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Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden 1 2 3

Effects of the application sequence of calcium-containing desensitising pastes during etch-and-rinse adhesive restoration

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Q1

Hongye Yang a,1, Dandan Pei a,b,1, Zhiyong Chen a, Jinmei Lei a, Liqun Zhou a, Cui Huang a,* a

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory for Oral Biomedical Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, People’s Republic of China b Department of Prosthodontics, Stomatology Hospital of Xi’an Jiaotong University College of Medicine, Xi’an, Shaanxi, People’s Republic of China

article info

abstract

Article history:

Objectives: To evaluate the effects of different application sequence of calcium-containing

Received 16 November 2013

desensitising pastes on bonding effectiveness and tubule occlusion during etch-and-rinse

Received in revised form

(E&R) adhesive restoration.

16 March 2014

Methods: Seventy molars were sectioned parallel to the occlusal plane, polished and randomly

Accepted 31 March 2014

divided into seven groups (n = 10). Group 1 was etched with 35% phosphoric acid for 15 s.

Available online xxx

Groups 2–4 were treated with different calcium-containing desensitisers, including an argi-

Keywords:

calcium phosphate (CPP-ACP)-containing paste and a calcium-sodium phosphosilicate (Nova-

Dentine hypersensitivity

min)-containing paste, respectively. Afterward, these groups were etched with 35% phospho-

Desensitising paste

ric acid for 15 s. Groups 5-7 were initially etched and then treated with Arg-CaCO3-, CPP-ACP-

nine-calcium carbonate (Arg-CaCO3)-containing paste, a casein phosphopeptide-amorphous

Application sequence

and Novamin-containing desensitisers, respectively. In each group, samples were equally

Dentine bonding

distributed into two subgroups (n = 5) to bond with either a two-step E&R adhesive Adper

Etch-and-rinse adhesive

SingleBond 2 (SB) or a three-step E&R adhesive Adper ScotchBond Multi-Purpose (SBMP). The microtensile bond strengths (MTBS) were tested and fracture modes were analyzed by stereomicroscopy and field-emission scanning electron microscopy (FESEM). Eight additional dentine disks were prepared to evaluate tubule occlusion prior to bonding using FESEM. Results: The application sequence of calcium-containing desensitising pastes did not significantly affect MTBS irrespective of two-step SB (P > 0.05) or three-step SBMP E&R adhesives (P > 0.05). Effective dentinal tubule occlusion was observed in the mode of etching-desensitising. Conclusions: Applying calcium-containing desensitisers (particularly Arg-CaCO3- and Novamin-based) after etching during E&R adhesive restoration could achieve effective tubule occlusion without affecting the bonding strength. # 2014 Published by Elsevier Ltd.

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12 * Corresponding author. Tel.: +86 27 87686130; fax: +86 27 87873260. E-mail address: [email protected] (C. Huang). 1

These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jdent.2014.03.018 0300-5712/# 2014 Published by Elsevier Ltd.

Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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1.

