Effect of Storage and Compressive Cycles on the Bond Strength After Collagen Removal
This study verified the influence of storage and compressive loads on the microtensile bond strength (μtbs) of an adhesive system when using the conventional technique or collagen removal treatment. Twenty bovine teeth were separated into four groups: G1) adhesive system Prime & Bond NT (PBNT) in accordance with the manufacturer's instructions, G2) PBNT after deproteinization with 10% sodium hypochlorite (NaOCl), G3) PBNT in accordance with the manufacturer's instructions + 50,000 compressive cycles and G4) PBNT after deproteinization + 50,000 compressive cycles. After 24 hours, the teeth were sectioned and half of the specimens were stored in water (37°C) for further evaluation (after 60 days). The failure mode was evaluated by scanning electron microscopy (SEM). The bond strength values were separately submitted to two-way ANOVA for each period and the differences among groups were determined by Tukey's test (p=0.05). Comparisons between 24 hours and 60 days were determined by multiple paired Student's t-tests (5%). The evaluation at 24 hours revealed that deproteinization did not affect μtbs for the non-cycled groups (G1 = 47.8 ± 4.7 and G2 = 52.4 ± 11.32). For the groups tested after the mechanical cycles, deproteinization produced higher μtbs (G3 = 32.7 ± 9.6 and G4 = 43.3 ± 16.7). At 60 days, deproteinization produced higher μtbs than the manufacturers' recommendations for the cycled and non-cycled groups (G1 = 31.5 ± 5.8; G2 = 48.8 ± 7.7; G3 = 17.1 ± 7.2 and G4 = 48.8 ± 12.2). In conclusion, the deproteinized groups were less susceptible to degradation than the groups restored with the conventional technique.SUMMARY
INTRODUCTION
Many studies have investigated the bonding area between restorative materials and dental structures. In spite of much progress, the region between dental adhesive and dentin is still susceptible to degradation.1 It has been speculated that hydrolytic degradation happens due to the presence of porosity, or voids resulting from poor monomer infiltration into the collagen fibrils.2–5 This poor infiltration occurs particularly at the bottom of the hybrid layer, creating a weak and degradable zone after long-term exposure to water.46–7
The role of the collagen and hybrid layer in the adhesion process remains unclear. Some studies affirm that collagen plays an important role in the bond strength between composites and tooth structure.8–9 In addition to providing retention, the hybrid layer would act as an inherent elastic buffering layer that is able to absorb the polymerization stress of the composite (resulting from shrinkage and the development of Young's modulus)10 and maintain bonding stability. On the other hand, it has been pointed out that the hybrid layer is not uniform. Moreover, porosities, voids and other types of defects have been identified in different regions of the hybrid zone. Such defects could be points of stress concentration and susceptibility to water degradation.46–7
The technique of collagen removal after the total etching procedure with sodium hypochlorite was proposed as a way to improve bond strength/stability between composites and dentin.11–12 However, the results of various studies are contradictory. While some studies showed that deproteinization did not affect the bond strength of the adhesive systems,13–14 other studies verified an increase in bond strength when the collagen was removed.15–18 Consequently, this effect may be attributed to alterations in the collagen-depleted dentin surface, such as higher mineral content, increased tubular aperture and the appearance of secondary tubules in intertubular dentin.19–21
The differences in the performance of adhesives in previous studies can be partially attributed to the variety of compositions tested (solvents and monomers, types and concentrations).1522–23 This assumption is supported by studies which demonstrated that acetone-based adhesives had a significant increase in bond strength when applied on deproteinized dentin surfaces, as opposed to what happened with ethanol/water-based adhesives, which showed significant reduction in bond strength.16–1724 Furthermore, a previous study concluded that NaOCl-treated dentin might be more compatible with hydrophobic resins than dentin treated by the conventional acid etching technique.25 Consequently, adhesive systems that do not have HEMA in their compositions would be more efficient.15 Since the presence of fewer hydrophilic monomers can be important for the formation of polymers that are less susceptible to hydrolytic degradation, the technique of collagen removal could be helpful in stabilizing bonding efficiency.
