Microleakage of pit and fissure sealings placed after enamel conditioning with phosphoric acid or with self‐etching primers/adhesives

Abstract Background It is still not fully understood what pretreatment is best for achieving maximum tightness for pit and fissure sealings (PFS). Aim This study investigated microleakage of PFS placed after etching with phosphoric acid or after the application of self‐etching primers/adhesives (SEPA). Design 131 third molars were assigned to ten groups. In Hel‐P, Helioseal® was applied after phosphoric acid etching. In the other groups, SEPA were used (Dyr‐AP: Adper™ Prompt™ L‐Pop™, Dyract® Seal; Bea: BeautiSealant Primer and Paste; Hel‐Exp: Experimental primer, Helioseal®; Hel‐Cl: Clearfil™ SE Bond1, Helioseal®). Specimens were stored in distilled water at 37°C (28 days), followed by 3500 thermocycles and staining with 5% methylene blue (M) or 5% silver nitrate (S). After methylene blue staining and sectioning, microleakage was assessed light microscopically. During silver nitrate staining, specimens were dissolved by 32% HCl and remaining PFS were evaluated under a SEM. Results Tightness, percentage of penetrated area, and maximum dye penetration were best for Hel‐P and Hel‐Cl (p < 0.05). Conclusions Phosphoric acid etching of enamel and Clearfil™ SE Bond resulted in the best sealing quality. Methylene blue staining allowed the evaluation of more criteria (fissure shape, voids, sealant penetration depth) compared to silver nitrate.


