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Abstract

This work aimed at characterizing the interface between dentin and the resin-infiltrated dentin made following a self-etch primer application. The commercial two-component, self-etch, self-cure primer primer used in this study contains functional monomers, HEMA and acrylophosphonic acid.

Results demonstrated that the primer reacted rapidly within the first tens of seconds and then stabilized above a pH value of 4. Infrared spectra illustrated chemical bonding and the elimination of some water. The measurement of fluid flow through the dentin-self-etch resin interface indicated that the bonded interfaces obtained with the primer resulted in a great decrease in dentin permeability. Microscopy revealed morphological relationships between the natural and synthetic structures and here showed a continuous and thick hybrid layer without droplets and cylindrical-conical resin tags which represents a stable interface.

The results show that the combination of HEMA and acrylophosphonic acid leads to the formation of a hybrid layer and stable interface, with no indication of interference between the monomers.

Keywords

adhesive dentistry, resin-dentin bonding, acrylophosphonic acid, hydroxyethylmethacrylate, self-etching monomers.

Introduction

Obtaining an adherent and impermeable hybrid layer seems to be a prerequisite to promote immediate satisfactory performances and to prevent early chemical and mechanical degradation of the bonded interfaces. However, the process of hybridization to dentin is still a difficult challenge to be accomplished under the dependence of many factors related to dentin substrate and the adhesive system [1,2]. Dental primers are complex mixtures containing different monomers whose association should be studied [3]. The self-etching adhesive systems like the studied primer contain high concentrations of active monomer acids. Hydrophilic monomers such as HEMA are also present to help cross the smear layer and bind to the dentin substrate [4]. The smear layer is then incorporated into the hybrid layer [5,6]. A major advantage of HEMA relies on its ability to improve the miscibility between hydrophilic and hydrophobic monomers. As a consequence, the incorporation of this monomer to one step self-etch adhesive can prevent phase separation and therefore enhance the adhesive performance [7].

Acrylophosphonic acid (H₂L) contains two acid protons on a phosphonate group, one polymerizable acrylate and one ethyl ester group [8]. H₂L reacts with biological apatite to form two new products: brushite and calcium diphosphonate. Dissolution of the biological apatite by the acid-etch releases phosphate and calcium ions that combine to form brushite. The remaining dissolved calcium ions neutralize the acrylophosphonic acid to form an ionic salt of formula Ca (HL)₂. These salts may resist hydrolysis and thus contribute to bond stability [9]. The comonomer HEMA, water soluble, cannot interact ionically with hydroxyapatite (Hap). The most appropriate way to describe the interaction of HEMA with demineralized dentin is a solvation mechanism whereby the HEMA not only entangles the collagen fibrils from the outside but also inside, by dissolving into the peptidic polymer network [10]. In a continuation of previous research having characterized individually the role of the functional monomer acrylophosphonic acid in the formation of hybrid layers and of HEMA, we now aimed to study the effect of association of the two monomers on the formation of the interface.

This study investigated the effect of the primer containing the two monomers on dentin by several methods: physico-chemical and microscopic.

Materials and methods

Products used and experimental design

 The primer, based on phosphonic acid, polyacrylic acid and HEMA, used for this study is Multilink Primer (Ivoclar Vivadent, Schaan, Liechtenstein). The primer’s composition is listed in Table 1.

Table 1. Primer’s composition

Tested product	Manufacturer	Composition (in wt%)		Batch
Multilink Automix Primer	IVOCLAR VIVADENT, Schaan, Liechtenstein	Part A Water Initiators      Part B Phosphonic acid acrylate Hydroxyethyl methacrylate Methacrylate mod. polyacrylic acid      Stabilizers	85.7 14.3   48.1 48.1 3.8 < 0.02	R71805   R68256

Dentin powder was prepared as described previously [11]. Briefly, the crowns were removed from the roots, the pulp removed and the enamel carefully eliminated with a diamond burr under water irrigation. Dentin blocks were ground at low temperature in a grinder (K Janke & Kunkel Ika^®, Labortechnik, Wasserburg, Germany) by adding nitrogen into the grinding chamber. The resulting dentin powder was sieved to obtain a powder with an average particle size of 0.67 µm and then stored in the absence of moisture.