Theoretically, the major advantage of the collagen removal technique would be that it maintains bonding stability. However, there are few data about the consequences of this technique in the long-term. Furthermore, data about the stability of the bonding area after application of mechanical forces is also scarce. Therefore, the aim of the current study was to verify the effect of 1) collagen removal, 2) mechanical forces and 3) water storage on the microtensile bond strength (μtbs) and failure pattern. The hypotheses of this study were that:
i) the bond strength would not be affected by the collagen removal technique;
ii) storage in water would not affect the μtbs, irrespective of the technique applied and
iii) mechanical forces would not affect the μtbs, irrespective of the technique applied.
METHODS AND MATERIALS
Microtensile Bond Test
Twenty bovine incisors were selected, cleaned and randomly separated into four groups. The crowns were sectioned 9 mm below the incisal edge with a double-face diamond disk (KG Sorensen, São Paulo, SP, Brazil) and mounted in a low-speed handpiece (air/water spray cooled) in order to obtain a flat surface. After sectioning, the surfaces were abraded with 600-grit silicon carbide (SiC) abrasive paper (Norton SA, São Paulo, SP, Brazil) in order to simulate the smear layer. The surrounding enamel was removed with a cylindrical diamond bur #2214 (KG Sorensen) at high speed and cooled with air/water. The teeth were embedded in acrylic resin to keep the bond interface perpendicular to the diamond saw, while cutting the beams. The periodontal ligament was simulated with polyether (Impregum Soft, 3M ESPE, St Paul, MN, USA) for the groups submitted to mechanical loading. This procedure was done to promote stress distributions closer to real clinical situations, as described by Soares and others.26
Tables 1 and 2 show the materials used and the groups tested, respectively. For all groups, the dentin surface was etched using a 37% phosphoric acid gel (Dentsply, York, PA, USA) for 15 seconds, rinsed with water for 30 seconds and blot dried, leaving a moist surface. For groups G1 and G3, Prime & Bond NT (PBNT, Dentsply) was applied in accordance with the manufacturer's instructions. For groups G2 and G4, 10% NaOCl solution (Proderma Ltda, Piracicaba, SP, Brazil) was applied for 60 seconds after the acid etching procedure, rinsed for 30 seconds, then blot dried before application of the adhesive. The adhesive system PBNT was then applied and light cured for 20 seconds with a halogen light source (VIP, BISCO, Schaumburg, IL, USA) with 600 mW/cm2 irradiance, as measured according to a calibrated power meter (Ophir Optronics Ltd, Jerusalem, Israel). Subsequently, a 4-mm layer of composite TPH Spectrum (Dentsply) was built-up on the flattened surface by the application of two 2-mm increments. After 24 hours storage (relative humidity at 37°C), the composite-dentin bonded specimens were sectioned with a water cooled diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA) in the x and y directions perpendicular to the adhesive interface to obtain several beams with cross-sectional areas of 1.0 ± 0.1 mm2 measured with a digital caliper with 0.01 mm of accuracy. Figure 1 shows representation of the specimens obtained. For each tooth, 12 beans were obtained. Half of the beams were subsequently fixed to the microtensile bond testing device with cyanoacrylate adhesive (Super Bonder Gel, Loctite, Diadema, SP, Brazil) and tested under tension at a crosshead speed of 0.5 mm/minute using a universal testing machine (Instron 4411, Instron Inc, Canton, MA, USA). The remaining beams were stored in distilled water at 37°C for 60 days, during which the water was changed every seven days. The beams were then tested under the same conditions as those tested after 24 hours.
Citation: Operative Dentistry 34, 6; 10.2341/08-064-L
Before the beams were sliced, groups G3 and G4 were subjected to 50,000 compressive cycles on the occlusal surface of the restored teeth (MSCM-2, São Carlos, São Paulo, Brazil), with a load of 30 N and a frequency of 2.0 Hz after 24 hours storage (relative humidity at 37°C).
The bond strength values were calculated in MPa and analyzed by two-way ANOVA (cyclic loading and adhesive technique). Differences among the groups were analyzed by the Tukey's test (p=0.05). The analyses were performed separately for each period (24 hours or 60 days). The comparisons between periods were made by multiple paired Student's t-tests (p=0.05).
