Clinical versus laboratory adhesive performance to wet and dry demineralized primary dentin

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1 Research Article Clinical versus laboratory adhesive performance to wet and dry demineralized primary dentin ANA CLAÚDIA CHIBINSKI, DDS, MS, RODRIGO STANISLAWCZUK, DDS, MS, DOUGLAS AUGUSTO RODERJAN, DDS, MS, ALESSANDRO DOURADO LOGUERCIO, DDS, MS, PHD, DENISE STADLER WAMBIER, DDS, MS, PHD, ROSA HELENA MIRANDA GRANDE, DDS, MS, PHD & ALESSANDRA REIS, DDS, PHD ABSTRACT: Purpose: To evaluate the influence of dentin moisture on bond strengths of an etch-and-rinse bonding agent to primary dentin clinically and in the laboratory. Methods: The sample consisted of two groups of 20 caries-free primary second molars: molars in exfoliation period (clinical group) and extracted molars (laboratory group). Class I cavities were prepared in all specimens leaving a flat dentin surface on the pulpal floor. A two-step etch-and-rinse adhesive was vigorously rubbed on either dry (n= 5) or wet demineralized dentin (n= 5) under clinical or laboratory conditions. After restorative procedures, the teeth from the clinical group were extracted after 20 minutes. All samples were processed and underwent microtensile bond strength test and silver nitrate uptake evaluation under scanning electron microscopy. Results: Statistically higher bond strength values were observed when the bonding was performed under laboratory conditions and on a wet demineralized dentin. Most of the failures were adhesive and mixed irrespective of the experimental condition. Silver nitrate uptake occurred in all groups irrespective of the experimental condition. Resin-dentin bond strengths produced in the laboratory in primary teeth may overestimate those produced under clinical circumstances. (Am J Dent 2011;24: ). CLINICAL SIGNIFICANCE: Wet bonding technique still seems to be required for primary teeth in order to achieve high immediate resin-dentin bond strength values for the etch-and-rinse adhesive system tested. : Dr. Alessandra Reis, Rua General Osório, Apto 422, Centro, Ponta Grossa, Paraná, Brazil E- : reis_ale@hotmail.com Introduction Most of the literature that supports our current knowledge about dentin bonding relies on laboratory investigations. Although they are relatively simpler, provide faster results and closer control of the variables involved in the bonding protocol, one should be careful when extrapolating their outcomes to clinical circumstances since laboratory tests using extracted teeth do not take into account the pulpal pressure and presence of dentin tubule fluid under realistic physiological conditions, which may adversely affect dentin bonding. Besides that, many other challenges imposed by the oral environment, such as thermal fluctuations, humidity, chemical challenges, loading stresses, etc., threaten the establishment of an ideal bonding. 1-3 Although some attempts have been made to validate the significance of laboratory data 4-12 every new technique should be re-evaluated under a clinical setting so as to provide clinical validation of new protocols. Etch-and-rinse adhesive systems require previous dentin demineralization with phosphoric acid in order to expose collagen fibrils for resin infiltration. Several investigators have reported that the demineralized dentin must be kept moist in order to maintain interfibrillar porosity for resin monomer infiltration and allow the achievement of high immediate bond strength values Air drying of the conditioned dentin surface has been shown to cause the unsupported collagen web to shrink and collapse, preventing monomers of the adhesive resin from efficiently wetting and infiltrating the conditioned surface. 17,18 However, the management of adequate moisture is not easily accomplished. The discrimination of the proper moisture degree for different solvent-based adhesive systems is still a challenge, since it depends on the solvent composition, 15 overall interpretation of instructions, the drying time, tooth-air syringe distance 19 and operator skills or handling by the operator. 