| INTRODUCTION
Recent systematic review shows that fissure sealing with resin-based materials has been an effective way to reduce caries formation by 11%-51% in 2 years compared to no sealing (Ahovuo-Saloranta et al., 2017). Pit and fissures of first permanent molars are the first to be affected by caries up to the age of 12 years, even in countries with mean DMFT <2 (Marthaler, 2004). It is this way clear that pit and fissure sealants are a very important tool in caries prevention. However, as with all resin materials, adhesion is the Achilles' ptern of long-term preservation of sealants and loss of it leads to microleakage (Kidd, 1976). When a resin-based material is placed, competition arises between polymerization shrinkage forces and bond strength to the subsequent dental structure (Van Meerbeek et al., 2011). If bond strength between sealant and enamel is weaker, a fracture is caused and a way through the gap is formed as the material separates itself from enamel, therefore resulting in microleakage and failure of the restoration (Kidd, 1976). A minimal degree of leakage can be tolerated and not cause a reaction. But in some cases it can become the source of postoperative pain and recurrent caries, leading to restoration failure (Kuhnisch et al., 2012).
Resin sealants exhibit the highest retention rate after 5 years (83.8%) compared to glass ionomer sealants (5.2%) (Kuhnisch et al., 2012). A disadvantage of resin sealants compared to glass ionomer cements is their technique sensitivity during clinical application, as excellent control of moisture is needed. According to their composition resin-based sealants can be conventional resins, compomers or giomers 2 . Compomers are polyacid-modified composites with fluoride-releasing silicate glasses (Kuhnisch et al., 2012). An acid-base reaction takes place as compomer absorbs water, which facilitates cross-linking structure and fluoride release. Giomers are composites with pre-reacted glass-ionomer fillers in their resin matrix (Ntaoutidou et al., 2018).
Pit and fissure sealing is described as a "micro-invasive treatment," as the conditioning of the tooth surface results in an irreversible removal of a small amount of dental hard tissue (Schwendicke et al., 2015). The outer enamel layer of permanent teeth is prismless and even when it's abraded due to mastication, the inner surface of the fissure, where sealants are placed, remains prismless (Schwendicke et al., 2015). For the conditioning of the prismless enamel layer, several treatment options have been suggested in the literature, like the application of phosphoric acid (Erdemir et al., 2014), the use of self-etching primers/adhesives (Ntaoutidou et al., 2018), air-abrasion (Kramer et al., 2008) or laser conditioning (Karaman et al., 2013). Etching the enamel with 35%-37% phosphoric acid for at least 30 s is the gold standard method to remove the prismless enamel surface layer prior to the application of resin-based pit and fissure sealants. Over the past years, self-etching primers/adhesives have been tested as pretreatment for pit and fissure sealing sacrificing the etching with phosphoric acid and rinsing with water-spray (Ntaoutidou et al., 2018). Self-etching primers/adhesives are supposed to condition the enamel with acidic functional groups and polymerizable acidic components (Pashley & Tay, 2001). Among their advantages, they reduce clinical steps (Perry & Rueggeberg, 2003) and the possibility of contamination of the occlusal surface with saliva, over-drying/wetting is avoided and they can be easily used in children with limited compliance (Ntaoutidou et al., 2018). On the one hand, systematic reviews show worse adhesion of sealants placed after the application of self-etching primers/adhesives to enamel compared to etching with phosphoric acid (Birlbauer et al., 2017;Botton et al., 2016;Pitchika et al., 2018). On the other hand, retention (Erbas Unverdi et al., 2017) and microleakage (Nahvi et al., 2018) of sealants used in combination with self-etching primers/adhesives have been shown to be equal to sealants with prior etching. However, marginal leakage of sealants with self-etching primers could also be due to unsuccessful adhesion at first place, as self-etching adhesives show reduced adhesion to enamel (Van Meerbeek et al., 2011). pH of the self-etching primer, as well as addition of functional monomers like 10-MDP in its composition, also play an important role on the adhesive performance of self-etching adhesives (Van Meerbeek et al., 2011).
In microleakage studies a penetrant is needed in order to mark the available areas for penetration around restorations or around sealants. The one mostly used is methylene blue (Agrawal & Shigli, 2012;Kramer et al., 2008), followed by fuchsine (Gillet et al., 2002;Hatirli et al., 2018;Heintze et al., 2008), and the more expensive silver nitrate (Heintze et al., 2008). A review by Heintze et al. (2008) showed no difference between the aforementioned substances (Heintze et al., 2008).
The aim of the present study was to compare the efficacy of a pretreatment with 37% phosphoric acid prior to pit and fissure sealing with the utilization of self-etching primers/adhesives as conditioners.
Furthermore, the results of two dye penetration tests, namely either with 5% methylene blue or with 5% silver nitrate, used for the assessment of the quality of preventive pit and fissure sealants in vitro should be checked against each other. The null hypotheses tested were: (i) different procedures of enamel conditioning have no impact on the sealing ability of pit and fissure sealants and (ii) there are no differences between the two dye penetration methods.

| Specimens selection and preparation
This study has followed the CRIS guidelines for in-vitro studies as discussed in the 2014 concept note (Krithikadatta et al., 2014). The conduction of the present laboratory study was approved by the local ethics committee of the Justus-Liebig-University Giessen, Germany (AZ 143/09).  Table 1). Helioseal ® was tested as a conventional resin sealant, Dyract ® Seal as a compomer and BeautiSealant as a giomer. Sealants were placed by a single calibrated operator and were polymerized with Bluephase lamp (Ivoclar Vivadent/light output of 1200 mW/cm 2 ± 10%). Half of the specimens proceeded to methylene blue penetration test (M) and the other half to silver nitrate penetration test (S). Specimens were stored in distilled water at 37 C for 28 days (Incubator Typ B20, Heraeus Holding, Hanau, Germany), and were then thermocycled for 3500 cycles (+5 C and +55 C, dwell time 30 s; TCS 30, Syndicad, Munich, Germany) to simulate an artificial aging.

| Methylene blue penetration test (M)
For the conduction of methylene blue penetration test, specimens' apices were sealed with glue wax (Chemical Dental Laboratory Oppermann-Schwedler, Bonn, Germany), and the roots were covered with acid resistant nail varnish (Manhattan, Stuttgart, Germany) to prevent a retrograde dye penetration. Followed to that, specimens were centrifuged for 5 min at 30g in 5% methylene blue solution (Heraeus Megafuge 8, Heraeus Holding). Upon completion, 1 mm thick slices were produced by a microtome (IsoMet™  inhomogeneity/voids within the sealant (in %), dye penetration depth (in μm) (Figure 1) (Kramer et al., 2008). Tightness was defined as the absence of dye penetration around the sealant and was calculated from the maximum percentage of dye penetration (see below). The even distribution of fissure shapes on the five different groups was tested by comparing the upper/lower fissure widths.

