IMPROVEMENTS ON THE QUALITY OF DENTIN BONDS

Transcription

1 IMPROVEMENTS ON THE QUALITY OF DENTIN BONDS Fernanda Tranchesi Sadek

2 UNIVERSITY OF SIENA SCHOOL OF DENTAL MEDICINE PHD PROGRAM: DENTAL MATERIALS AND THEIR CLINICAL APPLICATIONS PhD THESIS OF Fernanda Tranchesi Sadek TITLE Improvements on the quality of dentin bonds 2

3 ACADEMIC YEAR 2005 / December 2006 Siena, Italy Committee: Promoter: Prof. Marco Ferrari Co-Promoter: Prof. Franklin R. Tay Prof. Carl Davidson Prof. Egidio Bertelli Prof. Fernando Zarone Prof. Ivo Kreiji Prof. Hickel Prof. R. Grandini Prof. Polimeni Dott.ssa Cecilia Goracci Dott. Simone Grandini TITLE Improvements on the quality of dentin bonds CANDIDATE Fernanda Tranchesi Sadek December

4 CONTENTS Introduction Chapter I Bond strengths of current adhesive materials to dentin I 1 - Knowing the performance of current adhesive materials in the first hours after bonding.. 14 I 1 1 Coronal dentin I 1 1 a - The effect of immediate sectioning on microtensile bond strengths of current adhesive systems to dentin.. 16 I 1 1 b - Early and 24-hour bond strength and degree of conversion of current adhesive systems I 1 2 Radicular dentin I 1 2 a - Immediate and 24-hour evaluation of the interfacial strengths of fiber posts.. 77 Chapter II Efforts to improve bond strength and ultra structural characteristics of dentin bonds II 1 The use of filled bonding system II Comparative study of a filled and an unfilled version of a dentin bonding system to dental hard tissues 97 4

5 II 2 The use of hydrophobic resin adhesives II Application of hydrophobic resin adhesives to acidetched dentin with an alternative wet bonding technique II 2 2 Tubular occlusion allows the use of hydrophobic resin adhesives to deep acid-etched dentin with an ethanol wet bonding protocol. 176 Summary, Discussion, Conclusions and Future directions Complete list of References Acknowledgments. 231 Curriculum Vitae

6 INTRODUCTION The development and advent of resin-based adhesive materials has allowed numerous changes to occur in dental clinical practice, being the main change the ability to preserve health tooth structures. This ability is related to the micromechanical retention achieved my means of resin infiltration into the demineralized tooth structure (Buonocore, 1955; Nakabayashi et al., 1982). Effective adhesion to enamel has been reached with relative ease and has repeatedly proven to be a durable and reliable clinical procedure for routine applications in modern adhesive restorative dentistry (Van Meerbeek et al., 2001). Conversely, adhesion to dentin is not as reliable as adhesion to enamel, justifying the great number of studies in this area, including this thesis. The dentin adhesion strategy involves several procedures: a conditioner or acid etchant, followed by the primer or adhesion promoter agent, and eventually, by the application of the bonding agent or adhesive resin. Two main approaches are found in the 6

7 current market. The etch and rinse technique requires the use and rinse of an acid, usually phosphoric acid, as a separate phase to expose the microporous network of collagen. Simplified two-step version combines the second and the third step. On the other hand, on the self-etch approach there is no longer needs of an etch and rinse phase, since in this technique acidic monomers play the role of the acid agent. Independently to the approach chosen, bond to dentin is usually critical. The dentin smear layer produced during cavity procedures should be removed by the acid-etching phase, which concurrently results in the demineralization of the dentin surface (Tay et al., 2000). This procedure exposes a microporous network of collagen for the micromechanical interlocking of monomers (Tay et al., 1997; Van Meerbeek et al., 2001; Van Meerbeek et al., 2003). It was speculated that incomplete infiltration of the demineralized collagen network could result in a weak zone that would be susceptible to long-term degradation (Nakabayashi et al., 1991; Pashley et al., 1992). With the use of self-etch adhesives, in 7

8 which the infiltration of resin occurs, theoretically, simultaneously with the self-etching process, the risk of discrepancy between both processes would be low or non-existent. Nonetheless, morphological evidences were already provided showing discrepancies between the depth of demineralization and the depth of resin infiltration in some mild self-etch adhesives. The incomplete resin infiltration were associated to the reduced etching potential of the acidic monomers toward the base of hybrid layers, or the presence of acidic but non-polymerizable hydrolytic adhesive components, creating potential sites for the degradation of the bonded created by these self-etch adhesives (Carvalho et al., 2005). Moreover, it is known that the seal created by a bond to dentin is not always perfect, as demonstrated by micro and nanoleakage studies (Pereira et al., 2001; Sano et al., 1995; Tay et al., 2002; Tay and Pashley, 2003b). A failed seal of the margins can result in pos-operative sensitivity, marginal staining and recurrent caries, which are the most common reasons associated 8

9 with clinical failure of adhesive restorations (Van Meerbeek et al., 1999). Several factors can influence adhesive systems bonding performance to dentin. As already described, the incomplete infiltration of resin in the demineralized dentin is one of these factors (Carvalho et al., 2005; Nakabayashi et al., 1991; Pashley et al., 1992). Furthermore, there is a risk of collagen collapse during air drying after etching (Carvalho et al., 2003; Eddleston et al., 2003; Gwinnett, 1994; Pashley et al., 2001a; Tay et al., 1997; Van Meerbeek et al., 1998) and moisture control has been shown to be critical when using conventional adhesive systems (Pashley et al., 1993; Tay et al., 1996). Incomplete adhesive solvent removal can also interfere in dentin bond (Carvalho et al., 2003; Erdemir et al., 2004; Ferrari and Tay, 2003; Maciel et al., 1996; Nalla et al., 2005; Pashley et al., 2003; Tay et al., 1995). New materials and techniques are constantly introduced to the market to overcome these problems, however, it is still unclear if they can produce 9

10 strong and reliable bonds. Therefore, continual evaluations of their performances are necessary. REFERENCES Buonocore MG (1955). A simple method of increasing the adhesion of acrylic filling materials to enamel surfaces. J Dent Res 34(6): Carvalho RM, Mendonca JS, Santiago SL, Silveira RR, Garcia FC, Tay FR, et al. (2003). Effects of HEMA/solvent combinations on bond strength to dentin. J Dent Res 82(8): Carvalho RM, Chersoni S, Frankenberger R, Pashley DH, Prati C, Tay FR (2005). A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in selfetch adhesives. Biomaterials 26(9): Eddleston CL, Hindle AR, Agee KA, Carvalho RM, Tay FR, Rueggeberg FA, et al. (2003). Dimensional changes in aciddemineralized dentin matrices following the use of HEMA-water versus HEMA-alcohol primers. J Biomed Mater Res A 67(3): Erdemir A, Eldeniz AU, Belli S, Pashley DH (2004). Effect of solvents on bonding to root canal dentin. J Endod 30(8): Ferrari M, Tay FR (2003). Technique sensitivity in bonding to vital, acid-etched dentin. Oper Dent 28(1):

11 Gwinnett AJ (1994). Chemically conditioned dentin: a comparison of conventional and environmental scanning electron microscopy findings. Dent Mater 10(3): Kanca J, 3rd (1992). Resin bonding to wet substrate. 1. Bonding to dentin. Quintessence Int 23(1): Maciel KT, Carvalho RM, Ringle RD, Preston CD, Russell CM, Pashley DH (1996). The effects of acetone, ethanol, HEMA, and air on the stiffness of human decalcified dentin matrix. J Dent Res 75(11): Nakabayashi N, Kojima K, Masuhara E (1982). The promotion of adhesion by the infiltration of monomers into tooth substrates. J Biomed Mater Res 16(3): Nakabayashi N, Nakamura M, Yasuda N (1991). Hybrid layer as a dentin-bonding mechanism. J Esthet Dent 3(4): Nalla RK, Balooch M, Ager JW, 3rd, Kruzic JJ, Kinney JH, Ritchie RO (2005). Effects of polar solvents on the fracture resistance of dentin: role of water hydration. Acta Biomater 1(1): Pashley DH, Horner JA, Brewer PD (1992). Interactions of conditioners on the dentin surface. Oper Dent Suppl 5( Pashley DH, Ciucchi B, Sano H, Horner JA (1993). Permeability of dentin to adhesive agents. Quintessence Int 24(9): Pashley DH, Agee KA, Nakajima M, Tay FR, Carvalho RM, Terada RS, et al. (2001). Solvent-induced dimensional changes in EDTA-demineralized dentin matrix. J Biomed Mater Res 56(2): Pashley DH, Agee KA, Carvalho RM, Lee KW, Tay FR, Callison TE (2003). Effects of water and water-free polar solvents on the 11

12 tensile properties of demineralized dentin. Dent Mater 19(5): Paul SJ, Welter DA, Ghazi M, Pashley D (1999). Nanoleakage at the dentin adhesive interface vs microtensile bond strength. Oper Dent 24(3): Pereira PN, Okuda M, Nakajima M, Sano H, Tagami J, Pashley DH (2001). Relationship between bond strengths and nanoleakage: evaluation of a new assessment method. Am J Dent 14(2): Sano H, Takatsu T, Ciucchi B, Horner JA, Matthews WG, Pashley DH (1995). Nanoleakage: leakage within the hybrid layer. Oper Dent 20(1): Tay FR, Gwinnett AJ, Pang KM, Wei SH (1995). Variability in microleakage observed in a total-etch wet-bonding technique under different handling conditions. J Dent Res 74(5): Tay FR, Gwinnett JA, Wei SH (1996). Micromorphological spectrum from overdrying to overwetting acid-conditioned dentin in water-free acetone-based, single-bottle primer/adhesives. Dent Mater 12(4): Tay FR, Gwinnett AJ, Wei SH (1997). Ultrastructure of the resindentin interface following reversible and irreversible rewetting. Am J Dent 10(2): Tay FR, Carvalho R, Sano H, Pashley DH (2000). Effect of smear layers on the bonding of a self-etching primer to dentin. J Adhes Dent 2(2): Tay FR, Pashley DH, Yoshiyama M (2002). Two modes of nanoleakage expression in single-step adhesives. J Dent Res 81(7):

13 Tay FR, Pashley DH (2003a). Water treeing--a potential mechanism for degradation of dentin adhesives. Am J Dent 16(1):6-12. Tay FR, Pashley DH (2003b). Have dentin adhesives become too hydrophilic? J Can Dent Assoc 69(11): Van Meerbeek B, Yoshida Y, Lambrechts P, Vanherle G, Duke ES, Eick JD, et al. (1998). A TEM study of two water-based adhesive systems bonded to dry and wet dentin. J Dent Res 77(1):50-9. Van Meerbeek B, Yoshida Y, Snauwaert J, Hellemans L, Lambrechts P, Vanherle G, et al. (1999). Hybridization effectiveness of a two-step versus a three-step smear layer removing adhesive system examined correlatively by TEM and AFM. J Adhes Dent 1(1):7-23. Van Meerbeek B, Vargas M, Inoue S, Yoshida Y, Peumans M, Lambrechts P, et al. (2001). Adhesives and cements to promote preservation dentistry. Oper Dent Suppl 6: Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. (2003). Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 28(3):

14 Chapter I Bond strengths of current adhesive materials to dentin I 1 - Knowing the performance of current adhesive materials in the first hours after bonding In an attempt to simplify the clinical procedures and to solve the problems associated with dentin bonding, new materials are constantly developed. In order to document their efficacy, several tests should be performed. Due to the high costs in time and resources of clinical testing, laboratory evaluations are a reliable method to predict clinical efficacy (Ferrari and Garcia-Godoy, 2002; Van Meerbeek et al., 2003). In vitro bond strength and microleakage tests are often relied upon as predictors of clinical performances for new generation adhesive-restorative materials. However, in most of the in vitro bond strength tests, measurements were performed at 24 hours or longer after the bonding procedures (Armstrong et al., 2003; Asmussen and Peutzfeldt, 2003; Cardoso et al., 2002). This experimental setting does not exactly reflect the clinical situation, 14

15 since the bonded interface is subjected to stresses within seconds after its placement in vivo. Thus, it is of interest to examine the immediate bond strength potential of current adhesive systems in order to evaluate their performance before the onset of post-curing polymerization, stress relaxation or bond maturation. REFERENCES Armstrong SR, Vargas MA, Fang Q, Laffoon JE (2003). Microtensile bond strength of a total-etch 3-step, total-etch 2-step, self-etch 2-step, and a self-etch 1-step dentin bonding system through 15-month water storage. J Adhes Dent 5(1): Asmussen E, Peutzfeldt A (2003). Short- and long-term bonding efficacy of a self-etching, one-step adhesive. J Adhes Dent 5(1):41-5. Cardoso PE, Sadek FT, Goracci C, Ferrari M (2002). Adhesion testing with the microtensile method: effects of dental substrate and adhesive system on bond strength measurements. J Adhes Dent 4(4): Ferrari M, Garcia-Godoy F (2002). Sealing ability of new generation adhesive-restorative materials placed on vital teeth. Am J Dent 15(2): Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. (2003). Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 28(3):

16 I 1 1 Coronal dentin The effect of immediate sectioning on microtensile bond strengths of current adhesive systems to dentin. Sadek FT, Goracci C, Cardoso PEC, Tay FR, Ferrari M. Journal of Adhesive Dentistry 2005, 7(4): INTRODUCTION In vitro measurements of bond strength and marginal integrity are commonly referred to by manufacturers, researchers, and clinicians as indicators of the potential performances of new dentin adhesives (Pashley et al., 1995; Pereira et al., 2001; Van Meerbeek et al., 2003). However, in most of the in vitro bond strength tests, measurements were performed at 24 hours or longer after the bonding procedures (Armstrong et al., 2003; Asmussen and Peutzfeldt, 2003; Cardoso et al., 2002). This experimental setting does not exactly reflect the clinical situation, since the bonded interface is subjected to stresses within seconds after its 16

17 placement in vivo. These stresses may arise from polymerization shrinkage of resin composites, occlusal and contouring adjustment, as well as finishing and polishing procedures that are performed immediately after the placement of the restorations (Burrow et al., 1994). Staninec and Kawakami (Staninec and Kawakami, 1993) reported that some dentin adhesives exhibited an increase in bond strength over a short period of time (3 minutes, 1 hour and 24 hours). The authors suggested that these increases may be due to further polymerization at the interface, or to stress relaxation by hygroscopic expansion of the composite. Burrow et al. (Burrow et al., 1994) found a steady increase in tensile bond strength over a period of 24 hours, suggesting that bonds mature during this period. Barink et al. (Barink et al., 2003) also reported that stress is expected to decrease during the first hour after polymerization. According to these authors, it may be advisable to instruct the patient not to load the restoration for a certain period (Kuijs et al., 2003). 17

18 The microtensile test is considered to be a reliable adhesion testing technique (Pashley et al., 1999) that is capable of assessing the interfacial strength between an adhesive and the bonding substrate (Van Noort et al., 1989). It is also a testing technique that is conservative on the number of teeth employed, as virgin human teeth are becoming increasing difficult to harvest from some parts of the world (Pashley et al., 1999). To obtain microtensile specimens, a series of cuts are made on a single tooth that may induce the formation of internal defects. Similarly, under clinical conditions, flaws may develop as a result of the stresses acting on the newly created bonded interface. Some authors (Martin et al., 2003; Yap et al., 2003) believe that hygroscopic expansion caused by water sorption may help to relieve some of the internal stresses created during polymerization shrinkage or close marginal leakage gaps. However it is unclear if this phenomenon can heal these internal flaws (Huang et al., 2002; Momoi and McCabe, 1994). Self-etching primers and self-etching adhesives (all-inones) offer some advantages over total-etch adhesives, such as ease 18

19 of use and faster manipulation (Tay and Pashley, 2002), reduced technique sensitivity (Unemori et al., 2001), and limited postoperative sensitivity (Christensen, 2002). However, their etching potential is not as aggressive as that produced by phosphoric acid (Pashley and Tay, 2001; Tay and Pashley, 2001). The majority of 24-hour bond strength data available from peer-reviewed, nonmanufacturer supported studies revealed an impaired bonding potential of simplified systems, as compared with conventional 3- step total-etch adhesives. As it is imperative that the restoration supports initial masticatory loading and thermal fatigue, assessment of bond strengths immediately after the restoration procedure is important from a clinical perspective. Only limited information is available in the literature on the immediate or early bond strength of conventional 3-step total-etch and 2-step self-etch adhesives. Moreover, this type of information is totally missing for the more recently introduced simplified 2-step total-etch and 1-step self-etch adhesives. Thus, it is of interest to examine the immediate bond strength potential of current adhesive systems in order to 19

20 evaluate their performance before the onset of post-curing polymerization, stress relaxation or bond maturation. The objective of the present in vitro study was to compare the bond strengths of immediately sectioned specimens produced from four different classes of dentin adhesives (i.e. 3-step totaletch, 2-step total-etch, 2-step self-etch and 1-step self-etch) to crown dentin, with each class being representative of a current distinct approach to dentin bonding. The rationale behind the use of immediate sectioning was to simulate the creation of internal flaws within the bonded specimens (Callister, 1994; Griffith, 1920) in a way that may be comparable to the immediate stressing of bonded restorations in vivo (Frankenberger et al., 2003). The null hypotheses tested were that [1] the microtensile bond strengths of the adhesives are comparable regardless of the application procedure involved, and [2] there is no difference between early and 24-hour bond strengths when specimen sectioning is commenced immediately after bonding. 20

21 MATERIALS AND METHODS Thirty-three caries- and defect-free human molars were stored in 1% Chloramine T at 4 o C to prevent bacterial growth and used within one month after extraction. The teeth were cleaned, fixed with sticky wax on an acrylic resin cylinder support, which was mounted on an Isomet cutting machine (Buehler, Lake Bluff, IL, USA), and had their occlusal third removed using a slow-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA) under water cooling. The exposed coronal dentin surface of each tooth was polished manually with 180-grit silicon carbide paper under running water for 20 seconds, in order to create a thick, clinically relevant smear layer (Koibuchi et al., 2001). The teeth were drawn to be randomly distributed into eleven groups (n=3). Then, each group was assigned randomly according to one of the eleven adhesives tested, which were differentiated into four major categories (Table 1): A. 3-step total-etch: Adper Scotchbond MP (3M ESPE, St. Paul, MN, USA); 21

22 B. 2-step total-etch: Adper Scotchbond 1 (3M ESPE) and Optibond Solo Plus (Sybron- Kerr, Orange, CA, USA); C. 2-step self-etch: Clearfil SE Bond (Kuraray Medical Inc., Tokyo, Japan), AdheSE (Ivoclar-Vivadent, Schaan, Liechtenstein), Tyrian SPE + One Step Plus (Bisco Inc, Schaumburg, IL, USA) and Optibond Solo Plus self-etching (Sybron-Kerr); D. 1-step self-etch: One-Up Bond F (Tokuyama Corp., Tokyo, Japan), ibond (Heraeus Kulzer, Hanau, Germany), Adper Prompt L-Pop (3M ESPE) and Xeno III (Dentsply DeTrey, Konstanz, Germany). All adhesive systems were applied strictly according to the manufacturers instructions. A resin composite (Tetric Ceram, Ivoclar-Vivadent) was used incrementally to form a crown of approximately 6 mm in height. Each composite layer was light-cured for 40 seconds at a curing intensity of 600 mw/cm 2. 22

23 Immediately upon completion of the restorative procedure, each tooth was fixed with sticky wax on an acrylic resin cylinder support, which was mounted on an Isomet cutting machine (Buehler, Lake Bluff, IL, USA), in a way that the tooth remained parallel to the blade. By means of this water-cooled diamond blade, the tooth was sectioned occluso-gingivally into a serial slabs. Then, the slabs were further sectioned into 0.9 x 0.9 mm composite-dentin sticks, according to the non-trimming technique (Shono et al., 1999). Specimens that failed prematurely during sectioning were recorded in each group (Table 2), but were excluded in the statistical analyses. The intact sticks from each tooth were then randomly divided into two sub-groups. One subgroup was used for immediate testing of the bond strength. The remaining specimens were stored in distilled water at 37 C for 24 hours before being loaded to failure. For microtensile test, each specimen had its cross-sectional area precisely measured with a digital caliper and had its ends 23