20 This makes the maintenance of adequate moisture on demineralized dentin difficult to achieve in clinical practice, when treating pediatric patients. Interestingly, recent investigations reported that as long as etch-and-rinse adhesives are vigorously rubbed on the dentin surfaces, high immediate 21 and 1-year resin-dentin bonds 22 could be achieved when etch-and-rinse adhesives were applied on either wet or dry demineralized dentin. Considering that any simplification is of interest in pediatric dentistry, the benefits of dry bonding technique should be re-evaluated for primary dentition. Therefore this clinical study evaluated the influence of dentin moisture on resin-dentin bond strengths and silver nitrate uptake of an etch-and-rinse bonding agent to primary dentin under laboratory and clinical conditions. The null hypotheses tested were (1) there was no difference in bond strength and silver nitrate uptake produced clinically or in the laboratory and (2) there was no difference in the bond strength and silver nitrate uptake produced on wet or dry demineralized dentin. Materials and Methods Clinical sample preparation - After clinical and radiographic examination of approximately 210 subjects aging years old, 10 subjects were selected to take part in this study. Informed consent was received from the parents of these subjects, under a protocol approved by the Ethical Committee for Human Studies (#24/2009). These subjects had 10 caries-free primary second molars, with advanced root resorption and mobility, indicating activation of the physiological exfoliation process. Class I cavities were prepared in these teeth under local

2 222 Chibinski et al American Journal of Dentistry, Vol. 24, No. 4, August, 2011 anesthesia and rubber dam isolation, using a cylindrical diamond bur (#1092 a ) under water cooling. Each diamond bur was used for only three cavity preparations. The cavities were prepared in order to achieve: (1) the largest possible dimensions; (2) completely flat dentin cavity floor; and (3) complete enamel cavosurface margins. The sample was randomly divided in two groups, according to the adhesive procedures: wet (n=5) and dry bonding techniques (n=5). The restorative procedures consisted of cavity etching with 37% phosphoric acid gel for 15 seconds followed by water rinsing (15 seconds); bonding with a two-step etch-and-rinse adhesive system (XP Bond b ) and restoration with a nanohybrid composite resin (shade EA2; Opallis c ) in three increments. The bonding procedures differed in the study groups. For the wet bonding technique, teeth were rinsed with water for 20 seconds and completely air dried for 10 seconds after acid etching. Then the cavity was carefully re-wetted with water before adhesive application. For the dry bonding technique, dentin surfaces were kept dry before adhesive application. In both groups, two coats of the adhesive were applied under vigorous rubbing action. 21 Then, the adhesive layer was gently air-dried for 5 seconds at a tooth-air syringe distance of 5-10 cm and left undisturbed for 20 seconds before light curing with a lightemitting diode source for 10 seconds at 900 mw/cm 2 (Radii- Cal d ). The cavities were restored with three incremental layers of resin composite individually light-activated for 20 seconds with the light curing unit. Within 20 minutes after the completion of the bonding procedures, the tooth was extracted, immersed in distilled water for 24 hours before being prepared for the microtensile bond test and micromorphological analysis. Laboratory sample preparation - The same experiment performed clinically was repeated under laboratory conditions using 10 caries-free recently exfoliated primary second molars. Teeth had been stored in saline solution for no more than 3 months. Similar cavities were prepared and the same operator performed all restorative procedures. The sample was also divided into two groups: wet (n= 5) and dry bonding technique (n= 5). Teeth were kept in saline solution before restoration procedures, which were done as described for the clinical experiment. After restorative procedures, teeth were kept immersed in water for 24 hours and were prepared for the microtensile bond test and micromorphological analysis. Microtensile bond strength test (μtbs) - After storage of the bonded teeth in distilled water at 37 C for 24 hours, they were longitudinally sectioned in both x and y directions across the bonded interface with a diamond saw (Isomet 1000 d ) under water cooling at 300 rpm to obtain bonded sticks with a crosssectional area of approximately 0.8 mm 2. The cross-sectional area of each stick and the remaining dentin thickness were measured with the digital caliper (Absolute Digimatic f ) to the nearest 0.01 mm and recorded for subsequent calculation of the bond strength. Individual bonded sticks were attached to a device g for microtensile testing with cyanoacrylate resin (Super Bonder h ) in a way that tensile forces acted perpendicularly to the dentin/adhesive interface and subjected to a tensile force in a universal testing machine i at 0.5 