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Micromorphological characterization of adhesive interface of sound dentin and total-etch and self-etch adhesives/ Mikromorfoloska analiza odnosa adhezivnih sredstava sa potpunim nagrizanjem glebi identinaisamonagrizajucih adheziva sa zdravim dentinom.

Introduction

Adhesion is the interaction of the molecules at the interface of two different materials [1]. The sur faces of these materials are called adherents or substrates, and the substance that binds these two materials is called an adhesive. This interaction is defined as an adhesive bond. The most important step in the realization of adhesion is to make a close contact between the adhesive and the substrate. The ultimate goal in restorative dentistry has always been to achieve a strong and permanent bond between the dental hard tissues and restorations [2]. Dentin contains a large amount of water and organic structures, especially collagen type I, which makes it very difficult to bond to [3]. The structure and properties of dentin are different and depend on the location. The properties of dentin which are important for the realization of adhesive bond are: a) dentin permeability, b) number and orientation of dentin tubules, c) changes in dentin structure and d) mechanical properties [4].

One of the earliest and most widely used tools for analyzing the adhesive bond has been scanning electrone microscopy (SEM) [5]. The principle of SEM is based on pseudo three-dimensional picture that is formed point by point and line by line from secondary electrons [6]. Electrons are emitted in response to the collision of the electron beam and the test sample. The number of secondary electrons emitted from any one point on the surface depends on the difference in the structure, composition and texture of the surface.

Thanks to the short wavelength of the electron beams, SEM provides sufficient resolution to identify a few micrometers wide formations, such as a hybrid layer. Although the concept of hybridization was first introduced by Nakabayashi et al. in 1982 (using SEM) as the formation of a resin-reinforced dentin zone, it took other researchers almost ten years to accept this theory and examine other details of micromechanical bonding mechanism [7]. Thanks to the fact that SEM images of hybrid layer are of high magnification and of high quality, this test method has become the most popular in studying the mechanism of formation of adhesive bond. The most commonly used variant of SEM is low vacuum SEM [8, 9]. The low vacuum SEM is used for testing unfixed biological samples under low vacuum and wet conditions [10]. The most successful method for observing soluble samples using SEM technique is replication technique, which involves taking an impression of the test surface using silicon impressions, outpouring thus obtained impressions in epoxy resin, after which this replica is set in vacuum conditions in the SEM [11, 12]. Thus, the replica technique is used for semiquantitative analysis of marginal adaptation of adhesive restorations placed and followed up for several years in vivo [11]. The resolution obtained under the SEM is highly dependent on the accuracy of impression and preparation techniques of replicas. It is recommended to use a very low viscosity impression materials (light-body, polyvinyl siloxane based), and non-shrinking epoxy resin. The details obtainable with a replication technique do not allow examination of biomaterial-tooth interface, i.e. hybrid layer, because the clear detail can be seen up to 1000 x magnification.

Dentin is a vital, hydrated tissue, whose structure and properties vary depending on the location [3]. Instrumentation of dentin using burs results in smear layer production [13], which consists of hydroxyapatite, denatured collagen and dentin debris particles. It has a weak bond with underlying dentin, the bond being only 5 MPa [14]. The smear layer penetrates into the dentin tubules to form smear plugs, and thus reduces dentin permeability to 86% [15]. Contemporary adhesives can be classified according to their interaction with the smear layer. Consequently, there are two strategies for achieving bond and four types of adhesives:

1. Total-etch adhesives, 30-40% phosphoric acid is used to treat enamel and dentin simultaneously and to remove smear layer

a) three-step adhesives (conditioner + primer + resin)

b) two-step adhesives (conditioner + primer/resin)

2. Self-etch adhesives, without the conditioning step. They consist of non-rinsing acid monomer, which makes the smear layer permeable without its complete removal.

a) two-step self-etch adhesives (acid primer + resin)

b) one-step self-etch adhesives (all-in-one).

Bonding agents have constantly been improved in order to reduce the number of clinical steps and simplify their application [16]. The application of bonding agents results in the formation of resin reinforced layer of dentin (hybrid layer) and penetration of resin into dentin tubules (resin tags). Conditioning step (total-etch adhesives) results in complete removal of smear layer and wide opening dentin tubules, and improves adhesive penetration. In contrast, self-etch adhesives are not efficient enough to dissolve the smear plugs, and therefore remain in dentinal tubules as a part of a hybrid complex. Such hybrid complex is characterized by poorly formed resin tags, which result in inferior clinical performance of these adhesives [17].

The aim of this study was to apply SEM to analyze the interfacial micromorphology of total-etch and self-etch adhesives and sound dentin, the null hypothesis being that there are no important differences in the microstructure of the interfaces between the tested adhesive systems.