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Dentine becomes exposed because of wear, attrition, trauma, caries, non-caries cervical lesions or cavity preparation.1,2 When exposed, dentine contacts an external stimulus, such as chemical, thermal, evaporative, tactile or osmotic stimulus, causing a transient, sharp pain or dentine hypersensitivity, which has become a common complaint of adults.3,4 According to ‘‘hydrodynamic theory’’5 and Poiseuille–Hagen equation, the occlusion of dentinal tubules should be effective to reduce dentine permeability and thus manage dentine hypersensitivity.6 Among various tubule-occluding products, several new calcium-containing desensitising pastes have been introduced because these materials can simulate natural desensitising processes.7,8 Arginine-calcium carbonate (Arg-CaCO3)containing desensitising paste can be used to seal exposed dentinal tubules effectively and provide reliable relief for hypersensitivity.7,9 Arginine and calcium carbonate from ArgCaCO3-containing paste can work together to accelerate natural occlusion mechanisms by depositing dentine-like minerals in dentinal tubules and by forming a protective layer on dentine surfaces.10 Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complex, a milk protein derivative, can also remineralise dentine by buffering free calcium and phosphate ion activities.11–13 According to the manufacturer, CPP-ACP incorporated in commercially available paste can reduce tooth sensitivity by occluding dentinal tubules with remineralised dentine. Calcium-sodium phosphosilicate (Novamin) is a bioactive glass originally developed in advanced bone regeneration material.14 Novamin-containing desensitising paste can deposit hydroxycarbonate apatite and reduce sensitivity by releasing billions of calcium phosphate ions that would be available to the natural remineralisation in the mouth.15–17 In clinical practice, tooth sensitivity is observed as a result of mechanical tooth preparation during adhesive restoration. New calcium-containing desensitising pastes should be used before bonding to occlude dentinal tubules, thereby managing immediate sensitivity and preventing post-operative sensitivity during and after adhesive restoration.18–20 Etch-and-rinse (E&R) adhesive is by far a classic and reliable adhesive system preferred over self-etching adhesives; E&R adhesive is more suitable in handling complicated dentine surfaces than selfetching adhesives.21,22 Therefore, studies have focused on the compatibility of calcium-containing desensitising pastes used with E&R adhesive systems.23–28 The desired effect is to block dentinal tubules tightly and impede the occurrence of dentine hypersensitivity, including pulpitis and other adverse consequences, without affecting the final bond strength, which is a major problem in clinical applications. Researchers usually opt to apply calcium-containing desensitising paste after preparing the teeth and before etching of E&R adhesive.23–25 Because according to conventional thinking, the sequential application of etchant (primer or not) and bonding agent are taken for granted as an inextricable whole of bonding procedure for E&R adhesive system and this approach can minimise the negative effect of desensitisation on bonding strength at the highest extent.24 However, it is worth noting that this strategy (desensitising-etching) may not remove the smear

Introduction

layer produced by tooth preparation; such a layer is usually composed of small particles of mineralised collagen matrix, inorganic debris, blood, saliva and numerous microorganisms.29 These impurities spread over dentine surfaces or within dentinal tubules; this condition possibly induces pulp inflammation or infection and impedes the infiltration of desensitising paste. Another disadvantage of this application sequence involves the second step of 35% phosphoric acid after desensitisation; may dissolve and remove previous precipitated carbonate crystals on dentine surfaces and reopen the occluded dentinal tubules, resulting in an outward flow of tubule fluid. As such, desensitisation may fail to control and prevent postoperative sensitivity.30 What will happen if the application sequence of desensitising and etching is reversed? We speculated that, in this new strategy (etching-desensitising), the first step of etching involves the removal of the smear layer; therefore, the occurrence of bacterial infection and pulp disease is likely decreased. In the second step, calcium-containing desensitising pastes are applied; pastes can adequately penetrate and occlude open dentinal tubules, thereby impeding the outward flow of tubule fluid and therefore managing dentine hypersensitivity and preventing the occurrence of post-operative sensitivity. However, the speculation remains unconfirmed. Furthermore, in these two different application sequences (desensitising-etching or etching-desensitising) of calciumcontaining pastes, the smear layer is either retained or removed; different treatment then inevitably changes the property of dentine surfaces and possibly provides a different bonding substrate, which may elicit different effects on E&R bonding performance.31,32 Nevertheless, few studies have been conducted to compare their bonding strengths. Therefore, the study aimed to evaluate the effects of different application sequences of calcium-containing desensitising pastes (Arg-CaCO3-, CPP-ACP- or Novamin-based paste) on bonding effectiveness and tubule occlusion during E&R adhesive restoration. The following hypotheses were tested: (1) the application sequence (desensitising-etching or etchingdesensitising) of calcium-containing desensitising pastes has no significant influence on the microtensile bond strength (MTBS) of E&R adhesive systems and (2) the tubule-occluding effects are not different under these two application sequences.

2.

Materials and methods

2.1.

Experimental design

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A total of 78 caries-free human third molars were used in current study. All teeth were collected after the donors’ informed consents were obtained according to a protocol approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University, China. The teeth were cleaned thoroughly and stored in 1% chloramine at 4 8C and used within one month of post-extraction. Diagrammatic representation of the experimental grouping and treatment procedures used in the study was shown in Fig. 1. The materials, manufacturers, main compositions, classifications and application modes were listed in Table 1.

Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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Fig. 1 – Diagrammatic representation of the experimental grouping and treatment procedures used in the study.