mm/minute. The failure modes were evaluated at x400 (HMV- 2 j ) and classified as cohesive (C, failure exclusive within dentin or resin composite), adhesive (A, failure at resin/dentin inter- face), or adhesive/mixed (A/M, failure at resin/dentin interface that included cohesive failure of the neighboring substrates). Micromorphological analysis - Two representative sticks of each tooth from the clinical and laboratory samples were evaluated in scanning electron microscopy (SEM) (JSM 6360 LV l ). After 24 hours of storage in 50 wt% ammoniacal silver nitrate 23 the silver impregnated specimens were rinsed thoroughly in distilled water and placed in a photo-developing solution for 8 hours under a fluorescent light. The adhesive interfaces were polished with a descending grits of SiC papers (1,000; 1,200; 1,500; 2,000 and 2,500) and 1 and 0.25 μm diamond paste l using a polishing cloth. Specimens were ultrasonically cleaned and dried in a desiccator for 24 hours. Specimens were then mounted on stubs and sputter-coated with a 10 nm gold layer to be analyzed in the SEM using backscattered electron mode and dispersive X-ray spectrometry energy (EDX). The amount of silver nitrate inside the hybrid layer in each specimen was measured using EDX in three different sites (5 x 5 m left, medium and right) of the bonding area in each stick. The total length of the scanned hybrid layer used for silver nitrate penetration measurement was approximately 75 μm The silver nitrate uptake (SNU) was expressed in percentage of the total evaluated area. Statistical analysis - The sample size calculation was based on the pilot results from the laboratory experiment (mean and standard deviation (30.0 ± 3.0 MPa). In order to detect a significant difference of 20% of the means with a power of 90% and a level of significance of 5%, eight teeth were required per experimental condition. The μtbs values of all sticks from the same tooth were averaged for statistical purposes. Any prematurely debonded speci-men was included in the tooth mean as zero values. The mean SNU(%) of all specimens originated from the same teeth and was averaged for statistical purposes. The SNU of every test group was expressed as the mean of the five teeth used per group. The Kolmogorov-Smirnov test was performed to assess whether the data followed a normal distribution, and the Barlett s test for equality of variances was performed to determine if the assumption of equal variances was valid. After observing the normality of the data distribution and the equality of the variances, the μtbs (MPa) and SNU (%) data were submitted to appropriate data analysis. Results Microtensile bond strength - The mean cross-sectional areas of the bonded sticks ranged from 0.82 to 1.03 mm 2. No significant difference was observed between the groups (P> 0.05). The mean bond strength values and their respective standard deviations are summarized in Table 1. While the cross-product interaction (bonding condition vs. dentin moisture) was not statistically significant (P= 0.23) the main factors of bonding condition and dentin moisture were significant (P= and P= 0.011, respectively). Statistically higher bond strength values were observed when the bonding was performed under laboratory condition and on a wet demineralized dentin. The fracture pattern mode of the bonded sticks is shown in Table 2. Most of the bond failures were adhesive and mixed irrespective of the experimental condition. Silver nitrate uptake - The mean percentages and the respective

3 American Journal of Dentistry, Vol. 24, No. 4, August, 2011 Adhesive performance: Clinical vs. laboratory 223 Table 1. Means and standard deviations of resin-dentin bond strength values for the experimental conditions. Clinical 25.8 ± 3.4 A,a 28.2 ± 2.4 B,a Laboratory 28.7 ± 2.2 B,a 34.5 ± 0.9 B,b Different subscript uppercase letters indicate statistically different means within rows. Different subscript lowercase letters indicate statistically different means within columns ( = 0.05). Table 2. Number of specimens according to the fracture pattern mode of the resin-dentin beams for all experimental conditions. Clinical 14/1/1/0 15/0/1/0 Laboratory 19/1/2/0 21/1/0/0 Number of sticks with adhesive/mixed fracture mode/number of sticks with cohesive failure in dentin/number of sticks with cohesive failure in composite/number of premature debonded sticks. Table 3. Means and standard deviations of the relative percentage (%) of silver nitrate uptake within the adhesive and hybrid layers for the different experimental conditions (*). Clinical 16.9 ± 3.3 A 13.3 ± 6.2 A