The effect of combined application of new dentin desensitizing agent and deproteinization on dentin permeability of different adhesive systems

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ScienceDirect Tanta Dental Journal 10 (2013) 138e144 www.elsevier.com/locate/tdj

The effect of combined application of new dentin desensitizing agent and deproteinization on dentin permeability of different adhesive systems N.S. Farghal a,*, A.I. Abdalla b, S.M. El-Shabrawy c, E.A. Showaib d a Biomaterials Dept., Faculty of Dentistry, Tanta University, Egypt Conservative Dentistry Dept., Faculty of Dentistry, Tanta University, Egypt c Dental Bio-Materials Dept., Faculty of Dentistry, Alexandria University, Egypt d Mechanical Design & Production Engineering Dept., Faculty of Engineering, Tanta University, Egypt b

Abstract Purpose: To evaluate the effect of dentin desensitization using casein phosphopeptide amorphous calcium phosphate paste; CPPACP, with and without deproteinization (10% sodium hypochlorite), on dentin permeability of two types of self-etch adhesives and a total-etch one. Materials and methods: 90 freshly extracted, sound human molars were sectioned into 1 mm thick dentin disks, and randomly divided into three equal groups (S1, S2, and S3); 30 specimens each, according to the dentin surface treatment, as follows: (S1) adhesive system {AS} only, (S2) CPP-ACP þ AS, (S3) 10% NaOCl þ CPP-ACP þ AS. Each group was further divided into 3 divisions (D1, D2, and D3), 10 specimens each, according to the tested adhesive system (Solobond M, Clearfill SE and Futurabond DC) respectively. Dentin surfaces were treated Dentin permeability was measured at base line and after dentin treatment and storage under simulated pulpal pressure for 10 min. Results: CPP-ACP treatment significantly decreased the permeability of (S2D2) and (S2D3), but didn’t affect (S2D1). The combined NaOCl and CPP-ACP treatment decreased the permeability of the three tested adhesives significantly. Clinical significance: The combined application of 10% sodium hypochlorite and CPP-ACP paste may be recommended to improve the dentinal permeability before using dental adhesive systems. Ó 2013, Production and Hosting by Elsevier B.V. on behalf of the Faculty of Dentistry, Tanta University. Open access under CC BY-NC-ND license.

Keywords: Total-etch adhesives; Self-etch adhesives; CPP-ACP; Deprotinization; Permeability

* Corresponding author. E-mail address: [email protected] (N.S. Farghal). Peer review under the responsibility of the Faculty of Dentistry, Tanta University.

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1. Introduction Dentin bonding or adhesion refers to “the micromechanical coupling or union of restorative materials to dentin, particularly dental composites, via an intermediary adhesive resin layer” [1]. Hybrid layer formation, as a consequence of resin monomer penetration and impregnation in exposed dentin collagen, is a widely

1687-8574 Ó 2013, Production and Hosting by Elsevier B.V. on behalf of the Faculty of Dentistry, Tanta University. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.tdj.2013.11.006