24 glued with cyanoacrylate (Zapit, DVA, Corona, CA, USA) to the Geraldeli s device (Perdigao et al., 2002). This jig is made of two parts that are kept together by a couple of post-hole joints. The Geraldeli s device was placed in a Bencor unit, which was mounted on a universal testing machine (Triax digital 50, Controls, Milano, Italy). When the loading machine was activated in tension, the two rods of the Bencor device moved away from each other, and so did the two parts of the Geraldeli s device following the guidance of the post-hole joints, in such a way that purely tensile forces were applied to the microtensile stick. Failure modes were observed under an optical microscope (Nikon type 102, Tokyo, Japan), and recorded as adhesive or cohesive in either resin or dentin. Only the specimens that exhibited an adhesive failure mode were included for analysis. The distribution of microtensile bond strength data was first checked for normality with the Kolmogorov-Smirnov test and then statistically analyzed by a two-way ANOVA design to examine the effect of the factors adhesive and testing time on bond strength, both considering bond strength data per tooth (in 24

25 order to take into account the tooth-related variance) and polling all sticks coming from different teeth of the same group. The Least Significant Difference (LSD) test was applied for post-hoc comparisons. A Spearman rank correlation test was also performed to examine the correlation between premature failure percentages and the measured bond strength values. For all the analyses statistical significance was set at p < RESULTS The microtensile bond strengths and percentages of premature failure of the experimental groups are summarized in Table 2. Bond strength of the adhesive systems was not influenced by testing time (i.e. immediately vs 24-hour) when the specimens sticks were sectioned immediately after bonding (p>0.05). Conversely, the adhesive materials exhibited a highly significant influence on the measured bond strengths (p=0.0011). The interaction between adhesive material and testing time was not 25

26 significant (p>0.05). The all-in-one system ibond exhibited the lowest microtensile bond strength of all the materials tested, both for early testing ( MPa) and after 24 hours ( MPa). These differences were statistically significant (p<0.05). ibond also presented the highest percentage of premature failure (38%) during the pre-test phase. High frequencies of premature failure (>10%) were observed only with self-etching materials (Table 2), and the Spearman test revealed a highly significant negative correlation (p<0.01) between the percentage of premature failures and the bond strength. Adper Prompt L-Pop, Xeno III, Tyrian SPE + One Step Plus and One-Up Bond F, all being self-etching materials, developed a significantly weaker adhesion than AdheSE, Optibond Solo Plus, Adper Scotchbond 1, Optibond Solo Plus self-etching, Clearfil SE Bond, and Adper Scotchbond MP, which were all statistically equivalent. DISCUSSION 26

27 In this study, the bond strengths of eleven current adhesive systems were assessed following the non-trimming method of microtensile bond testing. This technique is currently considered to be a reliable adhesion test, for it allows the loading stress to be more uniformly distributed by the testing of small-sized specimens (Pashley et al., 1999; Van Meerbeek et al., 2003). Moreover, with this variant of the method, multiple specimens can be obtained from a single tooth, and the coefficients of variation associated with testing is usually between 10-25% (Pashley et al., 1999), providing more accurate results. Indeed, the coefficients of variation in this study were within the acceptable limits of frequency for all the tested groups (ranging between 2.5 to 24.7%) except for ibond, which exhibited a wide spread of the bond strength data, probably due to its poor adhesion, resulting in an interface more susceptible to the cutting procedures. We realized that the actual number of teeth employed in each group (vs. number of beams produced from each tooth) was rather small and that inter-tooth differences exist that may affect 27

28 our conclusion. However, the higher means and standard deviations obtained from specimens of smaller diameter enables the microtensile bond strength test to be discriminative enough for detecting differences arising from treatment variables with the use of a smaller number of actual tooth specimens (Tay et al., 2003b). Moreover, a previous paper has shown that the intra-tooth variation (i.e. the number of beams from each tooth) was either similar or even larger than inter-tooth variation (Loguercio et al., 2005). The trade-off for such an improvement in the reliability of adhesion testing is that the method is time consuming and technically demanding. Although we referred to the initial measurements in each tooth as early or even immediate bond strength, it should be noted that about one hour has elapsed between the time when bonding was performed and the time when the sticks were ready for microtensile bond testing. Moreover, to obtain the specimens for microtensile testing, serial cuts have to be made into the restored tooth, with the chance of creating internal flaws within the specimen. In this study, the 28

29 sticks were cut immediately, and then randomly divided into two similarly sized sub-groups; one for immediate testing and the other was aged for 24 hours in distilled water before being loaded. In this way, the same internal flaws within the sticks were created, irrespective of whether bond strengths were tested immediately or after 24 hours. Presumably, these flaws could not heal by themselves after this period. This is probably the reason why no differences between immediate and 24-hour microtensile bond strength were found in this study. Thus we have to accept the second null hypothesis, even though it is known that self-etch adhesives, which are acidic in nature, take a relatively longer time to achieve optimal polymerization (Suh et al., in press). Conversely, previous studies (Burrow et al., 1994; Staninec and Kawakami, 1993) that showed an increase in the bond strength were performed with tensile tests and the restored tooth were stored for 24 hours without any interference. These experimental settings do not exactly reflect the clinical situation, as the bonded interface is subjected to stresses within seconds after its placement. This is 29

30 the reason why all the specimens were obtained by an immediate cut in this study. Within the limits of this in vitro study, all the 1-step selfetching adhesives showed lower microtensile bond strengths than 2-step self-etching adhesives, with the exception of Tyrian SPE in combination with One-Step Plus (Table 2). Among the all-in-one systems, ibond exhibited the worst results, failing to produce a reliable adhesion to dentin. The poor performance of ibond may be caused by the hydrolysis of acidic monomer 4-META and the hydrophilic monomer HEMA, in the presence of water that is included within the adhesive. It has been shown that these monomers can be readily hydrolyzed by water upon storage at increased temperature (Nishiyama et al., 2004). As ibond is a water-containing, no-mix type 1-step self-etch adhesive, the 4- META can hydrolyze very rapidly under elevated temperature or prolonged storage. Even though all the materials tested were kept in the refrigerator after opening, one cannot be sure about the storage conditions during transportation or about the time interval 30

31 between production and delivery. Premature failures were frequently seen with all the 1-step self-etching systems, as well as with Tyrian SPE + One-Step Plus (Table 2). For example, only 64% of the sticks from the Adper Prompt L-Pop groups were useful for statistical calculations, as the rest failed prematurely during the cutting step (Table 2). Overall, the performance of 1-step self-etch systems in this study was inferior to the other adhesive categories. Similar findings have already been reported in the literature for this class of materials (Asmussen and Peutzfeldt, 2003; Brackett et al., 2002; Inoue et al., 2001) The satisfactory results of 2-step self-etch adhesives were in agreement with previous studies (Armstrong et al., 2003; Inoue et al., 2001). With the exception of Tyrian SPE + One-Step Plus, dentin bond strengths were comparable to those of total-etch adhesives. For Clearfil SE Bond and AdheSE, the bonding component creates a hydrophobic coat which prevents the adhesive layer from behaving as a permeable membrane after polymerization 31

32 (Tay et al., 2003c), that expedites water sorption within the adhesive layer (Carvalho et al., 2004). Also, the acidic primers of Clearfil SE Bond and AdheSE are not based on UDMA and TEGDMA. In addition AdheSE contains newly patented monomers that, according to the manufacturer, are hydrolytically stable at room temperature, retaining their quality throughout the storage time. The assessment of bond durability, however, remained beyond the scope of this investigation, which only focused on early and 24-hour bond strength. In conclusion, our results led us to the reject the first null hypothesis that the tested materials, representatives of different current approaches to dentin bonding, have similar adhesive potentials to dentin. The finding of significantly lower bond strength for 1-step self-etch adhesives suggests that simplification of the handling procedure still comes at the price of a reduction in bonding effectiveness. However, the results failed to reject the second null hypothesis. 32

33 ACKNOWLEDGMENTS The authors are grateful to Programme Al!an, European Union Programme of high level scholarships for Latin America for support (identification number E03D08091BR). We thank all manufacturers for the generous donation of the respective adhesives investigated. CLINICAL RELEVANCE The assessment of bond strengths immediately after the restoration procedure is important from a clinical perspective. The finding of significantly lower immediate bond strengths in one-step self-etch adhesives suggests that simplification of bonding procedures is achieved at the expense of reduction in bonding effectiveness. REFERENCES Armstrong SR, Vargas MA, Fang Q, Laffoon JE. Microtensile bond strength of a total-etch 3-step, total-etch 2-step, self-etch 2-33

34 step, and a self-etch 1-step dentin bonding system through 15- month water storage. J Adhes Dent 2003;5: Asmussen E, Peutzfeldt A. Short- and long-term bonding efficacy of a self-etching, one-step adhesive. J Adhes Dent 2003;5:41-5. Barink M, Van der Mark PC, Fennis WM, Kuijs RH, Kreulen CM, Verdonschot N. A three-dimensional finite element model of the polymerization process in dental restorations. Biomaterials 2003;24: Brackett WW, Covey DA, St Germain HA, Jr. One-year clinical performance of a self-etching adhesive in class V resin composites cured by two methods. Oper Dent 2002;27: Burrow MF, Tagami J, Negishi T, Nikaido T, Hosoda H. Early tensile bond strengths of several enamel and dentin bonding systems. J Dent Res 1994;73: Callister WDJ. Materials science and engineering. An introduction. New York: Wiley and Sons, Cardoso PE, Sadek FT, Goracci C, Ferrari M. Adhesion testing with the microtensile method: effects of dental substrate and adhesive system on bond strength measurements. J Adhes Dent 2002;4: Carvalho RM, Tay FR, Giannini M, Pashley DH. Effects of preand post-bonding hydration on bond strength to dentin. J Adhes Dent 2004;6:

35 Christensen GJ. Preventing postoperative tooth sensitivity in class I, II and V restorations. J Am Dent Assoc 2002;133: Frankenberger R, Strobel WO, Kramer N, Lohbauer U, Winterscheidt J, Winterscheidt B, et al. Evaluation of the fatigue behavior of the resin-dentin bond with the use of different methods. J Biomed Mater Res 2003;67B: Griffith A. The phenomena of rupture and flow in solids. Phil Trans Royal Soc London 1920;Series A: Huang C, Kei LH, Wei SH, Cheung GS, Tay FR, Pashley DH. The influence of hygroscopic expansion of resin-based restorative materials on artificial gap reduction. J Adhes Dent 2002;4: Inoue S, Vargas MA, Abe Y, Yoshida Y, Lambrechts P, Vanherle G, et al. Microtensile bond strength of eleven contemporary adhesives to dentin. J Adhes Dent 2001;3: Koibuchi H, Yasuda N, Nakabayashi N. Bonding to dentin with a self-etching primer: the effect of smear layers. Dent Mater 2001;17: Kuijs RH, Fennis WM, Kreulen CM, Barink M, Verdonschot N. Does layering minimize shrinkage stresses in composite restorations? J Dent Res 2003;82: Loguercio AD, Barroso LP, Grande RHM, Reis A. Comparison of intra and inter teeth resin-dentin bond strength variability. Journal of Adhesive Dentistry 2005;in press: 35

36 Martin N, Jedynakiewicz NM, Fisher AC. Hygroscopic expansion and solubility of composite restoratives. Dent Mater 2003;19: Momoi Y, McCabe JF. Hygroscopic expansion of resin based composites during 6 months of water storage. Br Dent J 1994;176:91-6. Nishiyama N, Suzuki K, Yoshida H, Teshima H, Nemoto K. Hydrolytic stability of methacrylamide in acidic aqueous solution. Biomaterials 2004;25: Pashley DH, Carvalho RM, Sano H, Nakajima M, Yoshiyama M, Shono Y, et al. The microtensile bond test: a review. J Adhes Dent 1999;1: Pashley DH, Sano H, Ciucchi B, Yoshiyama M, Carvalho RM. Adhesion testing of dentin bonding agents: a review. Dent Mater 1995;11: Pashley DH, Tay FR. Aggressiveness of contemporary self-etching adhesives. Part II: etching effects on unground enamel. Dent Mater 2001;17: Perdigao J, Geraldeli S, Carmo AR, Dutra HR. In vivo influence of residual moisture on microtensile bond strengths of one-bottle adhesives. J Esthet Restor Dent 2002;14:

37 Pereira PN, Okuda M, Nakajima M, Sano H, Tagami J, Pashley DH. Relationship between bond strengths and nanoleakage: evaluation of a new assessment method. Am J Dent 2001;14: Shono Y, Ogawa T, Terashita M, Carvalho RM, Pashley EL, Pashley DH. Regional measurement of resin-dentin bonding as an array. J Dent Res 1999;78: Staninec M, Kawakami M. Adhesion and microleakage tests of a new dentin bonding system. Dent Mater 1993;9: Suh BI, Feng L, Pashley D, Tay FR. Factors contributing to the imcompatibility between simplified-step adhesives and chemicalcured or dual-cured composites. Part III. Effect of acidic resin monomers. J Adhes Dent 2003;5(4): Tay FR, Pashley DH. Aggressiveness of contemporary self-etching systems. I: Depth of penetration beyond dentin smear layers. Dent Mater 2001;17: Tay FR, Pashley DH. Dental adhesives of the future. J Adhes Dent 2002;4: Tay FR, Pashley DH, Yiu CK, Sanares AM, Wei SH. Factors contributing to the incompatibility between simplified-step adhesives and chemically-cured or dual-cured composites. Part I. Single-step self-etching adhesive. J Adhes Dent 2003;5: Tay FR, Suh BI, Pashley DH, Prati C, Chuang SF, Li F. Factors contributing to the incompatibility between simplified-step 37

38 adhesives and self-cured or dual-cured composites. Part II. Singlebottle, total-etch adhesive. J Adhes Dent 2003;5: Unemori M, Matsuya Y, Akashi A, Goto Y, Akamine A. Composite resin restoration and postoperative sensitivity: clinical follow-up in an undergraduate program. J Dent 2001;29:7-13. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 2003;28: Van Noort R, Noroozi S, Howard IC, Cardew G. A critique of bond strength measurements. J Dent 1989;17:61-7. Yap AU, Shah KC, Chew CL. Marginal gap formation of composites in dentine: effect of water storage. J Oral Rehabil 2003;30:

39 Table 1: Materials used, components, batch number and manufacturers Materials Components Batch # Manufacturer Etch: 35% phosphoric acid, water, silica 3BC Adper Primer: water, HEMA, polyalkenoic 3AG 3M ESPE, St. Scotchbond acid copolymer Paul, MN, Multi-Purpose Bond: bis-gma, HEMA, CQ, 3NF USA EDMAB, DHEPT Adper Scotchbond 1 Optibond Solo Plus Clearfil SE Bond AdheSE Tyrian SPE + One Step Plus Etch: 35% phosphoric acid, water, silica Bond: water, ethanol, bis-gma, HEMA, DMA, photoinitiator, methacrylate functional copolymer, polyitaconic acids Etch: 35% phosphoric acid, water, silica Bond: ethanol, alkyl DMA resin, HEMA Primer: 10-MDP, HEMA, hydrophilic DMA, N,N-diethanol p-toluidine, CQ, water Bond: 10-MDP, bis-gma, HEMA, silanated silica, hydrophobic DMA, N,N-diethanol p-toluidine, CQ Primer: DMA, phosphonic acid acrylate, initiators, stabilizers in a aqueous solution Bond: HEMA, DMA, silicon dioxide, initiators, stabilizers Primer: 2-acrylamido-2-methyl propanesulfonic acid, bis(2- methacryloyloxy)ethyl)phosphonate, ethanol Bond: biphenyl DMA, HEMA, acetone, glass frit 3BC 3JB 3BC 207A F M ESPE 3M ESPE Sybron-Kerr, Orange, CA, USA Kuraray Medical Inc., Tokyo, Japan Ivoclar- Vivadent, Schaan, Liechtenstein Bisco Inc., Schaumburg, IL, USA 39

40 Optibond Solo Plus self-etch One-Up Bond F ibond Adper Prompt L-Pop Xeno III Resin composite Tetric Ceram Primer: ethanol, alkyl DMA resin Bond: ethanol, alkyl DMA resin, HEMA Methacrylate, water, fluoroaluminosilicate glass filler A Y03 Water, acetone, UDMA, 4-META, glutaric photoinitiators, stabilizers Liquid #1: methacrylated phosphate esters, Bis-GMA, initiators based on CQ, stabilizer Liquid #2: water, HEMA, polyalkenoic acid, stabilizer Liquid A: HEMA, water, ethanol, BHT, silicon dioxide Liquid B: phosphoric acid modified methacrylate resin, mono fluoro phosphazene methacrylate, UDMA, CQ, EDMAB Bis-GMA, UDMA, triethylene glycol DMA, inorganic fillers, catalyst, stabilizers, pigments HEMA: 2-hydroxyethyl methacrylate Bis-GMA: bisphenol A diglycigylmethacrylate CQ: camphoroquinone EDMAB: ethyl 4-dimethyl amino benzoate Sybron-Kerr Tokuyama Corp., Tokyo, Japan Heraeus Kulzer, Hanau, Germany M ESPE A B F55945 Dentsply DeTrey, Konstanz, Germany Ivoclar- Vivadent DMA: diglycidylmethacrylate 10-MDP: 10-methacryloxyloxydecyl dihydrogen phosphate BHT butylated hydroxytoluene UDMA urethane dimethacrylate resin 40

41 Table 2: Results of microtensile bond test ranked from the lowest to highest values and premature failure (PF) percentages. Microtensile Microtensile bond strength (MPa) bond strength (MPa) PF as determined by the as determined by the (%) number of beams (N= 18- number of teeth (N= 33) * 3) ** ibond c (18) Adper Prompt L-Pop b (20) b Xeno III (20) Tyrian SPE + One Step Plus b (29) One Up Bond F b (27) a AdheSE (33) Optibond Solo Plus a (32) a Scotchbond 1 (31) Optibond Solo Plus self-etch a (30) Clearfil SE Bond a (31) Scotchbond MP a (28) Early 24 hours Early 24 hours (19) (19) (20) (30) (26) (33) (31) (30) (30) (31) (29) step self-etch 2-step self-etch 2-step total-etch 3-step total-etch The groups with the same superscript letter are not significantly different (p>0.05), two-way ANOVA and LSD test. 41

42 * Values are means and standard deviations obtained by pooling all specimen beams from the same group. Number enclosed within the parenthesis represents the total number of beams in each group. ** Values are means and standard deviations obtained from the mean bond strength of each of the three teeth in a group. 42

43 Early and 24-hour bond strength and degree of conversion of etch-and-rinse and self-etch adhesive systems. Sadek FT, Calheiros FC, Cardoso PEC, Kawano Y, Tay FR, Ferrari M. American Journal of Dentistry, in press. INTRODUCTION In vitro measurements of bond strength and marginal integrity are commonly employed by manufacturers, researchers, and clinicians as indicators of the adhesive potential of new materials (Pashley et al., 1995; Pereira et al., 2001; Van Meerbeek et al., 2003). However, with most in vitro bond strength tests, measurements are performed at 24 hours or longer after bonding (Armstrong et al., 2003; Asmussen and Peutzfeldt, 2003; Cardoso et al., 2002). This experimental setting does not exactly reflect the clinical situation, since bonded interfaces are subjected to stress within seconds after its placement in vivo. Previous studies have shown that some dentin adhesives exhibited increases in bond strength over a short period of time 43

44 (Burrow et al., 1994; Staninec and Kawakami, 1993; Talic, 2003). Such increases may be due to further polymerization at the interface, or to stress relaxation by hygroscopic expansion of the overlying composite. Suh et al. (Suh et al., 2003) suggested that self-etch adhesives, which are acidic in nature, take a relatively longer time to achieve adequate degrees of conversion. Nonetheless, additional cure of the adjacent composite resin during restoration procedures may be sufficient to complete adhesive polymerization (Dickens and Cho, 2005). Higher stresses were observed along the bonded interfaces during the first few hours after polymerization (Barink et al., 2003). It was hypothesized that the lower conversion and higher stresses that occur immediately after setting or during initial stages of restoration could be responsible for restoration failure (Asmussen and Jorgensen, 1972). This hypothesis should be tested for current adhesive systems, since only limited information is available on the association between immediate bond strength and the degree of conversion of adhesives. 44