| Silver nitrate penetration test (S)
Sealed teeth were stored in 5% AgNO 3 solution for 24 h at 37 C (Incubator Typ B20, Heraeus Holding), followed by a storage in 0.

| RESULTS
No significant differences were noted between the shape of the fissures among the groups (p = 0.104, Kruskal-Wallis), showing that fissure forms were equally distributed.

| Methylene blue penetration test (M)
Tightness was significantly higher for control group compared to Dyr-    (Table 4).

| Silver nitrate penetration test (S)
Maximum silver nitrate penetration depth (in %) was increased for Dyr-AP-S compared to Hel-Cl-S (p = 0.013, Bonferroni) ( Table 4). The percentage of penetrated area by silver nitrate was significantly higher for the groups Dyr-AP-S and Bea-S in comparison to Hel-P-S and Hel-Cl-S (p < 0.05, Bonferroni) (Table 3). Statistically significant differences regarding the total area of the sealed fissure were observed among groups (Kruskal-Wallis, p < 0.001), with Dyr-AP-S and Bea-S having a higher amount of sealant applied to the pit and fissure system than the group Hel-P-S (p < 0.05, Bonferroni) (Table 3). Additionally, the total area of the sealed fissure was higher for the giomer group (Bea-S) compared to the resin composite groups applied with self-etching primers/ adhesives (Hel-Exp-S: p = 0.043, Hel-Cl-S: p = 0.009, Bonferroni).

| Comparison between the two methods
Parameters "maximum dye penetration depth (in %)" and "sealant penetration depth (in nm)" were compared for the two penetration methods tested (Table 4). Significant differences were noted between the two penetration tests regarding maximum dye penetration depth,

| DISCUSSION
The present study aimed to compare the use of phosphoric acid versus self-etching primers/adhesives as enamel pretreatment prior to sealants application. Three different resin materials were used as pit and fissure sealants, Helioseal as a conventional resin-based sealant, the compomer Dyract Seal, the giomer BeautiSealant; and two different dye penetration methods, methylene blue and silver nitrate penetration test, were compared.
Methylene blue penetration test is a reliable method for the evaluation of microleakage adjacent to pit and fissure sealants and has been used in several studies (Gillet et al., 2002). Further advantages of using the methylene blue penetration test are that the dye is not expensive and it is easy to use. On the basis of the microscopic images, many parameters may be measured and specimens can be investigated microscopically (Brocklehurst et al., 1992). The time-   consuming specimen preparation and measurements together with the toxicity of methylene blue (acute toxicity after oral intake, hazardous to the aquatic environment) are regarded as disadvantages (Van Meerbeek et al., 2011). For silver nitrate penetration test, the specimen preparation and evaluation may be completed faster. During microscopic evaluation, the entire pit and fissure sealing may be investigated, as dental hard tissues have been dissolved before by storage in hydrochloric acid. Compared to methylene blue penetration test, less parameters are measurable because only sealant material remains for evaluation after dissolution of the specimens' dental hard tissue.
All in all, specimens stained with methylene blue allow for the evaluation of additional parameters, as specimens are sectioned with a microtome enabling the assessment of the fissure shape, the number of voids, and the sealant penetration depth, as it was done in the present study. Moreover, the time-consuming and expensive manufactur- with methylene blue, evaluation is performed at a certain number of cuts, therefore at a limited percentage of the sealed area. Therefore, null hypothesis ii was rejected.
Regarding specimen preparation, prior to the sealant application it is recommended to clean the occlusal surface either with a lowspeed rotating bristle brush combined with pumice slurry or by air polishing, the later was used in the present study. Air polishing was chosen since it has been shown to increase bond strength of sealants and to enhance sealant penetration depth in vitro (Brocklehurst et al., 1992). Despite the fact that fissures demonstrate a variety of shapes (Nagano, 1961), homogenous distribution of specimens along the experimental groups allowed for an unbiased evaluation.
Thermocycling was performed in order to simulate the thermal changes which take place in the oral environment. In accordance with literature, temperature changed alternatively from 5 to 55 C (Heintze et al., 2008). Despite the fact that some published studies show no differences when thermocycling was performed, it was chosen in order to comply with the most of microleakage literature.

