Clinical relevance of tests on bond strength, microleakage and marginal adaptation

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1 d e n t a l m a t e r i a l s 2 9 ( ) Available online at jo u rn al hom epa ge : Clinical relevance of tests on bond strength, microleakage and marginal adaptation Siegward D. Heintze Head of Preclinical Research, Ivoclar Vivadent, Bendererstrasse 2, FL-9494 Schaan, Liechtenstein a r t i c l e i n f o Article history: Received 2 July 2012 Received in revised form 18 July 2012 Accepted 26 July 2012 Keywords: Dental adhesive Bond strength Microleakage Marginal adaptation Clinical trial Correlation a b s t r a c t Dental adhesive systems should provide a variety of capabilities, such as bonding of artificial materials to dentin and enamel, sealing of dentinal tubules, reduction of post-operative sensitivity and marginal sealing to reduce marginal staining and caries. In the laboratory, numerous surrogate parameters that should predict the performance of different materials, material combinations and operative techniques are assessed. These surrogate parameters include bond strength tests of various kinds, evaluation of microleakage with tracer penetration between restorative and tooth, two-dimensional analysis of marginal quality with microscopes and mapping of the micromorphology of the bonding interface. Many of these tests are not systematically validated and show therefore different results between different research institutes. The correlation with clinical phenomena has only partly been established to date. There is some evidence, that macrotensile and microtensile bond strength tests correlate better with clinical retention of cervical restorations than macroshear and microshear bond tests but only if data from different test institutes are pooled. Also there is some evidence that marginal adaptation has a moderate correlation in cervical restorations with clinical retention and in Class II restorations (proximal enamel) with clinical marginal staining. There is moderate evidence that microleakage tests with dye penetration does not correlate with any of the clinical parameters (post-operative hypersensitivity, retention, marginal staining). A rationale which helps the researcher to select and apply clinically relevant test methods in the laboratory is presented in the paper Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved. 1. Introduction Adhesive dentistry has generally revolutionized dentistry. With the possibility to bond artificial materials to enamel and dentin, there is no need for mechanical retention outlines to keep a restoration or a material in place, both for direct and indirect restorations [1]. This enables dentists to accomplish defect-oriented preparations, which means that they can limit the removal of sound tooth substance to that area of the defect that needs to be restored, whether it is a carious defect, an erosive defect, a tooth fracture or an esthetical defect. With the advent of adhesive dentistry, it has become possible to bond artificial materials to other artificial materials like composites, ceramics or metal alloys. This enables dentists to repair rather than replace restorations in the case of chippings, fractures, marginal caries or esthetic improvements required by the patient [2]. A prospective clinical trial over 7 years even showed that the repair of defective direct restorations is more successful than the replacement of such restorations [3]. In Tel.: ; fax: address: siegward.heintze@ivoclarvivadent.com /$ see front matter 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

2 60 d e n t a l m a t e r i a l s 2 9 ( ) prosthodontics, the mechanical retention of crowns and fixed partial dentures is less dependent on the macromechanical retention design of the prepared tooth structure or indirect restoration when adhesive technologies are applied [4]. In orthodontics, the movement of teeth with brackets and other appliances would not be feasible without bonding them to the enamel. Finally, in preventive dentistry the sealing of fissures or initial carious lesions has only become possible with the concept of adhesive bonding to enamel [5]. This short list of everyday dental applications illustrates the extent to which adhesive dentistry has penetrated different parts of dentistry and changed dental routines. The problem for dental professionals is to decide which adhesive technique or material/system is adequate for which type of bonding indication. Dentistry has seen ongoing changes and the launch of innovative products. However, innovation was mostly driven by reducing the time required to fabricate or place restorations. This has become possible by reducing the number of steps or materials required for bonding to either natural or artificial materials as well as by reducing the activation times and by bundling several adhesive indications into one universal product. The general practitioner, however, is rather confused with the variety of different materials on the market and the way they are promoted. The most common method to promote and market these products is by presenting data gained in laboratory tests. Yet, neither dentists nor laboratory researchers have a clue as to what these tests say on the possible clinical outcome in terms of predictability and longevity. Since conducting clinical studies is complicated and expensive and the results tend to be available only several years later, the easiest thing to do is to use results from laboratory tests or in vitro simulations. However, this begs the question as to how relevant the information from such in vitro tests is when it comes to predict the clinical performance of dental materials. Laboratory test results have consistently provided the basis for recommendations on how dentists should use composite materials in their daily clinical routine. Examples are special incremental and layering techniques for direct composite restorations [6,7], the use of a flowable composite under more viscous materials [8], soft-start polymerization [9] and selective bonding instead of total bonding [10]. But then, years later, clinical studies discovered that the restorations placed according to these recommendations did not perform better than those performed with other, usually simpler techniques. For instance, restorations placed with translucent wedges and transparent matrices do not better than those placed with wooden wedges and metal matrices [11]. Likewise, restorations bonded with a selective bonding technique perform equally well as those bonded with a total bonding technique [12,13]. To finish, cervical restorations polymerized with softstart polymerization are comparable to those polymerized without soft-start polymerization [14]. Two clinical studies even questioned the incremental technique in posterior composite restorations after the clinical results of fillings placed in bulk (one increment) in mid-sized cavities were comparable to the results of studies which used the incremental technique [15,16]. Thus, the purpose of this article is to critically analyze standard laboratory tests as a means of evaluating the adhesive properties of adhesive materials used for bonding to the natural tooth structure and artificial materials. Rather than looking at tests and then trying to correlate them to the results from clinical trials, this evaluation was conducted in the reverse order: First, we analyzed the evidence from clinical trials with respect to failures and their frequency and then looked at laboratory tests that may have simulated the phenomena observed in the clinic. This article does not focus on the chemical or other interactions of adhesive systems with the dentin/enamel or other substrates nor does it cover the adhesion to restorative materials like zirconia, glass ceramics or alloys. 2. When do dentists need adhesion? Adhesion to tooth substance is required in clinical situations if an inadequate amount of tooth substance has to be removed to produce enough mechanical retention. However, what does sufficient retention mean? Although not systematically analyzed, common sense in dentistry says that adhesion is required in the following clinical situations: - cervical restorations - build-up of fractured teeth (posterior/anterior) - short clinical die for full-coverage crowns or partial crowns - minimally invasive bridges like adhesive bridges with small lingual metal or ceramic retainers For restorations in the posterior region (Class I, Class II), the preparation itself and/or the caries removal normally generates enough mechanical retention, thus making adhesion to tooth substance less important. The guidelines of cavity design preparations published by Black [17] have been obsolete for several decades and were not even applicable for amalgam restorations [18]. However, adhesion is not only necessary for keeping the restoration in place but should also fulfill other tasks such as - sealing dentinal tubules to reduce post-operative sensitivity and - sealing restoration margins to reduce the risk of marginal staining and marginal caries. 3. Which failures do occur with restorative materials in clinical studies? 3.1. Cervical restorations (Class V) Class V non-carious non-retentive lesions are frequently used to clinically evaluate the effectiveness of adhesive systems. A meta-analysis on the clinical effectiveness of cervical restorations with glass ionomer derivates and composites revealed the following results [19]: - On average, 10% of the cervical fillings were lost and 24% exhibited marginal discoloration after 3 years. The variability ranged from 0% to 50% for retention loss and from 0% to

3 d e n t a l m a t e r i a l s 2 9 ( ) % for marginal discoloration. Hardly any marginal caries was detected. - The adhesive/restorative class had the most significant influence, with two-step self-etching adhesive systems performing best and one-step self-etching adhesive systems performing worst; three-step etch&rinse systems, glass ionomers/resin-modified glass ionomers, two-step etch&rinse systems and polyacid-modified resin composites were ranked in between. - Restorations placed in teeth with prepared dentin showed a statistically significant higher retention rate than those placed in teeth with unprepared dentin. - Beveling of the enamel and the type of isolation used (rubber dam/cotton rolls) had no significant influence Posterior direct restorations (Class I/II) As early as in the 1970s of last century, composite resins were placed in posterior teeth. Those resins were peroxide initiated macrofilled composites that were placed in bulk. In those days, the enamel was not etched with phosphoric acid and the cavities were drilled in the same way as for amalgam fillings [20,21]. Mostly calcium hydroxide or glass ionomer materials were placed as liners under the composite resin restorations. In the 1980s etching of the enamel became integrated into the operational procedure and it became common practice to use an unfilled, hydrophobic low-viscosity bonding material between resin and enamel. In the late 1980s the first dentin bonding agents were developed, but these materials still required separate etching of enamel. This method was later replaced by the so-called total etch technique, which involves the simultaneous etching of both enamel and dentin. In 1999 the first self-etching enamel dentin adhesive systems were released on the market. In the meantime, these systems either one-step or two-step have gained popularity amongst dental practitioners (dental professionals) because they shorten and simplify the operational procedures. A systematic review was carried out on composite posterior restorations [22]. Fifty-nine studies met the inclusion criteria with more than 7100 composite restorations at baseline and more than 700 of which had been placed without enamel etching and/or adhesive system. The following conclusions relevant for the adhesive system can be drawn: - Restorations that were placed without enamel etching and bonding showed significantly more post-operative sensitivity than restorations that were placed with enamel etching or self-etching adhesives; there was no significant difference between the latter two groups. However, the difference between the enamel etching and the no etching/no bonding group was less than 2% of the restorations affected. This is in contrast to other studies that assume a frequency of postoperative sensitivity in posterior composite restorations that varies between 1% and 29% [23,24]. Risk factors for the development of post-operative sensitivity include deep and large cavities, the removal of the smear layer and the type of tooth [25]. For pulpal reactions and hypersensitivity, a multifactorial etiology must be assumed, involving besides bacterial penetration due to gap formation contraction Fig. 1 Estimated median percentage of restorations across the studies and experiments without caries adjacent to the restoration in relation to the type of adhesive system [22]. stress of the composite at dentinal walls, residual caries, inadequate sealing of the opened tubules, etc. [26,27]. - The frequency of marginal caries was low in most studies with a median prevalence of about 4% after 10 years for restorations with etch&rinse systems. Statistically, the restorations that were placed without enamel etching and bonding experienced significantly more caries compared to those that were placed with enamel etching or self-etching. However, the difference between both groups was again very small (3% versus 2% after 5 years) (Fig. 1). - The increase in restorations with marginal staining was dependent on the tooth conditioning technique. When the enamel was not acid-etched with phosphoric acid and when no adhesive system was applied, the decline was rapid and after 4 years already 60% of the restorations showed marginal staining. By contrast, marginal discoloration was found in about only 10% of the restorations if the enamel was etched and in about 20% if a self-etching system was used (Fig. 2). - As for the outcome variable marginal integrity, the results were similar for restorations placed with etch&rinse and with self-etch techniques but not for restorations fabricated without enamel conditioning. For the latter group, the deterioration was not as rapid as in the case of marginal staining; after 4 years 35% of the restorations had detectable margins on average. In cross-sectional investigations, marginal caries was usually cited as the most frequent reason for replacing a restoration, irrespective of the restorative material used and irrespective of the type of cavity and location in the mouth [28 37]. There are several reasons for this discrepancy. Besides financial reasons due to the remuneration system, practitioners most often confound marginal staining with marginal caries.