45 It was previously observed that the bond strengths of contemporary dentin adhesives were not influenced by testing time when specimens were sectioned immediately after bonding (i.e. immediately vs 24 hours) (Sadek et al., 2005b). However, in that previous study, all the specimens were sectioned immediately after bonding without waiting for the bonds to mature. Thus, similar internal flaws were present within the microtensile testing beams irrespective of whether bond strength measurements were conducted immediately or after 24 hours. It is perceived that such an experimental design may be improved by using a split tooth approach, in which one half of the bonded tooth is sectioned immediately, while the half is bonded and aged for 24 hours to enable the resin-dentin bond to mature prior to sectioning. When bonded to dentin, adhesives are applied as thin films and the degree of conversion (DC) of these layers may be used as an indirect measure of inherent strength (Dickens and Cho, 2005). It is generally accepted that the mechanical properties of lightpolymerized dental materials are improved with increasing DC 45

46 (Ferracane and Greener, 1986; Lohbauer et al., 2005). Thus, would be of clinical significance to examine the DC of current dentin adhesives immediately upon polymerization to understand the effect of bond maturity on bond strengths of these adhesives. Although DC may be investigated using different methods, Fourier Transform Raman spectroscopy has been reported to be a simple and reliable and method (Lindberg et al., 2005; Truffier-Boutry et al., 2005; Witzel et al., 2005). Thus, the objective of this in vitro study was to compare the early and 24-hour bond strengths to dentin with the use of the aforementioned split tooth design, and DC of one adhesive system from each of the four different bonding strategies (i.e. 3-step totaletch, 2-step total-etch, 2-step self-etch and 1-step self-etch). The null hypotheses tested were that (1) there are no differences between early and 24-hour tensile bond strengths and DC, and (2) no correlation exists between bond strength and DC for the adhesives investigated. 46

47 MATERIALS AND METHODS Microtensile bond strength Forty caries- and defect-free human molars were stored in 0.5% chloramine T at 4 o C to prevent bacterial growth and used within one month after extraction. Prior to the testing procedures, they were removed from the disinfectant solution and stored in distilled water for 48 hours. The oclusal third of each tooth was removed using a slow-speed saw (Digital Isomet) a under water irrigation to expose the mid-coronal dentin. The latter was polished with a 180 grit silicon carbide paper under running water for 20 s to create a clinically-relevant smear layer (Koibuchi et al., 2001). The prepared surfaces were examined under a stereoscopical microscope (Nikon Type 102 b ) at 30x magnification to ensure that the bonding surfaces were free of enamel. Each tooth was sectioned occluso-gingivally into two halves using the Isomet saw under water irrigation, with the two halves from the same tooth comprising the immediate vs 24 hour components of the split tooth study design. 47

48 The teeth were then randomly divided into four groups (n=10) according to the adhesive system tested: one 3-step totaletch: Adper Scotchbond MP c ; one 2-step total-etch: Adper Scotchbond 2 c one 2-step self-etch: Clearfil SE Bond d and one 1- step self-etch: Adper Prompt L-Pop c. The chemical compositions of these adhesives are shown in Table 1. All adhesives were applied according to the manufacturers instructions. A resin composite (Tetric Ceram e ) was used incrementally to form a crown of approximately 6 mm in height. An incremental build up technique was employed with 1.5 mm-thick composite layers. Each composite layer was light-cured for 40 s using a quartz-tungsten-halogen light-curing unit (VIP f ) at an output intensity of 600 mwcm -2. Immediately upon completion of the restorative procedure, one bonded tooth-half from each tooth was immediately sectioned perpendicularly to the bonded interface with the Isomet saw into a series of slabs. Then, the slabs were further sectioned into 0.9 x 0.9 mm composite-dentin beams, according to the non-trimming 48

49 technique. Each stick was measured in width and thickness with a pair of digital calipers, and glued with cyanoacrylate (Zapit g ) to a Geraldeli s jig. The latter was placed in a Bencor Multi-T testing assembly h, which was mounted on an universal testing machine (Triax digital 50 i ). The specimens were stressed to failure at a crosshead speed of 0.5 mm min -1. The load at failure was recorded in Newtons and bond strength was expressed in MPa. The other bonded tooth-half of each tooth was stored in distilled water at 37 o C for 24 hours before sectioning and testing in the manner previously described. The failure sites after debonding were examined under the Nikon stereoscopical microscope b at 30x magnification and recorded as adhesive or cohesive in either resin or dentin. Only the specimen beams that exhibited an adhesive failure mode were included in the statistical analysis. Beams that exhibited premature failure in both the immediate and 24-hour periods were assigned null bond strength values and also included in the compilation of the mean bond strengths. 49

50 Degree of conversion Degree of conversion (DC) was measured using FT-Raman spectroscopy (RFS 100/S j ) by means of a Nd:YAG laser source at 1064 nm and a power of 50 mw. Fluorescence-free Raman spectra were acquired with the RFS 100/S spectrometer equipped with a liquid nitrogen-cooled Ge diode detector. The spectrometer was coupled to an Olympus BX series optical microscope (Raman Scope III j ). Iron molds, 3 mm diameter and 0.5 mm in thickness, were filled with the adhesives to be tested (N=3). The spectra of uncured adhesives were first obtained by evaporating the adhesive in each mold with oil- and moisture-free compressed air for 40 s. For the 2-step adhesives (Scotchbond MP and Clearfil SE Bond), both components were applied stepwise to the mold. The primers of these 2-step adhesives were evaporated also with oil- and moisture-free compressed air for 40 s prior to the application of the bonding resin components. New disc-shaped molds were then filled with adhesives that were air-dried as above, covered with a Mylar strip, and irradiated for the curing time recommended by the 50

51 respective manufacturer. After removal of the Mylar strip, the immediate spectra were collected. The disc-shaped specimens were stored in dark bottles for 24 hours at 37 º C. FT-Raman spectra were retaken at 1 and 24 hours after curing. All the spectra were obtained in front measurement channel with co-addition of 128 scans, at a resolution of 4 cm -1. The intensity ratio between aliphatic (1640 cm -1 ) and aromatic (1610 cm -1 ) carbon double bonds bands was used to calculate the DC (Figure 1), according to the formula DC% = [1 ((I C=C /I! ) cured specimen /(I C=C /I! ) uncured specimen)] x 100, where I C=C = intensity ratio of the aliphatic carbon double bonds, and I! = intensity ratio of the aromatic carbon double bonds. Although the initial measurements were referred to as early microtensile bond strength, it should be noted that about one hour has elapsed between the time when bonding was performed and the time when the specimen beams were ready for the initial measurements. Thus, the 1 hour DC measurement was performed to reflect the status of the bonds at the time of initial microtensile bond testing. 51

52 Statistical Analyses Bond strength data from microtensile bond strength and DC were normally distributed (Kolmogorow-Smirnoff test) and exhibited equal variances (Levene test). Thus, the data were analyzed separately using a two-way analysis of variance (ANOVA) for evaluating the effects of adhesive systems and testing times, and the interaction of these two factors on microtensile bond strength and DC. Tukey tests were used for posthoc comparisons. In addition, a linear regression analysis was performed to examine if correlation existed between the microtensile bond strength and degree of conversion of the adhesives. All the analyses were processed by the SPSS 11.0 software k, with the level of significance set at 95% probability level. RESULTS As the sectioning process took approximately 1 hour, the 52

53 early results were designated as 1-hour bond strengths. The microtensile bond strength results for the four adhesives are shown in Figure 2. The bond strength of the adhesives tested was not influenced by testing time (i.e. 1 h vs 24 h) (p>0.05). Conversely, the type of adhesives employed exhibited a highly significant influence on the bond strengths (p<0.001). The interaction between adhesive material and testing time was not significant (p>0.05). The 1-step self-etch system Adper Prompt L-Pop showed significantly lower microtensile bond strength both at 1-hour ( MPa) and 24 hours ( MPa). The results of DC are summarized in Figure 3. Although the DC of the adhesive systems tested increased slightly over time, the results were not statistically significant (p=0.219). However, significantly differences were observed among the adhesives (p<0.001). The DC, in percentage (immediate/1h/24h), were: SE (81/82/87) = MP (79/77/81) > SB2 (60/63/65) > L-Pop (39/37/42). The standard deviation for the DC ranged between 1.15 and Linear regression analysis showed that there was no correlation 53

54 between microtensile bond strengths of the adhesives and their degree of conversions (p > 0.05) (Figure 4). DISCUSSION In this study microtensile bond strength and DC of four adhesives from each of the four currently employed bonding strategies were evaluated at 1 hour and 24 hours after irradiation, with no significant differences identified between these testing times. Therefore, the first null hypothesis that there are no differences between early and 24-hour bond strengths and DC should be accepted. The microtensile results of this study are in agreement with our previous study (Sadek et al., 2005b), showing that bond strength do not increase significantly over 24 hours when the tested specimens were sectioned immediately after bonding. In the present study, however, we altered our study design by sectioning the teeth before bonding, using one half of each bonded tooth for immediate sectioning, while the other half was stored in distilled water for 24 hours prior to sectioning to enhance bond 54

55 maturation. Hence, the speculation that immediate sectioning would create the same internal flaws within the specimen beams, irrespective of whether bond strengths were measured immediately or after 24 hours should be discarded. The use of FT-Raman spectroscopy to assess DC provided additional chemoanalytical evidence to support the argument that bond maturation has limited effect on improving the microtensile bond strength of the resin-dentin bonds. Indeed, the DC results indicated that irrespective of the adhesive examined, very limited post-curing conversion (dark cure) occurs within 24 hours after the initial light curing of the adhesive (Figure 3), corroborating with the lack of differences between the 1h and 24h microtensile results. Even though bond strength was tested only 1 h after bonding procedures, immediate sectioning of the specimens tested at 1 h might simulate clinical early stress. Therefore, these findings suggest that with the initial bond strength and DC achieved in bonding to dentin, clinicians may perform contouring, finishing and polishing procedures or oclusal adjustments of the bonded 55

56 composites without adversely affecting the integrity of the restorations that was initially established. The corollary to these results suggests that bond maturation is unlikely to result in any additional improvement in bond integrity. As the present study was performed using flat dentin surfaces, this hypothesis should be further tested in cavity designs with different configuration factors. Thin adhesive films associated with sufficient light exposure probably contributed to the relative stable degree of conversion over time for each adhesive. The extent of the polymerization that is characteristic of each adhesive is achieved almost immediately after curing, with insignificant improvement after 24 hours of storage even for the acidic self-etch adhesives. These findings are in agreement with Bae et al. (Bae et al., 2005) showing that ph values of the adhesives have negligible effect on DC, flexural strength and microtensile bond strength of adhesive systems under weakly acidic conditions. Although microtensile bond strengths and DC of each adhesive did not alter significantly over time, the type of adhesive system employed exhibited a highly 56

57 significant influence on the measured bond strengths and DC. The microtensile bond strength obtained with the 2-step self-etch adhesive Clearfil SE Bond was not significantly different from the 2-step total-etch adhesives, and they were higher than the 3-step total-etch Scotchbond MP. The 1-step self-etch system Adper Prompt L-Pop showed the lowest microtensile bond strength values (Figure 2). These findings are in agreement with previous studies (Armstrong et al., 2003; De Munck et al., 2005a; Peumans et al., 2005; Sadek et al., 2005b). Factors such as incomplete wetting, insufficient thick adhesive layer, phase separation between hydrophilic and hydrophobic adhesive components and hydrolysis of the hydrophilic resin moieties have been used to explain this weaker bonding performance of Adper Prompt L-Pop to dentin (Peumans et al., 2005). The low resin concentration and/or low viscosity of this adhesive could also have resulted in composite resins displacing thin oxygen-inhibited adhesive layer from the surface of the hybrid layer and their penetration into dentin tubules. This 57

58 problem is further aggravated with a single layer application of the adhesive (Pashley et al., 2002b). Thus, incomplete coverage or wetting of the dentin substrate could have been the responsible for the low bond strength observed for Adper Prompt L-Pop (Figure 2). It has been speculated that low conversion of the adhesive resin monomer within a hybrid layer may lower its bond potential (Wang et al., 2001). Our DC data confirm this speculation. Indeed, the DC of Adper Prompt L-Pop was significantly lower than all other materials, achieving only 40% conversion (Figure 3). Using micro-raman spectroscopy with a different relative comparison protocol (vibrational band at 1640 cm-1 for C=C double bond, against the CH2 deformation band at 1453 cm -1 as an internal standard), Wang and Spencer reported that the Prompt L- Pop version employed in their study exhibited a DC of 77% in the absence of water, but increases to 93% with the addition of 20 vol% water. The DC values dramatically dropped from 93% to 36% when the water content increased further from vol%. The 36% DC with the inclusion of 60 vol% water, thus, is close to 58

59 the 40% conversion obtained in the present study. By mixing nonsolvated experimental resin blends with hydrogen bonding capacities (as measured by their Hoy's solubility parameter for hydrogen bonding " h ) with water, Yiu et al. reported that water retention within their experimentally formulated adhesives was highest in those containing high concentration of phosphatecontaining monomers, such as those present in Adper Prompt L- Pop (Yiu et al., 2005). These authors also reported that both solvent and water retention were greater in ethanol-based adhesives when compared with acetone-based adhesives. Moreover, with the increase in " h values of the adhesives, it was impossible to complete remove the solvent/water mixture from the resin monomers, so that water droplets could be identified in the adhesive after they were polymerized. The low degree of conversion of Adper Prompt L-Pop in our study, thus, may be attributed to the strong hydrogen bonding capacity of the phosphate-containing resin monomers. This could have precluded the complete removal of the solvent/water mixture even after blow- 59

60 drying for 40 s, a time-period that may be considered as unrealistically long by clinical standards. Our findings that DC did not extensive improved after 24 hours, thus, may be justified by the incomplete polymerization of the adhesive in the presence of water/solvent entrapment, as morphologically illustrated by Yiu et al. Such a low DC is further supportive of the findings by Wang and Spencer that their version of Prompt L-Pop continued to etch dentin beneath hybrid layers after the adhesive was light-cured. Another cause for the low DC of Adper Prompt L-Pop may be related to its bisacylphosphine oxide (BAPO) photoinitiator. The absorption band of BAPO is in the ultraviolet region, even though its absorption spectrum extends also to the visible light region (Neumann et al., 2005). In this study, the light-curing unit has an emission peak of 470 nm. The location of the absorption band within the visible light region of the spectrum could have additionally accounted for the low DC obtained for this adhesive. Improved DC with the use of LED over halogen light-curing units has recently reported for Adper Prompt L-Pop (Ye et al., 2006). 60

61 Even though a minimum DC has not yet been precisely established for optimal bond strength to be achieved for a particular adhesive (Dickens and Cho, 2005), the lower microtensile bond strength obtained by Adper Prompt L-Pop indicated that a 40% monomer conversion is inadequate to achieve high bond strength. However, no significant correlation between DC and microtensile bond strength was found (Figure 4) since the strength of an adhesive bond depends on additional parameters apart from its DC. Consequently, this led to the rejection of the second null hypothesis. The relative lower DC of Adper Scotchbond MP and Adper Scotchbond 2 found in this study, when compared to Clearfil SE, may also be related to the residual water (solvent) that was insufficiently removed and weakly bond by hydrogen bonding with 2-hydroxyethylmethacrylate (HEMA) (Peumans et al., 2005). Although the low DCs of Adper Scotchbond MP and Adper Scotchbond 2 did not influence their early or 24-hour bond strengths, it is prudent to point out that upon longer storage periods, 61

62 the presence of excessive unconverted double bonds within a polymerized adhesive network may expedite water sorption within the adhesive and result in bond strength deterioration over time (Carvalho et al., 2004). The assessment of bond durability, however, is beyond the scope of this investigation and should be duly investigated in future studies. CONCLUSIONS Within the limitations of this study, it may be concluded that neither bond strength nor DC increased significantly over a 24- hour period. In addition, the low bond strength and DC achieved for the 1-step self-etch adhesive suggest that it is not as reliable as 2- and 3-steps adhesives. ACKNOWLEDGMENTS This study was supported, in part, by R01 grants DE and DE from the NIDCR, USA (PI. David Pashley). We also thank all manufacturers for the generous 62

63 donation of the respective materials used. a Buehler, Lake Bluff, IL, USA b Nikon Corp., Tokyo, Japan c 3M ESPE, St. Paul, MN, USA d Kuraray Medical Inc., Tokyo, Japan e Ivoclar-Vivadent, Schaan, Liechtenstein f Bisco Inc., Schaumburg, IL, USA g DVA, Corona, CA, USA h Danville Engineering, San Ramon, CA, USA i Controls, Milano, Italy j Bruker Optik GmbH, Ettlingen, Germany k SPSS Inc., Chicago, IL, USA 63

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65 Asmussen E, Peutzfeldt A. Short- and long-term bonding efficacy of a self-etching, one-step adhesive. J Adhes Dent 2003; 5: Cardoso PE, Sadek FT, Goracci C, Ferrari M. Adhesion testing with the microtensile method: effects of dental substrate and adhesive system on bond strength measurements. J Adhes Dent 2002; 4: Talic YF. Immediate and 24-hour bond strengths of two dental adhesive systems to three tooth substrates. J Contemp Dent Pract 2003; 4: Burrow MF, Tagami J, Negishi T, Nikaido T, Hosoda H. Early tensile bond strengths of several enamel and dentin bonding systems. J Dent Res 1994; 73: Staninec M, Kawakami M. Adhesion and microleakage tests of a new dentin bonding system. Dent Mater 1993; 9: Suh BI, Feng L, Pashley DH, Tay FR. Factors contributing to the incompatibility between simplified-step adhesives and chemically- 65

66 cured or dual-cured composites. Part III. Effect of acidic resin monomers. J Adhes Dent 2003; 5: Dickens SH, Cho BH. Interpretation of bond failure through conversion and residual solvent measurements and Weibull analyses of flexural and microtensile bond strengths of bonding agents. Dent Mater 2005; 21: Barink M, Van der Mark PC, Fennis WM, Kuijs RH, Kreulen CM, Verdonschot N. A three-dimensional finite element model of the polymerization process in dental restorations. Biomaterials 2003; 24: Asmussen E, Jorgensen KD. A microscopic investigation of the adaptation of some plastic filling materials to dental cavity walls. Acta Odontol Scand 1972; 30: Sadek FT, Goracci C, P.E.C. C, Tay FR, Ferrari M. Microtensile bond strength of current dentin adhesives after immediate and 24 hours measurements. J Adhes Dent 2005; 7:

67 Ferracane JL, Greener EH. The effect of resin formulation on the degree of conversion and mechanical properties of dental restorative resins. J Biomed Mater Res 1986; 20: Lohbauer U, Rahiotis C, Kramer N, Petschelt A, Eliades G. The effect of different light-curing units on fatigue behavior and degree of conversion of a resin composite. Dent Mater 2005; 21: Lindberg A, Emami N, van Dijken JW. A Fourier transform Raman spectroscopy analysis of the degree of conversion of a universal hybrid resin composite cured with light-emitting diode curing units. Swed Dent J 2005; 29: Witzel MF, Calheiros FC, Goncalves F, Kawano Y, Braga RR. Influence of photoactivation method on conversion, mechanical properties, degradation in ethanol and contraction stress of resinbased materials. J Dent 2005; 33: Truffier-Boutry D, Demoustier-Champagne S, Devaux J, Biebuyck JJ, Mestdagh M, Larbanois P, Leloup G. A physico-chemical 67

68 explanation of the post-polymerization shrinkage in dental resins. Dent Mater Koibuchi H, Yasuda N, Nakabayashi N. Bonding to dentin with a self-etching primer: the effect of smear layers. Dent Mater 2001; 17: Bae JH, Cho BH, Kim JS, Kim MS, Lee IB, Son HH, Um CM, Kim CK, Kim OY. Adhesive layer properties as a determinant of dentin bond strength. J Biomed Mater Res B Appl Biomater 2005; 74: Peumans M, Kanumilli P, De Munck J, Van Landuyt K, Lambrechts P, Van Meerbeek B. Clinical effectiveness of contemporary adhesives: A systematic review of current clinical trials. Dent Mater 2005; 21: De Munck J, Van Landuyt K, Coutinho E, Poitevin A, Peumans M, Lambrechts P, Van Meerbeek B. Micro-tensile bond strength of 68