Laboratory 14.3 ± 7.3 A 11.9 ± 4.8 A (*) Groups identified by the same superscript uppercase letters are not significantly different (P> 0.05). standard deviations of silver nitrate uptake within the hybrid and adhesive layers are depicted in Table 3. Neither the cross product interaction (bonding condition vs. dentin moisture; P= 0.69) nor the main factors bonding condition and dentin moisture (P= 0.21 and P= 0.07, respectively) were statistically significant. Silver nitrate uptake occurred in all groups irrespective of the experimental condition. However the results show a trend towards less silver nitrate uptake in the groups bonded in the laboratory and following the wet bonding technique. Representative SEM images of the restorative interfaces can be seen in the Figure. Discussion Since the microtensile resin-dentin bond strengths of XP Bond bonded in the laboratory were higher than bonds made clinically, the first null hypothesis that there was no difference in bond strength produced clinically versus in the laboratory must be partially rejected. Although several studies have attempted to validate the laboratory data clinically, 4-12 there are few available studies 9-12 that compared the microtensile resindentin bond strengths produced under laboratory and clinical conditions. These studies, as well as the present one, concluded that for most of the materials evaluated, higher resin-dentin bond strengths were obtained when produced under laboratory conditions. One of the greatest differences between vital and extracted teeth is the presence of living odontoblastic processes, fluid in the dentin tubules and intrapulpal pressure. 2 The latter has been described as one of the factors able to negatively interfere with dentin adhesion. 2,3 Figure. Representative secondary electron SEM images of the resin-dentin interfaces for all experimental conditions. One can observe that most of the silver nitrate deposits are located in the bottom of the hybrid layer (white hand). (Co = composite; AL = adhesive layer; HL = hybrid layer and De = dentin). Under clinical conditions there is an outward fluid flow across exposed dentin in response to the low, but positive pulpal tissue pressure. 25 Although the use of local anesthetics with or without vasoconstrictors is capable of significantly reducing the intrapulpal pressure, 25,26 there is still an outward dentin fluid flow under clinical conditions that is completely absent in extracted teeth. This is the reason why lower bond strengths have been consistently reported in the presence of positive intrapulpal pressure, mainly for etch-and-rinse adhesives since this laboratory approach most resembles the clinical situation. 11,27-30 Under positive pulpal pressure, restricted diffusion of resin monomers into the dentin substrate is likely to occur. In fact, observations made under confocal laser scanning microcopy revealed that under simulated pulpal pressure a distinctly shallower penetration of etch-and-rinse adhesives into the dentin tubules occurred compared to the dentin treated without intrapulpal pressure. 31 This may well explain the tendency of higher silver nitrate uptake in groups bonded under clinical conditions. Besides, differences in the environmental conditions may also play a significant role on the differences observed between clinical and laboratory conditions. The relative humidity and temperature inside the oral cavity is significantly higher than that of the laboratory even under rubber dam isolation These factors may restrict the evaporation of water/solvent from the adherent surface to such an extent that it may limit the propagation of the copolymerization and the establishment of cross-linking between polymer chains. As more solvent is en-

4 224 Chibinski et al American Journal of Dentistry, Vol. 24, No. 4, August, 2011 trapped within the adhesive layer, the lower is the ultimate tensile strength of the adhesive itself 36,37 and the lower is the resultant resin-dentin bond strength Unlike previous primate studies, 41,42 the effects of functional stresses (fatigue) on clinical bond strength could not be appropriately assessed in the present human study due to the fact that the teeth were extracted 20 minutes after the restoration was finished. Despite the differences observed between clinical and laboratory conditions, one should emphasize that under both conditions the wet bonding technique was significantly better, which means that despite the lower laboratory bond strength values, the laboratory testing was capable of reaching the same conclusions than that observed clinically. On the other hand, no significant difference was reported in the silver nitrate uptake values under laboratory and clinical conditions, which seems to be in line with the finding of Donmez et al 10 who demonstrated a correlation between the micromorphological findings under clinical and laboratory conditions. Several studies 13,14 have already reported that a moist dentin surface is essential to allow resin infiltration with current adhesives. When water is present within the collagen interfibrilar and intrafibrilar spaces, it maximizes the expansion of the demineralized matrix. 