Failure Pattern Analysis
The specimens were sputter-coated with gold in a Denton Vacuum Desk II Sputtering device (Denton Vacuum, Cherry Hill, NJ, USA). Thus, the fractured surfaces were observed by scanning electron microscopy (SEM) (JSM–5600LV, JEOL Ltd, Tokyo, Japan) at 110× magnification for failure pattern classification. In addition, 300×, 500× and 1,000× magnifications were used in order to better observe the fracture surface.
The failure patterns were classified as:
ADF—adhesive failure—at least 75% of the bond area was in dentin, and it was possible to observe the dentin tubules, the grooves promoted by the silicon carbide papers and/or the resin tags on the resin side of the beam.
IAR—in adhesive resin—more than 75% of the fracture occurred inside the adhesive layers.
MAC—mixed failure simultaneously exhibiting remnants of adhesive failure and cohesive failure at the resin bond.
MIX—mixed failure exhibiting remnant areas of adhesive failure, failure in adhesive resin and parts of the restorative composite.
For the mixed failures, it is important to clarify that none of the substrates showed a predominance equal to or higher than 75% of the total failure area.
RESULTS
The mean μtbs values after 24 hours are shown in Table 3. No significant differences were found among the groups, irrespective of the mechanical cycle and dentin treatment applications.
Table 4 shows the mean μtbs values after 60 days. The mechanical cycle did not affect the results for the group treated with NaOCl before the adhesive application. On the other hand, the mechanical cycle had a negative effect over the μtbs values for the non-deproteinizated group.
In comparisons between the means obtained after 24 hours and 60 days for each dentin treatment, it can be observed that the use of NaOCl was responsible for maintaining the μtbs values after long-term storage in water (statistically similar). However, the use of PBNT alone resulted in a decrease in μtbs values after the 60-day period.
Figure 2 shows typical SEM images used to evaluate the failure mode. The failure pattern distributions obtained for the groups tested after 24 hours and 60 days are shown in Figure 3. The number of adhesive failures tended to increase, mainly in specimens that had the conventional adhesive technique applied and were subsequently submitted to compressive cycles and hydrolytic degradation.
Citation: Operative Dentistry 34, 6; 10.2341/08-064-L
Citation: Operative Dentistry 34, 6; 10.2341/08-064-L
DISCUSSION
The current in vitro study evaluated the influence of water storage and compressive loads on the μtbs values of an adhesive system when using the conventional adhesive technique or collagen removal treatment. The hypotheses evaluated were that: i) the bond strength would not be affected by the collagen removal technique, that ii) water storage and iii) the mechanical forces would not affect μtbs. The last hypothesis was accepted, but the first and the second hypotheses were partially rejected.
As shown in the Table 3, at 24 hours, the non-loaded specimens showed no significant difference in μtbs values between the conventional and deproteinized techniques. However, in all the other situations, deproteinization showed higher values than the conventional technique, which is in agreement with previous studies.16–1724 The evaluation performed after 60 days showed that the μtbs values obtained with the NaOCl treatment were higher than those obtained with the conventional technique. This can be considered the major finding of the current study, since there are scarcely any in vitro studies that verify the long-term durability of the aforementioned techniques and they do not reproduce clinical conditions.27
Examples of failure modes are shown in Figure 2. Figure 3 shows the distribution of each failure into the different groups, and these outcomes are probably related to the mechanical and hydrolytic mechanisms of degradation. In Group 1A, the number of mixed failures was higher than that of the adhesive failure mode. On the other hand, for Group 2A, the adhesive failure mode at the interface between the composite and dentin was predominant. Since the μtbs value was similar for both groups, perhaps the difference in failure modes between the two techniques (conventional x NaOCl treatment) lies at the points of stress concentration. For Group 1A, the hybrid zone would be more resistant than the cohesive strength of the adhesive resin due to defects created within the structure of the material, such as the well known presence of residual solvent.2228–29 However, for Group 2A, the bonding interface would be less resistant than the related structures. Thus, in a first moment, the difference might lie at the stress concentration points. These are some hypothetical explanations, and additional studies, probably using TEM images, are necessary to clarify the aforementioned phenomena.
