Apatite crystal protection against acid-attack beneath resin–dentin interface with four adhesives: TEM and crystallography evidence

Abstract

Objectives

Interaction between specific functional groups and apatite crystals may contribute to adhesion. The present study investigated effects of four adhesives with different compositions on protection of crystals beneath the hybrid layer against acid-attack using transmission electron microscopy (TEM) and selected area electron diffraction (SAED).

Methods

Human dentin was bonded with four adhesives; two with a carboxylic-based functional co-polymer (PAA): three-step etch-and-rinse Scotchbond Multi-Purpose (SMP, 3M ESPE) and one-step self-etch Adper Easy Bond (AEB, 3M ESPE), and two with a phosphate-based functional monomer (MDP): two-step etch-and-rinse Clearfil Photo Bond (CPB, Kuraray Medical) and two-step self-etch Clearfil SE Bond (CSE, Kuraray Medical). The specimens were the either left untreated (control) or subjected to acid–base challenge with demineralizing solution (pH 4.5) and 5% NaClO. All specimens were processed and observed by TEM. SAED was used to identify the presence or absence of apatite crystallites at the base or beneath hybrid layer before and after acid–base challenge.

Results

An apatite-rich zone was observed beneath the partially demineralized hybrid layer of CSE. The zone was thinner in AEB, but a demineralization-susceptible area was found beneath it. The etch-and-rinse adhesives (SMP and CPB) demonstrated completely or predominantly demineralized hybrid layers, which were devoid of the acid-resistant apatite-rich zone.

Significance

TEM/SAED evidence disclosed that the preserved dentin apatite crystals beneath the thin hybrid layer of the mild self-etch adhesives were protected against acid. Diffusion of reactive components beyond the hybrid layer, and their chemical bonding potential with the remaining crystals created the acid–base resistant zone.

Introduction

The ultimate goal in adhesive dentistry is to achieve a durable bond to dental tissues. Bonding to dentin was conventionally achieved by three main steps namely etching, priming and bonding, each applied as a separate agent . Self-etching primer systems combined the etching and the priming steps into one, while the bonding agent remained as a separate agent. More recently, one-step self-etch or all-in-one systems combined the self-etching primer and the bonding agent into one application . Adhesion to dentin substrate has been primarily attributed to micro-mechanical hybridization which involves infiltration and subsequent in situ polymerization of adhesive monomers within the demineralized microporous collagen scaffold . In addition, it was suggested that chemical interactions between carboxyl or phosphate ester groups of functional monomers in adhesives and apatite contributed to bonding .

Despite extensive morphological studies on the interface and small-scale destructive mechanical tests, the interactions of adhesive components with the structural units of hard-tissues ( i.e. biological apatite crystals in dentin) in situ , and the properties of the complexes formed to confirm actual intermolecular interactions have been investigated to a limited extent . In addition to ultrastructural observation, application of local analytical techniques on the interface of biomaterial-hard tissue would benefit further progress of the adhesive technology. Among those analytical techniques, selected area electron diffraction (SAED) is a crystallography method that provides information on the local crystalline structure of thin sections under transmission electron microscopy (TEM); however, only few studies to date have employed crystallography on the interface after demineralization challenge .

Recent studies revealed an acid–base resistant zone (ABRZ) beneath the visible hybrid layer when dentin was treated with specific functional monomers as part of mild self-etch adhesives . Based on the attributes of this zone, which was more resistant against acidic and basic attack compared to the underlying normal dentin, a dentin reinforcement concept was proposed . However, it is unknown whether apatite crystals remaining beneath the hybrid layer were acid-resistant after phosphoric-acid-etching step in the etch-and-rinse approach, with adhesives containing similar functional monomers, such as 10-methacryloyloxydecyl dihydrogen phosphate (MDP). In addition, little information is available on the acid-resistance of dentin treated with polyalkenoic acid (PAA)-based adhesives, considering the well-proven primary ionic interaction between the carboxyl groups of the PAA and the calcium (Ca) of apatite .

Therefore, the aim of the present study was to investigate the protection of crystals beneath the hybrid layer against acid-attack following application of commercially available PAA- or MDP-based etch-and-rinse or a self-etch adhesives. The null hypothesis tested was that the thickness of an apatite containing layer remaining after acid–base challenge was not different using various adhesive approaches containing different functional groups.

