INTRODUCTION

Dentine hypersensitivity is a common problem in daily dental clinics, and it affects approximately 8 to 35% of the population1). The condition has been defined as a transient and sharp pain caused by dentine exposure, typically when the dentine is exposed to chemical, thermal, tactile, evaporative and osmotic stimuli2).

Among several possible theories proposed to explain dentine hypersensitivity, hydrodynamic theory developed by Brannstrom3) has been widely accepted. According to this theory, any stimulant that causes the rapid movement of dentinal fluid can irritate nerve fibers and thus induce a pain response4). As a result, occluding dentinal tubules have been recommended to reduce dentine permeability and fluid flow, thereby alleviating the dentine hypersensitivity4,5).

Several recently developed calcium-containing desensitizers have attracted considerable attention because they can simulate natural desensitizing processes to occlude dentinal tubules by forming dentine-like minerals6,7). Specifically, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), derived from milk protein, was incorporated in commercially available paste to cure dentine hypersensitivity7). Previous studies showed that the CPP-ACP paste can simultaneously inhibit dentine demineralization and promote dentine remineralization by maintaining high concentrations of calcium and phosphate ions on dentine surface8). Calcium-sodium phosphosilicate (Novamin) is

a bioactive glass that was originally developed for bone regeneration9). The Novamin-containing paste has been reported to release calcium and phosphate ions that can quickly form a porous hydroxyapatite mesh-like structure on the dentine surface; consequently, its use promotes dentine remineralization and blocks dentinal tubules6,10).

Certain patients simultaneously suffer from dentine hypersensitivity and tooth defects. Simple sensitive treatment is insufficient for these patients. Aesthetic restoration with adhesive resins is a necessary complementary treatment to restore the desired color and shape of teeth. However, whether calcium-containing desensitizing paste, which is used to treat dentine hypersensitivity, can compromise the adhesive-dentine bonding strength remains unclear; the composition of desensitizers might hamper the interaction between dentine and the adhesive, as well as impede the subsequent infiltration of the adhesive resins11). Interestingly, our previous study showed that applying calcium-containing desensitizers does not have significant effects on the immediate bond efficiency of the etch-and-rinse (E&R) adhesive system12). However, the durability and stability of the adhesive-dentine bonds under this situation remain unknown and should be studied.

Long-term water storage is the most common artificial ageing model for bonding durability evaluation because it can simulate the moist intraoral environment13). Intraoral pH changes, depending on the acidic composition in tubule fluid, bacterial metabolism, saliva and dietary habits may trigger acid attack and

Effects of calcium-containing desensitizers on the bonding stability of an etch-and-rinse adhesive against long-term water storage and pH cyclingHongye YANG1*, Zhiyong CHEN2*, Huiyi YAN1 and Cui HUANG1

1 The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedical Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China

2 College of Stomatology, Guangxi Medical University, Nanning, ChinaCorresponding author, Cui HUANG; E-mail: huangcui@whu.edu.cn

This study aimed to evaluate the effects of two calcium-containing desensitizing pastes on the bonding stability of an etch-and-rinse (E&R) adhesive to dentine. After dentine hypersensitivity model established, dentine surfaces were assigned one of the following pretreatment: Group 1, no desensitizer; Group 2, CPP-ACP; and Group 3, Novamin. Specimens were then bonded with an E&R adhesive. Beams from each tooth were randomly divided into three subgroups and then subjected to microtensile bond strength (MTBS) test after 24 h; 12 months of water storage; or 15 runs of pH cycling. Failure modes, nanoleakage, and tubule-occluding effectiveness were analyzed. Results showed that CPP-ACP- or Novamin-pretreated specimens mainly preserved the bonding strength after 12 months of water storage, while effective tubule occlusion could be observed. The results suggested that the calcium-containing desensitizers were compatible pretreatment for bonding with E&R adhesives to obtain reliable long-term bonding strength and prevention of post-operative sensitivity.

