO and formalin, while the formalin treatment significantly affected the bond strengths of specimens stored in NaCl and NaClO. DISCUSSION
According to the CDC guidelines,1 it is necessary to sterilize dental specimens with either an autoclave or formalin for research purposes. In this study, we investigated the effects of various storage media and subsequent sterilization on the composite-to-dentin bond strength. The specimens were stored in each solution for 60 days to measure presumably strong effects of the storage condition within a relatively long storage time. SBS values. When specimens were stored in dH2O and were not sterilized, SBS values were significantly higher than they were when specimens were stored in NaCl or NaClO. It is possible that residual chlorine from these storage solutions might have negatively influenced bond strengths. However, when specimens were autoclaved after the initial storage period, we noted no differences between storage media. When specimens were treated with formalin after the initial storage period, the only storage medium that affected SBS negatively was NaClO. Autoclaving and formalin treatment. When we compared solutions as a function of
sterilization treatment (Table), autoclaving significantly reduced SBS values for specimens stored in dH2O (from 18.9 MPa to 11.0 MPa) and formalin (from 18.0 MPa to 10.6 MPa). On the other hand, autoclaving and formalin treatment increased SBS values significantly for NaClOstored specimens (from 3.6 MPa to 10.5 MPa and 7.8 MPa, respectively), while autoclaving and formalin treatment did not significantly affect the SBS values of chloramine T- and glutaraldehydestored specimens. Autoclaving increased the mean SBS values only for specimens stored in NaCl (not significantly) and NaClO (significantly), the two storage solutions that resulted in significantly lower SBS values when compared with dH2O when specimens were tested after storage only. For all other samples, autoclaving reduced mean SBS values. Consequently, autoclaving had an “equalizing effect” on SBS values, which resulted in no significant differences being noted for SBS values of specimens stored and autoclaved, regardless of the storage medium. We observed a similar trend for specimens sterilized with formalin, with the exception of specimens stored in NaClO followed by treatment with formalin; the mean SBS values for these specimens were significantly lower than those for the other samples treated with formalin. On the basis of these findings, we conclude that regardless of the initial storage solution, autoclaving the specimens tended to level the SBS values after the initial storage period. This effect was detrimental for specimens stored initially in dH2O or 10 percent formalin. NaClO storage also produced a change in the specimens’ color; they became a lighter hue of yellow in contrast to no color changes for the specimens stored in other solutions. This color change may be due to the oxidizing activity of NaClO or to residual chlorine; however, further investigation is required to determine the definitive reason for the color change. NaClO storage. Of all the storage solutions, NaClO appears to be the one that most negatively affects dentin bonding, especially if bonding is done immediately after storage or after sterilization with formalin. Therefore, we do not recommend NaClO as a storage solution for extracted teeth that will be used for mechanical tests of composite-to-dentin bonding. Results of previous evaluations of dentin bonding after short treatment with NaClO have been inconclusive.14-19 One clinical study that evaluated the two-year per-
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this technology is not easily available and Shear bond strengths for all groups. also is expensive, GROUP MEAN (SD*) SHEAR BOND STRENGTH IN MEGAPASCALS†‡ which probably puts it Storage Storage and Storage and 10% at a disadvantage Autoclave Formalin when compared with 18.9 (7.0)aA 11.0 (4.3)bA 12.6 (4.4)abA Control (Distilled Water) other alternatives, bB bA aA such as storage in 8.2 (4.4) 8.7 (4.2) 15.3 (6.9) 0.9% Sodium Chloride dH2O or 10 percent 14.1 (5.6)aAB 11.5 (5.3)aA 15.5 (4.1)aA 0.5% Chloramine-T formalin. 