Enamel microhardness and bond strengths of self-etching primer adhesives

Eur J Oral Sci 2010; 118: 191–196 Printed in Singapore. All rights reserved Ó 2010 The Authors. Journal compilation Ó 2010 Eur J Oral Sci European Journal of Oral Sciences Enamel microhardness and bond strengths of self-etching primer adhesives Olabisi A. Adebayo1, Michael F. Burrow1, Martin J. Tyas1, Geoffrey G. Adams1, Marnie L. Collins2 1 Melbourne Dental School, University of Melbourne, Vic., Australia; 2Department of Mathematics and Statistics, Statistical Consulting Centre, University of Melbourne, Vic., Australia Adebayo OA, Burrow MF, Tyas MJ, Adams GG, Collins ML. Enamel microhardness and bond strengths of self-etching primer adhesives. Eur J Oral Sci 2010; 118: 191–196. Ó 2010 The Authors. Journal compilation Ó 2010 Eur J Oral Sci The aim of this study was to determine the relationship between enamel surface microhardness and microshear bond strength (lSBS). Buccal and lingual mid-coronal enamel sections were prepared from 22 permanent human molars and divided into two groups, each comprising the buccal and lingual enamel from 11 teeth, to analyze two self-etching primer adhesives (Clearfil SE Bond and Tokuyama Bond Force). One-half of each enamel surface was tested using the Vickers hardness test with 10 indentations at 1 N and a 15-s dwell time. A hybrid resin composite was bonded to the other half of the enamel surface with the adhesive system assigned to the group. After 24 h of water storage of specimens at 37º°C, the lSBS test was carried out on a universal testing machine at a crosshead speed of 1 mm min)1 until bond failure occurred. The mean lSBS was regressed on the mean Vickers hardness number (VHN) using a weighted regression analysis in order to explore the relationship between enamel hardness and lSBS. The weights used were the inverse of the variance of the lSBS means. Neither separate correlation analyses for each adhesive nor combined regression analyses showed a significant correlation between the VHN and the lSBS. These results suggest that the lSBS of the self-etch adhesive systems are not influenced by enamel surface microhardness. Laboratory enamel–resin shear bond strength tests are often carried out using ground enamel surfaces finished with various abrasives (1–6), but it is often not disclosed how much enamel is removed in order to obtain the flat surfaces necessary for testing. This may be because the major objective is to achieve a flat surface. It is therefore impossible to establish the depth into enamel of the bonded surface. ÔSurface microhardness is a physical property of enamel and dentine surfaces and it is related to the mineral content of the dental structureÕ (7). Enamel hardness decreases with distance away from the surface (8–10) (i.e. towards the dentino–enamel junction), is related to changes in the chemistry of enamel (10), and varies with tooth side (buccal, central, and lingual) and between teeth (10). Enamel calcium and phosphate contents decrease towards the dentine (10). Enamel microhardness could therefore be an indirect indicator of enamel calcium and phosphate contents. It has been reported that some phosphoric acid ester monomers of some enamel–dentine adhesives bond to the calcium present in hydroxyapatite and that this may improve bond strengths (11, 12). A strong, positive correlation between shear bond strength and surface microhardness of acid-etched enamel and Prof. Michael F. Burrow, Melbourne Dental School, University of Melbourne, 720 Swanston Street, Vic. 3010, Australia Telefax: + 61–3–93411595 E-mail: mfburrow@unimelb.edu.au Key words: correlation; enamel; microshear bond strength; self-etching primer adhesives; Vickers hardness Accepted for publication January 2010 dentine bonded with a total-etch adhesive system has also been reported (13). Self-etching primer adhesives eliminate the Ôetch-andrinseÕ step of total-etch adhesives, thereby saving time. The self-etching primer etches and bonds directly to the finished flat enamel surface. Surface mineral loss is minimal compared with phosphoric acid etching (14, 15) and mineral removed is partly re-incorporated into the bond on curing. The self-etching primer adhesive therefore interacts directly with the finished enamel surface. The effect of enamel surface calcium content, assessed indirectly by the evaluation of surface microhardness, on bond strengths of adhesives containing phosphoric acid esters may probably be more readily observed with selfetching primer adhesives. A correlation between shear bond strength and enamel hardness may be another possible explanation for the variations in shear bond strengths reported between laboratories (16–18). The aim of this study was to determine if there is a relationship between enamel microhardness and the microshear bond strength (lSBS) of a two-step selfetching primer adhesive and an Ôall-in-oneÕ adhesive. The null hypothesis tested was that there is no relationship between enamel microhardness and the lSBS of two self-etching primer adhesives. 