Keywords: Dentine hypersensitivity, Desensitizing, Dentine bonding, Etch-and-rinse adhesive

*Authors who contributed equally to this work.Color figures can be viewed in the online issue, which is avail-able at J-STAGE.Received Jan 11, 2017: Accepted May 9, 2017doi:10.4012/dmj.2017-006 JOI JST.JSTAGE/dmj/2017-006

Dental Materials Journal 2018; 37(1): 122–129

Table 1 The materials, manufacturers, compositions and application modes

Material (category) Manufacturer Main composition Application mode

Etchant3M ESPE, St. Paul, MN, USA

35% phosphoric acid solution, water, synthetic amorphous silica, polyethylene glycol, aluminium oxide.

Applied, left in place for 15 s, rinsed for 30 s with water spray.

CPP-ACP-based desensitiser(Tooth Mousse)

GC, Tokyo, Japan

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-p-hydroxybenzoate

Applied with an applicator brush for 60 s, left for 3 min.

Novamin-based desensitiser(Repair&Protect)

Smithkline Beecham Consumer Healthcare, Berkshire, UK

Glycerin, PEG-8, silica, calcium sodium phosphosilicate (Novamin), sodium monofluorophosphate, aroma, titanium dioxide, carbomer, potassium acesulfame, limonene.

Applied with an applicator brush for 60 s, left for 3 min.

Adper ScotchBond Multi-Purpose

3M ESPEPrimer: HEMA, polyalkenoic acid copolymer, water; Bonding agent: Bis-GMA, HEMA, tertiary amines, photo-initiator

Apply one coat of primer, blow gently for 5 s; Then apply a layer of bonding agent and light-cured for 10 s.

Demineralising solution(pH=4.3)

Prepared according to literature16).

2.0 mM Calcium, 2.0 mM Phosphate, 75.0 mM Acetic acid.

Immersing specimens for 6 h per day (between treatment periods)

Remineralising solution(pH=7)

Prepared according to literature16).

1.5 mM Calcium, 0.9 mM Phosphate, 130.0 mM KCl, 20 mM Sodium cacodylate.

Immersing specimens for 18 h per day (overnight)

Bis-GMA, bisphenol-A-glycidyl methacrylate; HEMA, 2-hydroxyethyl methacrylate.

degrade the bonding interface14). Therefore, long-term water storage and pH cycling are necessary and feasible conditions for evaluating the stability of the adhesive-dentine interface.

This study aimed to evaluate the effect of dentine pretreatment with a calcium-containing desensitizing paste (CPP-ACP or Novamin) on the stability of the E&R adhesive-dentine interface, specifically against long-term water storage and pH cycling. The null hypothesis was that the microtensile bond strength (MTBS), failure modes and nanoleakage of desensitizer-treated specimens would not significantly differ after long-term water storage or pH cycling.

MATERIALS AND METHODS

Dentine sample preparationThirty extracted intact human third molars were used following the guidelines approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University. The teeth were cleaned thoroughly and maintained in 1% chloramine solution at 4°C within one month before used. These teeth were then sectioned parallel to the occlusal surface to expose the mid-coronal

dentine using a low-speed water-cooled diamond saw (Isomet, Buehler, Evanston, IL, USA). Each exposed dentine surface was carefully ground by a 600-grit SiC paper for 60 s to create a standardised smear layer. One percentage citric acid solution was used to immerse specimens for 20 s to mimic a dentine hypersensitivity model according to previous study15). After that, the specimens were rinsed thoroughly using water-spray.

Application of desensitizer and bondingThe materials, manufacturers, compositions, and application modes used in the present study are listed in Table 1. Two calcium-containing desensitizing pastes, casein phosphopeptide-amorphous calcium phosphate-containing paste (CPP-ACP, GC, Tokyo, Japan) and calcium-sodium phosphosilicate-containing paste (Novamin, Smithkline Beecham Consumer Healthcare, Berkshire, UK), were used in this study. Prepared teeth were randomly divided into three groups as follows (n=10 each group): Group 1: no treatment; Group 2 and 3 were respectively pretreated with CPP-ACP or Novamin for 60 s using an applicator brush.