3.6 (4.7)bC 10.5 (5.4)aA 7.8 (3.9)aB 5.25% Sodium Study limitations. Hypochlorite This study, however, 13.1 (4.0)aAB 11.2 (4.7)aA 12.2 (4.5)aA 2% Glutaraldehyde is not free from limitaaA bA § tions. Maintaining 18.0 (7.5) 10.6 (4.9) NA 10% Formalin even thickness of the * SD: Standard deviation. † The same lowercase superscript letters across groups (rows) indicate means that are not significantly phenolic rings loaded different at P = .05. with a bovine incisor ‡ The same uppercase superscript letters between groups (columns) indicate means that are not significantly different at P = .05. on grinding needs to § NA: Not applicable. be more carefully controlled, because a formance of resin-based composite restorations slanted surface may provide more surface area for placed with and without NaClO treatment dentin bonding, leading to error on shear strength showed that treating dentin with 10 percent testing. NaClO to remove exposed collagen after acidThere also has been a concern regarding the consistency of SBS testing owing to a possibility etching did not affect the clinical performance of that a portion of dentin may fracture off with an restorations placed with an acetone- or ethanolintact bonded interface6,7; however, further invesbased adhesive in teeth with nonretentive 20 noncarious cervical lesions. tigations of the surface morphology of ground Therefore, it appears that the negative effect of dentin and the shear-tested dentin surface are the NaClO solution used in our study likely was needed to verify this phenomenon. However, we due to the long-term storage. We can infer that did not observe fracture within the composite post long-term treatment of tooth structures with as a result of shearing force in any specimens, NaClO should be avoided if the teeth are to be indicating that the fracture occurred at the interused for bonding. NaClO also is commonly used face. This might be due, in part, to the relatively as an irrigant during endodontic treatment, slow crosshead speed used (0.5 mm/minute), because of its antibacterial activity and ability to which we selected on the basis of a previous study dissolve tissue. The results of this study suggest showing that this speed was less likely to induce that residual NaClO left on an endodontically cohesive fracture on the substrate.23 treated tooth might interfere with bonding of Although SBS tests commonly are used in in composite to the tooth. vitro bond strength studies,2,3,5 they have been Gamma irradiation. Another possible sterilcriticized for their lack of standardization and ization alternative not evaluated in this study is consistency.6,7 When compared with other bond the use of gamma irradiation. Sterilization via strength tests (that is, tensile and microtensile), gamma irradiation often is done for bone and for however, the SBS test has been shown to be banking extracted teeth. Researchers have invesappropriate and effective as a tool for evaluating tigated the bond strength of human dentin suband contrasting different bonding systems in mitted to gamma rays and the morphological vitro.8,9 Scanning electron microscopy also could 21,22 changes in dentin. The results of these studies provide more information than is obtainable with showed that gamma irradiation did not produce the SBS test with regard to the fracture site of structural changes in dentin. Scanning electron the bonding samples, as well as possible clues to micrographs also did not show alterations. Thus, the change in color caused by the 5.25 percent gamma irradiation neither affected the SBS nor NaClO solution. altered the dentin surface morphology; however, Finally, we used bovine teeth for consistency
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and convenience. Although human teeth ideally should have been used, it would be difficult to identify and collect a homogeneous sample of human teeth for this study. Studies have shown that bovine teeth are a suitable substitute for human teeth in bonding studies.11,12 CONCLUSIONS