Material and Methods

The teeth used for SEM analysis were non erupted third molars of the maxilla and mandibula, extracted by surgical procedure, at the Ward of Oral surgery of the Department of Dentistry of Vojvodina. The teeth damaged during surgery were not included in the study sample, which consisted of 20 teeth--10 teeth for each adhesive system. The residual parts of the soft tissues of the tooth were removed with a surgical curette after tooth extraction. The teeth were immersed in 0.5% solution of chloramine (bacteriostatic/bactericidal) at 4[degrees]C, and after seven days, they were rinsed with distilled water and stored in deionised water (ISO 3696) at 4[degrees]C. Deionised water was completely replaced every 48 hours. The time interval between tooth extraction and its being tested in the experiment was limited to one month. The use of human teeth for experimental purposes was approved by the Ethics Committee of the Department of Dentistry of Vojvodina and the Ethics Committee of the Faculty of Medicine in Novi Sad.

After removing enamel on occlusal surface with high-speed handpiece with copious air-water spray using diamond bur (Diamantsheibe, Edenta, Switzerland), a flat surface of dentin was achieved, which was then treated with silicon carbide abrasive papers (SiC 600-grit paper, 3M) under running water using custom made grinding cylinder [4]. The result was a flat surface of dentin. The depth was approximately halfway between the pulp chamber and enamel-dentine junction. Adhesives were applied on thus prepared dentin surface (Table 1) according to the manufacturer's instructions. Adhesive AdheSE One was applied and agitated for 30 s, then air was dispersed until there was no water movement, and treated for 10 s.

Adhesive Prime&Bond NT was applied on dentin surface after conditioning with Conditioner 36 (Dentsply DeTrey) for 15 s, which was followed by rinsing with water spray for 10 s.

The excess water was blotted with cotton pellet. Immediately after blotting, 2-3 consecutive coats of adhesive Prime&Bond NT for 20 s with gentle agitation were applied using fully saturated applicator. The moist surface was treated with a soft blow of air before light-polymerization for 10 s. AdheSE One F was directly applied to the dentin surface. When the dentin surface was completely coated, the adhesive was brushed into the entire surface for 30 seconds. Excess amounts of AdheSE One were dispersed with a strong stream of air until a glossy, immobile liquid film resulted. Light-cure AdheSE One was applied for 10 seconds.

The proprietary restorative resin composite of each adhesive was set on each sample tooth in one layer (2 mm thick). Adhesives and resin composites were light-cured with polymerization device (Smarlite IQ2, Dentsply, Caulk, DE Milford, Serial No.B 21581) in compliance with the manufacturer's instructions.

Thus formed samples were cut longitudinally through the tooth and composite to expose the adhesive interface. The adhesive interface was then treated with conditioner (Uni-Etch, Silica gel free, Bisco, Schaumburg, IL, LOT 0800012148) for 60 s, rinsed with copious air-water spray, and treated with 2% sodium-hypochlorite solution for 60 s to eliminate organic component of dentin [18]. Preparation of samples of biological tissues (teeth) for SEM was done in accordance with the conditions of low vacuum. The samples were then coated with gold 15-20 nm thick (SCD050 Sputter Coater; BAL-TEC,PA,USA), placed on aluminum carriers and examined using a scanning electron microscope (JEOL, JSM-6460 Low Vacuum, Tokyo, Japan) at 1000 x magnification [19].

After the identification and micromorphological analysis of hybrid layer, the hybrid layer thickness was measured as the distance between the top of the layer and height of the scalloped convexities at the base of the hybrid layer between adjacent resin tags, using microscope software (NIH Image Analyser) [18].

Results

The SEM analysis of the relations of composite systems and sound dentin was performed on a sample of human teeth in laboratory conditions. The samples were examined under magnification of 1000 x with scanning electron microscopy (JEOL, JSM6460 Low Vacuum). The thickness of the hybrid layer of the tested composite systems was measured with the software of the device itself (NIH Image Analyser) at five points on each sample (Figure 1). The thickness of the hybrid layer is shown in the microphotography as the distance between the arrowheads. Three measurements were made at the locations of approximately the same thickness, and the other two at the point of minimum and maximum thickness of the hybrid layer. The mean value of the measured thickness of the hybrid layer was then calculated and it was taken as a representative for the given specimen [26]. The obtained results are presented in Table 2.

The SEM photomicrography showing the sample with adhesive system Prime&Bond NT--Ceram XMono (Figure 2) clearly shows the presence of a hybrid layer, while the SEM photomicrography showing a sample with adhesive system AdheSE One--Tetric EvoCeram does not identify the hybrid layer (Figure 3).