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accepted effective method used to increase composite resin bond strength to dentin. However, several factors influence the stability of this layer over time, such as permeability of some adhesive systems and noneffective sealing of dentinal tubules, incomplete infiltration of adhesive system in all demineralized dentin depth, degradation of exposed collagen fibers which were not impregnated [2,3]. Some studies have reported that collagen fibers do not significantly contribute to bond strength, questioning their real role on resin adhesion and sealing efficiency. Dissolution and removal of collagen fibers with sodium hypochlorite after acid conditioning has been used with the attempt to increase the stability of restorative interface. This technique is known as deproteinization as a way to minimize the hybridization technique sensitivity, producing a more durable adhesion to dentin substrate through its hydroxyapatite component [4e6]. The incorporation of solvated hydrophilic resin monomers has substantially improved the initial bond strengths of contemporary etch-and-rinse and selfetching adhesives to intrinsically wet dentin substrates [7,8]. Despite this improvement, recent studies have shown that contemporary single bottle adhesive bond to dentin remains highly permeable after polymerization [9e11]. The hydrophilic nature of simplified adhesive systems permits transudation of water across its structure after polymerization [12e14]. The presence of water and solvents within the adhesive layer compromises its polymerization, mechanical properties, and durability [3,15]. During bonding procedures, most of the water that migrates to the adhesive layer originates from the underlying hydrated dentin. As current simplified adhesive systems are not able to create a perfect seal of exposed dentin surfaces, alternative treatments should be pursued in order to avoid transudation of water from dentin to the adhesive layer [16]. One way of reducing the increased adhesive permeability is the adjunctive use of dentin desensitizers on acid-etched dentin prior to adhesive application [17]. Management of hypersensitive dentin involves remineralization or occlusion of the patent dentinal tubules and agents used include fluoride containing solutions/compounds, oxalates and nitrates of potassium, potassium chloride or strontium chloride, amorphous calcium phosphates, resinbased bonding agents and the placement of restorations [18,19]. Recently, a nanocomplex of the milk protein casein phosphopeptide (CPP) and amorphous calcium phosphate (ACP) has been shown to prevent tooth sensitivity and enhance the obturation of dentinal tubules with remineralized dentin through maintaining a high

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concentration gradient of calcium phosphate on the tooth surface [20e25]. Dentin permeability was studied a as a function of hydraulic conductance. It is the ease with which fluid can move across a unit surface area under unit pressure per unit time [26]. On the other hand, the hydraulic conductance based on Poiseuille’s law is determined by a number of variables such as the pressure moving the fluid across dentin, length of the tubules, and viscosity of the fluid and the radius of the tubule. Dentin permeation is directly proportional to the radius of the tubule. Hence the most important variable in reducing the fluid flow (dentinal permeability) is with the reduction of the radius of the tubule (tubule occlusion) [27]. The effects of various desensitizers on dentin permeability of different adhesives have been well documented [27e29]. Still, the effect of CPP-ACP based desensitizing agent needs further investigations. Therefore, this study aimed to assess the effect of dentinal pretreatment with CPP-ACP based desensitizing agent with or without dentin deproteinization on dentin permeability, of three dentin adhesive systems to a light cured resin composite. 2. Materials & methods A total of ninety freshly extracted, sound human molars, gathered following informed consent approved by the Commission for Medical Ethics of the Faculty of Dentistry, Tanta University, were used in this study. After extraction, the teeth were cleaned of blood and soft tissue under running water and stored in 1% chloramine T solution at 4  C for no more than one month. It has been reported that from the second post extraction day, dentin permeability did not change over the following 3e4 weeks [30]. The teeth were sectioned parallel to the occlusal surface, 2.5e3 mm above the cementeenamel junction, and occlusally creating 1 mm thick deep dentin discs, using a diamond coated saw mounted on a cutting machine. After cutting, each disc was carefully inspected under a stereo-microscope, to ensure that it was free of coronal enamel or pulpal exposures. The prepared discs were stored in normal saline till bonding procedure, which was performed within 3 days. A fluid filtration system with split chamber device as described by Pashley and Galloway [31] was constructed for hydraulic conductance measurements Fig. 1. The apparatus was connected to an electric pump with a rubber tube to provide controlled pressure 15 cm H2O or 11 mm Hg, checked by using a sphygmomanometer, to simulate the physiological pulpal pressure [13]. The

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Fig. 1. Fluid transport apparatus assembly.

whole apparatus was immersed in distilled water bath to simulate the dentinal fluid [17]. All discs were treated with the phosphoric acid etching gel for 15 s, then properly rinsed. The discs were placed in the split chamber device (Fig. 2); the air bubble location was determined and recorded for each specimen. Then, the pressure pump was switched on, for 10 min, and the location of the air bubble was redetermined and recorded. Then the linear displacement of the air bubble in the glass pipette was calculated to the nearest 0.01 mm. Fluid flow (Q) was measured from the linear displacement of the air bubble, depending on the volume of the pipette, using the following equation [17]:-