Titrations

The primer was characterized by measurements of pH and by acid-base titrations. Two acids, which are 30% phosphoric acid and 30% lactic acid solutions, were used as references in order to prove the reliability of our study.

Primer and reference acids were dissolved under stirring in a 50% ethanol/water solution. The pH values were measured (Ω Metrohm^® model 827 pH meter, Herisau, Switzerland) after dissolution and then after addition of dentin powder (100 mg of dentin for 500 mg of product) during 15 min at constant 30 s time intervals.

The dentin/product acid reaction was stopped by addition of water. Aliquots were taken after contact times of 10, 20, 40, 60, 300, and 900 s and then titrated with a 0.1N sodium hydroxide solution verified against a 0.10224 mol/l potassium hydrogenphthalate solution (Ω Metrohm ion analysis 702 SM Titrino with Metrodata software TiNet, Herisau, Switzerland). The pKa of primer and reference acids were determined at the pH value at half equivalence points being found by the computer assisted titration curve derivatives.

Surface energy

Contact angle values vary with the level of interaction between liquid and solid. Thus the relative wetting characteristics of a liquid-solid system can be described by contact angle data. The wettability of a solid surface by a liquid is judged by the magnitude of contact angle: the lower the contact angle, the more wettable the surface [12,13].

From human teeth, a slice was cut at the level of the dental crown in the middle, parallel to the occlusal side and at the top of the pulp chamber, using a saw at low speed (IsoMet, Buehler Ltd., Evanston, USA) equipped with a diamond disk (11-4244-15HC, Buehler Ltd., Evanston, USA). These samples of dentin represent the deep dentin, area where the number and the diameter of the tubuli are most important. A drop of the primer was deposited on each sample of non-etched dentin. The room temperature was maintained at 22°C. The profile of the primer drop was analyzed after several contact times by a Digidrop instrument (contact angle measurement instrument with computer aided image analysis) GBX, France. The software of the Digidrop directly measured the contact angles to the right and left of the drop, then the average was calculated. Each contact angle was measured three times for each contact time.

Infrared spectroscopy

The FT-IR spectrum of pure dentin powder, dentin treated with primer and dentin/primer treated with dichloromethane solvent were recorded with a Nicolet Impact 410 spectrometer in the DRIFT mode. The FT-IR was used to record spectra from 500 to 4000 cm⁻¹ with no sample preparation. In the case of dichloromethane solutions, the solvent was evaporated under a stream of ambient air until a dried product was obtained. Freshly mixed dentin/primer was immediately agitated and well washed with dichloromethane until all components were dispersed. After washing, the solid obtained was filtered under vacuum, collected and dried under a stream of ambient air until the solvent completely evaporated. Finally, the solid was analyzed by IR on the diamond surface.

Microscopy

Ten non carious human third molars were used within one week following extraction and stored at 4°C in a 1% chloramine T solution. The teeth were divided into two groups and prepared for evaluation with optical microscopy or with scanning electron microscopy. Dentin disks were cut parallel to the occlusal face using the low-speed saw equipped with a rotating diamond impregnated copper disk (11-4244-15HC, Buehler Ltd., Evanston, USA) under water spray and in deep dentin. Deep dentin is an area where the number and diameter of tubuli increase, thus providing the best conditions for the resin-dentin interaction mechanism [14]. The chemical interaction of the adhesive can vary significantly according to the dentin region [15]. The primer was applied to each group following the manufacturer’s instructions (Table 2). The cement Multilink Automix (Ivoclar Vivadent, Schaan, Liechtenstein) was applied to each specimen and photopolymerized for 20 seconds.

Table 2. Protocol for the use of the primer

Tested product	Protocol
Primer (Parts A and B)	Rinse dentin with water and dry without excess; Mix the parts A and B (1 drop/1 drop), apply the mixture on the dentin surface and penetrate for 15-20 seconds using a micro-brush. Remove the excess of Multilink Primer using a flow of air until the liquid film is more visible.

