Dentine bond strength and antimicrobial activity evaluation of adhesive systems.

JJOD-2407; No. of Pages 10 journal of dentistry xxx (2015) xxx–xxx

Available online at www.sciencedirect.com

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

Dentine bond strength and antimicrobial activity evaluation of adhesive systems Carolina Bosso Andre´ a, Brenda Paula Figueiredo Almeida Gomes a, Thais Mageste Duque a, Rafael Nobrega Stipp b, Daniel Chi Ngai Chan c, Glaucia Maria Bovi Ambrosano d, Marcelo Giannini a,* a Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, Av. Limeira, 901, Piracicaba, SP 13414-903, Brazil b Department of Oral Diagnosis, Piracicaba Dental School, State University of Campinas, Av. Limeira, 901, Piracicaba, SP 13414-903, Brazil c Department of Restorative Dentistry, School of Dentistry, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-7456, USA d Department of Social Dentistry, Piracicaba Dental School, State University of Campinas, Av. Limeira, 901, Piracicaba, SP 13414-903, Brazil

article info

abstract

Article history:

Objectives: This study evaluated the dentine bond strength (BS) and the antibacterial activity

Received 3 November 2014

(AA) of six adhesives against strict anaerobic and facultative bacteria.

Received in revised form

Methods: Three adhesives containing antibacterial components (Gluma 2Bond (glutaralde-

1 January 2015

hyde)/G2B, Clearfil SE Protect (MDPB)/CSP and Peak Universal Bond (PUB)/chlorhexidine) and

Accepted 7 January 2015

the same adhesive versions without antibacterial agents (Gluma Comfort Bond/GCB, Clearfil

Available online xxx

SE Bond/CSB and Peak LC Bond/PLB) were tested. The AA of adhesives and control groups was evaluated by direct contact method against four strict anaerobic and four facultative

Keywords:

bacteria. After incubation, according to the appropriate periods of time for each microorganism,

Dentine

the time to kill microorganisms was measured. For BS, the adhesives were applied according to

Oral pathogens

manufacturers’ recommendations and teeth restored with composite. Teeth (n = 10) were

Adhesive systems

sectioned to obtain bonded beams specimens, which were tested after artificial saliva storage

Antibacterial activity

for one week and one year. BS data were analyzed using two-way ANOVA and Tukey test.

Bonding durability

Results: Saliva storage for one year reduces the BS only for GCB. In general G2B and GCB required at least 24 h for killing microorganisms. PUB and PLB killed only strict anaerobic microorganisms after 24 h. For CSP the average time to eliminate the Streptococcus mutans and strict anaerobic oral pathogens was 30 min. CSB showed no AA against facultative bacteria, but had AA against some strict anaerobic microorganisms. Conclusions: Storage time had no effect on the BS for most of the adhesives. The time required to kill bacteria depended on the type of adhesive and never was less than 10 min. Clinical significance: Most of the adhesives showed stable bond strength after one year and the Clearfil SE Protect may be a good alternative in restorative procedures performed on dentine, considering its adequate bond strength and better antibacterial activity. # 2015 Elsevier Ltd. All rights reserved.

* Corresponding author at: Department of Restorative Dentistry, Operative Dentistry Division, Piracicaba Dental School, State University of Campinas, Av. Limeira, 901, Bairro Areia˜o, Piracicaba, SP 13414-903, Brazil. Tel.: +55 19 21065340; fax: +55 19 21065218. E-mail address: [email protected] (M. Giannini). http://dx.doi.org/10.1016/j.jdent.2015.01.004 0300-5712/# 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Andre´ CB, et al. Dentine bond strength and antimicrobial activity evaluation of adhesive systems. Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.01.004

JJOD-2407; No. of Pages 10

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journal of dentistry xxx (2015) xxx–xxx

1.