After treatment with desensitizer, the specimens were rinsed with water-spray for 30 s to remove loosely

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bound pastes on dentine surface. All desensitizer-treated specimens were etched with 35% phosphoric acid for 15 s, applied with a classic three-step E&R adhesive system, namely, Adper ScotchBond Multi-Purpose (SBMP, 3M ESPE, St. Paul, MN, USA). Then adhesives were polymerized using an LED light-curing unit (Bisco, Schaumburg, IL, USA), followed by composite resin (Charisma, Heraeus Kulzer, Hanau, Germany) built up (thickness of 3 to 4 mm). All the bonding procedures were conducted by the same researcher. After storing in deionized water at 37°C for 24 h, the specimens were longitudinally sectioned to produce slabs with a thickness of 0.9 mm. Nine middle slabs from each group were left for nanoleakage observation, other slabs were continuously sectioned to produce beams with a dimension of 0.9×0.9 mm. After external beams were excluded, each bonded tooth yielded 9 beams, which were then randomly divided into three subgroups (n=3 each tooth) and suffered one of following treatments (n=30 for each subgroup):

Control: Beams without any artificial ageing treatment served as the baseline.

Twelve months of water storage: Beams were stored in deionized water in a biochemical incubator at 37°C for 12 months, replaced weekly.

pH cycling: Beams were stored in a demineralization solution (pH=4.3) for 6 h and then in a remineralization solution (pH=7.0) for 18 h, repeated 15 times. Between each alteration, beams were rinsed thoroughly with deionized water, the parameters (pH value and cycles) were mainly based on Stookey’s research16).

MTBS testThirty prepared beams from each subgroup were subjected to MTBS test. Each beam was fixed on a universal testing equipment (Microtensile Tester, Bisco) with cyanoacrylate glue (Zapit, Dental Ventures of America, Corona, CA, USA). The crosshead speed was set as 1 mm/min, and the real dimension of each beam was measured by a digital calliper (Taihai Measuring, Shanghai, China). MTBS in MPa were calculated.

Failure mode analysisAfter MTBS test, the failure modes of debonded beams were observed under a stereomicroscope (Stemi 2000-C, Carl Zeiss jena, Göttingen, Germany) at 50× magnification and classified as follows17): A, adhesive failure; CD, cohesive failure in dentine; CC, cohesive failure in composite; and M, mixed failure. Representative samples were air dried, coated with Au–Pd alloy and examined under a field-emission scanning electron microscope (FESEM) (Sigma, Carl Zeiss, Göttingen, Germany). The quantity of prematurely debonded beams before the MTBS test was also registered.

Nanoleakage evaluationNine residual slabs from each group were also randomly assigned to three different ageing process as mentioned above (n=3 each subgroup), then they were prepared

for interfacial nanoleakage evaluation according to Tay et al.18). Specifically, nail varnish was applied on slabs, leaving 1 mm space at the bonding interfaces. The slabs were then immersed in ammoniacal silver nitrate solution for 24 h, followed by a photo-developing solution for 8 h under fluorescent light to reduce [Ag(NH3)2]+. The silver-stained slabs were embedded, polished, clean ultrasonically, dehydrated, and finally examined using a FESEM (Sigma, Carl Zeiss) in the backscattered mode. Five images were randomly captured in each specimen, forming a total of 15 images for each subgroup. The nanoleakage percentage at the adhesive-interfaces was calculated by Image J (NIH, Frederick, MD, USA).