In this study, we investigated the effect of storage conditions and sterilization methods on the composite-to-dentin bond strength. Within the limitations of this study, we can draw the following conclusions. When storage solutions alone are compared, 0.9 percent NaCl and 5.25 percent NaClO resulted in significantly lower SBS values than those obtained with dH2O. Therefore, investigators should not use these solutions as the storage medium when teeth are to be used for dentin bonding studies. Sterilization with an autoclave negatively affected the SBS values of specimens stored initially in dH2O or 10 percent formalin, while sterilization with formalin alone had no significant effect. Although storage in dH2O resulted in the highest nominal SBS values among the sample studied, storage and simultaneous sterilization in 10 percent formalin appears to be the most logical treatment for extracted teeth that will be used in dentin bonding research. ■ The authors thank Dr. Ceib Philips and Mr. Michael Pannucci for assisting with the statistical analysis. 1. Kohn W, Collins A., Cleveland J, Harte J, Eklund K, Malvitz D; Centers for Disease Control and Prevention. Guidelines for infection control in dental health-care settings: 2003. MMWR Recomm Rep 2003;52(RR-17):1-61. 2. Triolo PT Jr, Swift EJ Jr. Shear bond strengths of ten dentin adhesive systems. Dent Mater 1992;8(6):370-4. 3. Triolo PT Jr, Swift EJ Jr, Barkmeier WW. Shear bond strengths of composite to dentin using six dental adhesive systems. Oper Dent
1995;20(2):46-50. 4. Fritz UB, Finger WJ, Uno S. Resin-modified glass ionomer cements: bonding to enamel and dentin. Dent Mater 1996;12(3):161-6. 5. al-Salehi SK, Burke FJ. Methods used in dentin bonding tests: an analysis of 50 investigations on bond strength. Quintessence Int 1997;28(11):717-23. 6. Van Noort R, Noroozi S, Howard IC, Cardew G. A critique of bond strength measurements. J Dent 1989;17(2):61-7. 7. Versluis A, Tantbirojn D, Douglas WH. Why do shear bond tests pull out dentin? J Dent Res 1997;76(6):1298-307. 8. Fowler CS, Swartz ML, Moore BK, Rhodes BF. Influence of selected variables on adhesion testing. Dent Mater 1992;8(4):265-9. 9. Cardoso PE, Braga RR, Carrilho MR. Evaluation of micro-tensile, shear and tensile tests determining the bond strength of three adhesive systems. Dent Mater 1998;14(6):394-8. 10. Eliades G. Clinical relevance of the formulation and testing of dentine bonding systems. J Dent 1994;22(2):73-81. 11. Nakamichi I, Iwaku M, Fusayama T. Bovine teeth as possible substitutes in the adhesion test. J Dent Res 1983;62(10):1076-81. 12. Schilke R, Bauss O, Lisson JA, Schuckar M, Geurtsen W. Bovine dentin as a substitute for human dentin in shear bond strength measurements. Am J Dent 1999;12(2):92-6. 13. Pashley DH, Tao L, Boyd L, King GE, Horner JA. Scanning electron microscopy of the substructure of smear layers in human dentine. Arch Oral Biol 1988;33(4):265-70. 14. Nikaido T, Takano Y, Sasafuchi Y, Burrow MF, Tagami J. Bond strengths to endodontically-treated teeth. Am J Dent 1999;12(4):177-80. 15. Vongphan N, Senawongse P, Somsiri W, Harnirattisai C. Effects of sodium ascorbate on microtensile bond strength of total-etching adhesive system to NaOCl treated dentine. J Dent 2005;33(8):689-95. 16. Saboia VP, Rodrigues AL, Pimenta LA. Effect of collagen removal on shear bond strength of two single-bottle adhesive systems. Oper Dent 2000;25(5):395-400. 17. Perdigão J, Lopes M, Geraldeli S, Lopes GC, García-Godoy F. Effect of a sodium hypochlorite gel on dentin bonding. Dent Mater 2000;16(5):311-23. 18. Pimenta LA, Amaral CM, Bedran de Castro AK, Ritter AV. Stability of dentin bond strengths using different bonding techniques after 12 months: total-etch, deproteinization and self-etching. Oper Dent 2004;29(5):592-8. 19. Pioch T, Kobaslija S, Schagen B, Götz H. Interfacial micromorphology and tensile bond strength of dentin bonding systems after NaOCl treatment. J Adhes Dent 1999;1(2):135-42. 20. Saboia Vde P, Almeida PC, Ritter AV, Swift EJ Jr, Pimenta LA. 2-year clinical evaluation of sodium hypochlorite treatment in the restoration of non-carious cervical lesions: a pilot study. Oper Dent 2006; 31(5):530-5. 21. Sperandio M, Souza JB, Oliveira DT. Effect of gamma radiation on dentin bond strength and morphology. Braz Dent J 2001;12(3):205-8. 22. DeWald JP. The use of extracted teeth for in vitro bonding studies: a review of infection control considerations. Dent Mater 1997;13(2): 74-81. 23. Hara AT, Pimenta LA, Rodrigues AL Jr. Influence of cross-head speed on resin-dentin shear bond strength. Dent Mater 2001;17(2): 165-9.
JADA, Vol. 138 http://jada.ada.org Copyright ©2007 American Dental Association. All rights reserved.