Specimens preparation for optical microscopy

The dentin disks were fixed in 10% formaldehyde (Rhone-Poulenc Ltd., Manchester, England) for 2 days, demineralized with trichloroacetic acid (Merck, Darmstadt, Germany) for 5 days and rinsed in water. The specimens were dehydrated in ascending grades of alcohol then cleared in toluene for 2 hours before being impregnated in paraffin (Merck, Darmstadt, Germany) for 2 days and cut in 5 µm sections with a microtome (Reichert–Jung NuBlock Germany). The sections were successively stained with Goldner’s trichrome and with crystal violet and picric acid [16]. The stained sections were observed and photographed using an optical microscope (020-507-101 Wild Leitz GmbH, Heerbrugg, Switzerland).

Specimens preparation for scanning electron microscopy

The dentin disks from each subgroup were used for scanning electron microscopy. They were fractured into two parts, decalcified in phosphoric acid for 20 s and deproteinised for 60 s in 2% sodium hypochlorite. The specimens were fixed in 2.5% glutaraldehyde, then rinsed and dehydrated in alcohol. They were dried by immersion in pure hexamethyl disilazane (HMDS) for 20 min. The HMDS was allowed to evaporate for 15 min in air before specimens were sputter-coated with gold-palladium. The specimens were examined and photographed with a SEM (S450, Hitachi, Tokyo, Japan) using an acceleration voltage of 15 kV. The structures analyzed were the architecture of the interface, the thickness and the homogeneity of the hybrid layer, the morphology of the tags. To standardize the observations, micrographs of the interface between the primer and dentin substrate were all taken at magnifications of 1500X and 2500X.

Hydraulic conductance

Change in dentinal permeability obtained after application of a primer or an adhesive was evaluated by measuring hydraulic conductance.

The hydraulic conductance is defined as the volume of fluid passing through a known surface per unit time under a constant pressure gradient. A fluid pressure on a given surface induces a value of dentinal flow. In this study, it was measured by the Flodec system (De Marco Engineering, Geneva, Switzerland) connected to a computer.

The fluid flow through a dentin slice was evaluated by the passage of physiological saline solution under a pressure of 200 cm H₂O over a surface of 0.28 cm². More precise details were reported previously [17].

Specimens preparation

Ten extracted non-carious human third molars from patients 18–25 years old were used for this part of the study. Dentin disks were cut from crown segments parallel to the occlusal surface at the top of the pulp chamber, using a low-speed saw (IsoMet, Buehler Ltd., Evanston, USA) equipped with a rotating diamond impregnated copper disk (11-4244-15HC, Buehler Ltd., Evanston, USA). The dentin specimens were considered to represent deep dentin, an area where the number and diameter of dentinal tubuli increase. Deep dentin was chosen to favor the infiltration of the dentin bonding systems into the demineralized dentin, thus providing ideal conditions for the resin-dentin interaction [18]. The specimens were reduced to a thickness of 1 mm by polishing with progressively finer abrasive disks (Pregrinder; Knuth-Rotor Struers, Copenhagen, Denmark) to create a smooth, uniform surface on both sides. All polishing procedures were performed by the same operator, who measured the thickness with electronic digital calipers (RS232C; Colombié-Cadet, Toulouse, France) with an accuracy of within 0.03 mm. Any specimens in which presence of the pulp horn or enamel could be detected were eliminated, the pulpal surface was centered on a polycarbonate disk made in the authors’ laboratory with a 6 mm diameter hole and glued using epoxy glue. The assembled elements were dried for 24 h before testing.

The technique compared the hydraulic conductance before and after the introduction of the primer. The application of the primer will close off a portion of the dentinal tubuli, resulting in a decrease of the initial permeability of dentin, depending on the level of sealing due to the bonding adhesive: the lower the permeability is, the better is the sealing. Every untreated dentin has a different permeability according to many factors, so all subsequent permeability measurements made on the same specimen were expressed as a percentage increase or decrease with respect to the initial hydraulic conductance of the untreated specimen. Thus each specimen served as its own control.