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2.2.

Specimen preparation

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Seventy teeth were sectioned parallel to the occlusal surface to expose the mid-coronal dentine by using a low-speed watercooled diamond saw (Isomet; Buehler, Evanston, IL, USA). The exposed dentine surfaces were ground using 600-grit silicon carbide paper under running water for 60 s to create a standardised smear layer. The prepared teeth were randomly divided into seven groups (n = 10 per group) as follows:

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Group 1 (control group): Specimens were not pretreated with any desensitising paste but simply etched with 35% phosphoric acid for 15 s. Groups 2–4 (desensitising-etching groups): Specimens were initially pretreated with Arg-CaCO3-, CPP-ACP- or Novamin-containing desensitising paste, respectively, according to the manufacturers’ instructions and subsequently etched with 35% phosphoric acid for 15 s. Groups 5–7 (Etching-desensitising groups): Specimens were etched with 35% phosphoric acid for 15 s and then treated with Arg-CaCO3-, CPP-ACP- or Novamin-containing desensitising paste, respectively, according to the manufacturers’ instructions.

After these procedures were conducted, the specimens were rinsed thoroughly with water. The specimens in each group were randomly divided into two subgroups (n = 5 per subgroup) to evaluate the effects of different E&R adhesive systems. The bonding agent of a two-step E&R adhesive Adper SingleBond 2 (SB) or the primer and bonding agent of a three-step E&R adhesive Adper ScotchBond Multi-Purpose (SBMP) were applied to dentine surfaces respectively according to the manufacturers’ instructions; these substances were then polymerised using an LED light-curing unit (Bisco Inc., Schaumburg, IL, USA). Afterward, a resin composite (Charisma, Heraeus Kulzer, Hanau, Germany) was formed at the interface in four increments (3–4 mm thick) and each increment was cured for 20 s.

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2.3.

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Microtensile bond strength test

The bonded teeth were stored in deionised water at 37 8C for 24 h and sectioned perpendicular to the bonding interfaces to produce a series of slabs with a thickness of 0.9 mm. These slabs were then sectioned vertically into beams with a dimension of 0.9 mm  0.9 mm. An average of 4 beams was obtained from one tooth after the external beams were

Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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Table 1 – Materials, compositions and application procedures in this study. Material

Manufacturer

Etchant

3M ESPE, St. Paul, MN, USA

Sensitive Pro-Relief

Colgate-Palmolive Company, NY, USA GC Corp., Tokyo, Japan

Tooth Mousse

Repair&Protect

Adper SingleBond 2

Adper ScotchBond Multi-Purpose

Smithkline Beecham Consumer Healthcare, Berkshire, UK 3M ESPE, St. Paul, MN, USA

3M ESPE, St. Paul, MN, USA

Main composition

Classification

35% phosphoric acid solution, water, synthetic amorphous silica, polyethylene glycol, aluminium oxide. Hydrated silica, calcium carbonate, glycerin, arginine, water, bicarbonate, flavour, cellulose gum, sodium saccharin, FD&C blue No. 1 Glycerol, 5–10% CPP-ACP, pure water, zinc oxide, CMC-Na, xylitol, D-sorbitol, silicon dioxide, phosphoric acid, titanium dioxide, guar gum, sodium saccharin, ethyl-p-hydroxybenzoate, magnesium oxide, propylene glycol, butyl-p-hydroxybenzoate, propyl-phydroxybenzoate Glycerin, PEG-8, silica, calcium sodium phosphosilicate (Novamin), sodium monofluorophosphate, aroma, titaniu m dioxide, carbomer, potassium acesulfame, limonene. Bis-GMA, HEMA, dimethacrylates, ethanol, water, photoinitiator system, methacrylate functional copolymer of polyacrylic and polyitaconic acids, silica nanofillers. Primer: HEMA, polyalkenoic acid copolymer, water; Bonding agent: Bis-GMA, HEMA, tertiary amines, photo-initiator

Application mode

CPP-ACP-containing paste

Applied, left in place for 15 s, rinsed for 30 s with water spray. Applied with a rotary cup at low speed for 3 s, then repeated for another 3 s. Applied with an applicator brush, left for 3 min.