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Using extracted teeth for research The effect of storage medium and sterilization on dentin bond strengths Jason Jonghyuk Lee, DDS; Anthony Nettey-Marbell, DDS; Archie Cook Jr, DDS; Luiz A.F. Pimenta, DDS, MS, PhD; Ralph Leonard, DDS, MPH; André V. Ritter, DDS, MS
ne of the ways in which clinicians select products for their practices is to compare the products’ performances in in vivo and in vitro studies. One method used often to assess the performance of dental adhesives is to test how well they bond to dentin by measuring their composite-todentin bond strengths. When dental specimens are used for in vitro adhesive studies, one of the factors that can affect the study outcome is how the specimens are stored and sterilized. The Centers for Disease Control and Prevention (CDC) has adopted guidelines for infection control of extracted teeth used for research and teaching.1 These guidelines require that teeth not containing amalgam be heat-sterilized by an autoclave cycle for 40 minutes before use. Teeth that contain amalgam should be stored in 10 percent formalin for two weeks before use. Although autoclaving does not seem to alter the tooth’s physical properties sufficiently for research purposes, it is unknown whether autoclaving affects the chemical and micromechanical relationship between dentin and dental
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ABSTRACT Background. The Centers for Disease Control and Prevention has adopted guidelines for infection control of extracted teeth used for research and teaching, requiring that teeth be sterilized before use. The authors conducted a study to test the null hypothesis that the storage medium and sterilization method have no effect on composite-to-dentin bond strengths. Materials and Methods. The authors collected 170 bovine incisors, cleaned them and placed them randomly into one of six storage media at 37°C for 60 days: distilled water (dH2O), 0.9 percent sodium chloride, 0.5 percent chloramine-T, 5.25 percent sodium hypochlorite (NaClO), 2 percent glutaraldehyde and 10 percent formalin. For sterilization, they autoclaved a subset of 10 specimens from every sample, while they stored another subset of 10 specimens from every sample (except for the 10 percent formalin sample) in 10 percent formalin for 14 days. The authors then embedded the specimens in epoxy and ground flat the facial surface to expose middledepth dentin, which they polished to 600 grit. They used a dental adhesive to apply composite to the exposed dentin. The authors tested the compositeto-dentin shear bond strength 24 hours after bonding. They analyzed the data using global analysis of variance and, when appropriate, multiple post hoc tests (P = .05). Results. Storage in NaClO resulted in significantly lower bond strength than that of the other treatment specimens. Sterilization with the autoclave negatively affected the bond strength of specimens stored initially in dH2O or 10 percent formalin, while sterilization with formalin alone had no significant effect on bond strengths. Conclusions. Storing bovine teeth in 5.25 percent NaClO may negatively affect composite-to-dentin bond strengths. Immersion in 10 percent formalin might be the best option for storage and sterilization of bovine teeth that are to be used in dental bonding studies in vitro. Key Words. Storage medium; extracted bovine teeth; sterilization of teeth; dentin bonding. JADA 2007;138(12):1599-1603.
At the time this study was conducted, Dr. Lee was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is a staff dentist and clinical supervisor, Tri-County Family Dental Center, Dunn, N.C. At the time this study was conducted, Dr. Nettey-Marbell was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is in private practice in Raleigh, N.C. At the time this study was conducted, Dr. Cook was a dental student, University of North Carolina at Chapel Hill School of Dentistry. He now is a captain in the U.S. Air Force, assigned to the 375th Dental Squadron, Scott Air Force Base, Ill. Dr. Pimenta is a clinical professor, Department of Dental Ecology, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, N.C. Dr. Leonard is a clinical professor, Department of Diagnostic Sciences and General Dentistry, University of North Carolina at Chapel Hill School of Dentistry, Chapel Hill, N.C. Dr. Ritter is an associate professor, Department of Operative Dentistry, University of North Carolina at Chapel Hill School of Dentistry, 441 Brauer Hall, CB#7450, Chapel Hill, N.C. 27599-7450, e-mail “[email protected]”. Address reprint requests to Dr. Ritter.
