Longitudinal bond strength evaluation using the deproteinized dentin technique
By Gleyce Oliveira Silva
Daphne Camara Barcellos, DDS
Cesar Rogerio Pucci, DDS, MS, PhD
Alessandra Buhler Borges, DDS, MS, PhD
Carlos Rocha Gomes Torres, DDS, PhD
Featured in General Dentistry, July/August 2009
Pg. 328-333
Posted on Thursday, July 02, 2009
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This study evaluated bond strength to dentin as a result of storage time for conventional adhesive systems (with or without collagen) that had been deproteinized with 10% sodium hypochlorite (NaOCl). For this study, 72 human molars were sectioned in a mesiodistal axial plane and embedded in acrylic resin; at that point, the vestibular and lingual surfaces were worn down with abrasive paper. Acid etching was performed for 15 seconds (using 37% phosphoric acid) and the specimens were divided into 12 groups (n = 6), depending on the adhesive system used, the dentin treatment performed, and the length of evaluation (24 hours or six months). A resin composite was inserted over the prepared area with the aid of a metal matrix. Following a mechanical shear test, fractured surfaces were analyzed by stereomicroscope and the data were submitted to ANOVA and Tukey’s test.
It was concluded that the dentin deproteinization treatment with 10% NaOCl improved the bond strength in five of the six groups. The bond strength after 24 hours was significantly higher than the bond strength measured after six months. Of the three adhesive systems tested in this study, DenTASTIC UNO demonstrated the lowest bond strength.
Received: October 24, 2008
Accepted: December 11, 2008
In 1955, Buonocore first suggested that the desired retention of restorative material in a cavity occurred as a result of acid etching the enamel.1 In 1979, Fusayama et al proposed the concept of etching enamel and dentin completely to remove the smear layer and expose the collagen fibers.2 Three years later, Nakabayashi et al reported the formation of an acid-resistant structure when the adhesive layer was bonded to etched dentin (a formation referred to as a hybrid layer or dentin hybridization).3
However, failures in hybrid layer formation have been observed. Excessive acid etching causes the formation of a very deep etched dentin surface. The adhesive cannot infiltrate the surface completely, resulting in a region where the exposed collagen fibers are weakened and more susceptible to hydrolysis.4,5 Overdrying causes the fiber network to collapse, resulting in inadequate hybridization, while overwetting causes interfacial defects to form.6
To avoid the problems observed with the hybridization technique, an alternative method has been described in the literature. This technique involves a combination of substances capable of decalcifying the dentin structure, followed by the application of sodium hypochlorite (NaOCl) after complete phosphoric acid etching.7 This decalcification promotes the appearance of a more porous dentin, with characteristics similar to those of enamel. It also results in widened dentinal tubules without the presence of collagen, which could inhibit adhesive diffusion into the dentin substrate due to drying or excessive humidity and thus compromise bonding effectiveness.
The use of these substances for the purpose of removing the organic dentin material was conceived as a different technique in dental substrate treatment. This technique does not require hybridization with micromechanical infiltration of the resin monomer among the collagen fibers to obtain effective bonding.7,8 Adhesive diffusion on the denatured surface becomes easier due to the absence of collagen, promoting bonding between the mineral structure of the dentin substrate and the resinous monomers and reducing the number of failures from regions with unprotected collagen.7,9
The longevity of the hybrid layer has been questioned due to hydrolysis of the bond interface over the course of time.10 This method for treating the dentin substrate has been evaluated in the literature. Separate studies followed acid-etching and exposition of the collagen network by applying a 10% sodium hypochlorite solution to the dentin to remove the exposed collagen, resulting in an extremely porous and mineral surface, similar to that of enamel.7,8 Both studies observed that applying the adhesive over this surface resulted in increased bond strength when compared to surfaces that had received collagen.
For that reason, this longitudinal laboratory study sought to evaluate the bond strength of selected adhesive systems to dentin and to verify whether using 10% NaOCl for deproteinization produced a similar bond to samples that were not treated with NaOCl.
Materials and methods
Seventy-two healthy human molars were cleaned and stored in distilled water and kept in a freezer (at –18°C) until their use.
Using a low-speed cutting machine (Labcut 1010), the roots of the specimens were cut off and the teeth were sectioned along a mesiodistal axial plane, separating them into two halves (vestibular and lingual). The sectioned teeth were embedded in colorless acrylic resin; each section measured approximately 40 mm x 20 mm x 10 mm. The vestibular and lingual faces were kept facing outward, maintaining the tooth surface parallel to the horizontal plane.