69 adhesives bonded to Class-I cavity-bottom dentin after thermocycling. Dent Mater 2005; 21: Pashley EL, Agee KA, Pashley DH, Tay FR. Effects of one versus two applications of an unfilled, all-in-one adhesive on dentine bonding. J Dent 2002; 30: Wang Y, McMann TR, Spencer P. Morphologic and chemical characterization of the dentin/self-etch interface. J Dent Res 2001; 80: Abstract Wang Y, Spencer P. Continuing etching of an all-in-one adhesive in wet dentin tubules. J Dent Res 2005; 84: Yiu CK, Pashley EL, Hiraishi N, King NM, Goracci C, Ferrari M, Carvalho RM, Pashley DH, Tay FR. Solvent and water retention in dental adhesive blends after evaporation. Biomaterials 2005; 26: Neumann MG, Miranda WG, Jr., Schmitt CC, Rueggeberg FA, Correa IC. Molar extinction coefficients and the photon absorption 69

70 efficiency of dental photoinitiators and light curing units. J Dent 2005; 33: Ye Q, Wang Y, Williams K, Spencer P. Characterization of photopolymerization of dentin adhesives as a function of light source and irradiance. J Biomed Mater Res B Appl Biomater 2006 [Epub ahead of print] Carvalho RM, Tay FR, Giannini M, Pashley DH. Effects of pre- and post-bonding hydration on bond strength to dentin. J Adhes Dent 2004; 6:

71 Table 1 Composition of the adhesives systems tested according to the manufacturers Adhesive Composition Batch number Primer: water; 2-hydroxyethyl methacrylate 8UK Adper Scotchbond MP (3M ESPE, St. Paul, MN, USA) Adper Scotchbond 2 (3M ESPE) Clearfil SE Bond (Kuraray Medical Inc., Osaka Japan) Adper Prompt L-Pop (3M ESPE) (HEMA); polyalkenoic acid copolymer. Bond: bisphenol A diglycigylmethacrylate (bis- GMA); HEMA; di-camphorquinone (CQ); ethyl 4- dimethyl amino benzoate (EDMAB); Dihydroxyethyl p-toluidene (DHEPT). Bond: water; ethanol; bis-gma; HEMA; hydrophilic dimethacrylate; photoinitiator; methacrylate functional copolymer, polyitaconic acids. Primer: 10-methacryloyloxydecyl dihydrogen phosphate (MDP); HEMA; hydrophilic dimethacrylate, CQ; N,N-diethanol-p-toluidine (DET); water Bond: MDP, HEMA, bis-gma, hydrophobic dimethacrylate, CQ, DET, silanated colloidal silica Red blister: Methacrylated phosphoric esters, bis- GMA, initiators based on CQ, stabilizers Yellow blister: water, HEMA, Polyalkenoic acid, stabilizers. 8KB 5BY 00558A 00788A

72 C=C A B Figure 1 A) Overview of a FT-Ramam spectrum obtained for a cured specimen of Scotchbond MP. B) Higher magnification of the detailed height in Figure 1A, showing the aliphatic (1640 cm -1 ) and aromatic (1610 cm -1 ) carbon double bonds bands. 72

73 B A A C Figure 2 Microtensile bond strength results (means and standard deviations). Subgroups that are statistically similar are indicated by the same alphabetical letter. 73

74 Degree of conversion 100 % of conversion Scotchbond MP Clearfil SE Scotchbond 2 Prompt 0 immediately 1 hour 24 hours Figure 3 Degree of conversion results according to the measurement period. 74

75 Degree of conversion (%) y = x R 2 = Bond strength (MPa) Figure 4- Regression analysis of the correlation between bond strength, in MPa, and degree of conversion, in percentage. 75

76 I 1 2 Radicular dentin Endodontically treated teeth are often severely damaged by decay, excessive wear or previous restorations, resulting in a lack of coronal tooth structure. Cast metal posts and cores have traditionally been used in these clinical situations to provide the necessary retention for the subsequent prosthetic rehabilitation. The recent developments in preventive dentistry and the advent of fiber posts significantly increased the demand for adhesive restorations on root dentin. Current adhesive systems used for adhesive coronal restorations can be used for fiber posts cementation in conjunction with different resin cements. However, little is known about their performance on the early stages of the bond process. 76

77 Immediate and 24-hour evaluation of the interfacial strengths of fiber posts. Sadek FT; Monticelli F; Goracci C; Grandini S; Cury HA; Tay F; Ferrari M. Journal of Endodontics 2006, in press. INTRODUCTION The materials available for the restoration of endodontically treated teeth have expanded considerably over the past decade (Brown and Hicks, 2003; Ferrari et al., 2000; Schwartz and Robbins, 2004). Prefabricated fiber posts offer several advantages over metal posts. They include simplification of chair side procedures (Ferrari et al., 2002b), reduction in the incidence of root fracture (Sirimai et al., 1999; Trope and Ray, 1992) and improvement in esthetics (Schwartz and Robbins, 2004). Despite these advantages, some debonding has been reported (Ferrari et al., 2000; Monticelli et al., 2003; Schwartz and Robbins, 2004). This is probably related to the highly unfavorable cavity configuration encountered within post spaces and the increased wall-to-wall contraction of thin resin films during 77

78 polymerization (Bouillaguet et al., 2003; Tay et al., 2005a). Moreover, it is impossible to employ a layering technique to reduce their polymerization shrinkage (Pirani et al., 2005). It has been demonstrated that post retention is largely contributed by friction along the canal walls (Goracci et al., 2005a), with the implication that materials with less technique sensitivity may be used for post retention. Hence, it is of clinical interest to evaluate the interfacial strength of fiber posts luted with a non-adhesive material such as zinc phosphate cement. Bond strength evaluations are conventionally performed at 24 hours or longer after restorative procedures (Bouillaguet et al., 2003; Goracci et al., 2005a; Sahafi et al., 2004; Sirimai et al., 1999). Clinically, however, the interfaces are subjected to stress immediately upon the completion of the restorations. Thus, the aim of this study was to compare the immediate and delayed (24h) interfacial strength of fiber posts luted with different resin cements and a zinc phosphate cement. The null hypotheses tested were: 1) there are no differences in the 78

79 retention of luted fiber posts with different cements; 2) there are no differences in the interfacial strengths of root sections tested immediately and after 24 hours. MATERIALS and METHODS Twenty-five anterior teeth were collected after the patients informed consent had been received under a protocol approved by the Institutional Review Board of the University of Siena, Italy. They were stored in 1% chloramine T at 4 o C to prevent bacterial growth, and used within one month after extraction. Teeth were decoronated using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, IL, USA), to create 17 mm long root segments. Canal patency was achieved with an ISO # 15 Flexo-file (Dentsply-Maillefer, Ballaigues, Switzerland). Working lengths were established 0.5 mm short of the apex. Instrumentation was performed with a crown-down technique using Profile rotary nickel-titanium instruments (Dentsply-Tulsa, Tulsa, OK, USA). All canals were prepared to ISO # 35, 0.06 taper, and irrigated 79

80 between instrumentation with 17% EDTA and 2.5% sodium hypochlorite. Prepared root segments were obturated with guttapercha and an epoxy resin-based canal sealer (AH-26, Dentsply DeTrey, Konstanz, Germany). Downpacking was performed with a System B heat source (SybronEndo, Orange, CA, USA) and backfilling was done using an Obtura II unit (Spartan, Fenton, MO, USA). After the filled roots were stored in physiological saline at 37 C for 24 hours, post spaces were prepared with a low-speed drill (size #3 drill for #3 FRC Postec post Plus, Ivoclar-Vivadent, Schaan, Liechtenstein) to a depth of 9 mm. The specimens were randomly divided into five groups (n=5). A silane coupling agent (Monobond-S, Ivoclar-Vivadent) was applied on the surface of the translucent glass fiber posts (size #3 FRC Postec Plus, Ivoclar-Vivadent), left for 60 sec, and gently air dried. The five experimental groups were: Group 1: AllBond 2/Duo Link (Bisco Inc., Schaumburg, IL, USA) 80

81 The post spaces were etched with 32% phosphoric acid (Uni- Etch) for 15 sec, irrigated with syringe and needle to rinse the surface, and gently dried with paper points. The AllBond 2 system, a conventional three-step etch-and-rinse adhesive, was mixed and applied with narrow microbrushes to the moist dentin and lightcured for 20 sec. Then, equal amounts of base and catalyst pastes of a dual-cured resin cement (Duo Link, Bisco) were mixed and applied to the post. The cement-coated post was seated into the canal. Light-curing was performed at an output density of 600 mw/cm 2 for 40 sec by placing the light-emitting tip of a halogen light-curing unit (VIP, Bisco) on top of the fiber post. Group 2: Optibond Solo Plus Dual Cure/Nexus 2 (Kerr Dental, Orange, CA, USA) The post spaces were etched with Kerr Gel Etchant (37.5% H 3 PO 4 ) for 15 sec, irrigated with syringe and needle to rinse the surface and lightly dried with paper points, leaving the surface moist. OptiBond Solo Plus and activator, a two-step etch-and-rinse 81

82 dual-cured adhesive, were mixed for 3 sec and two consecutives coats were applied with thin microbrushes. Excess adhesive was removed with paper points and the adhesive-coated dentin was light-cured for 20 sec. Then, a dual-cured resin cement (Nexus 2, Sybron-Kerr) was mixed and applied to the fiber post. The cementcoated post was inserted into the canal and light-cured for 20 sec. Group 3: Multilink (Ivoclar-Vivadent) The self-etching, self-curing luting system Multilink primer A and B were mixed, applied to the post space for 15 sec and gently dried with paper points. Multilink base and catalyst cement pastes were then mixed in a 1:1 ratio. The silanized fiber post was coated with the resin cement and seated into post space. Group 4: RelyX Unicem (3M ESPE, Seefeld, Germany) A capsule of the dual-cured self-adhesive resin cement was activated and triturated for 15 sec and applied to the fiber post, 82

83 which was then seated into the post space. Light-activation was performed similarly for 20 sec. Group 5: Zinc phosphate cement (Richter & Hoffmann Harvard Dental GmbH, Berlin, Germany) 1.5 g of powder were divided into four portions (", #, $, $). Beginning with the smallest portion, the powder was mixed with 1.0 g of liquid for 90 sec. The fiber post was coated with the mixture, and seated into the post space. Immediately upon the setting of the materials, each bonded post was sectioned transversely into five to six 1-mm thick slices using the Isomet saw under water cooling. The rationale behind immediate sectioning was to simulate the immediate stressing of in vivo bonded restorations. Slices from each tooth were alternately assigned for either immediate testing or stored in distilled water at 37 C for 24 hours prior to testing. Evaluation of the interfacial strength was performed using a push-out design. The thickness of 83

84 each slice was measured with a pair of digital calipers. The slice was then secured with cyanoacrylate glue to a loading fixture. A compressive load was applied to the slice via a cylindrical punch attached to a universal testing machine (Controls S.P.A., Milano. Italy). Each slice was secured with its apical aspect facing the punch tip that was aligned to contact only the post on loading. Loading was performed at a crosshead speed of 0.5 mm/min until the post segment was dislodged from the root slice. The retentive strength of the post segment was expressed in MPa, by dividing the load at failure (Newtons) by the area of the post fragment (S L ). The latter, being the lateral surface of a truncated cone, was calculated by the formula S L = #(%R+r)[(h 2 + (R-r) 2 ] 0.5, where #&= 3.14, R = coronal post radius, r = apical post radius and h = root slice thickness. The normally distributed data (Kolmogorov-Smirnov test) with homogeneous group variances (Levene test) were analysed with a two-way ANOVA with cement type and testing time as factors, followed by the Tukey test for post-hoc comparisons at '=

85 RESULTS Interfacial strengths for the five experimental groups are shown in Table 1. Both the cement type and the testing time significantly affected the interfacial strength of the fiber post luted to post spaces (p<0.05). However, the interaction between these two factors was not significant (p>0.05). Optibond Solo Plus/Nexus and the zinc phosphate cement attained the highest interfacial strengths. The interfacial strength of Multilink was comparable to the aforementioned groups and did not differ significantly from RelyX Unicem. All Bond 2/DuoLink had the lowest interfacial strength, which was not significantly different from that of RelyX Unicem (Table 1). Specimens from all resin-containing groups tested at 24 hours exhibited significantly higher interfacial strengths than those obtained immediately after luting (p<0.05, Table 1). No difference was found for specimens luted with the zinc phosphate cement when they were tested immediately or after 24 hours (p>0.05) 85

86 DISCUSSION The results of this in vitro study require the rejection of the null hypotheses that cement types and testing time have no effect on the interfacial strength of the luted fiber posts. The resin cements employed for this study differ in their bonding approach and activation mode. Bond strengths of dual-cured resin systems are highest when both adhesive and resin composite are dual polymerized (Oook et al., 2004), as reflected by the results obtained from the use of Optibond Solo Plus self-cured/nexus2 (Table 1). RelyX Unicem has a limited etching potential compared with the etch-and-rinse and self-etching adhesives (Behr et al., 2004; De Munck et al., 2004; Goracci et al., 2005b) and exhibited a low degree of conversion even after light-cured (Kumbuloglu et al., 2004). The lack of aggressiveness and suboptimal mechanical properties may account for the low interfacial strength results obtained when fiber posts were luted with this material (Table 1). 86

87 It is well accepted that light-curing from the top of post spaces is insufficient to optimally polymerize light-cured adhesives and resin cements (Pirani et al., 2005). The use of a light-transmitting glass fiber post has been claimed to improve polymerization through the depth of post spaces (Yoldas and Alacam, 2005). However, this concept was recently challenged (Roberts et al., 2004) and relatively low interfacial strengths (( 10MPa) obtained in this study provided further support to confirm this suspicion. Polymerization shrinkage stresses that were generated due to the highly unfavorable cavity configuration factor of the post spaces (Bouillaguet et al., 2003; Tay et al., 2005a) may also affect the interfacial strength of resin-based materials. The use of a slowsetting, self-curing luting cement, as exemplified by Multilink, provides a more favorable condition for the relief of these stresses along bonding interface (Bouillaguet et al., 2003; Monticelli et al., 2006). The relative high interfacial strength related to the zinc phosphate cement can be explained by the composition of the 87

88 material and can be correlated to the resistance to dislocation of the cement itself that contributed to frictional retention (Goracci et al., 2005a). Nonetheless, it must be pointed out that the clinical use of zinc phosphate cement, as luting material for fiber posts, needs further investigations. It has already been shown to have a low potential for sealing the root canal wall and a lack of adhesion to the surface of the post (Bachicha et al., 1998; Rogic-Barbic et al., 2006). The use of immediate testing time was to simulate clinical scenarios. Increases in interfacial strength over the 24 hour period may be related to further polymerization of the resin cement (Irie et al., 2004), enhanced bonding ability or setting during water storage (Feilzer et al., 1995), stress relaxation by hygroscopic expansion as a consequence of water sorption during storage (Feilzer et al., 1990; Huang et al., 2002; Irie and Suzuki, 2001; Watts et al., 2000) or hygroscopic expansion of luting materials (Chen et al., 2005; Iwami et al., 1998; Peutzfeldt, 1997). The latter, in particular, could have contributed to an adaptation of the cement 88

89 to the substrate. As already mentioned, a major contribution to retentive strength in push-out tests is expected to occur as a consequence of the interfacial sliding friction. Thus, the higher 24 hour interfacial strengths achieved with the resin cements may be caused by the increase in interfacial friction consequent to hygroscopic expansion. Within the limitations of this study, it is concluded that bond strength can increase during the first 24 hours and that interfacial strengths are predominantly contributed by frictional retention. However, further studies are still required to evaluate laboratory and clinical performance of the cements tested to indicate the optimal choice for clinical practice. REFERENCES Brown PL, Hicks NL. Rehabilitation of endodontically treated teeth using the radiopaque fiber post. Compend Contin Educ Dent 2003;24(4): , ; quiz

90 Schwartz RS, Robbins JW. Post placement and restoration of endodontically treated teeth: a literature review. J Endod 2004;30(5): Ferrari M, Vichi A, Mannocci F, Mason PN. Retrospective study of the clinical performance of fiber posts. Am J Dent 2000;13(Spec No):9B-13B. Ferrari M, Grandini S, Simonetti M, Monticelli F, Goracci C. Influence of a microbrush on bonding fiber post into root canals under clinical conditions. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94(5): Sirimai S, Riis DN, Morgano SM. An in vitro study of the fracture resistance and the incidence ofvertical root fracture of pulpless teeth restored with six post-and-coresystems. J Prosthet Dent 1999;81(3): Trope M, Ray HL, Jr. Resistance to fracture of endodontically treated roots. Oral Surg Oral Med Oral Pathol 1992;73(1): Monticelli F, Grandini S, Goracci C, Ferrari M. Clinical behavior of translucent-fiber posts: a 2-year prospective study. Int J Prosthodont 2003;16(6):

91 Bouillaguet S, Troesch S, Wataha JC, Krejci I, Meyer JM, Pashley DH. Microtensile bond strength between adhesive cements and root canal dentin. Dent Mater 2003;19(3): Tay FR, Loushine RJ, Lambrechts P, Weller RN, Pashley DH. Geometric factors affecting dentin bonding in root canals: a theoretical modeling approach. J Endod 2005;31(8): Pirani C, Chersoni S, Foschi F, Piana G, Loushine RJ, Tay FR, et al. Does Hybridization of Intraradicular Dentin Really Improve Fiber Post Retention in Endodontically Treated Teeth? J Endod 2005;31(12): Goracci C, Fabianelli A, Sadek FT, Papacchini F, Tay FR, Ferrari M. The contribution of friction to the dislocation resistance of bonded fiber posts. J Endod 2005;31(8): Sahafi A, Peutzfeld A, Asmussen E, Gotfredsen K. Effect of surface treatment of prefabricated posts on bonding of resin cement. Oper Dent 2004;29(1): Oook S, Miyazaki M, Rikut A, Moore BK. Influence of polymerization mode of dual-polymerized resin direct core foundation systems on bond strengths to bovine dentin. J Prosthet Dent 2004;92(3):

92 Behr M, Rosentritt M, Regnet T, Lang R, Handel G. Marginal adaptation in dentin of a self-adhesive universal resin cement compared with well-tried systems. Dent Mater 2004;20(2): Goracci C, Sadek FT, Fabianelli A, Tay FR, Ferrari M. Evaluation of the adhesion of fiber posts to intraradicular dentin. Oper Dent 2005;30(5): De Munck J, Vargas M, Van Landuyt K, Hikita K, Lambrechts P, Van Meerbeek B. Bonding of an auto-adhesive luting material to enamel and dentin. Dent Mater 2004;20(10): Kumbuloglu O, Lassila LV, User A, Vallittu PK. A study of the physical and chemical properties of four resin composite luting cements. Int J Prosthodont 2004;17(3): Yoldas O, Alacam T. Microhardness of composites in simulated root canals cured with light transmitting posts and glass-fiber reinforced composite posts. J Endod 2005;31(2): Roberts HW, Leonard DL, Vandewalle KS, Cohen ME, Charlton DG. The effect of a translucent post on resin composite depth of cure. Dent Mater 2004;20(7): Monticelli F, Osorio R, Albaladejo A, Aguilera FS, Ferrari M, Tay FR, et al. Effects of adhesive systems and luting agents on bonding 92

93 of fiber posts to root canal dentin. J Biomed Mater Res B Appl Biomater 2006;77(1): Rogic-Barbic M, Segovic S, Pezelj-Ribaric S, Borcic J, Jukic S, Anic I. Microleakage along Glassix glass fibre posts cemented with three different materials assessed using a fluid transport system. Int Endod J 2006;39(5): Bachicha WS, DiFiore PM, Miller DA, Lautenschlager EP, Pashley DH. Microleakage of endodontically treated teeth restored with posts. J Endod 1998;24(11): Irie M, Suzuki K, Watts DC. Marginal and flexural integrity of three classes of luting cement, with early finishing and water storage. Dent Mater 2004;20(1):3-11. Feilzer AJ, Kakaboura AI, de Gee AJ, Davidson CL. The influence of water sorption on the development of setting shrinkage stress in traditional and resin-modified glass ionomer cements. Dent Mater 1995;11(3): Feilzer AJ, de Gee AJ, Davidson CL. Relaxation of polymerization contraction shear stress by hygroscopic expansion. J Dent Res 1990;69(1): Huang C, Kei LH, Wei SH, Cheung GS, Tay FR, Pashley DH. The influence of hygroscopic expansion of resin-based restorative 93