192 Adebayo et al. Material and methods Ethics approval was obtained from the University of Melbourne Human Research Ethics Committee to use 22 whole adult molar teeth collected from the Royal Dental Hospital of Melbourne in this study. The teeth were stored in 1% chloramine T (pH 9.1) at 4 °C and used not more than 6 months after extraction. The teeth were cleaned of periodontal ligament, decoronated at the cemento–enamel junction using a slow-speed water-cooled diamond-bladed saw (230CA; Struers, Ballerup, Denmark), and sectioned into two along the long axes. The buccal and lingual midcoronal enamel surfaces were ground with 600-grit silicon carbide paper on a grinding/polishing machine (Tegrapol 25; Struers) and the specimens were mounted in dental stone in plastics moulds. The specimens were divided into two groups of 22 by random assignment of buccal and lingual enamel pairs from 11 teeth for the two adhesives used in the study: a two-step self-etching primer adhesive system, Clearfil SE Bond (CSE), and an Ôall-in-oneÕ adhesive system, Tokuyama Bond Force (TK). Material details are listed in Table 1. The ground enamel surface of each specimen was divided into two by the placement of a notch at the occlusal edge using a flat fissure diamond bur (D835 314 008; Komet, Lemgo, Germany). One-half of each enamel surface was used for hardness testing and the other half was used for lSBS testing. Vickers surface microhardness was assessed using a microhardness tester (MHT-10; Anton Paar, Graz, Austria) designed for optical microscopes (DM LP; Leica Microsystems, Wetzlar, Germany) and connected to a camera (DFC 320; Leica) and a computer. The Vickers indenter was applied to the enamel surface at a load of 1 N and with a dwell time of 15 s. Ten indentations were made on each enamel surface spaced a minimum distance apart of 80 lm. Images of the indentations were obtained using the corresponding microscope software (IM50 Image Manager; Leica) and analysed using ImageTool software (UTHSCSA ImageTool v. 3.00 for Microsoft Windows). The Vickers hardness number (VHN) for each enamel surface was calculated using the formula: (17) VHN ¼ 1854:4 P/d2 ; where P = load (g) and d = mean of diagonals of indentation (lm). The second half of each enamel surface was bonded with one of the two self-etching primer adhesive systems – CSE or TK – according to group. The CSE Primer was applied to the enamel in the first group, left undisturbed for 20 s, and air-thinned for 5 s. Then, CSE Bond was applied, gently airblown for 3 s, and translucent microtubes of 0.75 mm internal diameter and 1.5 mm height were placed on the adhesive-covered enamel and light-cured using a lightemitting diode light unit with an intensity of 800 mW cm)2 (Bluephase C8; Ivoclar Vivadent, Schaan, Liechtenstein). A hybrid resin composite, Clearfil Majesty Esthetic (Kuraray Medical, Okayama, Japan), was loaded into the tubes and cured for 20 s. Then, TK was applied to enamel in the second group, left in place for 20 s, air-thinned for 3 s, the microtubes placed, and the adhesive cured for 10 s. Bonding was carried out as described for CSE. Three to four microtubes were bonded to each enamel surface. After bonding, the specimens were placed in distilled water in an incubator at 37 °C for 24 h. Microshear bond strength test was carried out on a universal testing machine (Imperial 1,000; Mecmesin, West Sussex, UK) using the corresponding computer software (Emperor v. 01, Mecmesin, West Sussex, UK). The bonded specimens were placed in a jig with the enamel surface flush with the external surface of the jig, a wire loop (0.35 mm diameter) wound around the bonded cylinder at the composite–enamel interface at one end, and attached to a load cell connected to a computer. The bonds were stressed in shear at a crosshead speed of 1 mm min)1 until failure occurred, after which the maximum load at failure was recorded and converted to lSBS (MPa) by dividing the failure load by the bonded surface area. For