The thickness of the hybrid layer could not be measured for composite system AdheSE One--Tetric EvoCeram, so it is not included in the table that represents the basic statistical values (Table 3). As shown in Table 3, the thickness of the hybrid layer which forms the composite system Prime&Bond NT--Ceram X Mono with sound dentin was 2.68 [micro]m.

The SEM analysis of interface showed that the hybrid layer on sound dentin was well formed, with a large number of resin tags and a large amount of lateral branches (Figure 2) for samples with a composite system Prime&Bond NT--Ceram X Mono, while the presence of the hybrid layer could not be seen in the samples with composite system AdheSE One--Tetric EvoCeram and resin tags were poorly identified (Figure 3).

The SEM microphotography of samples with Prime&Bond NT--Ceram X Mono (PB/CXM) placed perpendicular to the direction of the dentinal tubules (Figure 2) shows the composite (K), the adhesive layer (A) that is not penetrated into the acid-etched dentin (D), the hybrid layer is visible as a bright area. Resin tags are large in number with numerous lateral branches.

The SEM microphotography of samples with AdheSE One--Tetric EvoCeram (AO/TEC) placed perpendicular to the direction of the dentinal tubules (Figure 3) shows the composite (K), thick layer of the adhesive (A) that has not penetrated into dentin. A hybrid layer is not visible. Resin tags are poorly present, and lateral branching is not evident.

Discussion

An ultimate goal in restorative dentistry has always been to achieve a strong and permanent bond between the dental hard tissues and restorations. A chemical bond with the dental hard tissues is formed with glass-ionomer cements and polycarboxylate cements, whereas other restorative materials achieve mechanical or micromechanical bond (amalgam, composite materials).

The study sample consisted only of human teeth-non erupted third molars as a substrate. Having been extracted, the teeth were kept according to the protocol ISO405 [20]. The analysis of adhesive bonds was performed using low vacuum SEM, which is the most commonly used method in similar studies [5].

The smear layer resulting from treating dentin with diamond or carbid burs is compact and thick and compromises the efficiency of self-etching adhesives [21]. The finding that the most important role in the realization of high-quality adhesive bonds is played by wettability and the degree of penetration of the adhesive into dentin, has resulted in the creation of adhesives which effectively penetrate into the demineralized dentin and partially or completely remove the smear layer [22]. Such systems form a hybrid layer, resin reinforced partially demineralized dentin [23].

The absence of a hybrid layer in self-etch adhesives can be explained with their weaker acidity, which results in less demineralization of dentin, and exposes a small number of dentinal tubules, leading to poorer penetration of adhesive into dentin [24]. The results of previous studies suggest that the ability of self-etching primers is weaker in sound dentin than total-etch adhesives [25]. Self-etching adhesive AdheSE One shows pH = 1.5 and it is one of mild self-etch adhesives (pH between 1 and 2). It has an interaction depth between 1 and 2 [micro]m [26]. Only strong self-etch adhesives, having pH [less than or equal to] 1, can form typical resin tags in dentin. Mild self-etch adhesives can hardly form resin tags, they mostly demineralize smear plugs slightly and subsequently infiltrate this area with the resin. The hybrid layer thickness for mild self-etch adhesives is between 0.5 and 1 [micro]m [26]. Erhardt MCG at al. examined adhesive AdheSE One and found a thin and irregular hybrid layer with small number of resin tags [27].

Mild self-etch adhesives are not efficient enough to dissolve the smear plugs, which close dentinal tubules, and therefore they remain in dentinal tubules as a part of a hybrid complex. That hybrid complex is characterized by sparse resin tags. In this case, the lateral penetration of the adhesive monomer into dentinal tubules does not contribute to the formation of the hybrid layer. On the other hand, in total-etch adhesives, the smear layer affects the penetration of adhesive monomers into dentin because dentinal tubules open after its removal in the etching phase, and promotes the penetration of the adhesive.

Pashley et al. have found that the smear layer in sound dentin reduces the effectiveness of self-etch adhesives in achieving the adhesive bond [23]. A smear layer is a product of burs activity in dentin, and it contains denatured collagen, hydroxyapatite and mineral submicron particles [28]. These structures can interfere with the infiltration of resin monomers, and prevent adequate formation of resin dentin interface [23].