94 materials on artificial gap reduction. J Adhes Dent 2002;4(1): Irie M, Suzuki K. Current luting cements: marginal gap formation of composite inlay and their mechanical properties. Dent Mater 2001;17(4): Watts DC, Kisumbi BK, Toworfe GK. Dimensional changes of resin/ionomer restoratives in aqueous and neutral media. Dent Mater 2000;16(2): Iwami Y, Yamamoto H, Sato W, Kawai K, Torii M, Ebisu S. Weight change of various light-cured restorative materials after water immersion. Oper Dent 1998;23(3): Peutzfeldt A. Resin composites in dentistry: the monomer systems. Eur J Oral Sci 1997;105(2): Chen XM, Wang NB, Niu L, He T, Yang Y, Zhang GJ, et al. [Effects of expansion fashion and expansion quantity on retention of cement-expanded composite screw post]. Hua Xi Kou Qiang Yi Xue Za Zhi 2005;23(5):

95 Table 1. Mean and standard deviation (SD) of the values of interfacial strength (MPa) for all the tested groups, immediately and after 24 hours. N= number of slices. CEMENT TIME N Mean SD Significance (p<0.05) Optibond Solo- Nexus early hrs Total A early Zinc-Phosphate 24 hrs Total A early Multilink 24 hrs Total AB early RelyX Unicem 24 hrs All Bond2- DuoLink Total BC early hrs Total C Testing time as a factor early * 24 hrs Cement was a significant factor for interfacial strength and mean values with the same alphabetic letter (A, B or C) were not statistically different (p<0.05). Testing time was also a significant factor for interfacial strength, increasing significantly from early to 24 hour-testing (p<0.05, different symbols (* and ) mean statistical significance). 95

96 Chapter II Efforts to improve bond strength and ultra structural characteristics of dentin bonds II 1 The use of filled bonding system Some authors reported that tensile bond strengths to dentin are significantly correlated with the mechanical properties of the adhesive resins as well as the resin composites (Hasegawa et al., 1999; Takahashi et al., 2002). Adhesives containing nanofillers show higher modulus than unfilled resin (Takahashi et al., 2002). Therefore, cured adhesive resins with higher mechanical properties could promote good initial bond performances with improved marginal sealing. Moreover, the incorporation of nanofillers in the adhesives improve their viscosity, so that the filled adhesive may be applied as a single layer, which can be an efficient shock-absorbing resin layer (Choi et al., 2000; Inoue et al., 2001; Miyazaki et al., 1995; Perdigao et al., 1996). 96

97 Comparative study of a filled and an unfilled version of a dentin bonding system to dental hard tissues. Sadek FT, Goracci C, Cardoso PEC, Tay FR, Ferrari M. Dentistry South Africa 2005, 7(6): INTRODUCTION Adhesive systems have been extensively used for esthetic restorations (Fukuda et al., 2003; Soderholm, 1991). They are classified into two main groups according to the etching procedure, which can utilize either phosphoric acid or acidic monomers. The former results in complete removal of smear layer and demineralization of the underlying tooth structures. These etchand-rinse adhesives may be applied either in three steps or two steps (Van Meerbeek et al., 2003). Two-step etch-and-rinse adhesives are preferred by clinicians due to their simpler and faster application in comparison with the three-step systems. However, to achieve satisfactory bonding, the adhesive should be liberally applied, in order to saturate the exposed collagen fibril network and establish a sufficiently thick resin layer on the top of the hybrid 97

98 layer. The presence of a thick resin layer acts both as a shockabsorber and prevents over-thinning of the adhesive that compromises its polymerization via oxygen inhibition (Choi et al., 2000; Pashley et al., 2002b; Van Meerbeek et al., 2001). The creation of a shock-absorbing resin layer is important not only to protect against the stress produced by the shrinkage of the composite resin, but also to absorb masticatory forces, tooth flexure effects, and thermal cycling effects (Van Meerbeek et al., 2001). Several clinical trials (Trevino et al., 1996; Van Meerbeek et al., 1994; Van Meerbeek et al., 1996) support this elastic bonding concept, with excellent results reported. A shock-absorbing layer may be produced by either applying several coats of an unfilled adhesive consecutively (Pashley et al., 2002b), or incorporating nanofillers in the adhesive to improve its viscosity, so that the filled adhesive may be applied as a single layer (Choi et al., 2000; Inoue et al., 2001; Miyazaki et al., 1995; Perdigao et al., 1996). The latter method has been perceived by the marketing divisions of some manufacturers to be an appealing approach that satisfy the goal of 98

99 creating adhesives with the objectives of being simpler and faster in their applications. Along this line of marketing strategy, a new filled version of the previously unfilled Adper Scotchbond 1 [also known as Adper Single Bond in USA] (3M ESPE (St. Paul, MN, USA) has recently been commercialized. This new filled one-bottle adhesive is known as Adper Scothbond 1 XT [also known as Adper Single Bond 2 in USA] (3 M ESPE), and is claimed by the manufacturer to possess desirable characteristics for simple and effective bonding to enamel and dentin. Similar to Adper Scotchbond 1, this product also contains VitreBond polyalkenoic acid copolymer. As a further improvement, 5 nm-diameter silica particles were incorporated in the amount of 10% weight, with the claim that the incorporation of nanofillers would further strengthen the bond and enhance the performance of the adhesive system. However, the benefits of the filled versus the unfilled version of this adhesive system have not been fully substantiated in the literature, apart from the information supplied by the manufacturer. 99

100 Thus, the objective of this study was to analyze, through bond strength and ultrastructural evaluation, the adhesive potential on enamel and dentin of the filled version of a simplified adhesive in comparison with its predecessor. The null hypothesis tested was that the incorporation of nanofillers in a simplified etch-and-rinse adhesive has no effect on in vitro bonding performance. MATERIALS AND METHODS Specimens preparation for microtensile bond strength evaluation Eighteen caries- and defect-free human third molars were stored in 0.5% chloramine T at 4 o C and used within one month after retrieval. They were cleaned to remove all debris and calculus and then randomly divided into two equally-sized groups, E and D, for enamel and dentin bond strength testing and ultrastructural examination. Prior to the testing procedures, they were removed from the disinfectant solution and stored in distilled water for

101 hours. For the teeth of group E, the most superficial portion of enamel on the buccal aspect of each tooth was removed using 180- grit silicon carbide paper under running water, in order to remove the surface aprismatic enamel layer and to create a flat, abraded enamel bonding surface. For the teeth of group D, the occlusal enamel of each tooth was removed using a slow-speed saw equipped with a diamond-impregnated blade (Digital Isomet, Buehler, Lake Bluff, IL, USA) under water irrigation, to expose a mid-coronal dentin surface. The dentin surface was polished with a 180 grit silicon carbide paper under running water for 20 seconds, to create a clinically-relevant smear layer. The prepared surfaces were examined under a stereoscopical microscope (Nikon type 102, Tokyo, Japan) at 30X magnification to ensure that no dentin was exposed in the teeth designated as Group E, and that the bonding surfaces of teeth designated as Group D were free of enamel. Adper Scotchbond 1, the unfilled version, and Adper Scothcbond 1 XT, the new filled version of the simplified etch-andrinse adhesive were bonded to the prepared surfaces (n=3) 101

102 according to the manufacturer s instructions and light-cured using a halogen light-curing unit (VIP, Bisco Inc., Schamburg, IL, USA), with the curing intensity set at 600 mw/cm 2. A resin composite (Tetric Ceram, Ivoclar-Vivadent, Schaan, Liechtenstein) was applied to each tooth incrementally to form a core 6 mm in height. Each increment was individually light-cured for 40 seconds. The restored teeth were then stored in distilled water at 37 o C for 24 hours. After this period, they were sectioned perpendicularly to the bonded interface with the Isomet saw into a series of slabs. Each slab was further sectioned into 0.9 x 0.9 mm sticks, yielding a cross-sectional area of approximately 0.8 mm 2. The use of three teeth for each subgroup resulted in a total of sticks in enamel and sticks in dentin for testing of microtensile bond strength. Sticks with premature failure were discarded and not included in the compilation of the bond strength means. Each stick was measured in width and thickness with a digital caliper, and glued with cyanoacrylate (Zapit, DVA, Corona, 102

103 CA, USA) to the Geraldeli s device (Perdigao et al., 2002). This jig is made of two parts that are kept together by a couple of post-hole joints. The Geraldeli s device was placed in a Bencor Multi-T testing assembly (Danville Engineering, San Ramon, CA, USA), which was mounted on an universal testing machine (Triax digital 50, Controls, Milano, Italy) at a crosshead speed of 0.5mm/min. Each specimen was stress to failure under tension. The load at failure was recorded in Newtons and bond strength was then calculated in MPa. Bond strength data from the four groups were statistically analyzed, with the number of sticks as the statistical unit. The data distribution was first analyzed for their normal distribution using the Kolmogorov-Smirnov test, and their equal variance using the Levene median test. A two-way ANOVA was subsequently applied to examine the effects of adhesive systems and substrates, and the interaction of these two factors on microtensile bond strength. All the analyses were processed by the SPSS 11.0 software (SPSS Inc., 103

104 Chicago, IL, USA), with the level of significance set at 95% probability level. Transmission electron microscopy Three additional teeth with bonding surfaces prepared in dentin were used for each adhesive version. Both the filled and unfilled versions of the simplified adhesive were applied as previously described and coupled with a 2 mm thick layer of resin composite. After storage in distilled water for 24 hours, a 2 mm thick longitudinal slab was prepared from each tooth using the slow speed saw under water cooling. These slabs were coated with nail polish that was applied as close to the bonded interface as possible. The coated slabs were subsequently immersed in a 50 wt% ammoniacal silver nitrate tracer solution (Tay et al., 2002), in the dark for 24 hours. The silver-impregnated slabs were rinsed thoroughly in running water for one hour and immersed in a photodeveloping solution for 8 hours under a fluorescent light to reduce the diamine silver ion complexes into metallic silver grains within potential 104

105 voids along the resin-dentin interfaces. The slabs were then processed for transmission electron microscopy (TEM). Undemineralized, epoxy resin-embedded, nm thick sections were prepared according to the TEM protocol of Tay et al. (Tay et al., 2002). Without further staining, the undemineralized slabs were examined using a TEM (Philips EM208S, Eindhoven, The Netherlands) operated at 80 kv. RESULTS The mean and standard deviation of the microtensile bond strength values measured for all of the tested subgroups and the percentages of premature failures are reported in Table 1. The twoway ANOVA indicated that there was no significant difference between the adhesive systems tested (p>0.05). However, the dental substrate had a significant effect on bond strength (p<0.01), with enamel specimens showing lower bond strengths ( MPa) than dentin specimens ( MPa). The interaction between adhesive material and substrate was not significant 105

106 (p>0.05). Pre-test failures occurred only on sectioning enamel specimens. Examination of unstained, undemineralized TEM micrographs of the resin-dentin interface, after immersion in ammoniacal silver nitrate, revealed that nanoleakage was abundant along the base of the hybrid layer when both Adper Scotchbond 1 and Adper Scotchbond 1 XT were used in dentin (Figure 1 and 2). However in Adper Scotchbond 1 interfaces, silver deposition occurred predominantly in the form of isolated silver grains and water tree channels unevenly distributed in the hybrid layer (Figure 1). Conversely, with Adper Scotchbond 1 XT silver nitrate infiltration presented in the form of reticular patterns within the hybrid layer (Figure 2). Higher magnification of these interfaces showed a phase separation of the polyalkenoic acid copolymer component, which could be seen in both the first and the second adhesive layer (Figure 3 and 4). However, with Adper Scotchbond 1 XT, in relation with the lower water content, phase separation was not so 106

107 evident in the first layer, being more readily recognized in the second adhesive layer (Figure 4). This spherical polyalkenoic acid copolymer phases were found to consist of both larger and very fine silver grains that were confined into nanofiller clusters (Figure 5). The filled resin matrix was predominantly devoid of these silver grains. DISCUSSION In the current study a newly introduced two-steps total-etch adhesive and its predecessor were evaluated in their bonding ability to enamel and dentin. The non-trimming variable of the microtensile test was chosen for been considered a reliable adhesion testing technique that is capable of assessing the interfacial strength between an adhesive and the bonding substrate, avoiding the occurrence of cohesive failures during bonding testing due to a better stress distribution during loading (Pashley et al., 1999). Another advantage of this technique is that multiple specimens can be obtained from a single tooth by means of series 107

108 of cuts made on a single tooth (Shono et al., 1999). However it has been speculated that these cuts may induce the formation of internal defects into the specimens. Some authors (Ferrari et al., 2002a; Goracci et al., 2004; Sadek et al., 2005a) have already demonstrated that structural defects are commonly observed in SEM analysis, being more frequent on enamel than on dentin specimens. It has been hypothesized that the inherent fragility of enamel in the small cross-sections of microtensile specimens may be the responsible for their higher frequency of defects, and also for their failure under relatively low loading levels in comparison to dentin. As a matter of fact, in the present trial, regardless of the adhesive system, enamel specimens constantly showed lower bond strength values than dentin specimens. This finding is in disagreement with the literature data provided by conventional tensile and shear bond strength tests (Armstrong et al., 1998; Inoue et al., 2001; Toledano et al., 2001), and years of clinical experience, which confirm that enamel offers bonding conditions more favorable and consistently reliable than dentin. Another issue 108

109 that should be considered is that pre-test failures occurred only on sectioning enamel specimens, emphasizing its fragility. Probably the values found for enamel are unreal measurements, since it is as a very brittle substrate and the defects created into the interface or within the substrate can quickly propagate (Griffith, 1920) weakening the bond interface, consequently decreasing bond strength values recorded (Sadek et al., 2005a). In order to measure the true bond strength in enamel, alternative cut equipments, which places the least possible stress on the substrate should be used in further researches to evaluate bond efficacy of adhesive materials in enamel. As regards in particular the effect of the improvement in the adhesive system, no statistical differences were found in the bond strength values between Adper Scotchbond 1 and Adper Scotchbond 1 XT, and concerning the TEM analysis, both adhesive systems revealed abundant nanoleakage along the base of the hybrid layer in dentin. Therefore it can be concluded within the limitations of this study that the incorporation of nanofillers in this 109

110 adhesive did not strengthen the bond nor enhanced its sealing performance. Since no difference was found between both systems and greatest number of factors that affect the quality of bond in dentin than on enamel due to it s intrinsically heterogeneity, in the present study only TEM micrographs of the resin-dentin interface were done. Isolated silver grains, water trees and reticular patterns of nanoleakage silver deposits were detected along the hybrid layer. It has been demonstrated that water trees represent regions in which minute quantities of water are retained within the adhesive-dentin interface (Tay and Pashley, 2003b). Accordingly to these authors, water trees formation is a common finding especially with ethanolbased total-etch adhesives, possibly in relation to the more difficult water displacement. Conversely, the reticular patterns of nanoleakage silver deposits were probably caused by collapsed demineralized collagen matrix that was not infiltrated by resin. This phenomenon was more clearly observed with Adper Scotchbond 1 XT (Figure 2), probably due to its lower water 110

111 content when compared to Adper Scotchbond 1. The collapsed matrix re-expanded when the specimen was immersed in silver nitrate, so that the matrix remains filled up with silver. The phase separation of the polyalkenoic acid copolymer component, also observed in this trial, had already been reported (Agee et al., 2003; Tay and Pashley, 2003b). Accordingly to Agee et al. (2003) the high degree of nanoleakage found on the hybrid layer of Adper Scotchbond 1 was probably due to the polyalkenoic acid copolymer, which may block adequate penetration of resin monomers inside the collagen fibril network. And since Adper Scotchbond 1 XT has also this copolymer, expectedly elevated degrees of nanoleakage on this novel product was found. The findings of the current study demonstrated that this novel filled bonding system did not improve bond strength and sealing ability in enamel nor in dentin. However, more research is needed to know the whole performance of this novel adhesive system, and also most reliable conclusions about its performance in the oral environment must be derived from long clinical trials. 111

112 ACKNOWLEDGMENTS The authors are grateful to Programme Al!an, European Union Programme of high level scholarships for Latin America (identification number E03D08091BR). REFERENCES Soderhölm KJ. Correlation of in vivo and in vitro performance of adhesive restorative materials: a report of the ASC MD156 Task Group on Test Methods for the Adhesion of Restorative Materials. Dent Mater 1991; 7: Fukuda R, Yoshida Y, Nakayama Y, Okazaki M, Inoue S, Sano H, Suzuki K, Shintani H, Van Meerbeek B. Bonding efficacy of polyalkenoic acids to hydroxyapatite, enamel and dentin. Biomaterials 2003; 24: Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P, Van Landuyt K, Lambrechts P, Vanherle G. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent 2003; 28:

113 Choi KK, Condon JR, Ferracane JL. The effects of adhesive thickness on polymerization contraction stress of composite. J Dent Res 2000; 79: Pashley EL, Agee KA, Pashley DH, Tay FR. Effects of one versus two applications of an unfilled, all-in-one adhesive on dentine bonding. J Dent 2002; 30: Van Meerbeek B, Vargas M, Inoue S, Yoshida Y, Peumans M, Lambrechts P, Vanherle G. Adhesives and cements to promote preservation dentistry. Oper Dent 2001; Supplement 6: Van Meerbeek B, Peumans M, Verschueren M, Gladys S, Braem M, Lambrechts P, Vanherle G. Clinical status of ten dentin adhesive systems. J Dent Res 1994; 73: Van Meerbeek B, Peumans M, Gladys S, Braem M, Lambrechts P, Vanherle G. Three-year clinical effectiveness of four total-etch dentinal adhesive systems in cervical lesions. Quintessence Int 1996; 27: Trevino DF, Duke ES, Robbins JW, Summitt JB. Clinical evaluation of Scotchbond Multi-Purpose adhesive system in cervical abrasions. J Dent Res 1996; 75:

114 Inoue S, Vargas MA, Abe Y, Yoshida Y, Lambrechts P, Vanherle G, Sano H, Van Meerbeek B. Microtensile bond strength of eleven contemporary adhesives to dentin. J Adhes Dent 2001; 3: Perdigão J, Lambrechts P, Van Meerbeek B, Braem M, Yildiz E, Yucel T, Vanherle G. The interaction of adhesive systems with human dentin. Am J Dent 1996; 9: Miyazaki M, Ando S, Hinoura K, Onose H, Moore BK. Influence of filler addition to bonding agents on shear bond strength to bovine dentin. Dent Mater 1995; 11: Perdigão J, Geraldeli S, Carmo AR, Dutra HR. In vivo influence of residual moisture on microtensile bond strengths of one-bottle adhesives. J Esthet Restor Dent 2002; 14: Tay FR, Pashley DH, Yoshiyama M. Two modes of nanoleakage expression in single-step adhesives. J Dent Res 2002; 81: Pashley DH, Carvalho RM, Sano H, Nakajima M, Yoshiyama M, Shono Y, Fernandes CA, Tay FR. The microtensile bond test: a review. J Adhes Dent 1999; 1: Shono Y, Ogawa T, Terashita M, Carvalho RM, Pashley EL, Pashley DH. Regional measurement of resin-dentin bonding as an array. J Dent Res 1999; 78:

115 Ferrari M, Goracci C, Sadek F, Cardoso PEC. Microtensile bond strength tests: scanning electron microscopy evaluation of sample integrity before testing. Eur J Oral Sci 2002; 110: Goracci C, Sadek FT, Monticelli F, Cardoso PEC, Ferrari M. Influence of substrate, shape, and thickness on microtensile specimens' structural integrity and their measured bond strengths. Dent Mater 2004; 20: Sadek FT, Cury AH, Monticelli F, Cardoso PEC, Ferrari M. The influence of the cutting speed on bond strength and integrity of microtensile specimens. Dent Mater; In press. Armstrong SR, Boyer DB, Keller JC. Microtensile bond strength testing and failure analysis of two dentin adhesives. Dent Mater 1998; 14: Toledano M, Osorio R, Leonardi G, Rosales-Leal JI, Ceballos L, Cabrerizo-Vilchez MA. Influence of self-etching primer on the resin adhesion to enamel and dentin. Am J Dent 2001; 14: Griffith A. The phenomena of rupture and flow in solids. Phil Trans Royal Soc London 1920; Series A: Tay FR, Pashley DH. Water treeing--a potential mechanism for degradation of dentin adhesives. Am J Dent 2003; 16:

116 24. Agee KL, Pashley EL, Itthagarun A, Sano H, Tay FR, Pashley DH. Submicron hiati in acid-etched dentin are artifacts of desiccation. Dent Mater 2003; 19:

117 Table 1. Means, standard deviations and percentage of premature failures (PF) of the tested groups. Mean Standard deviation PF (%) Scotchbond 1 Scotchbond 1 XT Dentin Enamel Dentin Enamel