Measurement of hydraulic permeability

The initial hydraulic flow through the slice of the dentin was evaluated by measuring the passage of physiologic saline (T0). An experimental, standardized smear layer was produced on the occlusal surface of each specimen by means of 50 consecutive abrasions executed by manually moving very fine grained sandpaper (P4000, Buehler Ltd., Evanston, USA) horizontally under water irrigation. The hydraulic conductance was measured again after this experimental abrasion at time T1. The dentin slides received the primer according to the manufacturer’s instructions (Table 2). The hydraulic conductance was measured after application at time T2.

Table 3. pH values of primer after different contact times with dentin

Contact time (s)	pH values near pKa 1	pH values near pKa 2
0	3.30	6.70
10	4.35	7.42
20	4.35	7.51
40	3.97	6.55
60	3.90	7.39
300	4.01	7.42
600	3.87	6.94
900	4.05	7.06

The statistical analysis comprised an analysis of the mean and standard deviation.

Results

Titrations

Upon dentin powder introduction into the primer and the references, the pH values went from acidic to neutral values. The initial pH values and its changes as a function of contact time with dentin powder were for the primer from 3.10 to 3.62, for 30% phosphoric acid, from 2.47 to 2.73 and for 30% lactic acid from 3.16 to 3.75. The pH values showed that the primer had almost the same behavior as lactic acid (Figure 1), while phosphoric acid had the highest acidity observed at all times compared to the lactic acid and primer.

Figure 1. pH variations as a function of contact time with dentin powder. With the primer, variation of pH is from 3.10 to 3.62.

Primer and reference acids titrations with 0.1N NaOH showed different titration curves corresponding to different proton numbers. The pKa values were calculated at the pH value of the half equivalence points (pH_eq) (Figure 2). The variation of pH values as a function of NaOH (0.1N) volume showed that the primer had two pKa values, pKa1 at 3.30 for the strong proton and pKa2 at 6.70 for the second proton. Similar results showed in the case of phosphoric acid that pKa1= 2.61 and pKa2= 7.09, whereas, the lactic acid has only one pKa at 3.81. This shows that the primer and lactic acid have low acidity compared to phosphoric acid.

Figure 2. pH_eq and pKa’s values of primer and reference acids as a function of sodium hydroxide (0.1N) volume. The pKa’s are determined at the pH value of half equivalence points (pH_eq).

The evolution of the pH values was evaluated as a function of time. From time 0 to time 900 s, the pH near the pH_eq1 varied from 3.30 to 4.05 and the pH near the pH_eq2 from 6.70 to 7.06. As for the 30% phosphoric acid, the pH near the pH_eq1 varied from 2.61 to 2.71 and the pH near the pH_eq2 decreased from 7.09 to 7.00; as for the 30% lactic acid, the pH values ranged from 3.81 to 4.02. Table 3 shows small fluctuations in pH values (with estimated errors of 0.1 unit) as a function of time near the pKa1 value of the primer. However these pH values follow a visible trend which illustrates the neutralization of the acid primer during the buffering action with hydroxyapatite. The results demonstrated that the acid reacted rapidly within the first tens of seconds and then stabilized above a pH value of 4. The pH values near the second pKa remained in the neutral zone.

Surface energy

The values of the contact angles as well as their evolution are shown in Table 4.

Table 4. Contact angles observed between primer and dentin, representing the average of 3 determinations for each contact time and the standard deviation

Liquid test	Support	Time (s)	Contact angle (SD)
Primer	Dentin	30	15.7 (0.7)
		60	13.6 (0.8)
		120	11.9 (1.1)
		180	11.5 (0.4)

When a drop of primer was left on the dentinal surface, the values of the contact angles changed from 45.8° to 11.5° after 180 s. Actually those values decreased rapidly between time 0 and 30 s, and afterwards did not change much.

Infrared spectroscopy

As the studied primer is made of a large number of diverse molecules, we indexed their spectra by attributing adsorption bands in order to characterize the main functional groups.