Introduction

In the past, some dental product ingredients were suggested as having cavity disinfecting properties. Such ingredients products include benzalkonium chlorite, sodium hypochlorite, hydrogen peroxide, iodine-based oral disinfectants and cetyl pyridinium chloride.1 Currently, other antibacterial components have been incorporated to restorative materials and some of them were also added in attempt to improve the bonding mechanism and its durability.2 Gluma adhesive produced by Bayer AG company was the first bonding agent that contained an aldehyde organic compound. The glutaraldehyde is able to stabilize the collagen fibres, which is an important part of hybrid layer.3 Additionally, glutaraldehyde has an inherently strong antibacterial activity4,5 and its concentration in adhesive solution has been approximately 5%.6,7 Chlorhexidine digluconate has been used as an irrigating agent during treatment of periodontal diseases, in endodontic treatment, to disinfect cavity preparations and as a matrix metalloproteinase inhibitor.8,9 The inhibition of matrix metalloproteinase is reportedly able to prevent degradation of collagen, increasing the bonding durability.10,11 The effects of addition of chlorhexidine into bonding agents still remain unknown. The literature mainly reported on the mechanical and adhesion properties of restorative materials. Methacryloyloxydodecyl pyridinium bromide (MDPB) is a monomer that is present in the composition of Clearfil SE Protect primer solution. The bonding resin also has fluoride releasing properties. The active ingredient of MDPB is actually not released but acts as a contact inhibitor against the bacterium that is in direct contact with the restoration. MDPB is a polymerizable quaternary ammonium methacrylate that destroys the bacterial cell membrane killing them, while polymerization group copolymerizes with other adhesive monomers.12,13 An effective bactericidal effect from adhesives systems could be an alternative to suppress residual contamination after caries removal and increase the longevity of the restored tooth.14 Also, these adhesives could prevent the colonization of bacteria in gaps formed by resin shrinkage and interface degradation, preventing secondary caries even in cases of adhesives with good bond strength results.5,14,15 This study compared the dentine bond strength of (glutaraldehyde, chlorhexidine antibacterial-containing digluconate and MDPB) adhesive systems to same adhesive versions without antibacterial agents after artificial saliva storage for one week and one year, and analyzed the antibacterial activity of these adhesives against strict anaerobic and facultative bacteria. The null hypothesis was that (1) the artificial saliva storage does not influence the bond strength of adhesive systems and that (2) antibacterialcontaining adhesive systems would not have antibacterial activity against strict anaerobic and facultative oral pathogens until 24 h.

2.

Materials and methods

2.1.