Materials and methods

Adhesives and bonding procedures

The chemical compositions and the manufacturers’ instructions of the four adhesives used in this study are listed in Table 1 . The three-step etch-and-rinse system, Scotchbond Multi-Purpose (SMP; 3M ESPE, St Paul, MN, USA) contains about 10–20 wt% carboxylic-based functional co-polymer of PAA. The two-step etch-and-rinse system, Clearfil Photo Bond (CPB; Kuraray Medical, Tokyo, Japan) contains about 5–10 wt% phosphate-based functional monomer of MDP. The two-step self-etch system, Clearfil SE Bond (CSE; Kuraray Medical) also contains about 25–30 wt% MDP. The one-step self-etch system, Adper Easy Bond (AEB; 3M ESPE) contains about 1–5 wt% PAA, and 5–15 wt% of a phosphoric acid monomer.

Table 1
The composition, pH, batch number and application procedure of the tested adhesives.
Adhesive systems Components a pH Batch number Applications b
Scotchbond multi-purpose; SMP 3-step etch-and-rinse (3M ESPE; St Paul, MN, USA) Etchant gel: 37% phosphoric acid 0.6 a (15 s), b, e, f, g, h (10 s)
Primer: PAA, HEMA, water 3.3 N211706 (primer)
Bond: Bis-GMA, HEMA, initiators 8.2 N207729 (bond)
Clearfil photo bond; CPB 2-step etch-and-rinse (Kuraray Medical; Tokyo, Japan) K-Etchant: 40% phosphoric acid 0.6 a (15 s), b, c, d, f, h (10 s)
Catalyst liquid: MDP, HEMA, Bis-GMA, hydrophobic DMA, BPO, CQ 00448A (catalyst)
Universal liquid: N,N-diethanol p-toluidine, sodium benzen sulfinate, ethanol 2.5 (mixture) 00544A (universal)
Clearfil SE bond; CSE 2-step self-etch (Kuraray Medical) Primer: MDP, HEMA, hydrophilic DMA, CQ, water 2.0 01049 A (primer) e (20 s), f, g, h (10 s)
Bond: MDP, HEMA, Bis-GMA, hydrophobic DMA, CQ, N,N-diethanol p-toluidine, silanated colloidal silica 2.0 01566 A (bond)
Adper easy bond; AEB 1-step self-etch (3M ESPE) PAA, phosphoric acid-methacryloxy-hexylesters, Bis-GMA, HEMA, 1,6 hexanediol DMA, silane-treted silica, CQ, ethanol, water 2.8 415031 d (20 s), f, h (10 s)

a Abbreviations : BPO, benzoyl peroxide; Bis-GMA, bisphenol A diglycidylmethacrylate; CQ, camphorquinone; DMA, dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; MDP, 10-methacryloxydecyl dihydrogen phosphate; PAA, polyalkenoic acid co-polymer.

b (a) acid-etch; (b) rinse and blot dry; (c) mix each drop of adhesive catalyst and universal liquid; (d) apply adhesive; (e) apply primer; (f) gently air dry; (g) apply bond; (h) light-cure.

The study set-up is illustrated in Fig. 1 . Twelve freshly extracted non-carious third molars were used following the guidelines approved by the Tokyo Medical and Dental University Ethical Committee. A 1.5-mm-thick dentin disk was prepared from mid-coronal dentin using a low-speed diamond saw (Isomet; Buehler, Lake Bluff, IL, USA) under water cooling. The disk was covered with acid-resistant nail varnish (Shiseido, Tokyo, Japan) except for a window (2.5 mm × 2.0 mm) in the center of the mid-coronal dentin surface. The discs were divided into two halves at the center of the window; the crosscut surface on each hemisection was wet-sanded with 600-grit SiC papers to create a standardized smear layer. Three pairs of half-disks obtained from three teeth were randomly assigned to each of the adhesives. The adhesives were then applied on the ground dentin surfaces of the teeth that were to be bonded with the etch-and-rinse adhesives were etched for 15 s with 37–40% phosphoric acid or treated with the self-etching adhesive according to the manufacturers’ instructions. The adhesive was light-cured using a visible light curing unit (Optilux 501; Demetrom, Danbury, CT, USA) at an output of 600 mW/cm 2 prior to the incremental placement of a hybrid composite (Z100; 3M ESPE).

Fig. 1
Schematic drawing for the sample preparation and visualization under the TEM and SAED analysis. Ultramorphological features and apatite crystallites at resin–dentin interface after acid–base challenge were studied by TEM and SAED.