The infrared spectrum of dentin (Figure 3a), showed absorption peaks attributed to the organic phase in dentin (amide peptide linkages near 1600 cm^-1) and to the mineral species (carbonates at 1450 cm^-1 and phosphates at 1100 cm^-1 and 600 cm^-1) and a broad water peak near 3300 cm^-1. The spectrum of the dentin was typical of a mixed organic and carbonated mineral composition.

Figure 3. FT-IR spectra of dentin (a), dentin mixed with primer (b), dentin with primer and washed with CH₂Cl₂ (c).

The addition of primer to the dentin powder (Figure 3b) resulted in the appearance of new peaks related to the phosphonic acid corresponding to the presence of the chemical bonds (C-H) at 2959 cm^-1 and (C=O) around 1715 cm^- 1; as well, the double bond (C=C) at 1636 cm^-1. Note the presence of a peak around 1200 cm^-1 characteristic of the (C=O) group related to the acrylate functional group. In spectrum 3c, the main idea of using dichloromethane as a solvent was to dissolve excess polymer. After dichloromethane evaporation, the solid obtained resulted in a spectrum with the same peaks occurring as in the spectrum 3a but with additional peaks near 2900 cm^-1, and a deceased water absorption at 3400 cm^-1. When dentin particles were suspended into the extracted dichloromethane solution, the organic molecules reacted with the calcium phosphate and the typical absorption bands were present in the spectra of the filtered and dried precipitate. They remained even following washing, indicating the formation of insoluble surface complexes.

Microscopy

Interface structure observed by optical microscopy : Representative optical micrographs using crystal violet and picric acid are shown in Figure 4 and 5. The mineralized dentin is yellow, the hybrid layer and the tags are bright pink, and the adhesive is beige. The observation showed a regular hybrid layer and numerous tags.

Figure 4. Representative light micrograph of the dentin with the self-etch primer. The hybridized complex (HL) is thick and continuous with numerous tags (original magnification 180X).

Figure 5. Representative optical micrograph showing the primer-dentin interface. Tags are numerous (T) and thick with V-shaped at the junction (original magnification 320X).

Interface structure observed by scanning electron microscopy: Representative SEM micrographs of the dentin interfaces obtained with the adhesive following the technique recommended by the manufacturer on normally humid dentin are shown in Figure 6 and 7. The interface obtained had a regular and thick hybrid layer which globally averaged to 1.1 µm and tags up to 50 µm long, without droplets.

Figure 6. SEM micrograph showing the primer-dentin interface. Note the uniform hybridized complex (HL) and the numerous tags (T) (original magnification 1500X).

Figure 7. SEM micrograph showing the primer-dentin interface. A thick hybrid layer (HL) can be seen, with numerous tags (T) (original magnification 2500X).

Hydraulic conductance: The creation of a smear layer entailed a mean decrease of permeability of 25.9% ± 9.9 with relatively high standard deviations with respect to the mean value, indicating differences among the dentin specimens used. The variability in dentin specimens justified the use of each specimen as its own control. Analyzing the ‘‘adhesive’’ factor by comparing the mean and standard deviation revealed that the primer led to a reduction in permeability of 48.1% ± 8.0.

Discussion

2-Hydroxyethylmethacrylate (HEMA) was a key ingredient in the development of the hybrid layer concept [10]. Acid etching of burred dentin is used to remove the smear layer and demineralize the exposed collagen fibrils. The subsequent water rinsing step effectively eliminates the water-soluble phosphate and calcium ions and leaves behind water-soaked collagen fibrils in the expanded state. The intended use of hydrophilic HEMA was then to infiltrate the demineralized dentin and prevent collagen collapse. Bis-GMA is acknowledged to form a composite material with collagen but is too hydrophobic to penetrate the wet demineralized zone. It was reported that the mixture of HEMA and bis-GMA monomers was operational in forming the hybrid layer. HEMA was thought to allow water sequestration at the dentin hybrid layer interface. Contemporary self-etch resins have low HEMA content, in an effort to minimize mobile water phases or lacunae. On the other hand several contemporary self-etch dental adhesives are associated with acrylophosphonic acid monomers to combine the effect of acidity in formation of the hybrid layer. Thence, in this study we showed the effect of combination of the adhesives previously cited and the reaction mechanism as a function of acidity. The pH and pKa values of an acid were generally considered as the major parameter that determines how molecules interact with mineralized tissues. The aggressiveness of monomers was evaluated by its ability to dissolve the HA and was correlated to acid strength and pH values [19,20]. Yoshida, et al. [21] and Salz, et al. [1] have studied acid monomers alone in order to point out self-etch aggressiveness. According to our results, we confirm dentin demineralization and demonstrate that the combination of phosphonic acid with HEMA allowed them to react together as they did when isolated.