Tubule-occluding observationAdditional one tooth was sectioned perpendicular to the long axis of the teeth to produce two consecutive dentine discs with a thickness of 1 mm. Then the opposite surfaces from two discs were polished under water spray. Each disc was then split carefully into two halves, and four similar dentine surfaces produced. Three sections were used, they were randomly treated according to experiment design: Group 1: no treatment; Group 2 and 3 were pretreated with CPP-ACP and Novamin, respectively. After loose deposition was removed using water spray, the prepared specimens were dehydrated, and subjected to atomic force microscopy (AFM, Veeco, Plainview, NY, USA) observation, and then the same specimen were observed under the FESEM.

Statistical analysisStatistical analysis was performed using SPSS 16.0 (SPSS, Chicago, IL, USA). For MTBS and nanoleakage data, the correlation between desensitizer types and artificial ageing methods was evaluated by two-way ANOVA factorial analysis, respectively. Post-hoc multiple comparisons were conducted using Tukey’s test with statistical significance set at a=0.05.

RESULTS

MTBS resultsThe MTBS result of each subgroup is list in Fig. 1. The interaction between the ageing methods and type of desensitizers was significant (p<0.05). The different ageing methods had a significant effect on the MTBS (p<0.05). However, no significant differences were observed on the MTBS among different desensitizer treatments (p>0.05). Specifically, pH cycling clearly reduced the MTBS of all groups regardless of the presence or absence of desensitizers. However, compared with control group (no desensitizer), 12 months of water storage maintained the MTBS of the CPP-ACP-treated specimens, and slightly reduced that of Novamin group.

Failure mode analysisThe failure frequency of all debonded specimens during the MTBS test is presented in Fig. 2. Adhesive failure was the most popular failure mode in all groups, followed by mixed failure. The cohesive failure in dentine

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Fig. 1 Means and standard deviations of MTBS for each group. (Groups with the same superscripts are not statistically significant (p>0.05)). Fig. 2 Distribution of failure modes after MTBS test.

Fig. 3 Representative FESEM images (2,500×, A–D) of dentine sides of fractured specimens after MTBS test. The insert at the top right corner indicates general condition of fractured surfaces (100×, a–d). (A)

Specimen of the control group (no desensitizing+no ageing) shows an adhesive failure. Uniform residual adhesive resin covered the dentine surface. (B) Specimen of group (CPP-ACP+no ageing) shows a mixed failure. Both dentine tubules with resin tags and large remnant adhesive existed. (C) Specimen of group (CPP-ACP+12 months water ageing) shows a cohesive failure in composite. Residual adhesive resin covered the dentine surface, leaving vague image of dentinal tubules with resin tags. (D) Specimen of group (CPP-ACP+pH cycling) shows a cohesive failure in dentine. Except for dentinal tubules with resin tags, lots of collagen fibrils presented, indicating the failure mainly located at the bottom of the hybrid layer. A circular region occurred outside of the fracture surface in (d), the low magnification image of this group. Circle: open dentin tubules; triangle: sealed dentin tubules; pointer: resin tags.

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Fig. 4 The statistical analysis of nanoleakage expression. (Groups with the same superscripts are not statistically significant (p>0.05).

Fig. 5 Representative FESEM images (1,000×) of interfacial nanoleakage expression from different groups. (A) Specimen of the group (no desensitizing+no ageing) shows minimal silver deposition at the bonding

interface. (B) Specimen of the group (Novamin+no ageing) shows sparse nanoleakage expression. (C) Specimen of the group (Novamin+water ageing) shows obvious and much more silver uptakes occurred at the bottom of adhesive layer. (D) Specimen of the group (Novamin+pH cycling) shows that extensive silver deposited along the whole adhesive layer, even in some dentinal tubules. Pointer: silver uptake.

increased in the pH cycling groups. The representative FESEM images are shown in Fig. 3. A circular region was present around the fracture interface in the pH cycling groups (Fig. 3d).