Novamin-containing paste

Brush twice with approximately 2 cm of paste.

Two-step E&R adhesive

Apply 2–3 coats for 15 s with gentle agitation, gently air thin for 5 s and light-cure for 10 s. Apply one coat of primer, blow gently for 5 s; Then apply a layer of bonding agent and light-cured for 10 s.

Etchant

Argin-CaCO3-containing paste

Three-step E&R adhesive

Abbreviations: CMC-Na, sodium carboxyl methyl cellulose; Bis-GMA, bisphenol-A-diglycidylether dimethacrylate; HEMA, 2-hydroxyethyl methacrylate.

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excluded from the periphery dentine, and a total of 20 beams were obtained from each subgroup. The prepared beams were fixed on a universal testing apparatus (Microtensile Tester; Bisco, Schaumburg, IL, USA) with cyanoacrylate glue (Zapit; Dental Ventures of America, Corona, CA, USA). Each beam was loaded in tension at a crosshead speed of 1 mm/min. The dimension of each beam was measured using a digital calliper, and cross-sectional interface area was calculated for the subsequent conversion of microtensile bond strength (MTBS) in MPa.

silicon carbide papers under running water. One disc was not pretreated; the seven remaining disks were pretreated as previously indicated (Groups 1–7). A slit was produced along the pulpal side of each dentine disc by using a low-speed water-cooled diamond to facilitate the fracture of the disks in two halves.34 One half was used for top surface observation and the other half was used for longitudinal surface observation. The specimens were rinsed, dehydrated, and sputtercoated with Au-Pd alloy. The tubule-occluding effects of each group were observed using FESEM (Zeiss, Sigma, Germany).

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2.6.

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After MTBS test, the debonded dentine specimens were assessed using a stereomicroscope (Stemi 2000-C; Carl Zeiss Jena, Go¨ttingen, Germany) at 50 magnification and classified as follows33: A, adhesive failure between dentine and resin; CD, cohesive failure in dentine; CC, cohesive failure in composite; and M, mixed failure involving a maximum of 50% each of the adhesive and cohesive resin composite failures. In addition, the representative failures of each subgroup were sputter-coated with Au-Pd alloy and observed under a field-emission scanning electron microscopy (FESEM) (Quanta 450 FEG; FEI, Eindhoven, the Netherlands).

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2.5.

Failure mode analysis

Statistical analysis

Statistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA). A two-way ANOVA factorial analysis was performed to evaluate the effect of interactions between desensitising sequence and type of desensitising pastes on the MTBS. One-way analysis of variance was used to compare mean MTBS between SB and SBMP adhesive systems for each group. Post hoc multiple comparisons were conducted using Tukey’s test. P values of 0.05 were set to indicate statistical significance.

3.

Results

3.1.

MTBS results

Tubule-occluding observation

Eight additional teeth were sectioned serially to produce dentine disks with a thickness of 1 mm. The dentine disks were sequentially wet-ground with 600-, 800- and 1000-grit

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MTBS values (in MPa) were shown in Fig. 2. Regardless of SB or SBMP adhesive systems, the desensitising sequence

Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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(desensitising-etching or etching-desensitising) did not significantly affect MTBS (P > 0.05), while there was no significant difference on MTBS among desensitising pastes (Arg-CaCO3-, CPP-ACP- or Novamin-based paste) (P > 0.05). The interaction of desensitising sequence and type of desensitising pastes was not significant (P > 0.05) either. However, one-way ANOVA showed that the specimens of SBMP showed higher MTBS than that of SB in each group (P < 0.05).

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The data obtained in the fracture pattern were analysed by frequency distribution (Fig. 3). The predominant failure mode of all debonded specimens was adhesive failure (50.4%), particularly in the control group (65.0%). Cohesive failure in dentine was common in desensitising-etching groups (Groups 2–4). Cohesive failures in composite increased in etchingdesensitising groups (Groups 5–7). By comparison, a few mixed failures (11.4%) were also observed. In the representative images of FESEM (Fig. 4), some specimens were fractured at the

Fig. 2 – Means and standard deviations of microtensile bond strength for each group.

Failure mode analysis

Fig. 3 – Distribution of failure modes following microtensile bond strength test.