each enamel surface, descriptive statistics (mean, standard deviation, coefficient of variation, and range) were calculated for the lSBS and VHN values. Weighted and non-weighted linear regression analyses were carried out to investigate possible relationships between the lSBS and the VHN using the statistical packages spss (spss 17 for Windows; SPSS, Chicago, IL, USA) and Stata (v. 10; StataCorp, College Station, TX, USA). Mean lSBS were regressed on mean VHN as the coefficients of variation were considerably less for the mean VHN values than for the mean lSBS values. The weights used for the weighted regression analyses were the inverse of the variances (variance)1) of the lSBS means. This assigns more weight to those specimens where there was low variability in the lSBS values and less weight to those specimens where there was high variability in the lSBS values. Regression diagnostics (residuals, leverage, and CookÕs distance) were used to assess the fit of the regression lines. The correlation analyses Table 1 Materials Adhesive Code Contents Manufacturer Batch no. Clearfil SE bond CSE Kuraray Medical, Okayama, Japan 51766 Tokuyama bond force TK Primer: 10-MDP; HEMA; hydrophilic dimethacrylate; di-camphoroquinone; N,N-diethanol-p-toluidine; water Bond: 10-MDP; Bis-GMA; HEMA; hydrophobic dimethacrylate; di-camphoroquinone; N,N-diethanol-p-toluidine; silanated colloidal silica Methacryloyloxyalkyl acid phosphate; C2-4 alkyl; HEMA, Bis-GMA; triethylene glycol dimethacrylate; camphoroquinone; purified water; alcohol Tokuyama Dental, Tokyo, Japan YT11407 Bis-GMA, bisphenol A diglycidylmethacrylate; HEMA, 2-hydroxyethyl methacrylate; 10-MDP, 10-methacryloyloxydecyl dihydrogen phosphate. Enamel microhardness and bond strengths 193 Table 2 Microshear bond strength (lSBS) and Vickers hardness number (VHN) data Clearfil SE bond (n = 22) Specimen no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Tokuyama bond force (n = 21) Mean lSBS (MPa) SD of mean lSBS (MPa) Mean VHN Specimen no. Mean lSBS (MPa) SD of mean lSBS (MPa) Mean VHN 20.1 17.4 22.0 15.7 21.6 16.8 23.4 22.8 17.4 20.1 21.9 20.4 22.5 18.6 14.6 15.8 17.5 15.5 19.8 16.6 18.6 21.8 4.25 7.10 4.82 4.45 4.52 5.54 5.68 3.50 7.71 1.39 3.45 2.67 2.44 3.86 7.15 1.94 2.16 5.97 2.30 2.63 7.92 9.95 344.1 357.4 380.9 368.1 343.0 344.6 384.0 355.3 352.4 357.3 379.4 356.4 323.7 364.3 403.7 358.6 375.5 323.8 360.7 369.4 359.3 366.1 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41* 42 43 44 7.00 11.1 7.65 11.7 12.4 11.4 10.8 8.33 7.87 8.53 6.28 4.23 7.90 6.24 6.35 5.90 8.85 5.95 3.20 3.44 5.83 8.99 3.57 5.18 2.13 6.15 4.88 3.12 7.76 2.52 2.60 1.65 1.50 2.34 3.16 1.17 0.53 1.92 1.56 0.65 0.24 1.25 2.51 1.79 343.7 313.0 365.0 332.4 363.8 349.4 324.8 313.2 376.6 365.7 398.3 380.1 338.8 314.1 353.2 336.2 340.4 362.0 387.0 331.9 381.8 384.2 * Specimen excluded from the weighted regression analysis for Tokuyama bond force. SD, standard deviation. were performed for each adhesive separately. The specimens from the two adhesives were combined for the regression analyses, and the regression models were fitted with (i) separate intercepts and separate slopes for each adhesive and (ii) separate intercepts and a common slope for each adhesive. The results from these models were compared using a likelihood ratio (LR) test. Correlations and regression coefficients were considered significant if their P-value was < 0.05. Results The lSBS and VHN results are shown in Table 2. For the 22 enamel surfaces assigned to each adhesive group, the VHN ranged from 255 to 481 VHN for CSE and from 221 to 450 VHN for TK. The mean VHN values for the enamel surfaces were 324–404 VHN for CSE and 313–398 VHN for TK. The coefficients of variation of the mean VHN were 4–14% for the 22 specimens bonded with CSE and 4–23% for those bonded with TK. The mean lSBS for the 22 enamel surfaces was between 14.6 and 23.4 MPa for CSE and between 3.2 and 12.4 MPa for TK. The coefficients of variation of the mean lSBS were between 7 and 49% for CSE-bonded specimens and between 8 and 72% for TK-bonded specimens. Whilst carrying out the weighted regression analysis, one observation from the TK adhesive group (specimen 41, Table 2) was found to have a much higher weight relative to the weights of the other observations and so was investigated in greater detail (Fig. 1). This observation was highly influential in determining the regression coefficients as a result of its very high leverage value coupled with its high mean VHN and low mean lSBS values. When