Within the cyclically loaded groups, adhesive failures tended to be predominant for Group 3A, and this might be related to the stress concentration points within the hybrid layer created by incomplete resin penetration resulting from the low surface energy of the collagen when compared with that of hydroxyapatite.30 These defects may be more susceptible to fracture caused by loading forces than fracture resulting from the aforementioned defects created within the resinous layer. On the other hand, for the NaOCl-treated group (Group 4A), mixed failures were predominant, and this effect is perhaps minimized due to 1) the lower amount of water (important to avoid the collapse of organic structure when using the conventional technique) caused by removal of the collagen fibrils,15 2) the higher homogeneity of the resultant bonding interface, since the risk of solvent trapping is probably reduced31–33 and 3) not using HEMA, known to be a very hydrophilic monomer.246–715 Furthermore, the higher amount of Ca ions from hydroxyapatite on the deproteinized dentin surface could increase the effectiveness of PENTA, as the interaction between these two components has already been described.30
The evaluation performed after 60 days reveals that hydrolytic degradation also plays an important role in the deterioration of bonding. It was observed that the number of adhesive failures increased for groups treated with the conventional technique (1B and 3B). This probably occurred because of degradation of the exposed collagen fibrils resulting from incomplete adhesive penetration.4–5 Consequently, the unprotected collagen may be penetrated by water, accelerating its degradation. On the other hand, the removal of collagen fibrils probably allows the adhesive to adapt better to dentin. This better adaptation is supported by a previous study that verified a good adaptation of PBNT using nanoleakage.15 These assumptions are also supported by two main outcomes in the current study. The first is related to maintenance of the μtbs values over time for the groups treated with NaOCl (comparing 2A and 2B; comparing 4A and 4B), which was not observed for the groups treated with the conventional technique (comparing 1A and 1B; comparing 3A and 3B). The second is that only the NaOCl-treated group was not affected by mechanical cycling (Table 4).
CONCLUSIONS
The first hypothesis was partially rejected, since the bond strength for the NaOCl-treated dentin was higher than that produced by the conventional technique after 60 days of water storage, irrespective of the loading conditions.
The second hypothesis was partially rejected, because the 60 days of water storage reduced the μtbs value only for groups treated with the conventional technique.
The third hypothesis was accepted, since the mechanical loading did not affect the μtbs value, irrespective of the adhesive technique applied. However, it seems that the mechanical loading affected the failure modes.
In general, the deproteinization technique promoted more stable bonding between composite and dentin.
Representation of sample preparation. (a) Tooth embedded in acrylic resin. (b) Crown sectioned to obtain a flat surface. (c) Surrounding enamel removed with a diamond bur. (d) Adhesive and restorative procedures. Tooth sectioned in the x (e) and y (f) directions perpendicular to the adhesive interface. (g) Diamond disc used to separate the specimens. (h) Samples ready for evaluation.
Failure modes of the tested specimens. Figures 2A and 2A' show the two sides of the adhesive failure (ADF). Figure 2B shows the fracture into the adhesive resin (IAR) and Figure 2B' shows a higher magnification. The mixed failure involving adhesive resin and dentin (MAC) is shown in Figure 2C and, Figure 2C' shows higher magnification; the details of the adhesive (a) and dentin (d) show an example of mixed failure in Figure 2D and, in Figure 2D', the details of fractured composite (c) and dentin (d).
Occurrence and mode of failures in the specimens tested after 24 hours and 60 days. (ADF = adhesive failure, IAR = in adhesive resin, MAC = mixed failure exhibiting remnants of adhesive failure as well as cohesive failure at the resin bond and MIX = mixed failure exhibiting remnant areas of the adhesive failure).
Contributor Notes
Luciano de Souza Gonçalves, DDS, MS, PhD student, Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, UNICAMP, Brazil
Simonides Consani, DDS, PhD, professor, Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, UNICAMP, Brazil
Mário Alexandre Coelho Sinhoreti, DDS, MS, PhD, professor, Department of Restorative Dentistry, Dental Materials Division, Piracicaba Dental School, UNICAMP, Brazil
Luis Felipe J Schneider, DDS, MS, PhD, professor, School of Dentistry, University of Passo Fundo, Passo Fundo, Brazil
Vincente de Paulo de Aragão Saboia, DDS, MS, PhD, professor, School of Dentistry, Federal University of Ceará, Fortaleza, Brazil