Fig. 4 – Representative FESEM images (10,000T) of dentine surface after fracturing of MTBS test. (A) Specimen of Group 1 bond with SB shows an adhesive failure. Dentinal tubules occluded by resin tags (triangles) demonstrate that the failure was at the top of the hybrid layer. (B) Specimen of Group 2 bonded with SB shows a cohesive failure in dentine. Several open dentinal tubules (pentagram) indicate that the failure was mainly at the bottom of the hybrid layer. (C) Specimen of Group 6 bonded with SBMP shows a cohesive failure in composite, lots of composite fillers can be observed. (D) Specimens of Group 7 bonded with SBMP shows a mixed failure. Residual adhesives (black arrow), intertubular dentine (white arrow) and composite all can be seen from the fracture interface. Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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Fig. 5 – FESEM top surface examination (2500T) and longitudinal surface examination (10,000T) of tubule-occluding effects in different groups. (A and B) Top and longitudinal surface views for specimens after grinding showing smear layer and smear plugs. (C and D) Top and longitudinal surface views of Group 1 (control) showing a smear-free layer with patent dentinal tubules (pentagram). (E and F) Top and longitudinal surface views of Group 2 (Arg-CaCO3 + Etchant) showing patent dentinal tubules (pentagram) and residual particles deposited on intertubular and intratubular dentine surfaces. (I and J) Top and longitudinal surface views of Group 3 (CPP-ACP + etchant) showing patent dentinal tubules (pentagram) and a slight intertubular deposition of small particles. (M and N) Top and longitudinal surface views of Group 4 (Novamin + etchant) showing patent dentinal tubules (pentagram) and exposed dentine surfaces. (G and H) Top and longitudinal surface views of Group 5 (etchant + Arg-CaCO3) showing occluded dentinal tubules (triangles) and small particles deposition on intertubular dentine. (K and L) Top and longitudinal surface views of Group 6 (Etchant + CPP-ACP) showing partly occluded dentinal tubules and a thin, membrane-like coating on dentine surface; the occlusion within dentine tubules are superficial (arrow). (O and P) Top and longitudinal surface views of Group 7 (etchant + Novamin) showing a homogeneous layer of crystal-like deposits on the dentine surface and fewer open dentinal tubules; deep particles infiltration into dentinal tubules and tight combination with tubular walls were observed (triangles).

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top of the hybrid layer (Fig. 4A). Cohesive failures in dentine (Fig. 4B) or in composite (Fig. 4C) and mixed failure (Fig. 4D) could also be observed.

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Fig. 5 reveals the top surface examination and longitudinal surface examination of tubule-occluding effects in different groups. For instance, a rough smear layer was formed when no

Tubule-occluding observation

desensitising paste or etchant was used on dentine surface (Fig. 5A and B). Almost all of the dentinal tubules were occluded by fragments generated in grinding. In Group 1 (Fig. 5C and D), a smear-free layer with patent dentinal tubules was observed on dentine surfaces etched with 35% phosphoric acid for 15 s. In Group 2 (Fig. 5E and F), dentinal tubules were reopened when Arg-CaCO3-containing paste-treated dentine was etched, although residual particles deposited on intertubular and intratubular dentine surfaces. In Group 3 (Fig. 5I

Please cite this article in press as: Yang H, et al. Effects of the application sequence of calcium-containing desensitising pastes during etch-andrinse adhesive restoration. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.03.018

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and J), the majority of dentinal tubules were reopened with a slight intertubular deposition of small particles. In Group 4 (Fig. 5M and N), Novamin-containing deposits in dentinal tubules were removed by 35% phosphoric acid challenge and the specimens had exposed dentine surfaces with few occluded tubules. In Group 5 (Fig. 5G and H), dentinal tubules were mostly occluded by large particles after Arg-CaCO3-containing paste was applied on etched dentine while these particles incorporated into tubules deeply. Small particles slightly deposited on intertubular dentine. In Group 6 (Fig. 5K and L), some dentinal tubules were occluded superficially, while small particles of CPP-ACP paste deposited on the intertubular dentine surface, forming a thin, membrane-like coating on dentine surface. In Group 7 (Fig. 5O and P), a homogeneous layer was formed by crystal-like deposits of Novamin-containing particles on etched dentine surface. Only a few open dentinal tubules were visible.