4 62 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 2 Estimated median percentage of restorations across the studies and experiments without marginal staining in relation to the adhesive technique, adhesive system and observation time [22]. This holds true for both amalgam and composite restorations. Studies have shown that most of the dentists are not able to clearly identify caries that is associated with restorations. In one study, 9 dentists evaluated 228 teeth with different types of restorations [38]. In only 37% of the cases, the diagnosis of the dentists corresponded with the histological analysis of the tooth with regard to caries and there was a considerable variability between the dentists. Special training and calibration can considerably increase the inter-examiner agreement [39]. Marginal caries does significantly occur more in patients who have a high caries activity. Unfortunately, only few clinical trials have evaluated the individual caries activity of the included subjects. In a prospective 5-year clinical study on posterior resin restorations that defined caries activity of a subject as having either more than 30 restored surfaces and/or high counts of mutans streptococci all the seven restored teeth with marginal caries out of the 51 available restorations were in the group of subjects with high caries activity [40]. Several in situ and clinical studies have shown that marginal staining is no indicator for marginal caries [41 43]. The meta-analysis that was cited above showed that after 10 years the number of teeth with marginal staining was five times higher than the number of teeth with marginal caries on average (acid-etch), which supports the findings of in situ studies. Another fact that is of paramount importance is that practitioners most often judge the quality of a restoration by looking at its easily visible parts most often the occlusal surfaces of posterior fillings or the labial surfaces of anterior fillings. These surfaces, however, are the areas where marginal caries rarely occurs. A clinical trial has shown that marginal caries is about 8 times more frequent at the gingival section of proximal surfaces in Class II composite fillings than at the occlusal site [44]. This can be explained by different facts: - In the occlusal part of Class II restorations, the formation of a biofilm at the interface between the restoration and the enamel is restricted or impossible because of selfcleaning mechanisms in the mouth (saliva, tongue, etc.) and oral hygiene measures conducted at home (toothbrush, toothpaste). In contrast, the biofilm can develop relatively undisturbed, differentiate itself and offer a biological habitat for cariogenic bacteria at the proximal box of Class II restorations. Initially, after a Class II restoration has been placed, the concentration of interdental Streptococcus mutans decreases quite dramatically because of the manipulations that have been taking place. It takes about 6 9 months before the S. mutans concentration regains the baseline value and starts to increase again [45]. Differences have been found among different restorative materials. In clinical and in situ studies, a significantly larger number of S. mutans was identified in the interdental plaque which had formed on the composite surfaces, compared to the interdental plaque adjacent to the unrestored enamel [46,47] or amalgam restorations [47,48] in the mouth of the same test subject. - The quality of the dentin and enamel bond in the gingival cervical area of Class II restorations is inferior to that in other areas of the cavity, even if a good adhesive system is used [49,50]. If the filling s margin lies just above the cemento enamel junction, the operator has to deal with enamel which becomes ever thinner toward the edge and breaks easily. Furthermore, there are hardly any enamel prisms to etch at this site. Furthermore, a filling s margin below the cemento enamel junction lies in an area of dentin which contains no dentinal tubules in an area of approx. 100 m, and further toward the pulp their number is still quite low; thus, the preconditions necessary for a good bond are not met [51]. - A possible other important reason for the predilection site of marginal caries at the gingival margin of Class II restorations is the inadequate polymerization of the composite at this location, which will not be discussed further Anterior direct restorations Another meta-analysis was carried out on anterior restorations (Class III, Class IV) and provided similar results to the analysis on posterior restorations, even if considerably fewer clinical studies could be retrieved in the literature and included in the review (21 studies, 2008 restorations at baseline) [52]: - Marginal caries was also infrequent in anterior restorations and accounted for about 2.5% of replacements after a period of service of 10 years, independent of the tooth conditioning system used. - Anterior restorations placed with self-etching systems were about two times more likely to show marginal staining than those placed with the phosphoric acid enamel etch technique after 5 years (30% versus 15%).

5 d e n t a l m a t e r i a l s 2 9 ( ) Full-coverage indirect restorations The luting agent should help to prevent the restorations from being dislodged. There is an inverse relationship between the adhesive properties of the luting agent and the macromechanical retention. The lower the macromechanical retention is the higher the adhesive strength has to be to compensate for the low degree of retention. Long-term clinical studies (up to 10 years) indicate that retention loss in single crowns occurred more often in crowns and FPDs that were cemented with zinc phosphate cement than with glass ionomer cements [53,54]; in both studies the retention loss frequency was 5% for zinc phosphate cement and 0% for glass ionomer cement. A meta-analysis of metal-ceramic FPDs (n = 1307) revealed an estimated 10-year risk of 6.4% for retention loss (CI ) [55]; for all-ceramic restorations the estimated 5-year risk for retention loss was calculated to be 2.3% (CI ) [56]. However, these studies did not differentiate between zinc phosphate and glass ionomer cements or other cements. A systematic analysis in the longevity of metal-ceramic FPDs compared to zirconia-supported FPDs included also the evaluation of debonding/decementation [57]. The frequency of debonding was higher for restorations luted with zinc phosphate cement than for restorations luted with any other type of luting agents, none of which revealed any difference in performance (RelyX Unicem, Panavia, glass ionomer cements, resin-modified glass ionomer cements). These data confirm the findings of the above mentioned studies on glass ionomer and zinc phosphate cements. Consequently, none of the luting agents, except for zinc phosphate cement, has a critical effect on debonding in standard routine preparations of single crowns and FPDs. In clinical situations with no or low mechanical retention, such as preparations for Maryland bridges or endo-crowns (endodontic crowns) or in situations with reduced stump height 3 mm and/or high angle of convergence (>30 ), the adhesive properties of the luting agents are crucial for the prevention of debonding. premature replacement of restorations because of suspected marginal caries. - Most post-operative sensitivities seem to be non-persistent and mostly resolve with time. - Retention of cervical restorations depends on dentin preparation and the adhesive system. - Retention of posterior restorations depends on the macromechanical preparation design of the teeth involved as well as the long-term bonding properties of the adhesive systems. 5. Which laboratory tests provide clinically relevant information? 5.1. Requirements for laboratory tests Laboratory tests are useful for testing new operative techniques and materials before they are clinically implemented. The methods employed, however, should meet the following requirements [58]: 1. The results must be reproducible, i.e., when the same test is repeated under the same conditions and with the same materials, the same results should be obtained. 2. The parameters which influence the test results must be known. 3. The variability of the measured values must be low and within an acceptable range. The coefficient of variation, that is, the ratio of the standard deviation to the mean, should be under 20%. The coefficient of variation determines the number of specimens per group. 4. If devices are employed for the test itself and/or to measure parameters and post-testing conditions of the specimens, then these devices must be suitable for the given purpose, that is, they must be qualified. This, in turn, must be proven and documented. A device may have to be calibrated before performing the test or measurement. 4. Conclusions from clinical trials The most important conclusions to be drawn from the clinical trials are as follows: - Marginal caries most often occurs at the proximal gingival margins of restorations. It is, however, infrequent and does occur practically independently of the sealing properties of the adhesive system. Patient-related factors like high S. mutans counts and sucrose-rich diet are promoting factors for marginal caries. - Marginal staining develops on the grounds of marginal irregularities (gaps, fractures). The frequency in relation to time of service depends on the type of adhesive system. Marginal staining per se is not linked to marginal caries. However, many practitioners confound marginal staining with marginal caries. Therefore, laboratory tests can help to identify products and mechanisms that show good in vitro performance with high external validity (correlation to marginal staining) and therefore also help to reduce the If all of these requirements are met, the test method is internally valid. Because the test method is used to provide information or prognoses about the clinical suitability of the material, the results must correlate with clinical findings. If this is the case, the test is also externally valid. These requirements were described for medical devices and compiled under the title Good Laboratory Practice by regulatory authorities such as the Food & Drug Administration (FDA) in Washington or the European authorities in Brussels in the 1970s and 1990s, respectively [58,59]. These specifications apply to medical devices in general and are not defined specifically for dental materials Bond strength tests Rationale Two materials are normally bonded together by applying an adhesive between them. To test the strength of such a configuration, a straightforward method is to try to separate both materials with an adequate testing machine after having bonded them and to measure the force required to separate