Nanoleakage evaluationThe statistical results of nanoleakage expression are shown in Fig. 4. The interaction between ageing methods and desensitizer types was significant (p<0.05). No significant differences were found among the different desensitizers (p>0.05), whereas the different ageing methods significantly affected nanoleakage expression (p<0.05). Specifically, long-term water storage and pH cycling obviously promoted the occurrence of nanoleakage in all groups. The respective FESEM images are presented in Fig. 5.

Evaluation of tubule occlusionThe FESEM (A–C) and AFM (A’–C’) images of tubule occlusion are presented in Fig. 6. Almost all dentinal tubules opened after the specimens were immersed in 1% citric acid solution for 20 s, thereby representing the dentine hypersensitivity model (Figs. 6A and A’). The CPP-ACP-treated dentine presented the formation of numerous crystals on the dentine surface, with occluded tubules (Figs. 6B and B’). Almost all dentinal tubules were occluded on the dentine surface when specimens were treated with Novamin (Figs. 6C and C’). The square average roughness (RMS) values were automatically presented by AFM; the RMS of A’, B’ and C’ in Fig. 6 were 368.5, 482.7 and 503.4 nm, respectively.

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Fig. 6 Representative FESEM images (8,000×, A–C) and AFM images (15.0×15.0 um, A’–C’) of dentinal tubules. (A and A’) Specimen of group 1 (no desensitizing) shows a smear-free layer with patent dentinal tubules.

(B and B’) Specimen of group 2 (CPP-ACP) shows sealed dentinal tubules by crystal deposition, forming a rough dentine surface. (C and C’) Specimen of group 3 (Novamin) shows occluded dentinal tubules, forming a lumpy-looking layer on dentine surface. Pentagram: open dentin tubules; triangle: sealed dentin tubules.

DISCUSSION

This study evaluated the effects of dentine surface pretreatment with calcium-containing desensitizing pastes on the bonding stability and durability of a commercial E&R adhesive. Our result showed that bonding strengths of desensitizer-treated groups were mainly maintained after long-term water storage, leading to a possible strategy to preserve bonding mechanical properties in adhesive restoration. Therefore, the null hypothesis was basically rejected.

Sensitive and defective teeth require the application of a desensitizer before bonding. Our previous study showed that CPP-ACP- and Novamin-containing desensitizing pastes can induce tubule plugging to some extent; both pastes can form a protective layer on the dentine surface12). This conclusion was supported and confirmed by the FESEM and AFM images (Fig. 6) in the present study. The possible mechanism by which CPP-ACP and Novamin seal the dentinal tubules mainly involves remineralization, as revealed by other studies6-10).

Although dentine adhesive systems have rapidly developed from the first to eighth generations; E&R adhesive is still reliable dentine bonding system, especially for complicated dentine surfaces, compared with self-etch adhesives19). Therefore, the compatible use of calcium-containing pastes and E&R adhesives should

be evaluated to provide guidance for dental clinical application.

The present study showed that the pretreatment of the dentine surface with a calcium-containing desensitizing paste does not affect the immediate bonding strength of E&R adhesives. The conclusion could be indirectly supported by the AFM results (Figs. 6A’–C’). The use of AFM has many advantages when evaluating the surface characteristics of biomaterial samples because it can be performed under naturalistic or aqueous conditions that are similar to the physiological environment of human beings20).

Furthermore, AFM can provide 3D images with more details; the physicochemical properties of specimens can be obtained, such as the RMS20). The wetness of the dentine surface can be enhanced by the presence of microsurface roughness, according to the Wenzel equation21). In the present study, the RMS of the CPP-ACP- or Novamin-treated dentine increased, compared with that of the control group. This finding indicated that the surface might become more wet and lower contact angles, which were in favour of mechanical interlocking and adhesion22). These factors might compromise the side effects of desensitizer-pretreatment on dentine bonding, such as collagen collapse and subsequent insufficient adhesive infiltration, thereby producing few changes on the immediate bonding strength.