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The results of the MTBS test showed that the application sequence (desensitising-etching or etching-desensitising) of calcium-containing pastes did not significantly affect MTBS during E&R adhesive restoration. When any of the three calcium-containing pastes was applied on etched dentine surface, dentinal tubules were mostly occluded with large particles, small particles slightly deposited on intertubular dentine and a protective layer was formed. However, the majority of dentinal tubules were reopened when the desensitising paste-treated dentine surface was etched. Therefore, the first hypothesis was accepted and the second one was rejected. The Arg-CaCO3-containing paste is a new desensitising product used mainly to prevent dentine hypersensitivity. During desensitisation, positively charged arginine is attracted to the negatively charged dentine surface; as such, CaCO3 becomes attracted and adheres to fibre networks on dentine surface and deeply infiltrate into dentinal tubules. The Arg-CaCO3 agglomerate creates an alkaline environment, where calcium and phosphate ions can precipitate, and further blocks dentinal tubules.10,35 The desensitisation of Arg-CaCO3-containing paste is similar to the natural desensitisation of saliva; for this reason, this procedure is commonly performed.36 The in vitro results of the present study indicated an effective occlusion performance. Considering the FESEM images (Fig. 5G and H), we found that dentinal tubules were effectively plugged and sealed after the paste was applied on etched dentine surfaces (etching-desensitising); a slight intertubular deposition of small particles was also observed. A calcium-containing layer might be formed to protect dentine surfaces and orifices of dentinal tubules. Calcium and phosphate are essential components of dentine in the form of highly insoluble complex (hydroxyapatite); in the presence of CPP, these minerals remain soluble and biologically available on dentine surfaces.37 The CPP-ACP-containing paste can also reduce dentine hypersensitivity successfully.18 The possible mechanism by which CPP-ACP seals the exposed dentinal tubules may involve remineralisation.38 Under acidic

Discussion

7

conditions, CPP-ACP can extract calcium phosphate; CPP-ACP can also increase and maintain high concentrations of calcium and phosphate ions on dentine surfaces, thereby impeding mineralisation and promoting demineralisation.11–13 In the current study, some dentinal tubules were occluded superficially; a thin, membrane-like coating was formed on etched dentine surface (Fig. 5K and L) (etching-desensitising). The presence of several patented tubules may be attributed to the short application time. The current study suggested that the recommended application time (3 min) of the manufacturer was short to induce sufficient remineralisation of CPP-ACP by forming substances that occlude dentinal tubules. The bioglass particles in Novamin-containing paste can deposited on dentine surfaces and form hydroxyapatite crystals with excellent biological activities and chemically similar structures to minerals found in dentine.39 In desensitisation, Na+ in Novamin immediately exchanges with H+ or H3O+ when the paste is exposed to an aqueous environment. At the same time, Ca2+ and PO43 in Novamin are released continuously.16 PH increases as a result of Na+ release, thereby inducing Ca2+ and PO43 from Novamin, along with the original hydroxyapatite crystals, to precipitate on dentine surfaces and form microcombination, thereby physically occluding dentinal tubules.16 As a result, dentine hypersensitivity is relieved. This condition could probably explain why Novamin-containing paste applied on etched dentine (etching-desensitising) can form a homogeneous layer covering the dentine surface with crystal-like deposits and tight combination with tubular walls (Fig. 5O and P). In the proposed application sequence (etching-desensitising), the calcium-containing paste-treated dentine provided different dentine topographies; the bonding strengths at adhesive –dentine interfaces should also vary at different degrees. However, the MTBS test in the present study indicated that the application of any of these three calcium-containing pastes on etched dentine did not significantly affect MTBS. This result possibly occurred because the proposed approach provides the same level of advantages and disadvantages for bonding. The possible reasons are manifold. For instance, one of the disadvantages of this strategy in bonding is the collagenous structures that were subjected to mechanical trauma during the polishing stage; another disadvantage includes that the formation of resin tags might be compromised by tubules occlusion with calcium-containing desensitising pastes, resulting in a certain damage to the mechanical lock of E&R adhesive restoration.18 By contrast, the advantages of this application sequence were evident. For instance, the smear layer formed during grinding was removed in the first step of etching; dentinal tubules were opened completely and some endogenous minerals within collagen networks were dissolved; as a result, calcium-containing desensitising pastes could penetrate dentinal tubules with great ease. We speculated that Arg-CaCO3, CPP-ACP or Novamin dissolved in residual water could reduce the liquid/vapour surface tension and increase the wettability of dentine surfaces, thereby creating a high energy surface and forming low contact angles, which are usually in favour of mechanical lock and adhesion.40 Furthermore, collapsed collagen fibres (during polishing process) might be rehydrated and re-expanded by HEMA