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December 2007
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materials.1 Teeth often are stored in a solution after they are collected and before sterilization, and it is not clear how storage solutions affect enamel and dentin. Storage solutions and sterilization methods might influence how these substrates respond to resin-based composite bonding. Investigators commonly use in vitro shear bond strength (SBS) tests to analyze quantitatively and rank the bonding performance of adhesive resins on enamel and dentin surfaces.2-4 Although SBS tests are used widely in vitro,5 they have been criticized for their lack of standardization and consistency, as well as for their potential to produce tensile failure rather than shear failure.6,7 However, when compared with tensile and microtensile bond tests, the SBS test has been shown to be appropriate and effective as a tool for the evaluation and comparison of different bonding systems in vitro.8,9 Nevertheless, it is unlikely that the results obtained in these mechanical tests can be extrapolated to predict the clinical performance of the materials tested,10 although they can be used as an effective screening tool. The purpose of our in vitro study was to evaluate the effect of storage medium and sterilization methods on composite-to-dentin SBS. The study tested the null hypothesis that storage medium and sterilization methods have no effect on composite-to-dentin bond strengths. MATERIALS AND METHODS
Storage medium. We used 170 bovine incisor crowns in this study (Randolph Packing, Asheboro, N.C.).11,12 We cleaned the specimens of debris and assigned them randomly to one of the following storage solutions: distilled water (dH2O) (n = 30), 0.9 percent sodium chloride (NaCl) (n = 30), 0.5 percent chloramine-T (n = 30), 5.25 percent sodium hypochlorite (NaClO) (n = 30), 2 percent glutaraldehyde (n = 30) and 10 percent neutral buffered formalin (n = 20). We stored the specimens at 37°C for 60 days. Sterilization. After the initial 60-day storage period, we selected 10 randomly chosen specimens from each sample and autoclaved them in dH2O for 40 minutes. Simultaneously, we randomly selected an additional 10 specimens from each sample (with the exception of the sample stored in formalin) and stored them in 10 percent formalin for 14 days. Bond strength testing. After completing the storage and sterilization treatments, we mounted 1600
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all specimens in phenolic rings (Buehler, Lake Bluff, Ill.) with epoxy resin (Buehler). We then ground flat the labial surface of each tooth with 120-grit silicon carbide abrasive paper (Buehler) under running water and using mild pressure to expose the middle-depth dentin. To create a standardized smear layer, we polished dentin surfaces with wet 240-, 400- and 600-grit silicon carbide abrasive paper (Buehler).13 We etched the exposed dentin surfaces with 35 percent phosphoric acid (Scotchbond Etchant, 3M ESPE, St. Paul, Minn.) for 15 seconds, rinsed the surfaces for 15 seconds and blot-dried them for two seconds. We applied to the dentin surfaces two consecutive coats of adhesive (Adper Single Bond Plus, 3M ESPE), dried them for two seconds and light-cured them for 10 seconds. We bonded resin-based composite (Filtek Z250 Universal Restorative, 3M ESPE) to the adhesivecoated dentin surface by using a split matrix. We inserted the resin-based composite into the matrix in two 2-millimeter thick increments and light-cured them for 20 seconds each. Immediately afterward, we opened the split matrix and light-cured the composite post for an additional 40 seconds. The matrix resulted in a uniform bonded area of 2.38 square millimeters. We chose these materials as representative examples of currently used adhesives and resin-based composites; however, they certainly do not represent every single category of dental adhesive and restorative material. For all light-curing procedures, we used a quartz-tungsten-halogen light-curing unit (XL 3000, 3M ESPE). We used a curing radiometer (Kerr, Orange, Calif.) to monitor the curing light intensity, which ranged from 450 to 500 milliwatts/square centimeter. After carefully examining each composite post for uniform diameter at the base, we immersed the specimens in distilled water at 37˚C immediately after bonding. After 24 hours, we tested the specimens using a universal testing machine (Instron, Canton, Mass.) at a crosshead speed of 0.5 mm/minutes. We placed the shear knife parallel to and approximately 0.2 mm from the dentin surface, perpendicular to the composite post.
ABBREVIATION KEY. CDC: Centers for Disease Control and Prevention. dH20: Distilled water. NaCl: Sodium chloride. NaClO: Sodium hypochlorite. SBS: Shear bond strength.