6 64 d e n t a l m a t e r i a l s 2 9 ( ) them. Various industrial sectors, such as the construction, aircraft or automotive industries, use established bond tests. For many of the different materials ASTM standard tests are published History If PubMed is searched, the first article on bond strength tests for dental materials was published in 1965 by Bowen [60]. In this publication, a tensile bond strength test with a restricted bonding area was elaborated and described. Since then, 4960 articles are listed in PubMed when searching for bond strength and dental and 2695 articles for bond strength and dentin and 1545 articles with the search terms bond strength and enamel (search period , search month April 2012). These articles advocate various test setups, such as the shear bond, microtensile, microshear, push-out and the fracture toughness test (see below). There is only a small degree of standardization, which explains the variation in results. Of the 4960 articles, there are only 12 relevant reviews which critically evaluate the different bond strength tests in terms of their strengths and weaknesses [61 72]. All the other thousands of studies on bond strength testing in dentistry investigated various modifications of test setups and substrates. Additionally, most of them focused on testing specific materials. The first publications on the correlation between bond strength tests and clinical trials appeared only in 2010 and 2011 (see below) Scientific evidence of bond strength tests A systematic multi-center analysis on the factors influencing the various bond strength tests has not yet been performed. However, several factors that affect the results and are crucial for the implementation of laboratory bond strength tests can be identified on the basis of reviews on bond strength tests as well as on the basis of 40 years of bond strength testing published in numerous papers and presented at dental congresses: Biological substrate: Studies indicate that bovine teeth is a good substitute for human teeth [73]. This has been proven for the microtensile test [74], the tensile test [75] and the shear bond test [76]. However, only coronal bovine dentin should be used and not root dentin. Cutting of teeth: Cutting should be performed with a qualified sawing equipment at low speed without producing cracks and fractures. Regular maintenance and calibration of the sawing equipment is a prerequisite. Cutting level: The gingiva-coronal height (human molars) or labio-oral depth (bovine incisors) should be similar for all teeth. Bond strength values are higher in superficial dentin than in deep dentin [77]. Non-carious versus carious dentin: Bond strength values are higher in non-carious dentin than in carious dentin [78]. Eroded versus non-eroded dentin: Bond strength to artificially eroded dentin has been proven to be significantly lower than to sound dentin [79]. Regional differences within the same tooth: By means of microtensile tests, significantly lower bond strength values were found at the gingival floor of Class II restorations than at the axial or occlusal wall [80,81]. This may be explained by the histological conditions which are less favorable to establish an adequate bond at this site (see above). Number of specimens: As the coefficient of variation is between 20% and 40% depending on the adhesive system to be tested and on the test method [69] it is necessary to use between 10 and 40 specimens per group provided that a difference of 25% in bond strength value is to be considered statistically significant. For Weibull statistics, at least 15 specimens are needed. Preparation of substrate: It has to be ensured that the embedded substrate is polished virtually 100% parallel to the level of the testing device and 100% perpendicular to the composite cylinder. Direction of force and size of bonded area: Bonded composite cylinders can be sheared (shear bond) or torn (tensile bond) away or pushed out of prepared cavities. The disadvantage of shear bond tests is that the composite cylinder is compressed while inadequate tensile forces are created in the tooth substrate due to the bending moment, as FEM calculations have shown [82,83]. This explains why many cohesive failures occur with shear bond tests. In contrast, no such forces occur with tensile bond tests if performed correctly. In 1994 the tensile test with specimens of a bonded area of 3 mm was modified by cutting small sticks of 0.7 or 1.0 mm from a single tooth [84]. Small bonded areas show higher values than large bonded areas [85]. The advantage of these microtensile tests is that fewer extracted teeth are necessary and that regional differences in the tooth can be taken into account. However, the intra-tooth variability is greater than the inter-tooth variability [86]. In 1998 another shear test with smaller specimens was proposed [85]. In 1993 the so-called fracture toughness test was introduced to characterize the fracture resistance of the adhesive interface between tooth substance and resin by paying attention to crack initiation and propagation [87]. Studies on the ranking of different adhesive systems in relation to the bond strength test carried out on them yielded conflicting results [69,88 91]. Restricted adhesion area: The adhesive area should be restricted to the area of the composite cylinder. Excess of adhesive creates higher bond strength than restricted areas and creates additional stress in the substrate [92]. Type of composite: In shear bond tests, the modulus of elasticity has an effect on the test result. The higher the modulus of elasticity, the higher is the bond value [93]. Therefore, the same composite material should always be used, at least for shear bond tests. Pre-test failures: If the bonded specimen falls (breaks) apart before testing, these specimens should receive a value 0. Test equipment: The universal testing machine has to be qualified for the intended use. The machine has to be calibrated on a regular basis by a qualified body [58]. Time interval between bonding and testing: Immediate bond tests do not give clinically meaningful results nor do they after 24 h. They should only be regarded as baseline values to interpret the decrease in bond strength after stressing and prolonged storage (see below). Thermocycling: Short-term thermocycling (500, 5 C/55 C) does not significantly alter the ultimate bond strength

7 d e n t a l m a t e r i a l s 2 9 ( ) values [93]. But also prolonged thermocycling (up to 10,000 cycles), has little effect on both the microtensile and macroshear bond tests [72]. Systematic research on the influence of thermal stress on the bond strength has not been carried out to date. Therefore, it remains unclear whether the decrease in bond strength was caused only by exposure to water for a prolonged time or indeed by thermal stress or by both [94]. There is no evidence that thermal stress alone leads to a clinically relevant deterioration or debonding of the adhesive interface. Storage of bonded specimens in water: The storage of specimens in water (37 C) results in a decrease in the bond strength compared with the baseline value depending on the adhesive system and the test [72,93]. Water can lead to a hydrophilic degradation of the dentin/enamel-composite interface. Statistics: When microtensile or microshear tests are applied, sticks originating from the same tooth are not statistically independent of one another. If this dependency is not taken into account, it is entirely possible that an erroneous product ranking may result [95]. Variability: Study results from different study groups on bond strength tests are not comparable [72,93,96,97] and even within the same study group great variability of the results have been reported. The intra-institute variability is about 20 40% (coefficient of variation) whereas the interinstitute variability is between 30% and 50%, with slight differences between the various test methods [69]. The microtensile bond strength of specimens from Class II restorations in vivo is lower than that of restorations in vitro [98] Standards on bond strength tests In 2012 an ISO standard on bond tests will be published (No : Dentistry Adhesion Notched-edge shear bond strength test). The test uses the Ultradent jig, which bears a notch which surrounds half of the specimen [99]. The adhesive is applied unrestrictedly on either bovine or human dentin. The composite cylinder with a diameter of 2.38 mm is sheared off after 24 h storage of specimens in water. A round-robin test has been performed amongst several dental manufacturers with three adhesive systems. The results of the round-robin test, however, have not been published yet. This standard will not replace the ISO Technical Specification (TS) with the title Testing the adhesion to tooth structure (No , first edition 1994, second edition 2003). The specification will, however, be revised, deleting e.g. the part of shear bond test. This specification represents still a useful approach to standardize some important variables for tensile bond strength [100]. Unfortunately, few research workers follow these recommendations [93]. Some important parameters listed in the ISO are: - Defined and limited bonding area - 6-month water storage before testing - Weibull statistics with a minimum of 15 specimens per group - Tensile bond strength: 90 angle alignment of the tensile forces acting on the specimen - If the coefficient of variation is above 50%, a thorough inspection of each process is recommended. Because no internationally recognized standardized test protocol for the testing of adhesive systems is yet available, completely different bond strength values can be found and published for the same product, depending on the test institute. A systematic review published in 2008 showed that the shear bond test was the most common type of test (used by 46% of the studies reviewed), human dentin was used in 77% of the investigations, and 24-h post-placement bond strength testing was the predominant time of specimen testing (67%) [66]. The dimensions of the contact surface area or the specimen diameter was stated in only 65% of the papers reviewed Validation of bond strength tests Few attempts have been made to validate the bond strength tests applied in dentistry. For the microtensile test several critical factors that influence the result have been identified, such as the diameter of the stick, type of jig, and trimming versus non trimming [101]. For the shear bond test, no systematic analysis of influencing factors other than the universally applicable ones (thermocycling, restricted bond area, operator variability) have been published. One study indicated that the crosshead speed influences the test result [102]. As far as the comparability of the different test methods is concerned, Scherrer et al. published a review paper on the correlation between different bond strength test methods [69]. The authors selected the following tests: shear, microshear, tensile and microtensile. The review revealed that a large variability for the same adhesive system evaluated with the same bond strength method was present not only at different test institutes (inter-institute variability) but also at the same test institute (intra-institute variability). The variability was similar for each test method. Then, the authors pooled the bond strength results for the individual adhesive system and the test method. The ranking between the six adhesive systems varied depending upon the test method chosen. However, this result must be interpreted with caution because the study pooled values which derived from the same test method but different test parameters. Only the data of adhesive systems that are tested with the same test parameters. This approach was applied in the following comparative analysis: In Fig. 3, the mean Log-transformed bond strength results after 24 h of water storage of 12 different dentin adhesive systems tested with the microtensile, tensile and shear bond strength test are shown in a scatterplot together with an interpolation curve for each test method. The studies were retrieved from PubMed with a search for specific material combinations (search period , search month April 2012). Only studies that applied the same test protocol (human teeth, midcoronal dentin, same diameter of test specimen according to test method, 24 h storage in water) were included; the included studies (n = 24) comprised at least 4 different materials. It can be easily noticed that the absolute values differ widely for the same material and the same test method, highlighting the influence of the test institute. In most cases, microtensile tests yield higher values than the other two tests. If the test results are pooled in relation to the test method, none of