this observation was included in the analysis, a significant weighted correlation (R = )0.68, P < 0.001) was found between VHN and lSBS for TK, whereas the non-weighted correlation was not significant (R = )0.32, P = 0.15). When the observation was removed from the analysis, neither the weighted correlation (R = 0.09, P = 0.69) nor the non-weighted correlation (R = )0.23, P = 0.31) were significant. Therefore, this observation was removed from the analysis. For CSE, neither the weighted correlation (R = )0.31, P = 0.15) nor the non-weighted correlation (R = )0.05, P = 0.83) were significant. Table 3 shows the results from the weighted regression analyses. Comparison of regression models shows that Model 1 (separate slopes and separate intercepts for each adhesive) is not significantly better than Model 2 (common slope and separate intercepts for each adhesive) (LR = 1.83, P = 0.18). Furthermore, the common slope parameter is not significantly different from zero (P = 0.97), indicating that there is no relationship between VHN and lSBS. Figure 1 also shows the regression lines of the mean lSBS against the mean VHN for Models 1 and 2. Also shown for comparison purposes is the regression line from Model 1 for adhesive TK with specimen 41 included. The regression results for Model 1 with specimen 41 included is also stated in Table 3. 194 Adebayo et al. Model 1 (CSE): µSBS = 37.09 – 0.050 VHN 25 Mean microshear bond strength (MPa) 7 8 13 20 19 10 21 9 6 14 17 2 20 16 18 15 11 12 1 Model 2 (CSE): µSBS = 19.04 + 0.00053 VHN 3 22 5 4 Model 2 (TK): µSBS = 6.29 + 0.00053 VHN 28 26 24 15 27 Model 1 (TK): µSBS = 4.03 + 0.0069 VHN 29 10 39 44 32 31 30 35 23 25 33 36 43 38 5 37 40 42 34 Model 1 (TK) (specimen 41 included): µSBS = 27.85 – 0.063 VHN 41 0 300 325 350 375 425 400 Mean Vickers Hardness Number (VHN) Fig. 1. Plot of mean microshear bond strength vs. mean enamel Vickers Hardness Number. The size of the circles is proportional to weights associated with each specimen for weighted regression analysis. Numbers beside each circle are the specimen numbers. Also shown are regression lines obtained from fitting Models 1 and 2. For comparison purposes, the regression line for Tokuyama Bond Force (TK) that was obtained from fitting Model 1 when specimen 41 was included is also shown. Model 1: Separate Intercepts, Separate Slopes; Model 2: Separate Intercepts, Common Slope. Specimen 1–22 = Clearfil SE Bond (CSE) Specimen 23–44 = TK. Discussion As the depth of cut into enamel and/or the amount of enamel removed in preparing flat surfaces for bond strength testing is often not disclosed, the enamel surfaces being bonded to may have different hardness properties. Cuy et al. (10) reported an 11% and a 12% decrease in CaO and P2O5, respectively, from the enamel surface to the dentino–enamel junction, which corresponded to a decrease in enamel hardness. Enamel and dentine microhardness have been positively correlated with calcium concentration (13). A strong, positive correlation between enamel and dentine microhardness and shear bond strength has been reported (13). Some phosphate ester-based monomers are reported to bond to the calcium of hydroxyapatite by their phosphate groups (11) and thus may further improve bond strengths, in addition to the mechanical interlocking of resin within the etched enamel. Therefore, using microhardness as an index of enamel calcium content, our study aimed to ascertain any possible correlation between Vickers hardness and the lSBS of two phosphate ester-containing self-etching primer adhesives. One-half of the ground enamel surface was used for the microhardness test and the other half was used for the lSBS test, in order to avoid the incorporation into the bond of possible subsurface cracks that may result from microhardness testing (8, 9) and which could potentially weaken the bond and thus predispose to early failure. The results of a weighted regression analysis make use of more information from the data collected (i.e. in addition to the means, it incorporates the values of the variance)1 and more completely represents the data population, and thus was preferred over the results of a non-weighted analysis). The results of our study, however, showed a weak, negative insignificant linear correlation between the lSBS of one of the adhesives used (CSE) and Vickers hardness. The observation of a negative correlation suggests that the lSBS of the CSE decreases with increasing enamel hardness and could be interpreted, based on the reported relationship