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Data analysis. We calculated the SBS by dividing the failure load by the bonded surface area and expressed the result in megapascals. We used software (TestWorks, MTS Systems, Eden Prairie, Minn.) to record the data. We carried out the statistical analysis by using the R software package (Free Software Foundation, Boston), subjecting the data to analysis of variance (ANOVA) at the .05 significance level. We performed a Fisher post hoc test when ANOVA revealed a statistically significant difference between the samples (P < .05). RESULTS
The mean and standard deviation SBS values are shown in the table. Raw mean SBS values varied greatly across all samples and ranged from 3.6 MPa (storage only, NaClO) to 18.9 MPa (storage only, dH2O). An ANOVA F test produced a P value of 2.755 × 10-12 (P < .05), indicating that significant differences existed between treatments. Bond strengths were influenced significantly by the storage medium. Autoclaving significantly affected the bond strengths of specimens stored in dH2O, NaClO and formalin, while the formalin treatment significantly affected the bond strengths of specimens stored in NaCl and NaClO. DISCUSSION
According to the CDC guidelines,1 it is necessary to sterilize dental specimens with either an autoclave or formalin for research purposes. In this study, we investigated the effects of various storage media and subsequent sterilization on the composite-to-dentin bond strength. The specimens were stored in each solution for 60 days to measure presumably strong effects of the storage condition within a relatively long storage time. SBS values. When specimens were stored in dH2O and were not sterilized, SBS values were significantly higher than they were when specimens were stored in NaCl or NaClO. It is possible that residual chlorine from these storage solutions might have negatively influenced bond strengths. However, when specimens were autoclaved after the initial storage period, we noted no differences between storage media. When specimens were treated with formalin after the initial storage period, the only storage medium that affected SBS negatively was NaClO. Autoclaving and formalin treatment. When we compared solutions as a function of
sterilization treatment (Table), autoclaving significantly reduced SBS values for specimens stored in dH2O (from 18.9 MPa to 11.0 MPa) and formalin (from 18.0 MPa to 10.6 MPa). On the other hand, autoclaving and formalin treatment increased SBS values significantly for NaClOstored specimens (from 3.6 MPa to 10.5 MPa and 7.8 MPa, respectively), while autoclaving and formalin treatment did not significantly affect the SBS values of chloramine T- and glutaraldehydestored specimens. Autoclaving increased the mean SBS values only for specimens stored in NaCl (not significantly) and NaClO (significantly), the two storage solutions that resulted in significantly lower SBS values when compared with dH2O when specimens were tested after storage only. For all other samples, autoclaving reduced mean SBS values. Consequently, autoclaving had an “equalizing effect” on SBS values, which resulted in no significant differences being noted for SBS values of specimens stored and autoclaved, regardless of the storage medium. We observed a similar trend for specimens sterilized with formalin, with the exception of specimens stored in NaClO followed by treatment with formalin; the mean SBS values for these specimens were significantly lower than those for the other samples treated with formalin. On the basis of these findings, we conclude that regardless of the initial storage solution, autoclaving the specimens tended to level the SBS values after the initial storage period. This effect was detrimental for specimens stored initially in dH2O or 10 percent formalin. NaClO storage also produced a change in the specimens’ color; they became a lighter hue of yellow in contrast to no color changes for the specimens stored in other solutions. This color change may be due to the oxidizing activity of NaClO or to residual chlorine; however, further investigation is required to determine the definitive reason for the color change. NaClO storage. Of all the storage solutions, NaClO appears to be the one that most negatively affects dentin bonding, especially if bonding is done immediately after storage or after sterilization with formalin. Therefore, we do not recommend NaClO as a storage solution for extracted teeth that will be used for mechanical tests of composite-to-dentin bonding. Results of previous evaluations of dentin bonding after short treatment with NaClO have been inconclusive.14-19 One clinical study that evaluated the two-year per-