Long-term water storage has been widely used to

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evaluate the stability of adhesive-dentine interfaces13,19). When desensitizing paste was not applied on the dentine surface in the present study, the MTBS significantly decreased after 12 months of water storage, which is a common problem of current dentine bonding systems. This problem could be mainly attributed to the inconsistency between the etching depth and adhesive infiltration depth23). During long-term water storage, the exposed fibrils and associated resins at this weak area are continuously attacked by hydrolysis, thereby leading to the disintegration and collapse of the hybrid layer24).

However, when the sensitive dentine surfaces were firstly treated with calcium-containing pastes, the long-term bonding strength of E&R adhesive was satisfying, which is what researchers have always wanted to achieve. Based on the function mechanisms of CPP-ACP and Novamin7,8,25,26), this phenomenon might be explained as follows: CPP could stabilize ACP on the dentine surface after CPP-ACP treatment; Ca2+ and PO4

3− gradually released during long-term water storage and maintained at high concentrations in the hybrid layer, thereby forming an ion osmotic gradient and promoting the remineralization of the hybrid layer and demineralized dentine7,8); When Novamin-treated dentine was subjected to long-term water storage, Na+ exchanged with H+ or H3O+, and the pH increased, which resulted in the continuous release of Ca2+ and PO4

3− from Novamin to form microcombination with the original hydroxyapatite crystals for reinforcing the hybrid layer6,10,26). As a result, the bonding strengths of desensitizer-pretreated specimens in the present study did not significantly decreased after 12 months of water storage.

pH cycling has been used to mimic cariogenic challenges and evaluate the bonding stability during such situations16,27). pH cycling obviously reduced the MTBS in the present study, regardless of the presence or absence of desensitizing paste. The influence of pH cycling on adhesive-dentine interfaces has not been clearly elucidated, but acid challenge is suspected to deteriorate the hybrid layer and degrade bulk adhesives28). The representative FESEM images of failure modes (Fig. 3d) showed that a degrading circle formed around the interfaces, which indicated that the erosion pathway of pH cycling was from the margins to the center of the adhesive-dentine interfaces. The observed circle also suggested that acid erosion from pH cycling was strong enough to prevent the calcium-containing pastes and remineralizing solution of pH cycling from functioning. The frequency of cohesive failure in dentine was higher in the pH cycling groups than that in the other groups, which also suggested that dentine could be eroded much easier than adhesive composite. Due to pH cycling resembling highly cariogenic conditions, novel calcium-containing desensitizers with good antimicrobial properties or/and excellent acid resistance should be developed in the future to prevent secondary caries and acid erosion at the adhesive interface.

Adhesive failure was the dominant failure mode in

almost all groups, which indicated that the observed MTBS could represent the actual adhesive-dentine bond strength29). The variation in nanoleakage expression in this situation was basically consistent with the changes in MTBS in the present study. For example, the MTBS of all groups significantly decreased after pH cycling, and the nanoleakage expression obviously increased. This trend echoes the general consensus that high bonding strengths usually correspond to low nanoleakage expression30,31).

The present study evaluated the effects of two calcium-containing desensitizing pastes on the bonding durability and stability of an E&R adhesive to dentine in vitro. The strategy will need to be verified in vivo through long-term clinical studies, while new anti-erosion desensitizing pastes are needed to develop in future studies. Furthermore, the effects of calcium-containing desensitizer on self-etching bonding systems will be evaluated in future studies to provide much more clinic guidance for dentists.

CONCLUSION

In conclusion, the calcium-containing paste (CPP-ACP or Novamin) used as a pretreatment was compatible with the use of E&R adhesives under the conditions of this study. The immediate and long-term bonding strengths of desensitizer-treated specimens were satisfactory. Therefore, the application of CPP-ACP or Novamin before E&R adhesive restoration was a feasible strategy for patients to obtain reliable long-term bonding strength and prevention of post-operative sensitivity.

ACKNOWLEDGMENTS

The authors declare that they have no conflict of interest. This work was financially supported by National Nature Science Foundation of China (No. 81371191).

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