The remaining dentin thickness was standardized to 2 mm (±0.1 mm) with the use of a thickness meter.11,12 The vestibular or lingual enamel was worn down using 80 grit silicone carbide abrasive paper (3M ESPE), adapted to a plaster cutting machine (Kohlbach) under water cooling (to avoid overwarming), until the predetermined thickness was approximated. The smear layer of the dentinal surfaces was standardized using 320, 400, and 600 grit abrasive water papers (Struers Inc.) sequentially in a polishing machine (Struers DP-10, Struers Inc.) under water cooling at all times.
At this stage, the dentin surface was cleaned with pumice stone and water, using a Robinson brush. The bond area was delimited with Teflon adhesive tape and hollowed in a circular shape, with a standardized diameter of 3 mm.12
The specimens were divided into 12 groups, each consisting of 12 specimens (six vestibular and six lingual surfaces, selected at random), according to the adhesive system, dentin treatment, and evaluation time. (Table 1 lists the composition of each material used in this study.) Evaluations were made at 24 hours and again at six months. At each instance, six groups were evaluated.
All samples were etched for 15 seconds with 35% phosphoric acid. Group 1 samples were treated with DenTASTIC UNO adhesive (Pulpdent), Group 2 samples were treated with Prime & Bond NT (Dentsply/Caulk), Group 3 samples were treated with Adper Single Bond (3M ESPE), Group 4 samples were treated with DenTASTIC UNO after receiving 10% NaOCl for 60 seconds, Group 5 samples were treated with Prime & Bond NT after receiving 10% NaOCl for 60 seconds, and Group 6 samples were treated with Adper Single Bond after receiving 10% NaOCl for 60 seconds. The adhesive procedures were performed in accordance with the manufacturers’ specifications. The NaOCl was applied for 60 seconds, after the acid etching stage, to the samples in Groups 4–6.
To standardize the resin composite area and volume, a prefabricated metal device was used to position the test specimens and created an orifice (3 mm in diameter and 4 mm high) over the dentin surface that received the adhesive treatment, thus preventing displacement while the resin composite (Z-100, 3M ESPE) was inserted and polymerized. The resin composite was inserted using the incremental technique with the photocuring appliance (XL 3000, 3M ESPE) calibrated to 500 mW/cm2.
The specimens were submitted to a shear bond strength test using a universal testing machine (DL-1000, EMIX) with a 50 Kg load call at a speed of 1 mm/minute, in accordance with the standards described in ISO TR 11405. The force on fracture was recorded. When the specimens fractured, they were submitted to optic microscopy analysis (Stemi 2000C, Carl Zeiss, Inc.) and classified into four types, according to the plane of section for the fracture: cohesive fracture in resin composite (Type 1), cohesive fracture in dentin (Type 2), fracture in adhesive at the dentin/adhesive or adhesive/resin interface (Type 3), and a combination of cohesive and adhesive fractures (Type 4). The data were submitted to ANOVA (factors included the type of adhesive, length of storage, and usage of NaOCl) and Tukey’s (5%) test.
Results
The behavior of the means for the different groups is listed in Table 2, while Table 3 lists the types of fracture found in each groups. Table 4 lists the results of ANOVA; all three factors produced statistically significant differences.
The significant interactions between the three factors are cited in Table 5. Each interaction resulted in statistically significant differences. The results of Tukey’s test for the three factors are presented in Tables 6–8.
Tables 9–11 apply Tukey’s test to the interaction between the three factors: type of adhesive, storage time, and the use of 10% NaOCl.