43 If demineralized dentin is severely airdried, most of the water is removed causing shrinkage of the matrix, 44 thus reducing its permeability for resins and ultimately compromising the bond. This concept was challenged by Dal-Bianco et al. 21 The authors reported that the mechanical pressure applied to the dry demineralized dentin surface by vigorous rubbing action during adhesive application might compress the collagen network to such an extent that when the pressure is relieved the compressed collagen expands and the adhesive solution may be drawn into the collapsed collagen mesh. Also, the vigorous rubbing action may decrease the thickness of unstirred layers whereby allowing better inward monomer diffusion, while solvents are diffusing outward. Although this application method was shown to be effective for a water/ethanol based system (Single Bond m ) and an acetone-based system (One Step n ), 21,22 it does not necessarily mean that it will be effective for all simplified etch-and-rinse adhesives. The molecules in different substance mixtures are attracted to each other with different strengths (e.g. hydrogen bonding, electrostatic attractions) in the liquid phase. The amount of kinetic energy needed to overcome these interactions and evaporate, or the ease with which they evaporate, depends on the strength of the bonds between similar and dissimilar molecules within the formulation. The solvent (tert-butanol) presented in the adhesive evaluated in this study b has a boiling temperature similar to ethanol (82 and 78 C, respectively); 45 however its molar mass is much higher (74.12 and g/mol -1, respectively) 45 and their four-carbon chain might increase the intermolecular attraction with the resin monomers. These factors altogether likely make this material less sensitive to application technique, reducing the solvent evaporation by vigorous rubbing action. One may also hypothesize that the differences observed in this study and the previous ones 21,22 may be due to the different experimental designs. While in the present investigation the bonding was performed in a cavity, the previous studies applied the adhesive in a flat dentin surface. It is worth mentioning the limitations of the present investigation. For ethical reasons, teeth selected for the clinical experiment presented advanced root resorption and it is unlikely that teeth under this circumstance would be clinically restored even with deep caries lesions. Therefore, the clinical samples do not resemble exactly the same scenario clinicians would face daily in their office. Another source of concern is the vitality of these teeth as they had advanced root resorption. Exfoliated primary teeth constitute a viable source of stem cells for dental pulp tissue engineering 46,47 and this may be used as evidence of their tooth vitality. Besides, cavity preparation required anesthesia in this study, otherwise subjects would feel pain during the operative intervention. Within the limitation of this study we can conclude that resin-dentin bond strengths produced in the laboratory may overestimate those produced under clinical circumstances for primary teeth and therefore the extrapolation of laboratory results to clinical conditions should be cautiously done. Wet bonding technique seems to be still required for primary teeth in order to reach high bond strength values for some adhesive systems. a. KG Sorensen, São Paulo, Brazil. b. Dentsply DeTrey, Konstanz, Germany. c. FGM Dental Products, Joinville, SC, Brazil. d. SDI, Bayswater, Australia. e. Buehler, Lake Bluff, NY, USA. f. Mitutoyo, Tokyo, Japan. g. Odeme Biotechnology, Joaçaba, SC, Brazil. h. Locitec, São Paulo, SP, Brazil. i. Kratos, São Paulo, SP, Brazil. j. Shimadzu, Tokyo, Japan. k. Jeol Ltd., Tokyo, Japan. l. Erios Prod. Odont. Ltda, São Paulo, SP, Brazil. m. 3M ESPE, St. Paul, MN, USA. n. Bisco, Schaumburg, IL, USA. Disclosure statement: None of the authors has any conflict of interest. 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