The primer had almost the same behavior as lactic acid (Figure 1,2) and the fact that lactic acid dissolves more calcium despite its low acid strength was related to the chelating effect of the hydroxycarboylic acid and the water solubility of its calcium salt. This result demonstrated that the primer demineralized dentin [22].

Previous investigations have concluded that primary and important chemical bonding of HEMA to dentin components does not occur [10]. However, some workers claimed that HEMA interacts with collagen both physically and chemically. Infrared spectra show the typical absorption bands for the acrylate functional group near 1715 cm^-1 and phosphonic stretching frequencies at 1091 cm⁻¹. Acrylophosphonic acid and HEMA, acting together as a primer, flowed into the open tubules and the inter-fibrillar spaces to envelop the collagen fibrils. The combination of acrylophosphonic acid and HEMA led to the homogeneous demineralization of the dentin and maintained the demineralized collagen in the expanded state. The main role of dichloromethane (CH₂Cl₂) as solvent was in the clarification of chemical bonding and in the elimination of the molecular water which was shown by the infrared spectrum (Figure 3c) with less intense absorption at 3400 cm^-1.

The goal was to create a hybrid layer and obtain a leak-proof interface. Microscopy revealed morphological relationships between the natural and synthetic structures and here showed a continuous and uniform hybrid layer and a forest of laterally branched cylindrical-conical resin tags which represent a stable interface. Hydraulic conductance, a non-destructive method that does not require dehydration of the samples and allows tests to be performed in close-to-physiological conditions with the implicit role of pulpal pressure has already been used successfully in studies of water fluid flow within resin–dentin bonds during and after bonding procedures with total-etch and self-etch adhesives [16-18,23-25]. The results of this study indicated that the bonded interfaces obtained with the primer resulted in a significant decrease in dentin permeability. Thus, the null hypothesis tested was validated.

The role of HEMA in the proper formation of a hybrid layer was established long ago. The essential characteristics if HEMA comprise dissolution potential for polar and non-polar solvents or molecules, infiltration through smear layers and compatibility with water saturated collagen. HEMA is thought to act as a carrier for more complex molecules that can flow together with it through the demineralized collagen. On the other hand, HEMA does not carry any acidic functional groups so cannot by itself demineralize dentin. The small molecular weight of HEMA is considered as an advantage for penetrating the substrate of demineralized dentin and was found by NMR analysis to be very mobile [17]. It is however an acrylic-substituted alcohol liable to favor water uptake from biological tissue, and this is why manufacturers try to avoid HEMA in the hopes of preventing hydrolysis of hybrid layers formed in dentin milieu [7]. Comparison of HEMA containing and HEMA-free primers found that the presence of HEMA in the self-etch adhesive systems evaluated was not a predominant factor that influenced their bond strength to dentin [26]. Another study reported that experimental conditions significantly reduced μTBS of the HEMA-free adhesives, while the HEMA-containing adhesives exhibited no significant differences [27]. In this work, HEMA was present together with acrylophosphonic acid and water. The need for water is to express the acidity of the monomer and dissolve free calcium ions and phosphates. The main role of acrylophosphonic acid is to etch dentin and participate in hybrid layer formation [4,8]. It may thus be regarded that HEMA, water and acrylophosphonic acid work in synergy. It is possible that the acrylic functional group of HEMA, co-polymerized with acrylophosphonic acid, forms a higher molecular weight entity, less prone to water uptake. In any case our results show that the combination of HEMA and acrylophosphonic acid work in synergy and leads to the formation of a hybrid layer, with no indication of interference between the monomers.

Conflicts of interest statement

No conflicts of interest

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