8 66 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 3 Log-transformed bond strength values (MPa) of 12 adhesive systems tested with 3 bond strength tests at 24 test institutes. At least 4 adhesive systems were tested at each institute (references of studies are available on request). Etch&rinse systems 3 steps: 1 = OptiBond FL, 2 = Scotchbond Multipurpose. Etch&rinse systems 2 steps: 3 = Excite, 4 = One Step, 5 = Prime&Bond NT, 6 = Single Bond. Self-etch 2 steps: 7 = Clearfil SE, 8 = AdheSE. Self-etch 1 step: 9 = ibond, 10 = Xeno III, 11 = Adper Prompt, 12 = G-Bond. the test methods is able to clearly differentiate between the different materials (see interpolation line in Fig. 3). It seems, however, that 1-step self-etch systems are associated with lower microtensile strength values than the other three types of adhesive groups (self-etch 2 steps, etch&rinse 2 and 3 steps). No such clear differentiation can be observed with the tensile bond strength and shear bond strength test. In another approach, the studies were further reduced to those that evaluated the same adhesive systems. In order to end up with at least 3 studies using a microtensile test, it was possible to include only 3 adhesive systems. The absolute figures differ widely, as can be seen in Fig. 4, but not the relative rankings between the materials except for one shear bond test (SB 2). The differences between the materials are more pronounced with the microtensile test method than with the macrotensile and macroshear bond strength test. These results confirm the results of a meta-analytic review of parameters involved in dentin bonding [72]. Other important findings of the review were as follows: - The microtensile test discriminates more effectively between different adhesive systems than the macroshear bond test. - Thermocycling has a negligible influence on the decrease of bond strength both for the microtensile and macroshear test. - Long-term storage in water significantly decreases the bond strength if tested with the microtensile method. However, if tested with the macroshear test, no significant decrease was detected. - The following parameters significantly affected the bond strength results (in decreasing order): research group/institute Fig. 4 (Top) Mean bond strength test results (MPa) of three adhesive systems tested after 24 h storage in water, using three test methods 3 studies with microtensile bond strength test (MT), 2 with shear bond test (SB) and one with tensile bond test (TB). The data of the 3 systems were published in the same article and for MT and SB the test parameters were the same. (Bottom) The same test results presented with relative ranks (1 3). MT 1 [184], MT 2 [185], MT 3 [186], SB 1 [187], SB 2 [188], TB [189]. adhesive system adhesive class (etch&rinse 3 step/2 step, self-etch 2 step/1 step) substrate preparation (bur-cut or SiC-cut) substrate origin flexural modulus of composite Correlation with clinical findings In most cavity and abutment preparation designs, the largest area exposed after preparation is dentin. Therefore, bond strength on dentin should have its clinical correlate with retention. The sealing of the dentinal tubules is another important function of adhesive systems. The bonding to enamel is less important for the retention of the restoration but plays a more essential role in reducing gaps at the restoration margins and in preventing subsequent marginal staining. In posterior restorations about 90% or even 100% of the restoration margin is in enamel. Only after 30 years of bond strength testing, efforts were made to relate the results of these tests to clinical findings. Even now, there are only a few publications which correlate the in vitro bond strength data with the clinical outcome of the tested adhesive systems. The clinical model most often used to test the effectiveness of adhesive

9 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 6 Scatterplot and regression curves of mean pooled bond strength data of six adhesive systems [69] in relation to the estimated pooled clinical data after 2 years for the same adhesive systems [190]. Fig. 5 Scatterplot of retention versus microtensile data for 15 adhesive systems, together with Spearman correlation rho and associated p-value [103]. The numbers refer to different adhesive systems. Estimates of the adhesive effects in this model were used to summarize the clinical performance of each adhesive between 12 and 36 months. They were inverted and centered in such a manner that a positive value corresponds to a performance above average, and a negative value to a performance below average (a zero value represents average performance). systems is the restoration of non-carious cervical defects. Such defects are especially suitable to test adhesive systems due to: (1) practically no macro-mechanical retention is present, (2) straightforward clinical placement of the restoration and evaluation of debonding, reducing operator and evaluator variability, and (3) high prevalence, which makes patient selection simple and enables properly designed studies. Only three publications on this topic have been found. In one of these studies, the microtensile bond strength data of 15 adhesive/restorative systems placed by the same operator were correlated with the clinical studies of non-carious cervical Class V restorations. No correlation was found between the retention rate of cervical restorations after 3 years and the microtensile test results after 8 h or 6 months of water storage (Fig. 5) [103]. There was, however, a very moderate correlation between marginal staining and bond strength values after 6 months of water storage. A comprehensive database of microtensile bond strength data and an equally comprehensive database on the retention rates of restorations placed in non-carious wedge-shaped defects at the same test institute (University of Leuven) found a moderate correlation for laboratory specimens that were submitted to artificial aging and the correlation was higher for 5-year data than for 2-year data [104]. Another attempt was made to correlate the bond strength data with the retention of cervical restorations. In 2010 Scherrer et al. published data of laboratory studies on six dentin adhesive systems, available in the literature, and four laboratory methods (macroshear, microshear, macrotensile and microtensile bond strength test) [69]. They pooled the data across the different studies in relation to the adhesive system and the bond strength test and calculated mean values and standard deviations. These data were correlated to estimated pooled 2-year retention rates of Class V restorations using the same adhesive systems and retrieved from the databank of the meta-analysis on cervical restorations [19]. The results of the regression analysis for the pooled data revealed that only the macrotensile (adj. R 2 = 0.86) and microtensile tests (adj. R 2 = 0.64), but not the shear and the microshear tests, correlated more accurately with the clinical findings (Fig. 6). This finding suggests two points: - Pooled data across different institutes may correlate more accurately with retention loss of cervical fillings than individual data from one test institute. The explanation may be that these pooled data characterize the variability and efficacy of a certain bonding system more appropriately. - Tensile tests correlate with the retention loss of cervical restorations, whereas the shear tests do not. Therefore, bond strength tests should be carried out by different operators and/or research institutes to determine the reliability and technique sensitivity of the material under investigation. The correlation of bond strength test values with the retention rate of cervical fillings might have been better if the tensile test had been performed with artificially eroded dentin rather than with non-eroded dentin. Eroded dentin reflects the clinical situation of non-carious cervical restorations more accurately, as in most clinical cases erosion is the cause for the cervical defect and most clinicians do not prepare the

10 68 d e n t a l m a t e r i a l s 2 9 ( ) dentin, but rather only clean it with pumice or prophylaxis paste. Adhesive systems perform significantly worse in eroded dentin than in non-eroded dentin, as an in vitro study has shown [79]. This finding is supported by clinical evidence as retention of fillings placed in unprepared cervical defects was significantly lower than those placed in prepared cervical defects as a meta-analysis has revealed [19]. In this analysis the percentage of studies that included a preparation of dentin was about 50%. Most of the bond-strength tests were carried out on dentin and to a smaller extent on enamel specimens. A good bond to enamel is important to reduce the risk of marginal gap formation and staining. However, studies investigating the correlation of bond strength values of cut enamel and the occurrence of marginal staining in posterior or anterior restorations have not been found in the literature. If the bond strength tests are applied to fissure sealants, the results are misleading when compared to clinical results. For example, the bond strength tests showed an equal or even higher bond strength for the self-etching Primer Adper Prompt L-Pop in uncut enamel compared to the phosphoric acid etching method. This was true for both shear bond tests [ ] and microtensile tests [108]. Light-polymerizable fissure sealants with phosphoric acid conditioning of the enamel produce a retention rate of about 80% after 2, 3 and 5 years, as a meta-analysis has proven [109]. For Adper Prompt L-Pop selfetching primer/sealants, however, the clinical trials showed poor retention rates. In one clinical study, the retention rate of the self-etching primer Prompt L-Pop/Clinpro was only 44% after 1 year [110] and in a school-based programme sealants placed with Prompt-L-Pop were 6 times more likely to show retention loss after 1 year than sealants placed with phosphoric acid conditioning of the enamel [111] Assessment of bond strength tests The value of bond strength tests to make a prognosis on the clinical performance of an adhesive system or dental material is limited. A bond strength test helps developers of new products to select between different material variants. The test should be standardized as much as possible and well documented. The universal testing machine should be qualified for the intended purpose. To assess the variability of the adhesive efficacy of adhesive systems, different operators with different levels of experience in adhesive testing should perform the test in the same institute or in different institutes. Pooling of results enhances significantly the correlation with the clinical performance. Shear bond tests (macro and micro) are inadequate for the evaluation of the bond strength to dental hard tissues as clinically unrealistic stress is produced within the reaction zone. Tensile tests avoid such internal stress formation and their results correlate more accurately with the retention rate of cervical restorations. Microtensile tests are laborious and technique-sensitive and offer no advantage over macrotensile tests. For carrying out tensile tests, the ISO TS should be followed [100]. Testing the fracture toughness of the bonding interface can be regarded as a supplementary test to the macrotensile test, because it provides additional information. As it is techniquesensitive, it is not a routine test and the correlation to the retention loss of cervical restorations has not yet been established Simulation of retention loss in extracted teeth As bond strength tests yield very variable results, it may be reasonable to simulate retention loss of cervical restorations in extracted teeth. A laboratory trial attempted to simulate restoration loss in non-retentive cervical cavities. For this purpose, premolars with wedge-shaped defects were selected (n = 12) and restored with an adhesive system (Prompt L-Pop). Prompt L-Pop and his successor Adper Prompt L-Pop have a low ph and a strong etching effect on dentin which may be together with other peculiarities responsible for low bond strength values on dentin [112]. Restorations placed with this adhesive system are characterized by a relatively high retention loss over a period of 2 years in clinical trials. In the laboratory, the restored teeth were centrically loaded 1.2 million times with simultaneous thermocycling, followed by another 1.2 million cycles of eccentric loading on the lingual cusp of the premolars to produce tensile stress on the buccal side of the tooth where the restoration was located [113]. The result was surprising. None of the fillings was lost after 2.4 million thermo-mechanical cycles. In a second approach, 11 different adhesive systems (self-etch and etch&rinse) and one glass-ionomer cement were applied in cervical restorations of extracted premolars (n = 12 per group) and submitted to a rigid programme of artificial aging including storing the teeth for 3 6 months in water, and subjecting them to various thermocycling and simulated mastication procedures [114]. None of the adhesive systems not even with the glass-ionomer cement showed a loss of restoration during the simulation trial. Restoration loss only occurred when the conditioner (polyacrylic acid) was omitted for the fillings with glass-ionomer cement or the phosphoric acid etching of dentin and enamel for the fillings with an etch&rinse system. In sum, retention loss could not be simulated in fillings of extracted teeth when the materials were applied according to the recommendations of the manufacturer. However, self-adhering composite materials that are applied without separate adhesive system (e.g. Vertise Flow) may show retention loss in cervical fillings of extracted teeth. But this has to be proven Microscopic evaluation of interface The interface between composite and dentin/enamel can be evaluated with a microscope either by cutting the specimen and evaluating the interface with a scanning electron microscope (SEM), a fluorescence microscope or a confocal laser scanning microscope (CLSM) after marking the adhesive with a fluorescent dye [115]. The depth to which the adhesive system penetrates the dental hard tissue and the quality and thickness of the hybrid layer can be examined under the microscope. However, because the fluorescent dye does not chemically react with the adhesive system, the dye molecules can penetrate further into the dentin tubules than the adhesive system itself, thus distorting the results [116,117]. Another method is to completely dissolve the tooth in hydrochloric acid and to measure thereafter the length of the