between enamel calcium and hardness (10, 13), to imply that the lSBS Table 3 Regression statistics Estimate Model 1: separate intercepts, separate slopes Intercept CSE 37.1 Intercept TK 4.03 Slope CSE )0.050 Slope TK 0.0069 Model 2: separate intercepts, common slope Intercept CSE 19.0 Intercept TK 6.29 Slope (CSE/TK) 0.00053 Model 1: separate intercepts, separate slopes (specimen 41 included) Intercept CSE 37.1 Intercept TK 27.9 Slope CSE )0.050 Slope TK )0.063 SE P-value Log-likelihood LR test )80.6 14.8 5.19 0.041 0.015 0.016 0.44 0.23 0.64 )81.5 5.03 4.93 0.014 0.001 0.21 0.97 20.0 4.39 0.056 0.012 0.071 < 0.001 0.38 < 0.001 CSE, Clearfil SE bond; TK, Tokuyama bond force. * Significance level for the likelihood ratio (LR) test of Model 1 vs. Model 2. 0.18* Enamel microhardness and bond strengths of the adhesive decreases with increasing enamel calcium content. Considering that the phosphate ester monomer of this adhesive has been reported to bind readily to the calcium of hydroxyapatite (11, 12), a higher bond strength was expected with increasing microhardness. However, the results suggest the contrary. As the correlation between Vickers hardness and lSBS was weak, at best, for CSE, a definitive inference must not, however, be made about the relationship between the two parameters for the adhesive. The results for the Ôall-in-oneÕ adhesive, TK, showed no correlation between Vickers hardness and lSBS. The findings of no relationship between enamel lSBS and VHN for the two adhesives was confirmed by the outcomes of both the correlation and the regression analyses. A combination of micromechanical and chemical factors affect the adhesion of enamel–dentine adhesives. The etching ability of the self-etching primer may become more important on enamel with differing hardness values. Enamel with higher hardness values may be more mineralized and etch less well using the weak acidic monomer of a self-etching primer. Studies have shown that etch patterns are less distinct on hypermineralized enamel and that bond strengths to hypermineralized (i.e. fluorosed) enamel are significantly lower than to nonfluorosed enamel (19, 20). The lower surface area available for bonding that results from the poorer etch may compromise micromechanical adhesion, and the bond formed may be more readily sheared. Another possible explanation for the findings of this study is that although intra-tooth variations in enamel hardness exist, enamel is a homogeneous tissue with 96% inorganic matter by weight and the enamel hardness values fall within a known, relatively fixed, range (as seen in the coefficient of variation of 4–23%). The decrease in enamel calcium and phosphate with depth into enamel (10) may not be great enough to cause a dramatic change in enamel hardness and thus influence the lSBS. In addition, intra-tooth differences in calcium content may not be significant enough to affect the lSBS. A direct assessment of the relationship between enamel calcium concentration and the lSBS of the adhesives is warranted. Few studies have attempted to ascertain any possible correlation between enamel surface microhardness and bond strengths of enamel–dentine adhesive systems (13, 21). The results of our studies are in contrast to those of Panighi & Gsell (13), who reported a strong, positive linear correlation between enamel Vickers hardness and shear bond strength of a total-etch adhesive. The possible explanations for the differing results between their study and this study may be in the method used, of acidetched enamel (13). Acid etching exposes the intrinsic microstructural details of enamel (i.e. prisms, prism sheaths and intra-rod substance). Enamel microstructural components that contain larger quantities of organic material have been reported to have lower contents of calcium and phosphate than the surrounding highly mineralized enamel rods (10) and may bond differently. Acid-etched enamel presents a microscopically 195 rougher surface to the sharp hardness indenter than unetched enamel and may influence the hardness values observed. In addition, bonding to acid-etched enamel has reportedly resulted in higher, more consistent, bond strengths than self-etching primers as a result of the greater surface area (22–26), which promotes micromechanical adhesion and thus retention. The null hypothesis that there is no relationship between enamel microhardness and lSBS of the two self-etching primer adhesives thus cannot be rejected. 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