Baseline fluid filtration represents the maximum fluid flow of each specimen, and was arbitrarily assigned to a value of 100% permeability. The discs were demounted from the split chambers. The occlusal surfaces of the discs were polished with 600 grit silicon carbide paper under water irrigation for 10 s, to create a standardized smear layer. Then, according to the occlusal surface treatment, the discs were divided to three groups, 30 specimens each, as follows [17,32]: Group 1 (S1): Samples did not receive any treatment (control). Group 2 (S2): CPP-ACP paste was applied for 5 min and then washed off with an air water blast for 15 s. Group 3 (S3): A drop of 10% NaOCl solution was applied for 60 s, followed by CPP-ACP paste. Then each group was further divided according to the tested bonding agents, which were applied according to the manufacturer instructions, as follows: Division 1 (D1): 10 sp two step total etch adhesive (Solobond M). Division 2 (D2): 10 sp two step self-etch adhesive (Clearfil SE Bond). Division 3 (D3): 10 sp single step self-etch adhesive (Futurabond DC). Each treated disc was remounted in its specific chamber, then fluid filtration of bonded dentin, was calculated exactly as the baseline measurement was performed. The dentin permeability percent was obtained using the following equation, with each specimen serving as its own control [17,32]: fluidfilteration rateof resinbonded dentin 100 baseline fluidfilteration rate of etched dentin

Q ¼ displacement X cross sectional area of the pipette:

%P ¼

Permeability was expressed in terms of fluid filtration (Jv) where:

3. Results

Jv ¼ Q/AT Jv ¼ fluid filtration rate in ml cm‾2 min‾1 Q ¼ fluid flow in ml. A ¼ dentinal (or O ring central hole) surface area in cm2. T ¼ time in minutes.

Fig. 2. Opened split Teflon chamber device with pair of rubber O rings and a dentin disk. A: female part, B: male part.

The collected numerical data was analyzed using One-way Analysis of Variance (ANOVA) testing significance, for the comparison between surface treatments and adhesive systems. Duncan’s post hoc test was used for comparison between the means when ANOVA test is significant. The significance level was set at P  0.05 (95% confidence interval). Statistical analysis was performed with SPSS 14.0Ò (Statistical Package for Scientific Studies) for Windows. The effect of different dentin surface treatment on dentin permeability mean percent of each tested adhesive system is shown in Tables 1and 2. When no surface treatment was applied, i.e. control (S1); the highest dentin permeability mean percent occurred in S1D1, while S1D2 showed lowest dentin

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Table 1 Statistical analysis of dentin permeability mean percent after different dentin surface treatment. Adhesive system

ANOVA

Surface treatment

Solobond M (two-step total-etch) D1

Clearfill SE (two-step self-etch) D2

Futurabond DC (one-step self-etch) D3

F-value

P-value

Control S1 CPP-ACP S2 NaOCl þ CPP-ACP S3

24.55  2.6a

15.21  1.9c

20.28  2.9b

118.9

0.000*

23.1  1.7a

9.69  1.05b

9.1  1.2b

125.7

0.000*

11.76  1.6a

5.09  1.13c

8.39  1.5b

109.9

0.000*

*Significant at P  0.05. SD: standard deviation. Means with different letters are significantly different.