A well-formed hybrid layer and long resin tags with large number of lateral branches are characteristics of adhesives with organic solvent: ethanol or acetone [29]. The organic solvent in the composition of adhesives for wet bonding technique dehydrates acid etched dentin chemically [30]. This leads to the lateral shrinkage of the collagen fibrils, resulting in an increase in the width of interfibrilar spaces and reduction of the hydrophilicity of collagen matrix. Lateral dentinal tubules get filled with organic solvent and the bonding agent easily penetrates into them. In that way, a very well-formed hybrid layer with long resin tags and an expressed lateral branching is obtained. Prime&Bond NT contains acetone as a solvent. The use of acetone and ethanol as a solvent in an adhesive is derived from the fact that the comonomers of the adhesive does not dissolve in water. The role of dimethacrylate in the composition of the adhesive is to enhance crosslinking of polymers; they cannot be mixed with water, but are soluble in ethanol and acetone. The total-etch adhesives contain a mixture of the primers (i.e. HEMA) and monomers of the adhesive (i.e. bis-GMA) in a solvent containing a low amount of water. These are applied in two layers. The first layer acts as a primer, and the second acts as an adhesive layer. Scott and Thomlinson have demonstrated that the organic solvents (ethanol, acetone) remove the watery gel of glycosaminoglycans, thus removing them from the connective tissue [31]. Thus, the collagen fibrils in the acid-etched dentin shrink laterally after the application of an adhesive with organic solvents, and the lateral dentinal tubules are filled with an organic solvent (acetone, alcohol), and adhesive resin penetrates easily into them [32]. This is seen as perfectly formed resin tags in SEM microphotographs.

After acid conditioning of dentin, the smear layer is completely removed, dentin is demineralized and collagen fibrils are exposed. In order to form a high quality hybrid layer, the resin monomers must penetrate into this demineralized zone. However, there is a discrepancy between the depth of demineralization and penetration of resin monomers. It has been found that more effective infiltration of resin monomers is achieved by applying adhesive on a partially wet dentin surface than to a dry one, which is reflected in the measured higher bond strength values [33]. In addition, the application of self-etch adhesives shows discrepancies between the depth of demineralized zone and the penetration of resin monomers because these two processes occur simultaneously.

Phosphoric acid used for total-etch adhesives has pH = 0.1-0.4, while the acidity of self-etch adhesives ranges from pH [less than or equal to] 1 to pH [approximately equal to] 2 [23]. Accordingly, dentin demineralization is more evident after the application of phosphoric acid because it completely removes the smear layer and opens dentinal tubules. The composition of adhesives and techniques of their application are also very important. Therefore, adhesives with organic solvents (ethanol, acetone) applied in wet bonding technique form long resin tags with large number of lateral branches [30]. In that way, a wide zone of resin infiltrated dentin is provided, as well as numerous and "branched" resin tags.

The SEM analysis of adhesive interface shows that a hybrid layer has not been observed on specimens with the self-etch adhesives and sound dentin, whereas it has been observed on specimens with total-etch adhesives. Accordingly, the null hypothesis has been rejected stating that there are no important differences in the microstructure of the interfaces between tested adhesive systems.

Conclusion

Total-etch adhesives form a clear zone of resin reinforced dentin (hybrid layer) and numerous and branched resin tags. Self-etch adhesives, due to their weak acidity, are not able to remove the smear layer and form a well-defined hybrid layer and long resin tags. The results of this study indicate that the total-etch adhesives bond better with sound dentin than self-etch adhesives.

In clinical conditions, this means that a better and stronger bond is made by total-etch adhesives. However, self-etch adhesives represent a step forward in the adhesive dentistry because the simplification of clinical procedures reduces the possibility of therapist mistakes. Still they have poorer adhesive interface than total-etch adhesives. Selective etching of enamel margins with phosphoric acid is highly recommended for better bonding of self-etch adhesives. Consequently, total-etch adhesives are still the gold standard in achieving the adhesive bond.

Abbreviations

SEM--scanning electron microscopy/skening elektronska mikroskopija


UDK 616.314-089.27:615.461

DOI: 10.2298/MPNS1502010D

Acknowledgements

This research was supported by the Ministry of Science Grant No 174005.

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Rad je primljen 15. IV 2014.

Recenziran 18. X 2014.

Prihvacen za stampu 21. X 2014.

BIBLID.0025-8105:(2015):LXVIII:1-2:10-16.

Milan DROBAC, Igor STOJANAC, Bojana RAMIC, Milica PREMOVIC and Ljubomir PETROVIC

University of Novi Sad, Faculty of Medicine Department of Dentistry of Vojvodina

Corresponding Author: Doc.dr Milan Drobac, Medicinski fakultet Novi Sad, Katedra za stomatologiju, 21000 Novi Sad, Hajduk Veljkova 12, E-mail: milandrobac74@gmail.com

Table 1. Chemical composition and instructions for use of the
tested adhesives

Tabela 1. Hemijski sastav i na?in aplikacije adhezivnih sredstava
uklju?enih u ispitivanje

Adhesive/            Chemical composition       Application
Manufacturer
Adheziv/               Hemijski sastav      Uputstvo za upotrebu
Proizvoda?