118 Figure 1 - Unstained, undemineralized TEM micrograph of the resin-dentin interface using Adper Scotchbond 1. The specimen was examined after immersion in ammoniacal silver nitrate, demonstrating the two modes of silver impregnation: isolated silver grains (open arrowhead) scattered along the hybrid layer (H) and reticular patterns of nanoleakage silver deposits (pointer). Phase separation of the polyalkenoic acid copolymer component could be seen (arrow) into the adhesive layer (A). D: mineralized dentin. 118

119 Figure 2 - Figure 2. Unstained, undemineralized TEM micrograph of the resin-dentin interface using the experimental version of Adper Scotchbond 1 that contains nanofillers. The specimen was examined after immersion in ammoniacal silver nitrate. Two layers of the adhesive (A1 and A2) were applied as in the original Adper Scotchbond 1. Nanoleakage (pointer) is abundant along the base of the hybrid layer (H). Phase separation of the polyalkenoic acid copolymer component could be seen from both the first (open arrows) and the second (arrow) adhesive layer (A1 and A2 respectively). C: resin composite; D: mineralized dentin. 119

120 Figure 3 - A higher magnification view of the hybrid layer (H) of Figure 1, showing isolated silver grains scattered along the hybrid layer and silver-filled water channels (water-trees). Phase separation of the polyalkenoic acid copolymer component (arrow) could also be seen all along the adhesive layer (A). D: mineralized dentin. C: resin composite. 120

121 Figure 4 - TEM of Adper Scotchbond 1 XT bonded to acid-etched dentin that was air-dried for 1 s. The demineralized collagen matrix collapsed and there was no resin infiltration. The collapsed matrix re-expanded when the specimen was immersed in silver nitrate, so that the entire matrix is now filled up with silver. Also, because there is also less water, phase separation was not so apparent in the first layer (A2), and phases of polyalkenoic acid copolymer (arrow) were more readily recognized in the second adhesive layer (A1). 121

122 Figure 5 - At higher magnification, the spherical polyalkkenoic acid copolymer phases (P) were found to consist of both larger (pointer) and very fine (open arrowheads) silver grains. The filled resin matrix (FM) was predominantly devoid of these silver grains. 122

123 II 2 The use of hydrophobic resin adhesives Unfortunately, the incorporation of nanofillers in this adhesive tested did not strengthen the bond nor enhanced its sealing performance. Our results were in agreement with Braga et al. and Takahashi et al. that also reported that the filled adhesive applied to a flat dentin surface did not show much difference compared to the unfilled materials (Braga et al., 2000; Takahashi et al., 2002). Moreover, the examination of unstained, undemineralized TEM micrographs of the resin-dentin interface, after immersion in ammoniacal silver nitrate, revealed that nanoleakage was abundant along the base of the hybrid layer when both filled and unfilled adhesives were used in dentin. Therefore, different efforts should be done to improve the quality of dentin bonds. 123

124 Application of hydrophobic resin adhesives to acid-etched dentin with an alternative wet bonding technique. Sadek FT, Pashley DH, Nishitani Y, Carrilho MR, Donnelly A, Ferrari M, Tay FR. Journal of Biomedical Materials Research Part A: in press. INTRODUCTION A number of important paradigm shifts have influenced the development of dentin adhesion since the successful synthesis of 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (Bis-GMA), a methacrylate ester-substituted epoxy resin monomer, by Dr. Rafael Bowen (Bowen, 1962). Although relatively hydrophobic resins containing Bis-GMA and a triethyleneglycol dimethacrylate (TEGDMA) have been successfully used for bonding to acid-etched enamel (Buonocore, 1963; Buonocore et al., 1968), they lack specific functional groups for wetting dentin (Bowen and Marjenhoff, 1992). As hydrophobic resin monomers bond poorly to dentin, hydrophilic and ionomer resin monomers have been developed and were used for almost 124

125 half a century as priming and surface-active agents for coupling resin composites to dentin (Bowen, 1965). Dentin adhesion is further improved by the use of acids to remove the smear layer and partially demineralise the intact dentin, creating hybrid layers for micromechanical retention (Nakabayashi et al., 1982). In the case of the etch-and-rinse technique, the introduction of the clinical technique of wet bonding (Kanca, 1992a) and subsequent research on the collapse and rehydration of air-dried, demineralised dentin collagen matrices (Pashley et al., 2002a; Van Meerbeek et al., 1998) have broadened our understanding of how to maximize hydrophilic resin infiltration in hybridised acid-etched dentin. In the case of the self-etching technique, increased concentrations of ionic resin monomers in water were incorporated into ethanol or acetone solvents, rendering them acidic enough to dissolve or integrate the hybrid layer with the underlying partially demineralised dentin as part of the resin-dentin interface (Watanabe et al., 1994). The use of increasing concentrations of hydrophilic resins 125

126 in contemporary dentin adhesives raises concern on whether such adhesives have become too hydrophilic (Tay and Pashley, 2003a; Van Meerbeek et al., 2005). Similar to the results derived from other biomedical fields (Manetti et al., 2004; Thimmegowda et al., 2002), incorporation of hydrophilic resin monomers results in increases in water sorption (Hashimoto et al., 2005; Hashimoto et al., 2006; Wadgaonkar et al., 2006; Yiu et al., 2006) and decreases in mechanical properties of these resins (El Zohairy et al., 2004; Ito et al., 2005). These hydrophilic resinous films are also highly permeable to water (Cadenaro et al., 2005; Sauro et al., 2006b) that further expedite their hydrolysis (Armstrong et al., 2004; De Munck et al., 2005b). Thus, the use of hydrophobic resins as dentin adhesives would, theoretically, avoid these problems and improve the durability of resin-dentin bonds. The technique of wet bonding of etch-and-rinse adhesives requires that water that is originally present within the interfibrillar spaces of the collagen network be displaced by the polar solvents in these adhesives, and ultimately be replaced by pure resins (Kato 126

127 and Nakabayashi, 1996). Professor Nakabayashi was one of the first to recognise the similarity between adhesive infiltration in acid-etched dentin and epoxy resin tissue embedding in transmission electron microscopy (Nakabayashi et al., 1992). However, important differences exist between the technique of wet bonding and tissue embedding. Recent studies indicate that resin replacement within dentin hybrid layers by etch-and-rinse adhesives is incomplete (Wang and Spencer, 2003; Wang and Spencer, 2005). This should not be surprising because one is attempting to replace the water present in dentin directly with resins dissolved in ethanol or acetone within a short period of time. On the contrary, in tissue embedding, the water in the hydrated tissues is gradually replaced over many hours by stepwise immersion of the latter in an ascending series of these solvents. This is followed by the use increasing concentrations of epoxy resin dissolved in non-aqueous transitional solvents, and with the subsequent use of pure epoxy resin as the embedding medium. In the broadest sense, tissue embedding is a form of wet bonding, in 127

128 which the tissue is constantly suspended in a liquid phase to avoid collapse of the tissue components. Whereas acid-etched dentin is suspended in water in dentin bonding, in tissue embedding, soft tissue is suspended in an ascending concentration of polar solvent. Thus, it should be possible to simulate the process of tissue embedding in dentin bonding by using a series of ascending ethanol concentrations for chemical dehydration (i.e. water replacement) (Eddleston et al., 2003). Although such a technique does not eliminate shrinkage of demineralised collagen matrices (Becker et al., 2006), it does avoid the collapse of the interfibrillar spaces that impedes resin infiltration. The ultimate goal of "ethanol wet bonding", thus, is to infiltrate the interfibrillar spaces and dentinal tubules with hydrophobic dimethacrylate resins that mimics the filling of tissue spaces with hydrophobic epoxy resins in tissue embedding. Based on these principles, we hypothesise that it is possible to bond acid-etched dentin with hydrophobic resins via the use of an alternative ethanol wet bonding protocol that involves the use of 128

129 a series of ascending concentrations of ethanol. Thus, the objectives of this study were: 1) to examine the feasibility of bonding acid-etched dentin with an experimental Bis- GMA/TEGDMA resin system of known solubility parameters that is similar in composition to unfilled pit-and-fissure sealants; 2) to determine the optimal concentration of this Bis-GMA/TEGDMAethanol mixture for the formulation of a water-free, ethanol-based, hydrophobic dentin adhesive; and 3) to compare the bond strengths of this hydrophobic dentin adhesive applied to dentin via an alternative ethanol wet bonding technique, versus those derived from conventional three-step etch-and-rinse adhesives via the use of a conventional "water wet bonding technique". The null hypothesis tested was that there are no differences among the microtensile bond strengths of the hydrophobic dentin adhesive in its optimal resin concentration and those obtained from commercially available, hydrophilic three-step etch-and-rinse adhesives. 129

130 MATERIALS AND METHODS One hundred and twelve sound human third molars were collected after the patients' informed consent was obtained under a protocol reviewed and approved by the Human Assurance Committee of the Medical College of Georgia. Teeth were stored in 0.02% sodium azide at 4 o C to prevent bacteria growth and were used within 2 month after extraction. Tooth Preparation Flat dentin surfaces were prepared perpendicular to the longitudinal axes of the teeth with a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) to remove occlusal enamel and superficial dentin. Each surface was ground with 180- grit silicon carbide paper under running water for 20 s to create a clinically relevant smear layer. The teeth were randomly divided into ten groups (n=8). Each tooth was etched with 37% phosphoric acid gel (Etch 37, Bisco Inc., Schamburg, IL, USA) for 15 s, rinsed thoroughly with deionized water and left moist until bonding. 130

131 Experimental Hydrophobic Adhesives A comonomer resin blend consisting of 70 wt% Bis-GMA, wt% TEGDMA, 0.25 wt% camphorquinone and 1 wt% ethyl N,N-dimethyl-4-aminobenzoate was used to formulate the experimental hydrophobic adhesives employed in this study. The degree of hydrophobicity of this comonomer blend has previously been defined in terms of its Hoy's solubility parameters (Table 1) (Eddleston et al., 2003; Ito et al., 2005; Wadgaonkar et al., 2006; Yiu et al., 2004). Six experimental hydrophobic adhesives were designed to simulate commercially available three-step etch-andrinse adhesives by formulating separate primer and adhesive components. They were prepared by diluting the neat comonomer blend with known concentrations of absolute ethanol. The wt% resin:ethanol ratio of these six primers were 20/80, 30/70, 40/60, 50/50, 75/25 and 100/0 respectively. The Hoy's solubility parameters of these primers are listed in Table 1. They were used within one week after preparation and stored at 4 o C after each use. The same neat comonomer resin blend was employed as the 131

132 adhesive component for all the six experimental hydrophobic adhesives. Ethanol Wet Bonding (Full Chemical Dehydration Protocol) In the first part of the study, a full chemical dehydration protocol that is commonly employed for tissue embedding with hydrophobic epoxy resins (Newman and Hobot, 1999) was adopted for determining the optimal hydrophobic primer resin concentration for ethanol wet dentin bonding. Accordingly, each acid-etched, wet dentin surface was treated with a series of increasing ethanol concentrations (50%, 70%, 80%, 95% and 100% for three times for 30 s each). The Hoy's solubility parameters of these solvents, expressed in units of (MPa) ", are also included in Table 1. The ethanol/water mixture or absolute ethanol was dispensed from transfer pipettes to cover the entire dentin surface, replacing the 100% water or preceding ethanol solution that saturated the demineralised collagen matrix. This procedure was meticulously performed to ensure that the dentin surface was always immersed in a liquid phase by keeping it visibly moist prior to the application 132

133 of subsequent solutions with higher ethanol concentration. The ethanol-based experimental adhesive primers were then applied to the visibly moist, ethanol-saturated dentin using a microbrush with agitation. For the 20/80 and 30/70 primer versions with low resin contents, five consecutive coats were employed to simulate the primer application protocol in a three-step etch-andrinse adhesive that uses a relatively solvent-rich primer (All-Bond 2, Bisco). For the remaining primer versions, two consecutive coats were found to be adequate to wet the dentin surfaces, simulating the primer application protocol in other three-step etch-and-rinse adhesive (Adper Scotchbond Multi-Purpose, 3M ESPE, St. Paul, MN, USA). In the event that a matte dentin surface was observed, additional coats of the primers were applied to insure that the dentin surfaces were completely covered by the primer. Excess ethanol solvent was evaporated with a gentle air stream for 10 s at a distance of 15 cm from the dentin surface. Then, a layer of the neat comonomer adhesive of the same BIS-GMA/TEGDMA blend was applied, spread thin with moisture-free air, and light-cured for 20 s. 133

134 The rationale for the application of this adhesive layer was to generate a resinous layer of sufficient thickness to avoid the effect of oxygen inhibition on incomplete polymerisation of the primer resin that infiltrated the underlying demineralised dentin. The hydrophobic adhesive-coated dentin was light-cured for 20 s using an Optilux 500 halogen light-curing unit (Demetron/Kerr, Danbury, CT, USA) with a power output of 600 mw/cm 2. Composite buildups were constructed with a light-cured resin composite (TPH 3, Dentsply Caulk, Milford DE, USA) in five 1 mm-thick increments. Conventional (Water) Wet Bonding Controls Positive controls Two commercially available three-step etch-and-rinse adhesives (i.e. All-Bond 2 and Adper Scotchbond Multi-Purpose) were applied to visibly moist, water-saturated demineralised dentin according to the manufacturers' instructions, so that bond strength measurements derived from the experimental hydrophobic resin groups could be compared. 134

135 Negative control The experimental hydrophobic adhesive that generated the highest bond strength results with the ethanol wet bonding protocol was selected as the negative control for bonding to acid-etched dentin using a conventional wet bonding protocol in which the demineralised dentin was saturated with 100% deionized water. All procedures were repeated as previously described, except that the selected hydrophobic primer was directly applied to visibly moist, water-saturated demineralised dentin, using the number of coats designated for that particular primer in the experimental group. Tensile Testing All bonded teeth were incubated in water at 37 C for 24 h. Each tooth was vertically sectioned into 0.9 mm thick serial slabs using an Isomet saw under water cooling. The central slab of each tooth was utilized for subsequent morphologic examination. The two slabs adjacent to the central slab were further sectioned into 0.9 x 0.9 mm composite-dentin beams, according to the "non- 135

136 trimming" technique of the microtensile test. Two beams from each slab with the longest attached dentin were selected, with 4 beams per tooth x 8 teeth = 32 specimens available for tensile testing in each experimental or control group. Testing was performed using a testing jig (Bisco Inc.) mounted in a Vitrodyne V1000 universal tester (Liveco Inc, Burlington, VT, USA) at a cross-head speed of 1 mm/min. The specimens were glued to the testing jig using cyanoacrylate (Zapit, Dental Ventures of American, Corona, CA, USA). The dentin side of each fractured beam was immersed in Harris modified hematoxylin with 4% acetic acid (mercury-free) (Fischer Scientific, Fair Lawn, NJ, USA) for 20 min, rinsed with running water for 10 min and then immersed in eosin Y (Fischer Scientific) for 3 min to stain the area of exposed dentin for failure mode examination. The beam was then immersed in an ascending ethanol series (70%, 90% and 100 % for 1 min each). The failed bonds were examined at 30 X magnification using a stereoscopical microscope, with the mode of failures classified as adhesive (A), 136

137 cohesive in resin (CR), cohesive in dentin (CD) or mixed failures (M). Statistical Analysis To improve the robustness of the statistical testing with parametric methods, results from the negative control group that were much lower than those of the experimental and positive control groups and included many premature failures, were excluded from the statistical analysis. Since a preliminary data regression analysis showed the bond strength was not significantly affected by the origin of the teeth from which the prepared beams were derived, each beam was considered as independent unit within an experimental or control group. Bond strength data from the other eight groups were normal in their distribution and exhibited equal variances. They were analyzed using a one-way analysis of variance design and Tukey multiple comparison test, with statistical significances set at '=

138 Transmission Electron Microscopy (TEM) To determine the quality of hydrophobic resin infiltration in ethanol wet bonding, three central slabs from each of the six experimental groups were randomly selected for TEM examination of nanoleakage along the dentin hybrid layers. These slabs were immersed for 24 h in a tracer solution containing 50 wt% ammoniacal silver nitrate, according to the method employed by Tay et al. (Tay et al., 2002). The tooth slabs were varnish-coated, except for 1 mm along the bonded interface. The silverimpregnated slabs were then rinsed thoroughly in distilled water and placed in Kodak photodeveloping solution for 8 h under a fluorescent light to reduce the diamine silver ion complexes into metallic silver grains within potential voids along the resin-dentin interfaces. Undemineralized, epoxy resin-embedded, nm thick sections were prepared according to a previously described TEM protocol (Tay et al., 2002). Without further staining, the sections were examined using a TEM (JEM-1010, JEOL, Tokyo, Japan) operated at 60 kv. 138

139 Partial Chemical Dehydration Protocols Partial ethanol dehydration has also been employed since the early eighties with the revival of acrylic resin tissue embedding, using different formulations containing mixtures of hydrophilic and hydrophobic acrylates/methacrylates (Newman and Hobot, 1999). As epoxy resin embedding of partially dehydrated soft tissues does not produce satisfactory results (Idelman, 1964), equivalent outcomes were anticipated with the use of hydrophobic dimethacrylates to bond dentin. Hence, the second part of the study was performed to examine the detrimental effect of contamination of 100% ethanol-saturated acid-etched dentin with water. This should provide evidence of the necessity to reduce dentin permeability via tubular occlusion (Tay et al., 2003a; Tay et al., 2005b) prior to the use of the ethanol wet bonding technique on vital dentin. Accordingly, the ethanol wet bonding and tensile testing experiments were repeated with the experimental hydrophobic adhesive that generated the highest bond strength, but 139

140 using different partial dehydration protocols. Four additional groups were designed in which the water-saturated acid-etched dentin (n=8) was treated with a series of increasing ethanol concentrations up to 50%, 70%, 80% and 95%, respectively. To eliminate the time variable, repeated rinsing with the same ethanol concentration was performed once the designated concentration was reached. For example, in the "70% ethanol" group, the acidetched dentin was rinsed with 50% ethanol for 30 s and thereafter, with 70% ethanol six times for 30 s each. The tensile strength results of these partial dehydration groups were compared with that obtained from the full dehydration group. Regression analysis was performed to examine the correlation between their mean tensile bond strengths and the Hoy's solubility parameter for hydrogen bonding () h ) for the different ethanol/water concentrations (Table 1) at '=0.05. RESULTS No premature failures occurred during the preparation of 140

141 the composite-dentin beams except for the negative control group. The tensile testing results for the experimental hydrophobic adhesives bonded using the ethanol wet bonding technique and the positive controls of the two commercially available hydrophilic adhesives and the negative control bonded using the water wet bonding technique are shown in Figure 1. For the experimental hydrophobic primers, the 40/60 and 50/50 wt% resin/ethanol versions exhibited the highest tensile strength of 40.3±6.1 MPa and 43.6±5.1 MPa, respectively. These strengths were not significantly different from the Adper Scotchbond Multi-Purpose positive control (39.5±4.8 MPa; p>0.05). The 75/25 wt% resin/ethanol version of the experimental hydrophobic primer exhibited significantly lower bond strength (34.9±6.3 MPa; p<0.05) than the previously mentioned groups, which, in turn, was not significantly different from that of the All-Bond 2 positive control (31.4±5.8 MPa; p>0.05). The lowest bond strengths were obtained with the 20/80, 30/70 primers and the neat experimental hydrophobic primer/adhesives (26.5±5.7 MPa, 25.1±5.2 MPa and 27.0±

142 MPa, respectively), which were not significantly different from each other (p>0.05, Fig. 1). The 50/50 version of the experimental hydrophobic primer exhibited the highest tensile strength to ethanol-saturated acidetched dentin and hence was selected as the negative control. When this experimental adhesive was applied to water-saturated acidetched dentin using the conventional (water) wet bonding technique, 12 out of the 32 beams exhibited premature failure. When these failures were expressed as null bond strengths and included in the compilation of the mean bond strength, the resultant tensile strength (5.6±5.7 MPa) was lower than those obtained from all the ethanol wet bonding experimental groups and from the two positive control groups of commercial adhesives (Figure 1). Mixed failure that involved partial dentin failures along the surface (stained red by hematoxylin and eosin stain) and partial failures within the adhesive/resin was predominantly identified in the six hydrophobic primer groups and the two positive control groups. By contrast, adhesive failures were exclusively observed in 142