11 d e n t a l m a t e r i a l s 2 9 ( ) resin tags and the thickness of the hybrid layer with SEM. Adhesive systems which condition the dentin and/or enamel with phosphoric acid demonstrate both better microretention than self-etching systems and thicker hybrid layers. The thickness of the hybrid layer, however, is not correlated with the bond strength, the marginal integrity or the clinical performance of the adhesive system. A qualitative microscopic analysis of the adhesive interface is a valuable tool to get an insight into the bonding mechanism of a dental adhesive. For instance, a study using silver nitrate penetration and TEM impressively showed where the paths of water infiltration occur at the adhesive interface [118]. However, such investigations do not allow to make a prognosis on the clinical performance of a given material Evaluation of the marginal seal in vitro Tracer penetration test Rationale. The hypothesis is that the penetration of different markers along the interface between the restoration and dental hard tissues in extracted teeth is a surrogate variable for the in vivo penetration of bacteria, fluids and other liquids, which may provoke hypersensitivity, pulpitis, marginal staining and/or marginal caries. Normally, restorations (Class V, Class II) are placed in extracted teeth, which are then subjected to a variety of different agents, such as organic dyes (fuchsine, methylene blue, rhodamine, erythrosine, eosin, etc.), silver nitrate, radioactive markers ( 45 CaCl 2 ) or bacteria. Alternatively, hydrodynamics may be used to measure the movement of liquids such as saline. The application of dyes represents the most commonly used method because of its simplicity. Penetration of dyes/tracers involves the cutting of teeth with sawing equipments History. According to PubMed, the first article on tracer penetration along the restorative hard tissue interface was published by Going et al. in 1960 [119]. This study used radioactive tracers. Twelve years later, in 1972, the first author of this study summarized the knowledge available on tracer penetration/microleakage around dental restorations and concluded that more in vivo studies were necessary to validate the large amount of data collected in vitro. However, it was only 26 years later that efforts were made to correlate tracer penetration data to clinical findings (see below). In the meantime, several reviews on microleakage studies had been published [ ]; many of them complaining on the nonstandardization of the tests. The ISO standard on testing the adhesion to tooth structure describes a microleakage test in the cavities of third molars (mid-part of the buccal surface) with a diameter of 3 mm and a depth of at least 1 mm and a sample size of at least 10 [100]. No specific dye tracer is recommended. An ordinal scale is used for the measurement of the tracer depth. No values for the acceptance of a given material had been postulated Scientific evidence on dye penetration studies. Type of substrate: Dye penetrations tests are carried out in both bovine and human teeth. However, a systematic analysis on the literature available was inconclusive on whether the study results achieved in bovine teeth can be compared to those achieved in human teeth [124]. Type of dye tracer: The type of dye plays a negligible role in dye penetration studies, except for methylene blue [125]. The chemical solution of methylene blue is not stable at room temperature and under exposure to ambient light. Furthermore, hydroxyl ions can easily be reduced to leucomethylene blue, which is colorless. In the presence of strong acids, the dye is not stable either. Number of cuts: The more cuts are performed, the better is the level of agreement between test centers that have applied the same methodologies and materials [126]. Measurement device: The light microscope used for the evaluation of dye penetration should be qualified for this purpose as should be the software program designated to measure the length of the penetration. Results according to substrate: Practically all the laboratory studies have shown that within the same tooth and the same restoration dye penetration is larger in sites where the margin is located in the dentin compared to sites where the margin is located in the enamel, independent of the adhesive system investigated [125,127]. Artificial aging: Unsystematic research on the influence of thermocycling and occlusal loading and the combination of both these stress factors has generated conflicting results [94,122,123]. There is no evidence that thermal stress alone causes clinically relevant deterioration of the adhesive interface. Comparability of results between test institutes: A systematic review on dye penetration studies for restorative dental materials concluded that a comparison of study results was not possible due to great variability in methodologies and parameters [123]. The studies differ with regard to the substrate used (human/bovine), the dimensions of the prepared cavity, storage period until testing with dye tracer, type of stress and number of stress cycles (thermocycling, load cycling or a combination of both), the type and concentration of dye tracer, the length of immersion in dye liquid, the type of analysis of the dye penetration (cutting teeth), number of cuts and the evaluation method (metrically, score system). Variability of results: The variability of test results is very high with a coefficient of variation up to 50% and more. In order to prove a statistical significant difference between test groups, many specimens per group are needed. One of the studies on dye penetration in Class II restorations revealed that a sample size of 12 would discriminate in the range a dye penetration of 1.0 mm at enamel margins and a dye penetration of 2.2 mm at dentinal margins [125]. Acceptance level: A validated acceptance level does not exist [ ] Correlation with other laboratory test methods. Dye penetration and bond strength. A systematic review on the correlation of bond strength tests and dye/tracer penetration did not reveal any significant correlation [131]. Dye penetration and quantitative marginal analysis with SEM. A correlation between dye penetration and the occurrence of marginal gaps is only partially proven, if at all [125].

12 70 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 7 Correlation between penetration index in vitro and percentage of clinical restorations with loss of retention (left) or marginal staining (right) using the same adhesive system with Spearman correlation coefficient rho. The numbers in these diagrams refer to the number of the adhesive systems (n = 18) (unpublished data by the author). Penetration index: 0 = no penetration, 1 = penetration up to one-third of the cavity depth, 2 = penetration greater between one-third and two-third of the cavity depth, 3 = penetration greater than two-third of the cavity depth, 4 = penetration extending to the axial wall of the cavity Correlation with clinical findings. In vivo and in situ studies have shown that microleakage as such (alone) or the existence of marginal gaps does not correlate with the occurrence of hypersensitivity or the formation of marginal caries [27,43,132,133]. However, no systematic study has been conducted so far to correlate dye tracer or microleakage data to clinical findings on hypersensitivity, retention, marginal staining or marginal caries. It is not possible to extract data from the literature, because the methodologies of the dye penetration tests vary wildly from one study to another, thus making it impossible to correlate standardized laboratory data to clinical data. However, the author found microleakage data from the Clinical Research Associates test institute in Provo/USA (now: G Christensens Clinicians Report). In this study, cervical fillings were placed in extracted teeth using 18 different adhesive systems and the dye penetration was measured on an ordinal scale (published on an internet site in 2002, which is no longer available). The statistical analysis of these data in relation to the clinical results of Class V fillings revealed no correlation whatsoever for the variables of retention and marginal staining (Fig. 7) Assessment of microleakage studies. Several reasons allow us to reject this laboratory method as a procedure that should enable to predict the clinical performance of direct and indirect restorations: - Vast variability of test results - Validation of method not possible - No correlation with clinical findings Presumably, as the dyes used in the tests consist of very small particles (<1 nm) they penetrate any marginal discrepancy that may be present. Obviously, such a penetration pattern and penetration depth is clinically irrelevant. The only indication left for this method may be the assessment of the effectiveness of pit & fissure sealants, especially if it is taken into consideration that some self-etching primers or sealants are promoted for this indication. Although a systematic evaluation of dye penetration tests for this indication or a systematic correlation with clinical findings has not been carried out to date, the dye penetration test seems to be the only method to evaluate material variants in the laboratory and to gain some safety before clinical trials begin. Self-etching primers have to compete with the gold standard, i.e. etching the uncut enamel with phosphoric acid prior to the application of a light-cured unfilled or flowable resin. As mentioned above, retention rate of light-cured fissure sealants applied after phosphoric acid conditioning is about 80% after 2, 3 and 5 years respectively [109]. In contrast, 56% of the sealants placed with the self-etching primer Adper Prompt L-Pop were partially or completely lost after 1 year [110]. The performance of this self-etching system is unacceptable compared to phosphoric acid conditioning and the poor results were confirmed by several studies which applied microleakage/dye penetration tests of sealants in extracted teeth [ ] Quantitative marginal analysis with a microscope Rationale. The hypothesis is that gaps occurring at the interface is a surrogate variable for the penetration of bacteria, fluids and other liquids, which may provoke hypersensitivity, pulpitis, marginal staining, debonding with retention loss and/or marginal caries. The rationale is that the irregular