Acid–base challenge

After 24-h storage at 37 °C in water, the specimens were either left untouched (control) or subjected to the acid–base challenge as follows. First, 100 ml buffered demineralizing solution (pH 4.5, 2.2 mmol/l CaCl 2 , 2.2 mmol/l NaH 2 PO 4 and 50 mmol/l acetic acid) was used for 90 min to create artificial secondary caries . After the acid-challenge, the specimen was subjected to 5% NaClO for 30 min with ultrasonic vibration, in an attempt to remove any denatured dentin collagen fibrils, and finally rinsed with running water for 30 s . The specimens were subjected to the acidic and basic solutions (25 °C) in a beaker with a magnetic stirrer to ensure complete access of acid to all of the exposed surfaces.

Transmission electron microscopy

The interface of the adhesive and dentin in the control and acid–base challenged specimens was observed under transmission electron microscope (TEM). To facilitate ultramicrotomy, the specimens were trimmed into rectangular blocks (1 mm 1.5 mm 2 mm) using the diamond saw. The blocks were processed for TEM according to the procedure described in detail previously . Non-demineralized, epoxy-resin-embedded sections 90 nm in thickness were prepared and observed under TEM (H-7100; Hitachi, Tokyo, Japan) operating at 75 kV. Selected area electron diffraction (SAED) patterns were performed using an aperture that selected a 200-nm-diameter area of the TEM section; the d -spacings of the diffraction patterns were calibrated using the d -spacings of gold determined under identical conditions . SAED provided corroborative evidence for the presence or absence of apatite crystallites at the base and beneath hybrid layer before and after acid–base challenge.

Statistical analysis

The thicknesses of the hybrid layer and apatite-rich zone were measured using image analysis software (ImageJ, NIH, Bethesda, MD, USA). Ten representative images were considered for each adhesive. Data were statistically analyzed by one-way ANOVA and Tukey’s multiple comparison tests with statistical significance set at α = 0.05.

Materials and methods

Adhesives and bonding procedures

The chemical compositions and the manufacturers’ instructions of the four adhesives used in this study are listed in Table 1 . The three-step etch-and-rinse system, Scotchbond Multi-Purpose (SMP; 3M ESPE, St Paul, MN, USA) contains about 10–20 wt% carboxylic-based functional co-polymer of PAA. The two-step etch-and-rinse system, Clearfil Photo Bond (CPB; Kuraray Medical, Tokyo, Japan) contains about 5–10 wt% phosphate-based functional monomer of MDP. The two-step self-etch system, Clearfil SE Bond (CSE; Kuraray Medical) also contains about 25–30 wt% MDP. The one-step self-etch system, Adper Easy Bond (AEB; 3M ESPE) contains about 1–5 wt% PAA, and 5–15 wt% of a phosphoric acid monomer.

Table 1
The composition, pH, batch number and application procedure of the tested adhesives.
Adhesive systems Components a pH Batch number Applications b
Scotchbond multi-purpose; SMP 3-step etch-and-rinse (3M ESPE; St Paul, MN, USA) Etchant gel: 37% phosphoric acid 0.6 a (15 s), b, e, f, g, h (10 s)
Primer: PAA, HEMA, water 3.3 N211706 (primer)
Bond: Bis-GMA, HEMA, initiators 8.2 N207729 (bond)
Clearfil photo bond; CPB 2-step etch-and-rinse (Kuraray Medical; Tokyo, Japan) K-Etchant: 40% phosphoric acid 0.6 a (15 s), b, c, d, f, h (10 s)
Catalyst liquid: MDP, HEMA, Bis-GMA, hydrophobic DMA, BPO, CQ 00448A (catalyst)
Universal liquid: N,N-diethanol p-toluidine, sodium benzen sulfinate, ethanol 2.5 (mixture) 00544A (universal)
Clearfil SE bond; CSE 2-step self-etch (Kuraray Medical) Primer: MDP, HEMA, hydrophilic DMA, CQ, water 2.0 01049 A (primer) e (20 s), f, g, h (10 s)
Bond: MDP, HEMA, Bis-GMA, hydrophobic DMA, CQ, N,N-diethanol p-toluidine, silanated colloidal silica 2.0 01566 A (bond)
Adper easy bond; AEB 1-step self-etch (3M ESPE) PAA, phosphoric acid-methacryloxy-hexylesters, Bis-GMA, HEMA, 1,6 hexanediol DMA, silane-treted silica, CQ, ethanol, water 2.8 415031 d (20 s), f, h (10 s)
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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Apatite crystal protection against acid-attack beneath resin–dentin interface with four adhesives: TEM and crystallography evidence
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