Discussion
The literature has discussed the concept that adhesive systems have the ability to infiltrate into the collagen network of the demineralized zone, encapsulate the collagen and hydroxyapatite crystals, and form the hybrid layer.3 This concept has been explored primarily in longitudinal studies, as the resin-dentin bond structure could degrade over time and result in failures.10,12,13 NaOCl is a deproteinizing agent that can be used to remove collagen from demineralized dentin and provide adhesion.14-16 This step opens the dentinal tubules to facilitate the access pathways for resinous monomer and favors the formation of extremely porous mineral surfaces, whose characteristics are similar to those of enamel.17
The speed of diffusion is another important factor in resin monomer permeation. The adhesive systems that contain Bis-GMA and triethylene glycol dimethacrylate (TEGDMA) have a slow diffusion speed, as these monomers are highly hydrophobic. By contrast, adhesives containing hydroxyethyl methacrylate (HEMA), which is hydrophilic and has low molecular weight, offer easy and fast diffusion.18
Diffusion can be affected by the substance that is used to dilute the resinous monomer; for example, water evaporates more slowly than acetone solvent, harming the diffusion process and interfering with monomer polymerization. Alcohol- and acetone-based solutions have greater volatility and are more appropriate for an adequate pattern of diffusion; however, adhesive monomers are highly soluble in acetone.18 In addition, alcohol and acetone are unable to expand the collagen fibers with the same precision as water.19
Various studies have reported that the removal of collagen increases dentin bonding, depending on the adhesive’s composition.20,21 In the present study, Prime & Bond NT demonstrated a statistically higher mean bond strength value compared to DenTASTIC UNO (see Table 6). This result echoes the 2005 article by de Souza et al, who reported that the high concentration (70%) of acetone solvent in Prime & Bond NT provided high diffusion, allowing better contact between the monomer and the dentin structure and increasing the penetration into dentin.22 Perdigao et al reported that removing collagen fibers in an NaOCl solution could increase the bond strength between dentin and Prime & Bond NT.23 Nevertheless, the results of DenTASTIC UNO demonstrated that not all acetone-based adhesive systems present significant improvement in bond strength.
The present study demonstrated that the bond strength of all three adhesives improved when 10% NaOCl was used, with the exception of samples in Group 4 at six months. Groups 4–6 showed significantly higher means than Groups 1–3, which echoes previous findings in the literature.16,20
By contrast, other studies have reported that using NaOCl reduced the bond strength of conventional adhesive systems.11,24,25 These different results concerning bond strength may result from different methodologies used in the studies, particularly a lack of standardization with regard to application time and the concentration and viscosity of NaOCl.26 A 2005 study by Correr et al found that the concentration and application time of NaOCl interfered in the complete removal of dentin collagen. In their study, using 5% NaOCl for 120 seconds or 10% NaOCl for 30, 60, or 120 seconds removed dentin collagen completely, while using 5% NaOCl for 30 or 60 seconds did not.27
When storage time alone was evaluated, groups evaluated after six months demonstrated significantly lower means than the groups evaluated at 24 hours. Furthermore, it was verified that the DenTASTIC UNO obtained a significantly lower mean bond strength at six months than the other groups of adhesive systems, regardless of whether 10% NaOCl was used, which confirms that dentin bonding depends on the composition of the dentin adhesive. The samples in Groups 5 and 6 demonstrated statistically higher means than the other groups, in keeping with previous studies.22,23
In terms of the type of fracture that occurred, the majority of fractures were classified as Type 3 (adhesive), with small variations depending on the adhesive system and period of evaluation. No cohesive fractures occurred in either resin or dentin.11 The use of NaOCl did not affect the fracture pattern.
The longevity of the hybrid layer is considered to be critical, because hydrolysis occurs at the adhesive interface over time. Based on the literature concerning increased bond strength, the deproteinization technique appears very promising. Even though incorporating another step may represent an increase in technical complexity, the authors believe that the technique should be applied to increase the service life of restorations and minimize the need for periodic replacement. However, because degradation occurs over time due to the presence of dentin collagen at the bond interface, clinical longitudinal studies are necessary to confirm the efficiacy of NaOCl use, so that a technique that favors a stable bond to dentin may be developed.
Conclusion
Adhesive systems that subjected dentin to deproteinization treatment with 10% NaOCl demonstrated higher bond strengths than those that did not, with the exception of the DenTASTIC UNO adhesive system, which displayed the lowest bond strength of all systems tested, regardless of whether NaOCl was used. For all three systems, the bond strength at six months was shown to be significantly lower than the bond strength evaluated at 24 hours.
Acknowledgements
The authors thank the Sao Paulo State Research Support Foundation, Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), for the financial support (Process No. 06/60924-8).
Author information
Ms. Silva is a trainee of the Clinical Research Academic Group (GAPEC),
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Manufacturers
Carl Zeiss, Inc.,
Dentsply/Caulk,
EMIX, Sao Jose dos Pinhais, PR,
Extec,
Kohlbach, Jaragua do Sul, Santa Catarina, Brazil; 47.3481.3800
Pulpdent,
Struers Inc.,
3M ESPE,
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General Dentistry, July/August 2009 , Volume 57 , Issue 2
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