13 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 8 Cylindrical filling in bovine enamel; (left) specimen, (middle) SEM picture of irregular margin in enamel (300 ), (right) imperfect margin of cylindrical restoration in enamel resulting from interaction between water and impression material (300 ). margins or gaps at the outline between the restoration and the dental hard tissue allow researcher to predict the clinical performance of adhesive systems and/or material combinations or modifications of the operational procedure (cavity design, layering technique, flowable composite under high-viscous material, bulk technique, polymerization protocol, etc.). The marginal integrity is evaluated with different types of microscopes (Fig. 8). The advantage of this method is that it allows the investigator to look at the same longitudinal section of the margin after storage and after stressing the specimens. The disadvantage of this method is that the interface can only be examined two-dimensionally History. Although already proposed by Going in 1972 in his review article on microleakage tests, the technique with replicas and the subsequent quantification of the marginal characteristics by means of SEM was first described by Roulet in 1978 [138]. In this study, cylindrical fillings were placed in bovine enamel. In 1989, a computer-aided quantitative marginal analysis which can also be applied to replicas of clinical trials was presented by the same author [139]. Different types of cavities can be used for this purpose. Besides cylindrical cavities of various diameters (1 4 mm), Class V and Class II cavities both in bovine and human teeth have also been proposed. The ISO standard on testing the adhesion to the tooth structure describes a gap measurement test in cavities of third molars (mid-part of the buccal surface) with 3 mm diameter and at least 1.5 mm depth and a sample size of at least 10 [100]. Long-term water storage is listed as one of the options. Gaps are measured with a stereo microscope and an appropriate measuring device Scientific evidence on the evaluation of restorative margins. Type of substrate: No systematic study has been found on the topic of substrate influence (bovine/human) on marginal adaptation. Type of cavity: The type of cavity (cylindrical, Class V, Class I, Class II) has an important effect on marginal integrity. Therefore, the results of the marginal integrity of different cavity configurations are not necessarily comparable with each other. In a study, for instance, the marginal integrity measured in the dentin of cylindrical cavities did not reflect the marginal integrity measured in the cervical dentin of Class II restorations using the same materials [131]. Likewise, the dentinal/enamel marginal integrity of Class V restorations did not reflect the enamel marginal integrity of Class I/II restorations using the same materials [125, ]. Type of composite: The type of composite does have a significant effect on gap formation. A systematic evaluation found that shrinkage, shrinkage stress and flowability had an effect on gap formation, but not the modulus of elasticity of the composite [143]. Measurement equipment: The light microscope or scanning electron microscope used for the evaluation of marginal integrity should be qualified for this purpose, and so should the software program designated to measure the length of the margins under investigation. Results according to dentin/enamel: As with the data on microleakage, marginal integrity at the dentinal margin is consistently worse than that at the enamel margin both in Class II and Class V restorations when etch&rinse systems are used [ ]. When self-etching systems are evaluated, the result may be vice versa, depending on the enamel etching capability of the system [144]. Artificial aging: Unsystematic research about the influence of thermocycling, occlusal loading and the combination of both these stress factors has generated conflicting results [94]. There is no evidence that thermal stress alone causes clinically relevant deterioration of the adhesive interface. There are indications that mechanical loading deteriorates the marginal integrity (with or without simultaneous thermocycling) to a larger extent than thermocycling alone [ ]. Measurement of gaps/margins: The occurrence of gaps may be measured by various methods. Either the entire margin is evaluated by SEM or a light microscope (with or without dye for better visibility of the gap under the light microscope) and the percentage of continuous or gap-free margin in relation to the entire margin is calculated [ ], or the maximum gap is measured [148]. In another method, the width of the contraction gap is measured at eight points every 45 along the cavity margins, presenting the results as percentage of the gap width to the cavity diameter [149]. Still another method measures both the length of margins with gaps and the maximum gap width and then converts the results into a formula called a marginal index MI

14 72 d e n t a l m a t e r i a l s 2 9 ( ) [150]. One laboratory study used fluorescence microscopy for the evaluation of the marginal integrity of cervical fillings. It improves the detection of non-bonded areas beneath the filling because a special filter covering ultraviolet and blue light enhances the optical effect of entrapped air in marginal gaps and renders it visible [151]. However, also white lines and/or white areas indicate gaps and nonbonded areas. There was a close relationship between white lines seen in the stereo microscope and gaps in the SEM analysis. To reduce operator-related factors like bias or lack of experience and to reduce operator time, an automated system with an optical sensor and the corresponding software was developed for the detection of gaps around cylindrical cavities [152]. This system takes a profile of the surface by scanning the measuring point radially from the center of the restoration over the restoration margins. In a study, the results of the quantitative marginal analysis by means of the optical sensor were comparable to those obtained by SEM and an experienced evaluator. The method additionally allows the gap width and gap depth to be automatically measured, however, the latter two were less discriminatory between different adhesive systems compared to the percentage of gap-free profiles [153]. The system has been successfully applied to examine the margins in dentin. With margins in the enamel, however, it overestimates the marginal integrity, as small gaps and fractures that frequently occur at enamel margins are not measured by the sensor. With self-etch adhesive systems that contain water, it may happen that gaps are masked on replicas due to the interaction of water with the impression material (Fig. 8c). A more realistic approach for assessing the potential of adhesive systems and composite materials at a laboratory level before clinical use could be to evaluate the marginal seal of restorations placed in extracted teeth in the same way as the clinician does, namely with magnifying glasses and sharp explorers. By doing so, the same restorations can be evaluated at different intervals (after prolonged storage in water, after mechanical loading, etc.) and the possible deterioration can be observed. It is very likely that the differences between the materials or operational techniques observed on the microscopic level are no longer detectable on the macroscopic level during an evaluation of the restorations with an explorer and magnifying glasses. Furthermore, the results obtained in the process may correspond more closely with the clinical outcome. This approach is less timeconsuming and does not depend on expensive analytical equipment. To compare SEM evaluations with clinically simulated evaluations, the following brief study was carried out. Three-surface Class II cavities were prepared in lower first or second molars with the entire margin in the enamel (coronalapical depth: 5 mm, bucco-oral width: 80% of intercuspal distance, 8 restorations per group). In the group of the self-etch adhesive system beveling of the margin was not performed while for the etch&rinse system the margins were beveled in one group and not beveled in another group. After applying the adhesive systems (etch&rinse and self-etch) according to the manufacturer s instructions and after a single fine-particle hybrid composite material was inserted in three layers and cured, the excess material was removed and the restoration polished 1 day after placement with flexible disks. The teeth were then thermocycled for 10,000 times. Replicas for SEM analysis (200 ) were fabricated using epoxy resin and evaluated by one examiner. Another examiner evaluated the teeth with an explorer and loupes (2.5 ). The marginal defects were semi-quantitatively assessed according to the SQUACE system, which uses sketches of Class V or Class II restorations to quantify marginal defects [154]. Although the results were not directly comparable between the individual teeth, the mean values of the different groups showed a similar picture (Fig. 9). The mean frequency of marginal irregularities was compared for both evaluation methods. The values obtained with loupes and explorers were consistently higher than those from the SEM evaluation. The most striking difference was that the effect of margin beveling on the marginal integrity was only visible with SEM but not with loupes: marginal integrity of a etch&rinse system without enamel beveling was significantly worse compared to enamel beveling, as seen with SEM. However, the examination with loupes did not reveal any difference. This result is in line with clinical studies [22,155]. Reproducibility of microscopic analysis: As the microscopic analysis is performed subjectively by human operators, a certain variability is unavoidable. The inter-operator variability is greater than the intra-operator variability. When the same calibrated operator evaluated the same specimens twice, the difference in the results was in the range of 2 8% [139], whereas when two calibrated operators evaluated the same specimens, the difference was in the range of 10 20% [156]. Therefore, specimens should always be examined by the same operator. Variability: The variability can be very different from test center to test center. Based on the data of an in vitro/in vivo comparison of two test centers that evaluated the marginal integrity of cervical restorations of extracted teeth, the median coefficient of variation was 10% (range 2 94%) at dentinal margins in test center I (36 adhesive systems) and 48% (range 0 200%) in test center II (52 adhesive systems) [131]. For margins in enamel, the values were 4% (range 2 14%) in test center I and 17% (range 0 124%) in test center II. For both centers the values after stressing (thermocycling or mechanical loading) were used. If 8 specimens are used, the method of test center I can discriminate on the level of 6% for enamel margins and 14% for dentinal margins, while these values are 24% and 66% respectively for test center II. Also high numbers of specimens are needed to discriminate between different luting agents used for three-surface ceramic inlays placed in extracted teeth [157] Correlation with other laboratory test methods. Marginal integrity and bond strength. A systematic review on the correlation of bond strength tests and marginal integrity did not reveal any significant correlation [131] Correlation with clinical findings Clinical relevance of marginal gaps. Before trying to correlate laboratory results with the results from clinical trials, it is necessary to collect the evidence that is available on the clinical relevance of marginal gaps alone. Gaps seen in laboratory studies can be regarded as a surrogate parameter as