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hydrophilic monomer present in E&R adhesives because HEMA could decrease water vapour pressure and prevent evaporation.41 Another important factor is that the calciumcontaining layer formed by the calcium phosphate deposition at the bottom of exposed dentine collagen might fill the weak areas of the hybrid layer formed at a later stage.26 These factors contributed to the balance between advantages and disadvantages; hence, MTBS in etching-desensitising groups was not different from that of the control group. Researchers usually apply desensitisers before etching during E&R adhesive restoration. The present study showed that this traditional application sequence (desensitisingetching) did not affect MTBS either. However, effective tubule occlusion was not observed and the majority of dentinal tubules were reopened (Fig. 5E, I and M) under this situation. This result could be mainly attributed to 35% phosphoric acid. The combination of Arg-CaCO3, CPP-ACP or Novamin deposits and retained smear layer might be unstable. The acid resistance of calcium-containing desensitising pastes might also be limited.30,42 Therefore, phosphoric acid likely dissolves loose calcium and phosphate deposits in dentinal tubules until it encounters mineralised dentine matrix, thereby reopening the tubules. This result could be observed in the tubuleoccluding FESEM images in the present study. The reopened dentinal tubules could not limit dentinal fluid transudation and manage sensitivity during and after bonding.3 As a result, previous desensitising efforts were unsuccessful. Therefore, this traditional strategy could not effectively result in an acceptable tubule occlusion during E&R adhesive restoration compared with the new desensitising sequence of applying calcium-containing desensitising pastes after etching (etching-desensitising). Considering the E&R adhesive systems used in the current study, we found that the three-step SBMP adhesives demonstrated a better MTBS than the two-step SB adhesives. The result was mainly attributed to the separate application of primer and bonding agent of SBMP that could induce the complete hybridisation of treated dentine surfaces especially complicated dentine surface.22 One advantage of MTBS test is that the percentage of adhesive failures exceeds that of cohesive failures. Hence, realistic bonding strength can be examined.43,44 However, more adhesive failures do not correspond to a more effective bonding,45 which has also been proven by the failure percentage obtained in the present study. In this study, adhesive failure was the main fracture type of all groups, particularly in the control group. Cohesive failure in dentine was more commonly observed in desensitising-etching groups (Groups 2–4); this result may echo the reasoning that the combination between desensitising paste and retained smear layer was unstable; as such, this area is considered as the weakest part of a hybrid layer. Cohesive failures in composite increased in etching-desensitising groups (Groups 5–7), indicating that the dentine substrate was protected by the integrity of the subjacent adhesive layer.23 This integrity may be attributed to calcium salt precipitate filling the gap at the bottom of the hybrid layer. The frequency distribution of failure modes supported the explanation of MTBS to some extent.

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Conclusions

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The application sequence (desensitising-etching or etchingdesensitising) of calcium-containing desensitising pastes (Arg-CaCO3-, CPP-ACP- or Novamin-based) did not affect the bonding effectiveness of E&R adhesive systems. The proposed method, in which calcium-containing pastes were applied after etching, could provide a new potential strategy to achieve effective tubule occlusion without affecting bonding effectiveness during E&R adhesive restoration in clinical practice.

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Conflict of interest

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The authors declare that they have no conflict of interest.

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Acknowledgment

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This work was financially supported by National Natural Q2 Science Foundation of China (No. 81371191).

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references

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Effects of the application sequence of calcium-containing desensitising pastes during etch-and-rinse adhesive restoration.

To evaluate the effects of different application sequence of calcium-containing desensitising pastes on bonding effectiveness and tubule occlusion dur...
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