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J Am Dent Assoc, Vol 138, No 12, 1599-1603.
© 2007 American Dental Association |  |
RESEARCH |
The Effect of Storage Medium and Sterilization on Dentin Bond Strengths
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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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 middle-depth dentin, which they polished to 600 grit. They used a dental adhesive to apply composite to the exposed dentin. The authors tested the composite-to-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
Abbreviations: CDC: Centers for Disease Control and Prevention • dH20: Distilled water • NaCl: Sodium chloride • NaClO: Sodium hypochlorite • SBS: Shear bond strength
One 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-to-dentin 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 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.
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 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 adhesive-coated 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.
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).
MATERIALS AND METHODS
TOP
ABSTRACT
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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.
RESULTS
TOP
ABSTRACT
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
REFERENCES
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 x 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.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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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 NaClO-stored 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 glutaraldehyde-stored 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 performance of resin-based composite restorations placed with and without NaClO treatment showed that treating dentin with 10 percent NaClO to remove exposed collagen after acid-etching did not affect the clinical performance of restorations placed with an acetone- or ethanol-based adhesive in teeth with nonretentive noncarious cervical lesions.20
Therefore, it appears that the negative effect of the NaClO solution used in our study likely was due to the long-term storage. We can infer that long-term treatment of tooth structures with NaClO should be avoided if the teeth are to be used for bonding. NaClO also is commonly used as an irrigant during endodontic treatment, because of its antibacterial activity and ability to dissolve tissue. The results of this study suggest that residual NaClO left on an endodontically treated tooth might interfere with bonding of composite to the tooth.
Gamma irradiation. Another possible sterilization alternative not evaluated in this study is the use of gamma irradiation. Sterilization via gamma irradiation often is done for bone and for banking extracted teeth. Researchers have investigated the bond strength of human dentin submitted to gamma rays and the morphological changes in dentin.21,22 The results of these studies showed that gamma irradiation did not produce structural changes in dentin. Scanning electron micrographs also did not show alterations. Thus, gamma irradiation neither affected the SBS nor altered the dentin surface morphology; however, this technology is not easily available and also is expensive, which probably puts it at a disadvantage when compared with other alternatives, such as storage in dH2O or 10 percent formalin.
Study limitations. This study, however, is not free from limitations. Maintaining even thickness of the phenolic rings loaded with a bovine incisor on grinding needs to be more carefully controlled, because a slanted surface may provide more surface area for dentin bonding, leading to error on shear strength testing.
There also has been a concern regarding the consistency of SBS testing owing to a possibility that a portion of dentin may fracture off with an intact bonded interface6,7; however, further investigations of the surface morphology of ground dentin and the shear-tested dentin surface are needed to verify this phenomenon. However, we did not observe fracture within the composite post as a result of shearing force in any specimens, indicating that the fracture occurred at the interface. This might be due, in part, to the relatively slow crosshead speed used (0.5 mm/minute), which we selected on the basis of a previous study showing that this speed was less likely to induce cohesive fracture on the substrate.23
Although SBS tests commonly are used in in vitro bond strength studies,2,3,5 they have been criticized for their lack of standardization and consistency.6,7 When compared with other bond strength tests (that is, tensile and microtensile), however, the SBS test has been shown to be appropriate and effective as a tool for evaluating and contrasting different bonding systems in vitro.8,9 Scanning electron microscopy also could provide more information than is obtainable with the SBS test with regard to the fracture site of the bonding samples, as well as possible clues to the change in color caused by the 5.25 percent NaClO solution.
Finally, we used bovine teeth for consistency 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
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CONCLUSIONS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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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.
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FOOTNOTES |
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REFERENCES |
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