permeability mean percent. There was a significant difference among the three tested adhesives. After CPP-ACP treatment (S2); the dentin permeability of the S2D1 was significantly the highest, followed by S2D2 and S2D3 with no significant difference between the two later divisions. After NaOCl and CPP-ACP (S3) treatment; again the two-step total-etch adhesive (S3D1) showed the highest dentin permeability, followed by the one-step self-etch adhesive S3D3. The lowest dentin permeability mean percent occurred in the two-step self-etch adhesive S3D2, with a significant difference between the three tested divisions. Regarding each of the tested adhesive systems (Table 2), for the two-step total-etch adhesive (D1); the highest dentin permeability occurred when no surface treatment was applied (S1D1), and in the CPP-ACP treated division (S2D1), with no significant difference between them. The lowest dentin permeability occurred after application of both NaOCl and CPP-ACP (S3D1), with a significant difference with the other divisions. For the two-step self-etch adhesive (D2), the highest dentin permeability was when no surface treatment was applied (S1D2), and decreased significantly when CPP-ACP was applied (S2D2). The lowest dentin

permeability occurred when the dentin was treated with both NaOCl and CPP-ACP (S3D2), there was a significant difference among the three tested adhesives. The dentin permeability mean percent provided by the one-step self-etch adhesive (D3) was highest when no surface treatment was applied (S1D3), being significantly higher than the other divisions. This was followed by the CPP-ACP treated division (S2D3). Then the NaOCl and CPP-ACP treated division (S3D3), with no significant difference between the two later divisions. The dentin permeability mean percent ranking of different divisions Fig. 3 shows that the highest permeability was provided by the two-step total-etch without any surface treatment (S1D1), while the lowest dentin permeability occurred when the two-step selfetch adhesive was preceded with combined deproteinization and CPP-ACP treatment. 4. Discussion The integrity of bond between dentin and resin adhesive systems has an important implication for clinical dentistry in improving the success of composite resin restorations. The eventual longevity of

Table 2 Statistical analysis of dentin permeability mean percent of each tested adhesive systems. Adhesive system Surface treatment

Control S1 CPP-ACP S2 NaOCl þ CPP-ACP S3 F-value P-value

Solobond M (two-step total-etch) D1

Clearfill SE (two-step self-etch) D2

Futurabond DC (one-step self-etch) D3

Mean  SD

Mean  SD

Mean  SD

24.55  2.6

15.21  1.9

20.28  2.9a

23.1  1.7a

9.69  1.05b

9.1  1.2b

11.8  1.6b

5.09  1.13c

8.39  1.5b

34.016 0.000*

349.8 0.000*

55.71 0.000*

a

a

*Significant at P  0.05. SD: standard deviation. Means with different letters are significantly different.

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Fig. 3. Dentin permeability mean percent ranking produced by different subgroups of the study.

such bonded restorations and their bond strength depends upon the extent to which polymerized resin will penetrate into the demineralized dentin. The currently employed dentin bonding agents may not completely penetrate into the demineralized dentin leaving behind an un-infiltrated weak collagenous layer of dentin, that is susceptible to long term hydrolytic degradation [33,34]. This problem may be minimized or eliminated by the use of a proteolytic agent, like sodium hypochlorite treatment. It removes dentin organic components and changes its chemical composition, so that it becomes similar to etched enamel. This substrate is also rich in exposed hydroxyapatite crystals and may result in stable interface overtime because it is essentially made of mineral [5]. In the current study, all the contemporary investigated adhesives applied on deep dentin were subjected to water permeation, induced by simulated pulpal pressure after polymerization. This means that the fluid flow across bonded dentin could not be totally eliminated, although it could be reduced to an extent. Inability of the adhesives to properly seal the dentinal surface may be, generally, due to defective bonding mechanism as well as their polymeric hydrophilic nature which undergoes water sorption and polymerization shrinkage. It has been reported that in order to restore a tooth in such a way that is leak proof, there must be no dimensional mismatch at the toothrestoration interface [35]. Permeability of simplified total-etch adhesives was apparent even when wet bonding was performed with meticulous solvent removal in vital deep human dentin, incomplete water removal from the deeply etched dentin may have resulted in “over-wetting phenomena”