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this technology is not easily available and Shear bond strengths for all groups. also is expensive, GROUP MEAN (SD*) SHEAR BOND STRENGTH IN MEGAPASCALS†‡ which probably puts it Storage Storage and Storage and 10% at a disadvantage Autoclave Formalin when compared with 18.9 (7.0)aA 11.0 (4.3)bA 12.6 (4.4)abA Control (Distilled Water) other alternatives, bB bA aA such as storage in 8.2 (4.4) 8.7 (4.2) 15.3 (6.9) 0.9% Sodium Chloride dH2O or 10 percent 14.1 (5.6)aAB 11.5 (5.3)aA 15.5 (4.1)aA 0.5% Chloramine-T formalin. 3.6 (4.7)bC 10.5 (5.4)aA 7.8 (3.9)aB 5.25% Sodium Study limitations. Hypochlorite This study, however, 13.1 (4.0)aAB 11.2 (4.7)aA 12.2 (4.5)aA 2% Glutaraldehyde is not free from limitaaA bA § tions. Maintaining 18.0 (7.5) 10.6 (4.9) NA 10% Formalin even thickness of the * SD: Standard deviation. † The same lowercase superscript letters across groups (rows) indicate means that are not significantly phenolic rings loaded different at P = .05. with a bovine incisor ‡ The same uppercase superscript letters between groups (columns) indicate means that are not significantly different at P = .05. on grinding needs to § NA: Not applicable. be more carefully controlled, because a formance of resin-based composite restorations slanted surface may provide more surface area for placed with and without NaClO treatment dentin bonding, leading to error on shear strength showed that treating dentin with 10 percent testing. NaClO to remove exposed collagen after acidThere also has been a concern regarding the consistency of SBS testing owing to a possibility etching did not affect the clinical performance of that a portion of dentin may fracture off with an restorations placed with an acetone- or ethanolintact bonded interface6,7; however, further invesbased adhesive in teeth with nonretentive 20 noncarious cervical lesions. tigations of the surface morphology of ground Therefore, it appears that the negative effect of dentin and the shear-tested dentin surface are the NaClO solution used in our study likely was needed to verify this phenomenon. However, we due to the long-term storage. We can infer that did not observe fracture within the composite post long-term treatment of tooth structures with as a result of shearing force in any specimens, NaClO should be avoided if the teeth are to be indicating that the fracture occurred at the interused for bonding. NaClO also is commonly used face. This might be due, in part, to the relatively as an irrigant during endodontic treatment, slow crosshead speed used (0.5 mm/minute), because of its antibacterial activity and ability to which we selected on the basis of a previous study dissolve tissue. The results of this study suggest showing that this speed was less likely to induce that residual NaClO left on an endodontically cohesive fracture on the substrate.23 treated tooth might interfere with bonding of Although SBS tests commonly are used in in composite to the tooth. vitro bond strength studies,2,3,5 they have been Gamma irradiation. Another possible sterilcriticized for their lack of standardization and ization alternative not evaluated in this study is consistency.6,7 When compared with other bond the use of gamma irradiation. Sterilization via strength tests (that is, tensile and microtensile), gamma irradiation often is done for bone and for however, the SBS test has been shown to be banking extracted teeth. Researchers have invesappropriate and effective as a tool for evaluating tigated the bond strength of human dentin suband contrasting different bonding systems in mitted to gamma rays and the morphological vitro.8,9 Scanning electron microscopy also could 21,22 changes in dentin. The results of these studies provide more information than is obtainable with showed that gamma irradiation did not produce the SBS test with regard to the fracture site of structural changes in dentin. Scanning electron the bonding samples, as well as possible clues to micrographs also did not show alterations. Thus, the change in color caused by the 5.25 percent gamma irradiation neither affected the SBS nor NaClO solution. altered the dentin surface morphology; however, Finally, we used bovine teeth for consistency
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and convenience. Although human teeth ideally should have been used, it would be difficult to identify and collect a homogeneous sample of human teeth for this study. Studies have shown that bovine teeth are a suitable substitute for human teeth in bonding studies.11,12 CONCLUSIONS