AdheSE One           Derivatives of bis-     Apply and agitate
(Ivoclar Vivadent,    acrylamide, water,       for 30 s; air
Schaan,               bis-methacrylamide    disperse until there
Liechtenstein)            dihydrogen            is no water
LOT M50900             phosphate, amino       movement; light-
                       acid acrylamide,      curing for 10 s.-
                        hydroxy alkyl        Aplikovati adheziv
                       methacrylamide,      na povrsinu dentina
                       silicon dioxide,        30 s koristeci
                          catalysts,        pokrete utrljavanja,
                         stabilizers-          visak adheziva
                     Bis-akrilamid, voda,    odstraniti ja-kom
                      bis-metakrila-mid      strujom vazduha do
                      dihidrogen fosfat,     dobijanja sjajnog
                          amino-acid        te?nog filma koji se
                     akrilamid, hidroksil   ne pokrece, polimer-
                     alkil metakrilamid,     izovati svetloscu
                      visokodispergo-van            10s.
                       silikon dioksid,
                        katalizatori,
                        stabilizatori.

Prime&Bond NT          PENTA, UDMA1, T-       Condition dentin
(Dentsply Caulk,        resin (cross-           surface with
Milford, DE,USA)     linking agent), 1 D-      Conditioner 36
LOT 0905000886           resin (small        (Dentsply DeTrey)
                         hydrophilic        for 15 s, rinse for
                         molecules),          10, remove water
                      butylated hydroxy      excess with cotton
                       toluene, 4-ethyl        pellet, apply
                           dimethyl          adhesive for 20 s
                        aminobenzoate,         using rubbing
                          cetylamine        movements, air blow
                        hydrofluoride,       gently 5 s, light
                       acetone, silica       cure 10s. ?etkati
                      nanofiller-pENTA,     povrsinu dentina sa
                        UDMA1, T-smola         Conditioner 36
                         (sredstvo za       (Dentsply DeTrey) u
                           unakrsno            trajanju 15 s,
                      povezivanje), 1 D-       ispirati 10 s,
                         smola (mali          odstraniti visak
                          hidrofilni        vode kuglicom vate,
                      molekuli), butila-     adheziv aplikovati
                           tizirani              pokretima
                      hidroksitoluen, 4-    utrljavanja tokom 20
                         etil dimetil        s, blagom strujom
                        aminobenzoat,       vazduha u tra-janju
                          ketilamin          5 s istanjiti sloj
                        hidrofluorid,            adheziva,
                        aceton, silika        polimerizo-vati
                          nanopunilo          svetloscu 10 s.

Table 2. Hybrid layer thickness

Tabela 2. Debljina hibridnog slojaucim

          1      2      3      4      5

PB/CXM   2.02   3.23   3.53   3.03   1.61
AO/TEC    0      0      0      0      0

          6      7      8      9      10

PB/CXM   1.92   2.83   4.44   2.49   1.69
AO/TEC    0      0      0      0      0

PB/CXM-Prime&BondNT-Ceram X Mono; AO/TEC-AdheSE One-Tetric EvoCeram

Table 3. Hybrid layer thickness-basic statistics parameters for
composite systems

Tabela 3. Osnovni statisti?ki pokazatelji kompozitnih sistema za
debljinu hibridnog sloja

KS       n    [bar.x]   Med.   Min.   Max.   [sigma]   Sk.     Ku.

PB/CXM   10    2.68     2.66   1.61   4.44    0.91     0.62   -0.18

KS-composite system, n-number of specimens, [bar.x]-mean, Med.-
median, Min.-inimum, Max.-maximum,, [sigma]-standard deviation,
Sk.-skewness, Ku.-kurtosis-KS-hompozitni sistem, n-broj uzoraha,
[bar.x]-srednja vrednost, Med.-mediana, Min.-minimalna vrednost,
Max.-maksimaln vrednost, [sigma]-standardna devijacija, Sk.-
skewness, Ku.-kurtozis PB-CXM-Prime&Bond NT-Ceram X Mono
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Title Annotation:Original study/Originalni naucni rad
Author:Drobac, Milan; Stojanac, Igor; Ramic, Bojana; Premovic, Milica; Petrovic, Ljubomir
Publication:Medicinski Pregled
Article Type:Report
Date:Jan 1, 2015
Words:4624
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