143 the negative control group, including specimens that exhibited premature failures (Figure 2). TEM of the resin-dentin interfaces in the 20/80 and 30/70 versions of the hydrophobic adhesive revealed two modes of nanoleakage (viz. reticular networks of silver deposits and isolated silver grains) (Tay et al., 2002) within the 5 µm thick hybrid layer. Additional silver deposits were identified in the 8-10 µm thick adhesive layer (Figure 3A). By contrast, only the reticular pattern of nanoleakage could be seen in the hybrid layers of the 75/25 and 100/0 versions of the hydrophobic adhesives (Figure 3B). These reticular networks of silver deposits were denser than those identified from the 20/80 and 30/70 versions. No silver deposits were seen within the adhesive layers of the 75/25 and 100/0 versions. Minimal nanoleakage was present in the hybrid layers and adhesive layers of the 40/60 and 50/50 versions of the hydrophobic adhesives. These were exclusively fine, isolated silver grains that were localised to the periphery of the dentinal tubules (Figure 3C). 143

144 Tensile strengths obtained from the partial dehydration groups with the 50/50 version of the hydrophobic adhesive showed a sequential reduction in tensile strengths with increases in water that remained within the partially dehydrated dentin (Figure 4). When these results were combined with those obtained from the 100 % water hydrated negative control and the full hydration group, a significant negative power correlation was found between the increases in tensile strength and the decrease in Hoy's solubility parameter for hydrogen bonding () h ) of the ethanol/water mixtures with increasing ethanol content (R 2 =0.91; p<0.05, Figure 4). DISCUSSION In principle, a new paradigm for dentin bonding may emerge that is compatible with past scientific practices. The result of the negative control in this study clearly illustrates a central dogma in the lexicon of dentin bonding - that hydrophobic resins bond poorly to dentin (Brudevold et al., 1956). Paradoxically, when used in conjunction with the stepwise ethanol wet bonding 144

145 protocol, it is plausible for the commonest of dental hydrophobic dimethacrylates (70% BisGMA/30% TEGDMA) to bond to dentin and attain tensile strengths that are comparable with contemporary three-step etch-and-rinse hydrophilic adhesives (Figure 1). While this necessitates the acceptance of the proposed null hypothesis, the emergence of the new ethanol wet bonding concept may only be undertaken with the advantages of hindsight, for it has been known for more than half a century that hydrated soft tissues can be successfully embedded with Araldite-type hydrophobic epoxy resins via the use of full chemical dehydration albeit over many hours (Glauert and Glauert, 1958). One aspect of the parallelism must already be apparent; both hydrophobic resin embedding of soft tissue and hydrophobic resin for dentin bonding are achieved at the expense of time, by slow stepwise replacement of water with a chemical dehydrant, and subsequently with increasing concentrations of solvated resins, followed by pure resins. From a microscopist's perspective, dentin bonding may be regarded as an abridged version of tissue embedding in which the processes of 145

146 partial chemical dehydration and hydrophilic resin infiltration are performed simultaneously under rigorous time constraints. Indeed, current marketing strategies in dentin bonding are targeted at promoting simpler and faster adhesive systems. However, in view of the recent work on the in vivo degradation of resin-dentin bonds created with hydrophilic adhesives (Hashimoto et al., 2003; Hebling et al., 2005), there is a need for a reappraisal of bonding to dentin with hydrophobic resins in order to preserve the longevity of these bonds. For the sake of discussion, the ethanol wet bonding technique may be divided into two separate phases: a water replacement phase by ethanol, and an ethanol replacement phase by pure hydrophobic resins. The principle of utilising a series of increasing ethanol concentrations in tissue embedding is to remove water slowly from fragile animal tissues that are devoid of cell walls, so as to minimize the effects of osmotic shock on the delicate cellular ultrastructures. In the context of dentin bonding, as dentin collagen is highly crosslinked and contains no cell 146

147 membranes, it is envisaged that the same efficacy of water removal may be achieved by using 100% ethanol as the sole chemical dehydrant (Nishitani et al., 2006). However, a mild, gradual transition of increasingly concentrated chemical dehydrants may be of benefit when the technique is employed in vital deep dentin, by minimizing the aspiration of odontoblasts into the dentinal tubules during the process of abrupt desiccation(lilja et al., 1982). Moreover, the use of a slow regime that ends with three rinses of 100% ethanol may confer an additional advantage of reduced technique sensitivity, as will be discussed subsequently with the clinical implication of the partial dehydration results. Similar to other biomedical fields (Geckeler et al., 2003; Navarro-Lupion et al., 2005), solubility parameter theory(barton, 1991; Roy, 1970) has been successfully used to study the relative affinities among different polar solvents and dentin, and resin monomer blends of variable hydrophilic characteristics (Nalla et al., 2005; Pashley et al., 2003). It could be seen from Table I that although the solubility parameters ()) for nonpolar (dispersive) 147

148 interaction () d ) are not very much different for water and the hydrophobic resin blend, the ) differ widely in both the solubility parameters for polar interaction () p ) and hydrogen bonding () h ). The solubility parameters of a material may be represented as a point in three-dimensioned space with ) h, ) p and ) d represented as vectors along the orthogonal axes. When predicting the compatibility (miscibility) of two organic materials A and B, the distance in this three-dimensional space between the components is calculated as: *(A-B) 2 = [) d (A) - ) d (B)] 2 + [) p (A) - ) p (B)] 2 + [) h (A) - ) h (B)] 2. A good solubility level is typically assigned when *(5(MPa) " (van Krevelen, 1990). That is, a sphere with a diameter of 5 MPa " can be constructed in the 3-D space. Solution mixture that fit within that space are miscible with each other. These that fall outside the sphere are not completely miscible. As can be seen from the bottom row of Table II, the *value &between the neat hydrophobic resin blend and water is 35.7, making the two liquids mutually immiscible. When water in the demineralised dentin is replaced by 100% ethanol, the *&value is reduced to 11.8, 148

149 making it closer to the ideal value of 5. A large part of this reduction is contributed by the lower ) h of ethanol (20 MPa " ) compared with that of water (40.4 MPa " ) (Table I), thereby bringing the hydrophobicity of the ethanol-saturated dentin closer to that of the neat hydrophobic resin. It is well known that in tissue embedding that shrinkage occurs with the use of chemical dehydrants (Boyde and Maconnachie, 1981). Likewise, as ethanol is less capable of breaking spontaneously formed hydrogen bonding with adjacent collagen fibrils due to its reduced ) h value, replacement of water in demineralised dentin induces 15-17% shrinkage in the collagen matrix (Becker et al., 2006; Eddleston et al., 2003). The increased ability of the ethanol-saturated collagen fibrils to form interpeptide bonds also stiffens the collagen matrix, by reducing the plasticizing effect of water (Maciel et al., 1996). Thus, in spite of slight shrinkage that occurs during ethanol replacement, the ethanol-stiffened collagen matrix is prevented from collapsing while being suspended in the polar but less hydrophilic chemical dehydrant. This is a prerequisite for resin 149

150 infiltration in the second resin replacement phase (Eddleston et al., 2003). Ethanol is not really the ideal solvent for the Bis- GMA/TEGDMA resin comonomer, as indicated by the relatively large *&value (11.8 MPa " ) between the neat resin and 100% ethanol (Table II), However, ethanol is selected in lieu of acetone for patient compliance purpose and because acetone causes larger matrix shrinkages (ca %) (Nakajima et al., 2002). Thus, the objective of the second ethanol replacement phase is, to achieve in a stepwise manner, a gradual transformation of the solubility parameter characteristics of the ethanol-saturated collagen matrix to those that approximate the hydrophobic characteristics of the neat hydrophobic resin. The principle of sequential transition is also adapted from the use of a transitional solvent (propylene oxide) for epoxy resins in tissue embedding, as the latter is insoluble in ethanol. From Tables II and IIIA, when a 50% hydrophobic primer version is mixed with ethanol, the *&value is reduced to 6.9 (MPa) ". The mixing of the neat resin with the 50% 150

151 hydrophobic primer version additionally reduces the *&value to 7.0 (MPa) " (Table IIIA). Although these two *&values are still higher than the ideal value of 5 (MPa) ", a two-step primer/adhesive application would enable better miscibility than when the neat hydrophobic resin is directly applied to the ethanol-saturated dentin (* = 11.8, Tables II and IIIA). In Table II, it can also be seen that mixing the 20/80 and 30/70 hydrophobic primer/ethanol versions with 100% ethanol generated *&values of 2.8 and 4.1 respectively, making them more ideal than the 40/60 and 50/50 versions (* 5.6 and 6.9, respectively). The fact that the former two versions produced low tensile strengths compared with the two more concentrated versions (even though they had lower * values) may be interpreted as being due to less resin that was applied to the demineralised dentin. However, every attempt was made to ensure that the primer-saturated dentin remained glistening in appearance prior to the application of the neat resin. This phenomenon may alternatively be elucidated by referring to Table IIIA. Whereas a 151

152 *&value of 4.1 is initially achieved when a 30% hydrophobic primer is mixed with 100% ethanol, the subsequent application of the neat resin to the 30% hydrophobic primer resulted in a final *&value of 9.8. This probably accounts for the two modes of nanoleakage observed within hybrid layers created with this protocol (Figure 3A), with the reticular patterns representing incomplete resin infiltration and the isolated silver grains representing locations where remnant moisture still remained within the collagen fibrils of the demineralised dentin. It is possible that some of this moisture was trapped within the hydrophobic adhesive (Figure 3A). Likewise, the low bond strengths obtained with the use of the 75% hydrophobic primer version (Figure 1), appearing physically as the inability of the latter to spread uniformly on the surface of the ethanol-saturated dentin, may be explicated by the higher *&value of 10.4 (MPa) " in the primer application step (Tables II and IIIA). The use of the 100% adhesive version as the primer probably resulted in minimal moisture retention (no isolated silver grains) (Figure 3B vs 3A). However, direct application of the more viscous 152

153 non-solvated resin to demineralised dentin resulted in extensive areas of incomplete resin infiltration within the hybrid layer, as depicted by the heavy reticular type of silver deposits (Figure 3B). It appears therefore, for effective infiltration of neat hydrophobic resin into ethanol-saturated dentin, a sequence of steps is required in which the corresponding *&values remain consistently below 5 (MPa) ". Ideally, this may be achieved using initial primer coats that consist of 33.3% (i.e. a 1:2 dilution) of the neat hydrophobic resin in ethanol (Table IIIB). This is followed by the application of primer coats that consist of 66.7% (i.e. a 2:1 dilution) of the neat hydrophobic resin in ethanol, prior to the application of the neat resin as the final adhesive coat. Incidentally, this hypothetical protocol, based on solubility parameter approximations, corresponds well with the various dilutions of epoxy resins in transitional solvents employed in tissue embedding (published calculations). It is appropriate to perform an initial assessment of the technique sensitivity of the new ethanol wet bonding protocol prior 153

154 to the implementation of more elaborate dentin perfusion studies. From Figure 4, it is evident that partial dehydration protocols are not compatible with the use of even the 50/50 hydrophobic primer version that generated the best tensile strength results with a full dehydration protocol, and that the deterioration in tensile strengths correlates well with the increases in ) h values of different partial dehydration protocols. The corollary to these results is that the ethanol wet bonding protocol is technique sensitive, in that the presence of as little as 5% water in the demineralised dentin will result in an approximate 25% decrease in tensile strength of the hydrophobic adhesive to dentin. While this may not be a problem in non-vital dentin due to the use of a stepwise chemical dehydration sequence, it is anticipated that occluding dentinal tubules with calcium oxalate (Tay et al., 2003a; Tay et al., 2005b) may be necessary to minimise fluid transudation prior to the use of the ethanol wet bonding protocol. This should be tested in future studies before the technique can be recommended for clinical use. As the current ethanol wet bonding technique is more time 154

155 consuming and involves more steps than conventional three-step dentin bonding protocols, further work should also be performed to assess the longevity or in vitro degradation of hydrophobic resinbonded dentin to ensure that this new bonding concept confers real advantages over conventional hydrophilic adhesives that are currently being used for dentin bonding. CONCLUSIONS Within the limits of this study, it may be concluded that: 1) It is possible to bond hydrophobic resins to dentin using a stepwise sequence of replacement of water in acid-etched dentin with ethanol followed by a stepwise sequence of ethanol replacement with increasing concentrations of ethanol-solvated BisGMA/TEDGMA. 2) The design of such hydrophobic adhesive protocols may be assisted using Hoy's solubility parameters of the respective solvents and resins. This method may be used in the future for designing self-etch adhesives that become hydrophobic after sequential 155

156 replacements of solvated self-etching primer with non-solvated comonomer mixture to decrease the ) p and ) h values of the final resin. 3) The new ethanol wet bonding protocol is technique-sensitive to moisture contamination of the 100% ethanol-saturated dentin. This requires additional steps to circumvent water contamination when bonding to deep vital dentin in the presence of positive pulpal pressures. ACKNOWLEDGEMENTS The hydrophobic neat resin was a generous gift from Bisco Inc. This study was supported by R01 grant DE from NIDCR, USA (PI David Pashley). The authors graciously acknowledge the technical support of Penny Roon and Kelli Agee and the secretarial support provided by Michelle Barnes. REFERENCES 156

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166 Table I - Hoy's solubility parameters for the Bis-GMA/TEGDMA comonomer resin blend and the experimental ethanol-based hydrophobic primers prepared from this resin blend. The solubility parameters of ethanol and water are included for comparison. Hoy's solubility parameters (MPa) " Category Composition " d " p " h " t Water % ethanol, 50% water Solvents 70% ethanol, 30% water % ethanol, 20% water % ethanol, 5% water % ethanol Experimental 14% Bis-GMA; 5.75% TEGDMA; primer 0.2% EDMAB; 0.05% CQ; /80 80% ethanol Experimental 21% Bis-GMA; 8.62% TEGDMA; primer 0.3% EDMAB; 0.08% CQ; /70 70% ethanol 166

167 Experimental 28% Bis-GMA; 11.5% TEGDMA; primer 0.4% EDMAB; 0.1% CQ; /60 60% ethanol Experimental 35% Bis-GMA; 14.37% TEGDMA; primer 0.5% EDMAB; 0.13% CQ; /50 50% ethanol Experimental primer 75/ % Bis-GMA; 21.56% TEGDMA; 0.75% EDMAB; 0.19% CQ; 25% ethanol Neat resin 70% Bis-GMA; 28.75% TEGDMA; 1% EDMAB; 0.25% CQ Abbreviations. Bis-GMA: 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane; TEGDMA: triethyleneglycol dimethacrylates; EDMAB: ethyl N,N-dimethyl-4-aminobenzoate; CQ: DL-camphorquinone. All Hoy's solubility parameters were calculated using commercially available software (Computer Chemistry Consultancy < " d - Hoy's solubility parameter for dispersive forces; " p - Hoy's solubility parameter for polar forces; " h - Hoy's solubility parameter for hydrogen bonding forces; " t - Hoy's solubility parameter for the total cohesive forces, that is equivalent to Hildebrand's solubility parameter (! t =! d +! p! h ). 167

168 Table II - Table depicting the differences in Hoy's " parameters (*) (in MPa " ) based on the solubility parameters of the final solvent employed in the full or partial ethanol dehydration protocols and those of the various ethanolic dilutions of the hydrophobic adhesive comonomer resin blend. *values that are ( 5 (MPa) " are highlighted by asterisks. Experimental 100% water 50% ethano l 70% ethano l 80% ethano l 95% ethano l 100% ethanol primer 20/ * 2.8 * Experimental primer 30/ * Experimental primer 40/ Experimental primer 50/ Experimental primer 75/ Neat hydrophobic Resin The solubility parameters of a material may be represented as a point in three-dimensioned space with ) h, ) p and ) d represented as vectors along the orthogonal axes. When predicting the compatibility (miscibility) of two organic materials A and B, a sphere with a diameter of * in this threedimensional space between the components is calculated as: * (A-B) 2 = [) d (A) - ) d (B)] 2 + [) p (A) ) p (B)] 2 + [) h (A) - ) h (B)] 2. A good solubility level is typically assigned when * ( &5(MPa) ". 168

169 Table III-A Calculated Hoy's " differences * (MPa " ) between tissues or dentin equilibrated with 100% ethanol and hydrophobic resin diluted in different ethanol concentrations to be used as a primer vs. neat hydrophobic resin used as an adhesive. Initial [EtOH] % resin in primer! MPa " between 100% EtOH & primer % resin in adhesive! MPa " between primer & adhesive 100% 20% % % 30% % % 33% % % 40% % % 50% % % 67% % % 75% % % 100% % 11.8 The hypothetical optimal sequence of sequential hydrophobic resin application to 100% ethanol-saturated acid-etched dentin should be one in which all the * &values within a single row are consistently below 5(MPa " ). The use of an initial 50% resin followed by 100% resin generated * &values of 6.9(MPa " ) and 7.0(MPa " ), respectively. These values are close to, but still higher than 5(MPa " ). 169

170 Table III-B Calculated Hoy's " differences, * (MPa " ) for tissues (dentin) equilibrated with 100% ethanol and then infiltrated (primed) with increasing concentrations of resin in ethanol, applied as sequential primers 1 and 2 or primers 1 and 2 followed by 100% resin as an adhesive. % resin! MPa " % resin! MPa "! MPa " Initial [EtOH] in ethanol in between 100% EtOH and in ethanol in between Primer 1 and % resin in adhesive between Primer 2 & Primer 1 Primer 1 Primer 2 Primer 2 adhesive 100% 30% % % % 30% % % % 30% % % % 33% % % % 33% % % % 33% % %

171 Figure 1 - Tensile strength results obtained with the six experimental hydrophobic adhesives with different primers of increasing resin concentration. These adhesives were bonded to acid-etched dentin using the ethanol wet bonding technique. Positive and negative controls, bonded with the conventional water wet bonding technique, were included for comparison. The positive controls consisted of two commercially available, hydrophilic three-step adhesives. The negative control consisted of the 50/50 hydrophobic primer version. 171

172 Figure 2 - Distribution of failure modes in the experimental, positive and negative control groups described in Figure 1. AB2: All-Bond 2; SBMP: Scotchbond Multi-Purpose. 172

173 Figure 3 - TEM micrographs taken from unstained, undemineralised, silverimpregnated sections of acid-etched dentin bonded with different versions of the hydrophobic adhesives using the ethanol wet bonding technique. A: adhesive layer; Between open arrows: hybrid layer; D: mineralised intertubular dentin; T: dentinal tubule. A. A representative section from the 20/80 adhesive version. Pointer: reticular network of silver deposits within the hybrid layer; Open arrowhead: isolated silver grains that occurred randomly within the hybrid layer; Arrow: additional silver deposits that were localised to the adhesive layer. B. A representative section from the 100/0 adhesive version. Only thick reticular pattern of silver deposits (pointer) were identified within the hybrid layer. C. A representative section from the 50/50 adhesive version. Nanoleakage was minimal and only fine, isolated silver grains (open arrowheads) were found along the periphery of the dentinal tubules. Fig. 3A 173

174 Fig. 3B Fig. 3C 174

175 Figure 4 - A composite chart showing 1) the differences in tensile strengths obtained when the same 50/50 hydrophobic adhesive version was applied to acid-etched dentin that was chemically dehydrated to different extents. The ethanol/water ratio of the last chemical dehydrant employed in these groups were 0/100, 50/50, 70/30, 80/20, 95/5 and 100/0, respectively. Tensile strengths values shown are means ± standard deviations; 2) the correlation between the tensile strengths of various partial/total dehydration groups with the Hoy s solubility parameter for hydrogen bonding (" h ) of the last chemical dehydrant used in each group. A significant negative power correlation was observed between the tensile strengths and " h values. 175

176 Tubular Occlusion Optimizes Bonding of Hydrophobic Resins To Deep Dentin. Sadek FT, Pashley DH, Ferrari M, Tay FR. Submitted to Journal for Dental Research. INTRODUCTION The hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) is often used as a solvent for sparingly water-soluble resin monomers employed in contemporary dentin adhesive formulations. It increases the ability of these adhesives to wet dentin and reduces their sensitivity to moisture contamination (Tay and Pashley, 2003). Although the incorporation of hydrophilic and acid resin monomers has substantially improved the initial bonding of contemporary adhesives to intrinsically wet dentin substrates (Kanca, 1992), it also resulted in increases in water sorption (Hashimoto et al., 2005; King et al., 2005) and decreases in mechanical properties of the polymerized adhesives (El Zohairy et al., 2004; Yiu et al., 2004), thereby compromising the longevity of resin-dentin bonds (De Munck et al., 2003, 2005; Koshiro et al., 176