After the introduction of phosphoric acid conditioning by
Buonocore in 1955, enamel etching has been regarded as the gold standard in order to achieve adhesion via micromechanical retention of resin composites and therefore resin pit and fissure sealants (Buonocore, 1955). The application of phosphoric acid exposes prismatic enamel, creates microporosities into which resin-based sealants penetrate and once polymerized, resinous tags and mechanical anchoring are produced (Gwinnett, 1973). According to a recently published systematic review and meta-analysis, phosphoric acid etching combined with the use of resin-based sealants exhibited the best clinical long-term performance in terms of favorable retention rates.
All in all, the longevity of sealants placed in combination with primers/ adhesives was substandard, though depending on the type of primer/ primers. An in vitro study by Kanemura et al. (1999) comparing the application of either phosphoric acid or self-etching primers to intact enamel showed that self-etching primers demineralized the enamel insufficiently and resulted in swallower adhesive penetration, shorter resin tags as well as lower bond strength (Kanemura et al., 1999).
Short resin tags were also found by Torii et al. (2002) after the application of self-etching adhesives on ground bovine enamel, although tensile bond strength was comparable to etch-and-rinse adhesives (Torii et al., 2002). As a consequence, the inability of self-etching primers/adhesives to etch and penetrate deep into intact enamel of the occlusal surface may be a reason for the increased microleakage observed for Dry-AP and Bea in both dye penetration methods (Table 4). This finding is also in agreement with the microleakage study by Perry and Rueggeberg (2003).
In group Dyr-AP, the self-etching adhesive Adper™ Prompt™ L-Pop™ was combined with the compomer Dyract ® Seal. The application of Adper™ Prompt™ L-Pop™ was conducted in three layers, as multi-coating has been shown to increase microtensile bond strength (Frankenberger et al., 2001). Furthermore, the combination of this adhesive with a compomer increased bond strength in vitro (Frankenberger et al., 2001) 0.2%-31.8%) after 5 years for compomers used as pit and fissure sealants. In the present study, the tightness of sealings in group Dyr-AP (63% [54%-74%]) was low, though the sealant penetration depth was high (99% [93%-100%]) indicating an insufficient adaption of this dental material to aprismatic enamel (Table 2).
Giomer BeautiSealant, a resin-based sealing material additionally containing inorganic surface pre-reacted glass ionomer cement (S-PRG) fillers based on fluoroboroaluminosilicate glass (Ntaoutidou et al., 2018), was applied in group Bea. It is used in combination with the self-etching BeautiSealant Primer having a mild pH of 2.3. Ntaoutidou et al. (2018) assessed a total retention rate of as low as 6.9% for Beautisealant after 18 months in a randomized controlled clinical trial. A possible explanation was the limited demineralization potential of its self-etching primer (Ntaoutidou et al., 2018), which may also be an explanation for the higher microleakage of BeautiSealant (Table 4).  (Table 2), showing that the material tightly seals the pit and fissure system.

| CONCLUSION
Within the limitations of this in vitro study, enamel etching with 37% phosphoric acid, or application of Clearfil™ SE Bond as a self-etching adhesive prior to sealing pit and fissures with resin-based sealants, resulted in the lowest microleakage, and therefore tightest sealing.
The use of the methylene blue penetration test facilitated the evaluation of more parameters compared to the silver nitrate penetration test and was less expensive. Therefore, methylene blue penetration test may be recommended for microleakage studies in the laboratory.