In this study, we investigated the effect of storage conditions and sterilization methods on the composite-to-dentin bond strength. Within the limitations of this study, we can draw the following conclusions. When storage solutions alone are compared, 0.9 percent NaCl and 5.25 percent NaClO resulted in significantly lower SBS values than those obtained with dH2O. Therefore, investigators should not use these solutions as the storage medium when teeth are to be used for dentin bonding studies. Sterilization with an autoclave negatively affected the SBS values of specimens stored initially in dH2O or 10 percent formalin, while sterilization with formalin alone had no significant effect. Although storage in dH2O resulted in the highest nominal SBS values among the sample studied, storage and simultaneous sterilization in 10 percent formalin appears to be the most logical treatment for extracted teeth that will be used in dentin bonding research. ■ The authors thank Dr. Ceib Philips and Mr. Michael Pannucci for assisting with the statistical analysis. 1. Kohn W, Collins A., Cleveland J, Harte J, Eklund K, Malvitz D; Centers for Disease Control and Prevention. Guidelines for infection control in dental health-care settings: 2003. MMWR Recomm Rep 2003;52(RR-17):1-61. 2. Triolo PT Jr, Swift EJ Jr. Shear bond strengths of ten dentin adhesive systems. Dent Mater 1992;8(6):370-4. 3. Triolo PT Jr, Swift EJ Jr, Barkmeier WW. Shear bond strengths of composite to dentin using six dental adhesive systems. Oper Dent
1995;20(2):46-50. 4. Fritz UB, Finger WJ, Uno S. Resin-modified glass ionomer cements: bonding to enamel and dentin. Dent Mater 1996;12(3):161-6. 5. al-Salehi SK, Burke FJ. Methods used in dentin bonding tests: an analysis of 50 investigations on bond strength. Quintessence Int 1997;28(11):717-23. 6. Van Noort R, Noroozi S, Howard IC, Cardew G. A critique of bond strength measurements. J Dent 1989;17(2):61-7. 7. Versluis A, Tantbirojn D, Douglas WH. Why do shear bond tests pull out dentin? J Dent Res 1997;76(6):1298-307. 8. Fowler CS, Swartz ML, Moore BK, Rhodes BF. Influence of selected variables on adhesion testing. Dent Mater 1992;8(4):265-9. 9. Cardoso PE, Braga RR, Carrilho MR. Evaluation of micro-tensile, shear and tensile tests determining the bond strength of three adhesive systems. Dent Mater 1998;14(6):394-8. 10. Eliades G. Clinical relevance of the formulation and testing of dentine bonding systems. J Dent 1994;22(2):73-81. 11. Nakamichi I, Iwaku M, Fusayama T. Bovine teeth as possible substitutes in the adhesion test. J Dent Res 1983;62(10):1076-81. 12. Schilke R, Bauss O, Lisson JA, Schuckar M, Geurtsen W. Bovine dentin as a substitute for human dentin in shear bond strength measurements. Am J Dent 1999;12(2):92-6. 13. Pashley DH, Tao L, Boyd L, King GE, Horner JA. Scanning electron microscopy of the substructure of smear layers in human dentine. Arch Oral Biol 1988;33(4):265-70. 14. Nikaido T, Takano Y, Sasafuchi Y, Burrow MF, Tagami J. Bond strengths to endodontically-treated teeth. Am J Dent 1999;12(4):177-80. 15. Vongphan N, Senawongse P, Somsiri W, Harnirattisai C. Effects of sodium ascorbate on microtensile bond strength of total-etching adhesive system to NaOCl treated dentine. J Dent 2005;33(8):689-95. 16. Saboia VP, Rodrigues AL, Pimenta LA. Effect of collagen removal on shear bond strength of two single-bottle adhesive systems. Oper Dent 2000;25(5):395-400. 17. Perdigão J, Lopes M, Geraldeli S, Lopes GC, García-Godoy F. Effect of a sodium hypochlorite gel on dentin bonding. Dent Mater 2000;16(5):311-23. 18. Pimenta LA, Amaral CM, Bedran de Castro AK, Ritter AV. Stability of dentin bond strengths using different bonding techniques after 12 months: total-etch, deproteinization and self-etching. Oper Dent 2004;29(5):592-8. 19. Pioch T, Kobaslija S, Schagen B, Götz H. Interfacial micromorphology and tensile bond strength of dentin bonding systems after NaOCl treatment. J Adhes Dent 1999;1(2):135-42. 20. Saboia Vde P, Almeida PC, Ritter AV, Swift EJ Jr, Pimenta LA. 2-year clinical evaluation of sodium hypochlorite treatment in the restoration of non-carious cervical lesions: a pilot study. Oper Dent 2006; 31(5):530-5. 21. Sperandio M, Souza JB, Oliveira DT. Effect of gamma radiation on dentin bond strength and morphology. Braz Dent J 2001;12(3):205-8. 22. DeWald JP. The use of extracted teeth for in vitro bonding studies: a review of infection control considerations. Dent Mater 1997;13(2): 74-81. 23. Hara AT, Pimenta LA, Rodrigues AL Jr. Influence of cross-head speed on resin-dentin shear bond strength. Dent Mater 2001;17(2): 165-9.
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