15 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 9 Mean percentage of continuous margin and standard deviation of mod-class II adhesive resin restorations made in extracted teeth in relation to the applied adhesive system; (left) evaluation by operator 1 with SEM (200 ); (right) evaluation of the same restorations by operator 2 with loupes (2.5 ) and explorer using the SQUACE method (unpublished data by the author). Fig. 10 SEM pictures of the occlusal margin of a composite restoration 6 months after placement. The composite material was placed in one layer (bulk) (left) occlusal overview (10 ), (right) detail (50 ). marginal discrepancies may promote post-operative sensitivity, marginal staining, retention loss and eventually marginal caries. If one had clinical studies that included the taking of impressions and the fabrication of replicas to look at the margins the same way as the researcher is looking at them in the laboratory with replicas of fillings in extracted teeth, then one would be able to evaluate whether marginal gaps or other discrepancies are predictive for any of the above-mentioned clinical phenomena that occur years later (Fig. 10). The fabrication of accurate replicas by means of intraoral impressions is a challenge. Cleaning the teeth with pumice or cleaning paste is essential but not enough; some researchers suggest even the placement of a rubber dam [139]. Oral biofilm or debris can be trapped in marginal gaps and mask them on the replica. Therefore, some researchers suggest that two impressions should be taken (the first one should be discarded) or that the margins should be cleaned with sodium hypochlorite, which can be disagreeable for patients. Another obstacle is difficult to circumvent: The most interesting area the proximal margins and especially the gingival margin of the proximal box can rarely be replicated, unless the tooth does not have an adjacent tooth. One research institute has developed an h- shaped impression tray with hinges that can be split in two parts after impression taking [139]. This tray helps to reach axio-proximal margins but it does not provide a solution for the replication of the entire gingival margin. Due to these difficulties, most studies can only report on the marginal integrity of the occlusal part of the restoration [158]. This is, however, the site where marginal caries rarely occurs. Several clinical studies took impressions for a SEM evaluation of the margins in addition to the clinical examination. Impression taking was performed for Class V restorations [159,160], composite inlays [161], ceramic inlays [ ] and inlay-fixed partial dentures with fiber-reinforced core [166]. The majority of the studies, however, were conducted with direct Class II composite restorations [158, ]. Most of the authors did not describe the exact technique of impression taking. Of the 17 studies identified, only 6 showed an

16 74 d e n t a l m a t e r i a l s 2 9 ( ) intact margins stained margins margins with gaps soft dentin hard dentin Fig. 11 Data of a clinical study on posterior composite restorations [175]. Mean percentage of a defined marginal criterion as evaluated by SEM at baseline (0) and after 4 years of clinical service and by clinical examination at baseline (0) and after 4 years of clinical service. observation period of 4 years or more. Most of the studies reported that the majority of the restorations had marginal deficiencies already at baseline. This number increased over time. The clinical correlate is, however, less pronounced. In one study, the margins of inlay-retained FPDs were rated as perfect in the SEM evaluation even after 5 years of service [166]. Another study carried out a more detailed analysis of the clinical and microscopical behavior of the margins for both clinical parameters and SEM evaluation [175]. The authors differentiated between gap, negative step and overhang in both the SEM and clinical evaluation. Additionally, staining was evaluated in the clinical rating. In general, the agreement between SEM and clinical evaluation was weak or only moderate, even when refined parameters were applied (Fig. 11). Interestingly, the percentage of restorations with perfect margins at baseline was similar to the mean percentage of restorations showing perfect margins in the SEM. However, it has to be noticed that the mean percentage of a given parameter across all restorations is measured quantitatively in the SEM evaluation, whereas the clinical evaluation is related to the percentage of restorations with a certain parameter. Another study followed up 46 direct posterior restorations over a period of 10 years [170,171]. As early as at the 1-year recall, more than 90% of the restorations showed margins which were not perfect on more than two-thirds of their length, as seen with SEM on replicas, while clinically less than 5% of the restorations had detectable margins at a length of less than one third. At the 3-year recall, stained margins were seen in about 25% of the restorations, whereas more than 90% of them had imperfect margins on the replicas. After a period of 0 3 years, microcracks and marginal fractures began to appear. After 5 years, a reduction of the microcracks was evident. The determined marginal imperfections, either those seen with SEM on replicas or those observed clinically, did not influence the survival rate of the restorations or the development of marginal caries after a period of 10 years. Another study with 45 adhesively luted ceramic inlays followed up for a period of 8 years revealed similar results: Although after Fig. 12 Percentage of restorations with a clinically rated phenomenon in relation to soft or hard dentin after removal of restoration. Data compiled according to [42]. 8 years 77% of the inlays had marginal fractures seen with the SEM, only 29% had marginal defects detectable with explorers; 40% showed marginal discoloration and none of them showed marginal caries [164]. The conclusion of both studies is that there is a major discrepancy between the microscopic and the clinical findings. Based on the microscopic findings, many more restorations should show marginal problems, such as marginal staining and eventually marginal caries. Restorations with microscopically impeccable margins cannot be produced under clinical conditions. In a study in which Class II restorations were placed in premolars and extracted because of orthodontic reasons after 4 6 months, the SEM analysis showed that 43% of the cavities had been overfilled and 25% had been insufficiently filled [177]. This result was obtained independent of the adhesive, the layering technique and the matrix system used, or the experience of the operator. As there is no correlation between the presence of gaps and e.g. marginal caries, it may be assumed that the width and depth of the marginal gap represents a decisive factor rather than the percentage of margins with gaps. However, there is a lack of clinical studies which have systematically examined the relationship between the width of the marginal gap and the occurrence of marginal caries and investigated the factors of caries activity, location in the mouth and the type of restorative material used. Nevertheless, some indications seem to suggest that even the width of the marginal gap per se is not a prognostic indicator for the occurrence of marginal caries. In a clinical study on composite restorations, the restorations with imperfections, discoloration and marginal gaps were carefully removed and the dentin beneath was judged as being hard of soft. Furthermore, bacterial samples were taken [42]. The clinical judgment of the margin (intact, stained, gap) was then correlated with the presence of soft or hard dentin beneath the restoration (Fig. 12). If one compares the distribution of marginal phenomena with soft or hard dentin one sees an almost even distribution. However, there were about 20% more cases of hard dentin which were linked with intact margins and about 20% more cases of soft dentin that were linked with stained margins. Gaps as such were not indicative for caries only if a carious lesion could have been probed already at the margin [42].

17 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 13 Correlation between percentage of profiles with gap in vitro [152] and percentage of clinical restorations with loss of retention (left) or marginal staining (right) using the same adhesive system, with Spearman correlation coefficient rho. The numbers in these diagrams refer to the number of the adhesive systems (unpublished data by the author). In areas such as the proximal gingival floor of Class II restorations, which are more difficult to reach with oral hygiene measures and self-cleaning mechanisms, the marginal gap width associated with marginal caries is most probably smaller. But as mentioned in the introduction, the individual caries activity or risk of the patient will probably be of far greater importance than the actual marginal gap width Cylindrical fillings in bovine teeth. Although widely used for the evaluation of the marginal adaptation of adhesive materials, there is no study found in PubMed that has correlated the findings of marginal adaptation with the results of clinical studies. Therefore, data obtained with the automatic detection of marginal gaps in bovine dentin as described above were correlated with the database of clinical studies on cervical restorations. Data of 15 adhesive systems were included into the analysis. However, there was no correlation whatsoever, neither for retention loss nor for marginal staining (Fig. 13) Class V. Two research institutes (University of Berlin, University of Zurich) have established standardized test methods to evaluate adhesive systems in extracted teeth in the laboratory. Since the late 1980s, adhesive systems have been routinely tested at both institutes. Therefore, both institutes can claim a multitude of data on various adhesive systems; only parts of these data have been published in dental journals or books to date [142,178]. Both test methods involve the quantitative analysis of the restorative margins of Class V cervical fillings using SEM at high magnification. However, both test methods differ with regard to the type of tooth, cavity preparation, artificial aging of restorations, evaluation criteria and number of operators. The data were made available by both institutes so that they could be related to the clinical findings of Class V fillings. Forty-four clinical studies matched the inclusion criteria, for which also laboratory data were present. For Berlin, in vitro data after thermocycling on dentin were available for 32 adhesive systems, for which also data of in vivo studies were available; for Zurich data of 28 adhesive systems after thermomechanical loading in the chewing simulator could be included into the analysis. The comparative analysis revealed that the correlation between in vitro and in vivo variables was weak and only present if studies which used the same composite both in the in vitro and the in vivo trials were compared. However, one laboratory method (Berlin) showed a more consistent and more significant correlation with the in vivo findings than the other method (Zurich) (Fig. 14). No sizeable correlation was found between both in vitro methods. As already described above, the results from cervical restorations cannot be transferred to results from posterior Class II restorations. This holds also true for clinical results. Adhesive systems that perform well in cervical cavities do not necessarily perform well in Class II cavities. Less favorable conditions exist in Class II restorations, especially with regard to marginal integrity, which is mainly influenced by the bonding effectiveness of the adhesive system on dentin and enamel to compensate for the shrinkage and shrinkage stress of the composite material Class I restorations. A study tried to prove that the marginal integrity of occlusal restorations placed in patients can be compared to that of restorations placed in extracted teeth in vitro [179]. For this purpose, 40 Class I restorations with 5 different adhesive systems were placed in 20 subjects (8 per group) using the same composite material. The same materials were used for the placement of the restorations in vitro (8 per group). The in vitro restorations were submitted to 100,000 cycles of occlusal loading and 2500 thermocycles. The marginal integrity (percentage of continuous margin) of both in vitro and in vivo restorations was evaluated with SEM (200 ) on replicas. The marginal integrity of the clinical cases after 2 years was worse compared to that of the in vitro cases. However, there was a close relationship. The Pearson correlation coefficient was 0.84 for the baseline