Microtensile bond strength test

Sixty caries-free human third molars were used in this study. Teeth were stored in 0.5% thymol solution for no more than 3 months. These teeth were obtained under a protocol approved by the review board of the Institutional Ethics Committee (#139/2010). The teeth were sectioned 2 mm beneath the cementoenamel junction with a diamond saw (Buehler, Lake Bluff, IL, USA) under water-cooling to remove the roots. Their occlusal enamel surfaces were wet abraded with silicon carbide paper (180-grit) using a polishing machine (APL-4, Arotec, Cotia, SP, Brazil) to expose a flat dentine surface with a residual thickness of 4.0–5.0 mm. The dentine surfaces were then polished with 600-grit silicon carbide paper under water for 10 s and teeth were randomly divided into six groups (n = 10). Six commercially available adhesives were used in this study: three adhesives containing antibacterial components (Gluma 2Bond, Heraeus Kulzer GmbH, Hanau, Germany; Clearfil SE Protect, Kuraray Noritake Dental Inc., Kurashiki, Japan and Peak Universal Bond, Ultradent Products Inc., South Jordan, UT, USA) and the same adhesive versions without antibacterial agents (Gluma Comfort Bond, Heraeus Kulzer GmbH; Clearfil SE Bond, Kuraray Noritake Dental Inc., Kurashiki, Japan and Peak LC Bond, Ultradent Products Inc.). The chemical composition and lot number of each adhesive are summarized in Table 1. The commercial adhesive systems were applied according to the manufacturer’s instructions (Table 1). After application of adhesive, 6 mm high resin composite blocks were incrementally built-up over dentine using three 2-mm-thick layers of composite resin (shade A2, Filtek Supreme, 3M ESPE, St Paul, MN, USA). A light-curing unit (Optilux 501 Demetron/Kerr Corp., Orange, CA, USA) with an output of 660 mW/cm2 was used to polymerize the adhesives and the composite. Restored teeth were stored in distilled water at 37 8C for 24 h and then vertically, serially sectioned into 1.0-mm-thick slabs, using a diamond blade. Each slab was further sectioned perpendicularly to produce beams of approximately 1.0 mm2 in cross section and 10.0 mm in length. After one week immersed in artificial saliva, ten of the bonded sticks (beams) were immediately tested and the other ten were tested after one year of storage in artificial saliva at 37 8C. Each bonded stick was attached to the grips of a microtensile testing device with cyanoacrylate glue (Super Bonder gel, Henkel/Loctite, Diadema, SP, Brazil) and tested in tension in a universal testing machine (EZ Test, Shimazu, Kyoto, Japan) at a crosshead speed of 1.0 mm/min until failure. After testing, the specimens were removed from device and the cross-sectional area was measured to the nearest 0.01 mm with a digital calliper (mod. 727-/150, Starret, Itu, SP, Brazil). The cross-sectional area of each specimen was divided by the peak tensile load at failure to calculate stress of fracture (MPa). A single failure stress value was calculated for each group, according to the evaluation time (by averaging 10 specimens for each tooth). The specimens that did not fail at the dentine–adhesive interface were included in data. These bond strength data



3


Table 1 – Adhesive systems composition used in this study (information supplied by the manufacturer), batch number and application technique. Adhesive systems (lot number)

Application technique

Composition

Gluma 2Bond (010031)

Gluma Comfort Bond (010205)

Clearfil SE Protect (Primer: 00075A and Bond: 00125A)

Ethanol, 2-hydroxyethyl methacrylate, poly(methacrylic-oligo-acrylic acid), 4methacryloxyethyltrimellitic acid anhydride, glutaral, amorphous silica.

Acid etching (15 s); rinse (20 s); remove excess moisture; apply adhesive; wait for 15 s gentle air stream (10 s); light-cure (20 s).

Ethanol, 2-hydroxyethyl methacrylate, poly(methacrylic-oligo-acrylic acid), 4methacryloxyethyltrimellitic acid anhydride.

Acid etching (15 s); rinse (20 s); remove excess moisture; apply adhesive; wait for 15 s gentle air stream (10 s); light-cure (20 s).

Primer: 2-hydroxyethyl methacrylate, 10methacryloyloxydecyl dihydrogen phosphate, 12-methacryloyloxydodecylpyridinium bromide, hydrophilic aliphatic dimethacrylate, water, initiators, accelerators, dyes, others.

Apply primer; wait (20 s); gentle air stream; apply bond; gentle air stream; light-cure (10 s).

Bond: bisphenol A diglycidylmethacrylate, 2hydroxyethyl methacrylate, sodium fluoride, 10methacryloyloxydecyl dihydrogen phosphate, hydrophobic aliphatic methacrylate, colloidal silica, DL-camphorquinone, initiators, accelerators, others. Clearfil SE Bond (Primer: 01090A and Bond: 01089A)

Primer: 2-hydroxyethyl methacrylate, 10methacryloyloxydecyl dihydrogen phosphate, hydrophilic aliphatic dimethacrylate, DLcamphorquinone, water, accelerators, dyes, others.

Apply primer; wait (20 s), gentle air stream; apply bond; gentle air stream; light-cure (10 s).