177 2005). Moreover, it has been demonstrated that resin-dentin bonds created by contemporary adhesives are susceptible to fluid permeation, with the HEMA-based adhesives being more severely affected, which significantly reduces their bond strengths (Sauro et al., 2006). Theoretically, the use of comparatively hydrophobic resins as dentin adhesives would avoid these problems and improve the durability of resin-dentin bonds. However, it is impossible to wet or infiltrate acid-etched dentin with hydrophobic adhesives (Brudevold et al., 1956). Using concepts of tissue embedding for electron microscopy, it was recently shown that it is possible to bond hydrophobic resins blends to acid-etched dentin with an ethanol wet bonding technique (Nishitani et al., 2006; Sadek et al., 2006), attaining tensile strengths that were comparable with contemporary hydrophilic adhesives and with minimal nanoleakage within the hybrid layers and adhesive layers. In this technique, the water in the acid-etched dentin is slowly replaced by an ascending concentration of ethanol, avoiding the collapse of the interfibrillar 177

178 spaces within the collagen matrix. Preservation of these spaces enables the replacement of the ethanol with increasing concentrations of hydrophobic monomers dissolved in ethanol, and finally with neat hydrophobic resin. Although promising results were obtained when dentin specimens were bonded in the absence of fluid contamination from dental pulp, the ethanol wet bonding protocol was found to be technique sensitive in the presence of water. As hydrophobic monomers are immiscible with water, contamination of these resin monomers with as little as 5 vol% water on the ethanol-saturated dentin substrate resulted in a 25% reduction in tensile strength of the experimental hydrophobic adhesive to dentin (Sadek et al., 2006). This renders the ethanol wet bonding technique impractical when it is used in vital deep dentin (Lopes et al., 2006). Oxalate desensitizers have been shown in both in vitro (Gillam et al., 2001; Tay et al., 2003; Tay et al., 2005) and in vivo studies (Chang et al., 1996; Gillam et al., 2004; Muzzin and Johnson, 1989) to be effective in reducing the permeability of deep, 178

179 acid-etched dentin. When oxalate desensitizers are applied to acidetched dentin, the oxalate reacts with the calcium ions within the dentin tubules to form sparingly-soluble precipitates of calcium oxalate that occlude the tubules and minimize outward fluid movement to the dentin surface (Gillam et al., 2001; Tay et al., 2003). Similar to the use of oxalate desensitizers (Pashley et al., 2001), a poly(glutamic) acid-modified diluted ceramicrete (PADC) was recently found to be able to reduce dentin permeability by occluding dentinal tubules with very fine agglomerates of MgKPO 4.6H 2 O crystallites (Tay and Pashley, unpublished results). Rinsing of the ceramicrete-treated dentin surface for bonding procedures may remove enough of these crystallites from the surface, while blocking the tubules to prevent fluid contamination during the application of hydrophobic adhesives to acid-etched dentin. Although dentin bonding with hydrophobic resins using the ethanol wet bonding technique shows initially favorable results (Nishitani et al., 2006; Sadek et al., 2006), the protocol must be 179

180 further tested in vitro when bonding is performed under dentin perfusion before it may be recommended for clinical testing. Thus, the objectives of this study were: (1) to compare the microtensile bond strength of an experimental hydrophobic adhesive to midcoronal and deep-dentin, when bonding was performed under physiological pulpal pressure using the ethanol wet bonding technique; (2) to identify the effect of subsurface tubular occlusion with oxalate and PADC desensitizer during the ethanol wet bonding technique; and (3) to examine, with transmission electron microscopy, the ultrastructure of the bonded interfaces. The null hypothesis tested was that there is no difference in the bonding of an experimental hydrophobic adhesive to oxalate or PADC desensitizer-treated acid-etched mid-coronal and deep dentin with the use of an ethanol wet bonding technique under simulated physiological pulpal pressure. MATERIALS AND METHODS Tooth Preparation 180

181 Forty-eight human third molars were collected after the patients' informed consent was obtained under a protocol reviewed and approved by the Human Assurance Committee of the Medical College of Georgia. The teeth were sectioned 3 mm below the cementoenamel junction with a slow-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water-cooling. They were initially divided into two groups accordingly to the remaining dentin thickness: mid-coronal dentin [MD] or deep dentin [DD]. Flat dentin surfaces were then prepared perpendicular to the longitudinal axes of the teeth with the diamond saw under water cooling, leaving mm of dentin in MD group, and mm in the DD group. Each surface was ground with 180-grit silicon carbide paper under running water for 20 s to create a clinically-relevant smear layer. The final thickness of the remaining dentin was precisely measured with a dial caliper gauge (0.01mm, Renfert GmbH, Hilzingen, Germany). Experimental Design 181

182 Two experimental tubular occlusion materials were prepared: 3% potassium tetroxalate (JT Baker, Phillipsburg, NJ, USA) and PADC (0.05 µl of 10% phosphoric acid, 0.05 µl of poly(glutamic) acid (16 µg/ml of deionized water) and 50 µg of ceramicrete. The latter is a chemically-bonded phosphate ceramic consisting of MgO and KH 2 PO 4 (Wagh et al., 1999). A comonomer resin blend comprising 70 wt% Bis-GMA, wt% TEGDMA, 0.25 wt% camphorquinone and 1 wt% ethyl N,N-dimethyl-4- aminobenzoate was used to formulate the experimental three-step, etch-and-rinse, hydrophobic adhesive. The primer solution was prepared by diluting the neat comonomer blend with 50 wt% of absolute ethanol. The neat comonomer resin blend was employed as the adhesive component. Bonding Procedures A Plexiglass platform was constructed by inserting 18- gauge stainless steel tubing into hole created in a 2x2x0.6 cm piece of Plexiglass. After removing pulpal tissues from crown segments, 182

183 they were attached to the Plexiglass with a cyanoacrylate adhesive (Zapit, Dental Ventures of America, Anaheim Hills, CA, USA). Each Plexiglass-crown segment assembly was attached via a polyethylene tubing (Fisher Scientific, Pittsburg, PA, USA) to a syringe barrel filled with deionized water. The latter was raised to deliver 20 cm of water pressure. The MD and DD groups were each divided into three subgroups (n=8) accordingly to the dentin tubule occluding material employed prior to adhesive application (3% potassium tetroxalate; PADC or deionized water as the control group). Each tooth was etched with 37% phosphoric acid gel (Etch 37, Bisco Inc., Schamburg, IL, USA) for 15 s, rinsed thoroughly with deionized water and left moist. The tubule occluding materials were applied using a microbrush with agitation for 30 s, left undisturbed for 60 s, rinsed meticulously and the substrate was left moist with deionized water. A chemical dehydration protocol previously described by Sadek et al. (2006) was used for ethanol wet bonding. Briefly, 183

184 acid-etched and occluded wet dentin surfaces were treated with a series of increasing ethanol concentrations (50%, 70%, 80%, 95% and 100% for three times for 30 s each). This procedure was meticulously performed to ensure that the dentin surface was always immersed in a liquid phase by keeping it visibly moist prior to the application of the subsequent solution with a higher ethanol concentration. Two consecutive coats of the experimental hydrophobic primer were then applied to ethanol-saturated dentin. Excess ethanol solvent was evaporated with a gentle air stream for 10 s. Then, a layer of the neat comonomer adhesive was applied, spread thin with moisture-free air, and light-cured for 20 s using an Optilux 500 halogen light-curing unit (Demetron/Kerr, Danbury, CT, USA) with a power output of 600 mw/cm 2. Composite buildups were constructed with a light-cured resin composite (Clearfil APX, Kuraray Co., Okayama, Japan) in five 1 mm-thick increments. Tensile Testing After storage in deionized water at 37 C for 24 h, each 184

185 tooth was vertically sectioned into 0.9 mm thick serial slabs by means of the Isomet saw under water cooling. The central slab of each tooth was utilized for subsequent morphologic examination. The adjacent slabs were sectioned into 0.9x0.9 mm beams, according to the "non-trimming" technique of the microtensile test. Each beam was stressed to failure under tension using a testing jig (Bisco Inc.) mounted in a Vitrodyne V1000 universal tester (Liveco Inc, Burlington, VT, USA) at a cross-head speed of 1 mm/min. Bond strength data from the six subgroups were statistically analyzed with a two-way ANOVA design (tubular occlusion vs dentin depth). Post hoc multiple comparisons were performed with Tukey test, with statistical significance set at '=0.05. Transmission Electron Microscopy (TEM) Three central slabs from each of the six subgroups were used for TEM examination of nanoleakage along the dentin hybrid layers. These slabs were immersed for 24 h in a tracer solution 185

186 containing 50 wt% ammoniacal silver nitrate, according to the method employed by Tay et al. (2002). The silver-impregnated slabs were then rinsed thoroughly in distilled water and placed in photodeveloping solution for 8 h under a fluorescent light. Undemineralized, epoxy resin-embedded, nm thick sections were prepared and examined without further staining using a TEM (Philips CM-100, Eindhoven, The Nederlands) operated at 80 kv. RESULTS The bond strength data were normally distributed (Kolmogorow-Smirnoff test) and exhibited equal variances (Levene test). Two-way ANOVA revealed significant differences in the factors tubular occlusion (p<0.001), dentin depth (p<0.001) and their interactions (p<0.001). Post hoc comparisons revealed that significant difference between bond strengths of midcoronal vs deep dentin (p<0.05), between oxalate pre-treatment with the control (p<0.05) and between PADC pre-treatment with the control (p<0.05) (Figure 1). Under TEM examination, the 186

187 resin-dentin interface in the DD control subgroup (i.e. deionized water, no tubular occlusion material) separated and resin tags pulled out of the tubules. Extensive silver leakage was present (Figure 2). Unlike DD, the MD control subgroup was not sensitive to the effect of dentin perfusion (not shown). With the use of oxalate and PADC pre-treatments, only minimal nanoleakage could be identified within the hybrid layers created in deep dentin (Figures 3 and 4). For the oxalate pretreatment subgroups, the dentin surfaces were completely devoid of calcium oxalates crystals and the latter could only be identified within the dentinal tubules, at 4-5 +m from the tubular orifices (Figure 3). For the PADC pre-treatment subgroups, fine electrondense ceramicrete crystallites could be seen attached to the lamina limitans of the dentinal tubules (Figure 4). DISCUSSION The present work confirmed the results of our previous studies (Nishitani et al., 2006; Sadek et al., 2006) that is possible to 187

188 bond to mid-coronal dentin and achieve high tensile strengths when experimental hydrophobic adhesives are used with the ethanol wet bonding technique. However, tensile bond strength was severely compromised when the same technique was applied under dentin perfusion to deep dentin. Thus, we have to reject our null hypothesis. As the number and the diameter of dentinal tubules in deep dentin were much greater than those of mid-coronal dentin (Garberoglio and Brännström, 1976), more outward fluid movement may be expected during ethanol wet bonding procedures. Water derived from the dental pulp probably contaminated the chemically dehydrated dentin surfaces and resulted in their poor wetting by hydrophobic adhesives. The rationale of water contamination during the bonding of hydrophobic resins to chemically dehydrated acid-etched dentin may be explained in terms of the solubility parameter concept. Replacement of the water in a demineralized dentin matrix with ethanol results in lowering the solubility parameter for hydrogen bonding () h ) of the dentin matrix (Miller et al., 1998). Water, 188

189 having the highest ) h (40.4 J/cm 3 ) 1/2, has a better affinity for dentin () h = 23.6 J/cm 3 ) 1/2 than for hydrophobic monomers () h = 6.5 J/cm 3 ) 1/2. Consequently, water contamination of the ethanol-dehydrated dentin matrix will result in increase in ) h values of the matrix, rendering the matrix less compatible with the hydrophobic primer solution () h = 13.3 J/cm 3 ) 1/2. Thus, it is critical to avoid water contamination in the ethanol wet bonding technique after the demineralized collagen matrix is suspended in absolute ethanol, so as to optimize the infiltration of hydrophobic resins. The use of tubular occlusion materials represents a possible solution to avoid water re-contamination. The bond strengths in MD were not compromised by the adjunctive use of these products prior to ethanol dehydration. Conversely, higher bond strengths were observed in deep acid-etched dentin when these products were used. The potassium tetroxalate used is acidic enough (ph 2.5) to etch subsurface dentin, reacting with calcium further down in the dentinal tubules and forming calcium oxalate dihydrated crystals that block water movement. Its potential effectiveness in 189

190 occluding dentinal tubules and reducing dentinal permeability has previously been reported (Gillam et al., 2001; Pashley et al., 2001; Pereira et al., 2005) and may be readily appreciated in Figure 3. Ceramicrete is classified as a ceramic material in which the ceramic phase is not derived from powder sintering, but from acid-base reaction. The one utilized in this study is based on the reaction of magnesium oxide with dibasic potassium phosphate (Wagh et al., 2003). The ceramic phase in ceramicrete usually contains large crystals and when they are present on the acid-etched dentin surface, would prevent infiltration of adhesive resins into the partially-demineralized dentin matrix. By using poly(glutamic) acid to control crystal nucleation and growth in diluted ceramicrete formulations, very fine MgKPO 4.6H 2 O crystallites may be produced for dentin desensitization purpose (Tay and Pashley, unpublished results). Within the limitations of this study, it may be concluded that the technique sensitivity of the ethanol wet bonding technique in deep dentin mat be resolved with the adjunctive use of tubular 190

191 occlusion materials. This renders the bonding of hydrophobic monomers to dentin an achievable goal for shallow, mid-coronal and deep dentin substrates. Further studies should be performed to evaluate the longevity of hydrophobic resin-dentin bonds created by the ethanol wet bonding technique. ACKNOWLEDGEMENTS This study was supported by R01 grant DE from NIDCR, USA (PI David Pashley). The authors graciously acknowledge the technical support of Penny Roon and the secretarial support provided by Michelle Barnes. 191

192 REFERENCES Brudevold F, Buonocore M, Wileman W (1956). A report on a resin composition capable of bonding to human dentin surfaces. J Dent Res 35(6): Chang LC, Auyeung L, Lin YT (1996). In vivo study of potassium oxalate gel in tooth hypersensitivity. Changgeng Yi Xue Za Zhi 19(4): De Munck J, Van Meerbeek B, Yoshida Y, Inoue S, Vargas M, Suzuki K, et al. (2003). Four-year water degradation of total-etch adhesives bonded to dentin. J Dent Res 82(2): De Munck J, Van Landuyt K, Peumans M, Poitevin A, Lambrechts P, Braem M, et al. (2005). A critical review of the durability of adhesion to tooth tissue: methods and results. J Dent Res 84(2): El Zohairy AA, De Gee AJ, Hassan FM, Feilzer AJ (2004). The effect of adhesives with various degrees of hydrophilicity on resin ceramic bond durability. Dent Mater 20(8): Garberoglio R, Brännström M (1976). Scanning electron microscopic investigation of human dentinal tubules. Arch Oral Biol 21(6): Gillam DG, Mordan NJ, Sinodinou AD, Tang JY, Knowles JC, Gibson IR (2001). The effects of oxalate-containing products on the exposed dentine surface: an SEM investigation. J Oral Rehabil 28(11): Gillam DG, Newman HN, Davies EH, Bulman JS, Troullos ES, Curro FA (2004). Clinical evaluation of ferric oxalate in relieving dentine hypersensitivity. J Oral Rehabil 31(3):

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194 Pereira JC, Segala AD, Gillam DG (2005). Effect of desensitizing agents on the hydraulic conductance of human dentin subjected to different surface pre-treatments--an in vitro study. Dent Mater 21(2): Sadek FT, Pashley D, Nishitani Y, Carrilho MR, Donnelly A, Ferrari M, et al. (2006). Application of hydrophobic resin adhesives to acid-etched dentine with an alternative wet bonding technique. J Biom Mater Res Part A (in press). Sauro S, Pashley DH, Montanari M, Chersoni S, Carvalho RM, Toledano M, et al. (2006). Effect of simulated pulpal pressure on dentin permeability and adhesion of self-etch adhesives. Dent Mater (in press). Tay FR, Pashley DH (2003). Have dentin adhesives become too hydrophilic? J Can Dent Assoc 69(11): Tay FR, Pashley DH, Mak YF, Carvalho RM, Lai SC, Suh BI (2003). Integrating oxalate desensitizers with total-etch two-step adhesive. J Dent Res 82(9): Tay FR, Pashley DH, Hiraishi N, Imazato S, Rueggeberg FA, Salz U, et al. (2005). Tubular occlusion prevents water-treeing and through-and-through fluid movement in a single-bottle, one-step self-etch adhesive model. J Dent Res 84(10): Wagh A, Strain R, Jeong S, Reed D, Krouse T, Singh D (1999). Stabilization of rocky flats Pu-contamined ash within chemically bonded phosphate ceramics. J Nucl Mat 265: Wagh AS, Jeong SY, Lohan D, Elizabeth A (2003). Chemically Bonded Phospho-silicate ceramics. US: Patent number 6,518,212 B1. 194

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196 Figure 1. Microtensile bond strength (means and standard deviations) obtained from the six experimental groups. An experimental, three-step, etch-and-rinse hydrophobic adhesive was bonded to mid-coronal and deep acid-etched dentin using the ethanol wet bonding technique under simulated pulpal pressure. The acid-etched dentin was pre-treated with deionized water (control), 3% potassium tetroxalate (oxalate) or poly(glutamic) acid-modified, diluted ceramicrete (PADC) prior to ethanol dehydration. Groups identified with the same letter are not significantly different (p > 0.05). 196

197 Figure 2. A representative TEM micrograph taken from an unstained, undemineralized, silver-impregnated section in the control-deep dentin subgroup. Acid-etched deep-dentin was bonded with the hydrophobic adhesive using the ethanol wet bonding technique. A: adhesive layer; C: resin composite; D: mineralized intertubular dentin; E: space created when the adhesive-resin interface separated from the dentin, showing some voids (*) that are indicative of incomplete polymerization of the embedding epoxy resin due to release of water from the interface during polymerization. Arrow: resin tags that pulled out of the tubules. Open arrowhead: silver deposits. The demineralized collagen matrix could not be identified clearly. 197

198 Figure 3. TEM micrograph taken from an unstained, undemineralized, silver-impregnated section from the oxalate-deep dentin subgroup. A: adhesive layer; C: resin composite; D: mineralized intertubular dentin; H: hybrid layer. A. A low magnification view showing the presence of calcium oxalate crystals (open arrows) in the dentinal tubules, about 5-8 µm from the tubular orifices, blocking water movement during dentin perfusion. B. At a higher magnification, the dentin surface was completely devoid of oxalate crystals. Most of oxalate crystals inside the dentinal tubules dislodged during ultramicrotomy, leaving empty spaces. Some of them, however, remained and appeared as electron-dense aggregates (open arrowhead). Only minimal nanoleakage could be identified as isolated, round silver grains (arrows) with the hybrid layer. A B 198

199 Figure 4. TEM micrograph taken from an unstained, undemineralized, silver-impregnated section of the poly(glutamic) acid-modified, diluted ceramicrete-deep dentin subgroup. A: adhesive layer; C: resin composite; D: mineralized intertubular dentin; H: hybrid layer. A. An overall view of the resin-dentin interface, showing the generalized absence of silver nanoleakage. The dentinal tubules were coated along their peripheries with a layer of material (arrows) that has dislodged during ultramicrotomy, leaving electron-lucent spaces that were neither infiltrated with the adhesive resin nor epoxy resin. The hybrid layer was only 2 µm thick, probably reflecting shrinkage that occurred during the stepwise chemical (ethanol) dehydration regime. B. At a higher magnification view, spaces occupied by the ceramicrete crystallite aggregates were seen on the dentin surface and inside the dentinal tubules (arrows). The use of poly(glutamic acid) resulted in the reduction in the sizes of these crystallites to the dimensions that were smaller than the diameters of the dentinal tubular orifices. Hence, they did not prevent resin infiltration into the tubules and demineralized collagen matrix. A fine layer of electron-dense ceramicrete crystallites could be seen within a tubule and could be readily discerned from the peritubular dentin (pointer). Only minimal nanoleakage could be identified as isolated, round silver grains (open arrowhead) with the hybrid layer. C. At a very high magnification, electron-dense ceramicrete crystallites (open 199

200 arrowheads) approaching the dimensions of dentin apatites could be seen attaching to the lamina limitans (arrows) of the dentinal tubules and trapped by resin. 200