18 76 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 14 Top: Cervical cavity on an extracted upper central incisor (Photo courtesy U. Blunck, Charité Berlin) [191]. Bottom: Correlation between marginal index in vitro and percentage of clinical restorations with loss of retention (left) or marginal staining (right) using the same adhesive system and composite with Spearman correlation coefficient rho. The numbers in these diagrams refer to the number of the adhesive systems. Marginal index = (1 % of perfect margin + 2 % of irregularities + 3 % of gap <2 m + 4 % of >2 m)/100. and even 0.99 for the follow-up correlation. This study really proved that it is possible to simulate the marginal integrity seen with high magnification in laboratory conditions. The study, however, does not give an answer to the question whether the marginal deficiencies seen under high magnification have any clinical relevance. In essence, the same authors denied the significance of the laboratory test when they wrote that all restorations were clinically acceptable despite different ratings in marginal integrity Class II restorations. We have learned that there is some weak correlation between the marginal integrity of cervical fillings in extracted teeth and the retention rate of cervical fillings in vivo. But what about the prognostic value of the marginal integrity of Class II restorations placed in extracted teeth and the performance of Class II restorations in vivo? As true marginal caries is a rare event and as marginal staining does appear quite more often and is related to gaps, it may be speculated that the gaps or irregularities discovered in vitro allow a prognosis with regard to the occurrence of marginal staining in vivo. It has been tried to establish a correlation with 8 adhesive systems that were placed with the same composite material in lower molar teeth and stressed with 10,000 episodes of thermocycling. The results were compared with clinical trials on the same adhesive systems. The data derive from the meta-analysis on Class II restorations [22]. Fig. 15 shows that a weak correlation with regard to marginal staining could be established after 2 years and it can be speculated that if the fillings have less than 40% of the margins with gaps in vitro (mean value) less than 20% of the restorations should show marginal staining after 2 years in vivo. However, this result needs to be substantiated with large-scale multi-center studies Retention of indirect restorations Adhesive technology has enabled the clinician to prepare the teeth according to the necessities of the selected materials rather than the requirements of macromechanical retention to keep the restoration in place. The adhesive properties refer to both the bond to the prepared tooth as well as to the bond to the indirect material, e.g. resin, ceramic or metal alloy, covering the tooth. The luting agent should help to prevent the restoration from being dislodged. There is an inverse relationship between the adhesive properties of the luting agent and the macromechanical retention. The lower the macromechanical retention, the higher the adhesive properties have to be to compensate for the low degree of retention. The smaller the convergence angle of the prepared tooth is and the larger the height and surface area of the prepared tooth are, the higher the macromechanical retention will be. Other material factors

19 d e n t a l m a t e r i a l s 2 9 ( ) influencing crown dislodgement were stump height and convergence angle as well as the luting agent. Adhesive resin cements generally produced the highest values, followed by glass ionomer and zinc phosphate cements. Seating force, surface roughness, type of cutting bur and use of a desensitizing agent had all a negligible effect on the test results. Artificial aging like thermocycling had no influence on glass ionomer cements, while in resin-based cements thermocycling combined with prolonged water storage generated a similar failure stress as thermocycling alone. The comparison with clinical results did not reveal conclusive evidence that the results of the laboratory methods completely reflect the results of prospective clinical trials in conjunction with single crowns and fixed partial dentures. In view of the results of the review, the following experimental setup can be recommended: Fig. 15 Correlation of the mean percentage of proximal enamel margin with gaps of Class II restorations placed in extracted teeth with 8 different adhesive systems and the percentage of Class II restorations with marginal staining after 2 years placed with the same adhesive systems (adj. R 2 = 0.6) (unpublished data by the author). of the luting agent include the flexural strength of the material (the higher the flexural strength, the lower the risk), the shrinkage (the lower the shrinkage, the lower the risk) and the expansion (slight expansion due to water absorption can increase the retention as it enhances the interlocking between restoration and prepared tooth). How can adhesive cements be tested for their effectiveness to hold a restoration in place? The most common laboratory tests to assess the adhesive properties of a luting agent are bond strength tests (see above). Numerous studies on these tests to assess luting agents have been published. Currently, an ISO standard on polymer luting agents with adhesive properties is being elaborated that proposes amongst many other tests such as flexural strength, solubility, and water sorption either a macroshear or a macrotensile bond strength test to dentin (ISO Working Draft No , 2011: Polymerbased luting materials containing adhesive components). The advantages of bond strength tests, if designed properly, are the reproducibility of the results within the same test lab and the ease of conducting the test. The main criticism, especially from clinicians, is that the bond strength test does not reflect the clinical situation. In response, laboratories developed crown pull-off tests to simulate the clinical procedure as early as in the 1970s. In 2009 a systematic review on this topic was published [180]. The studies varied largely with regard to tooth type (molars, premolars), number of specimens (9 25), stump height (3 6 mm), angle convergence ( ), standardization and measurement of preparation surface, seating force ( N), artificial aging, crosshead speed for tensile force and statistical analysis. The coefficient of variation of the test results varied from 3% to 100%. The most important factors - at least 20 specimens per group - stump height 3 mm - convergence angle 20 - thermocycling of specimens (5000 ) - avoidance of shearing forces during dislodgement - failure probability statistics (Weibull). Another approach may be to subject ceramic or metal crowns to cyclic/dynamic loading in a chewing simulator or another fatigue test device and record the number of cycles until debonding/decementation occurs [181]. As the bonding area is crucial to the test and to the variability of the results, natural teeth should be prepared in a standardized method. Today, this can best be achieved with 5-axis CNC machines New research methods and devices As new methods for the non-destructive evaluation are becoming available, they will also used in dentistry. For instance, it has been suggested that micro-computer tomography (Micro-CT) should be used to assess the internal bond of restorations in extracted teeth. These devices are useful to detect porosities in the restorative material, visualize root morphology and generate 3D models for finite element analysis [182,183]. However, to date they have failed to appropriately show the adhesive interface with high resolution (Fig. 16). 6. Rationale for meaningful laboratory testing of the effectiveness of adhesive materials The scientific evidence about the correlation between laboratory tests and clinical findings with dental adhesive materials allows us to establish a few rationales with regard to the meaningful testing of dental adhesive materials. If the currently established laboratory tests are used, a prognosis on the following clinical phenomena is not possible: 1. Post-operative hypersensitivity 2. Caries at the restorative margins If the currently established laboratory tests are used, a prognosis on the following clinical phenomena is possible:

20 78 d e n t a l m a t e r i a l s 2 9 ( ) Fig. 16 Two-surface composite restorations in a molar tooth; (left) Micro-CT (resolution 7 m; picture courtesy by Scanco Medical, Switzerland); (middle) microscopic picture of transversal cut in the same region of the same tooth as seen on the CT-picture of the left side; (right) SEM picture of internal adaptation with gaps. 1. Bond strength tests on dentin Retention 2. Marginal integrity in cavities with enamel margins Marginal staining 6.1. Bond strength tests Shear bond tests should be abandoned due to the critical and inadequate stress generation at the reaction zone and the resultant unreliable correlation with retention loss in cervical restorations. Instead tensile tests should be applied according to ISO TS. Tensile tests produce a more uniform stress distribution in the reaction zone and show a more reliable correlation with clinical retention loss. Measurements at baseline (24 h) and after 3 months water storage should be carried out to assess the possible degradation of the adhesive interface due to water infiltration. The bond strength should be measured at the coronal dentin of molars both after grinding with abrasive paper with and without eroding the dentin thereafter. Eroding of dentin can e.g. be simulated by means of a ph cycling process involving the demineralization of dentin with citric acid and remineralization with a mixture of specific minerals and ions as described by Zimmerli et al. [79]. To assess the variability of test results, specimens should be fabricated by various operators or should be carried out at various test institutes that apply the same test protocol. Microtensile tests are laborious and technique-sensitive and do not provide substantial advantages over tensile tests. However, microtensile tests can be used to further investigate the localized bond strength at critical areas such as the cervical gingival floor of Class II restorations. This area is the benchmark for improving the effectiveness of adhesive systems Marginal integrity The marginal integrity of cervical restorations involving margins in the dentin has a moderate clinical correlate with retention loss of Class V restorations, provided that the whole procedure is as much standardized as possible (always same operator for the fabrication of specimens and same operator for the evaluation). Eroding the dentin after tooth preparation could further simulate the clinical situation (see above). The marginal integrity of posterior restorations with enamel margins (either occlusal in Class I or axio-proximal in Class II) have a moderate clinical correlate with marginal staining of posterior restorations, provided that the whole procedure is as much standardized as possible (always same operator for the fabrication of specimens and same operator for the evaluation). The clinically simulated evaluation of restorations in vitro (Class V and Class II) with loupes and explorers may be a reasonable alternative to the microscopic evaluation with SEM and needs further investigation. The evaluation of marginal integrity with tracer penetration does not show any correlation with clinical parameters (retention loss, marginal staining, postoperative hypersensitivity) and should therefore be abandoned Retention effectiveness of luting agents The crown-pull off test is a suitable test method to predict the retention capability of luting agents for indirect restorations. However, the protocol has to be standardized as much as possible. The most critical points are (1) standardized tooth preparation, and (2) avoidance of shear forces during the pulloff of the crown. 7. Recommendations for dental journals Dental journals should no longer accept manuscripts with test methods that do not show any clinical relevance, such as e.g. dye tracer penetration tests. Since 2008, the Journal of Endodontics has ceased to accept papers that apply the dye tracer penetration method. Other dental journals should follow this example. As far as bond strength tests are concerned, the scientific journals should define a clear acceptance policy with regard to the principles laid down in the ISO Technical Specification on testing the adherence to tooth substance.