water that would fill the inter-fibrillar spaces which would consequently dilute the resin and interfere with its infiltration, as well as competing with the monomers for binding sites on collagen [36]. When the CPP-ACP-containing paste is applied to dentin, the protein component readily binds to the tooth surface and consequently provides a reservoir of calcium and phosphate ions. High concentration gradients of these ions on the tooth surface have been reported and, hence, have been correlated with an increase in surface hardness due to mineral deposition [20,37]. Since water is the main causative factor in degradation of resin dentin interface, complete or partial blockage of the dentinal fluid flow from the underlying dentin may improve dentin sealing. The CPP-ACP containing paste probably did not occlude the dentinal tubules with the currently tested total-etch adhesive, because of the bonding mechanism of total etch adhesive, which removes the smear layer, this makes it difficult for the calcium or phosphate ions to precipitate inside the dentinal tubules [38,39]. The current significant decrease in dentin permeability of the two-step self-etch adhesives when the CPP-ACP paste was applied is probably due to the partial occlusion of the dentinal tubules and subsurface deposition of CPP crystals inside the dentinal tubules. The peptide complex may bind to hydroxyapatite providing a localized source of bio-available calcium and phosphate for dentin remineralization and dentinal tubules occlusion [39e43]. Besides, the primer of the two-step self-etch adhesive utilized in this study contains 10-MDP as a functional monomer dissolved in water, to result in a pH around 2, it doesn’t remove the smear layer, but it impregnates the smear plugs, fixing them at the tubules, thus decreasing the dentin permeability and providing a better seal [40], The application of CPP-ACP paste before application of the one-step self-etch adhesive in the present research also decreased its permeability; it may has counteracted the demineralizing effect of the phosphoric and carboxylic acid esters of the adhesive, so the calcium and phosphate ions were precipitated over the smear layer. When the self-etch adhesive was applied, the acids in the bonding agent are neutralized by the hydroxyapatite in the tooth structure, and this process only runs until the acid is exhausted [44]. Dentin sealing improved even better with combining the CPP-ACP paste with deproteinization with sodium hypochlorite before using the total etch and two-step self-etch adhesive, It has been reported that not only the exposed collagen plays a role in rapid bond degradation, but also the properly infiltrated one.

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As part of the demineralized dentin collagen may be in a denaturized unstable state, which is highly sensitive to hydrolysis [45,46]. Smear layer composition is similar to the originating tissue (50% vol. minerals and 30% vol. collagen). The application of NaOCl on smear layer would eliminate its collagen phase which would facilitate the penetration of the self-etch primer through water-filled channels between particles of smear layer, enlarging them to reach the underlying dentin more easily. Studies have reported that application of NaOCl prior to self-etch primer might have improved the marginal quality since it facilitated the action of the acidic primer to demineralized the underlying matrix, exposing new collagen fibrils and forming a new hybrid layer with better resin penetration [35,47]. On the other hand, the dentin permeability was not significantly different when CPP-ACP and NaOCl were applied to one-step self-etch adhesive compared to CPP-ACP subgroup, this may be attributed to the production of a relatively porous smear layer due to removal of its organic content and some of the organic content of the underlying dentin. Such effect might have facilitated resin penetration into dentin after the application of CPP-ACP, improving the micromechanical retention [48,49]. 5. Conclusions 1 None of the tested adhesives could provide an impervious seal of the dentin surfaces, after storage for 10 min under simulated pulpal pressure. 2 CPP-ACP treatment improved the sealing of the two-step and the one-step self-etch adhesive, however, it didn’t improve the permeability of the two-step total-etch adhesive. 3 Dentin deprotinization may constitute an effective modality for reducing the bonded interface permeability. References [1] Fawzy AS, Amer MA, El-Askary FS. Sodium hypochlorite as dentin pretreatment for etch-and-rinse single-bottle and twostep self etching adhesives: atomic force microscope and tensile bond strength evaluation. J Adhes Dent 2008;10:135e44. [2] Baseggio W, Consolmagno EC, de Carvalho FL, Ueda JK, Schmitt VL, Formighieri LA, et al. Effect of deproteinization and tubular occlusion on microtensile bond strength and marginal microleakage of resin composite restorations. J Appl Oral Sci 2009;17:462e6.

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