Bond: bisphenol A diglycidylmethacrylate, 2-hydroxyethyl methacrylate, 10-methacryloyloxydecyl dihydrogen phosphate, hydrophobic aliphatic methacrylate, colloidal silica, DL-camphorquinone, initiators, accelerators, others. Peak Universal Bond (DO63)

Ethyl alcohol, 2-hydroxyethyl methacrylate, methacrylic acid, 0.2% chlorhexidine di(acetate).

Acid etching (15 s); rinse (20 s); remove excess moisture; apply adhesive; air stream (10 s); lightcure (20 s).

2-Hydroxyethyl methacrylate, methacrylic acid, ethyl-4-dimethylamino benzoate, ethyl alcohol.

Acid etching (15 s); rinse (20 s); remove excess moisture; apply adhesive; air stream (10 s); lightcure (20 s).

Peak LC Bond (CO63)

were analyzed using a two-way analysis of variance (ANOVA) to determine the effect of adhesive systems and storage times and their interaction on bond strength. Tukey post hoc test was used to detect differences among experimental groups. All statistical testing was performed at a preset alpha of 0.05. Fractured surfaces of tested specimens were allowed to airdry overnight at 37 8C after which they were sputter coated with gold (MED 010, Balzers, Balzer, Liechtenstein) and examined using a scanning electron microscope (JSM-5600, Jeol Inc., Peabody, MA, USA). Failure patterns were classified as: (1) cohesive within the composite resin, (2) adhesive between bonding agent and composite resin, (3) adhesive along the dentine surface, (4) mixed when simultaneously involving the adhesive failure and cohesively in dentine, adhesive layer, and/or composite, (5) cohesive within the adhesive layer, (6) cohesive within the hybrid layer and (7) cohesive within the dentine. Representative areas of the failure patterns were photographed at 400 (Fig. 2).

2.2.

Direct contact method

The same adhesives used in bond strength test were analyzed in this part of this study; however, some control groups were added: two negative controls (Inoculum and 0.9% physiological saline solution) and four positive controls (0.2% and 2% chlorhexidine, 5% glutaraldehyde and the primer of Clearfil SE Protect). Adhesives and controls were tested against four facultative bacteria: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Lactobacillus casei (ATCC L324M) and Streptococcus mutans (ATCC 25175) and four strict anaerobic microorganisms: Porphyromonas gingivalis (ATCC 49417), Prevotella intermedia (ATCC 25611), Prevotella nigrescens (ATCC 33563) and Fusobacterium nucleatum (ATCC 25586). Facultative bacteria were subcultured onto Brain Heart Infusion Agar medium (BHIA – Hi-Media Laboratories, Mumbai, India) under 5% CO2 at 37 8C for 24 h. Afterwards, bacteria strains were individually inoculated into tubes containing



4


5 mL of Brain Heart Infusion (Hi-Media Laboratories) medium, which were adjusted spectrophotometrically to 530 nm to match the turbidity of 1.5 108 CFU/mL (equivalent to 0.5 McFarland standard). Strict anaerobic microorganisms were subcultured onto Fastidious Anaerobic Agar (FAA – Lab M Ltd., Bury, UK) 5% blood agar plates under anaerobic atmosphere (Don Whitley Scientific – 80% N2, 10% CO2, 10% H2) for 48 h. Afterwards, anaerobic bacteria were individually inoculated into tubes containing 5 mL Fastidious Anaerobe Broth (Lab M Ltd.) sterile suspension, which was adjusted spectrophotometrically to 800 nm to match the turbidity of 3.0 108 CFU/ mL (equivalent to 1 McFarland standard). Thirty microlitre of each adhesive was placed in a cylindrical matrix (2.8 mm high and 4.4 mm diameter) and light-cured for 20 s (output of 660 mW/cm2) (Optilux 501). For two-step self-etching systems (Clearfil SE Protect and Clearfil SE Bond), it was mixed 10 mL of primer with 20 mL of bonding resin. When the primer of Clearfil SE Protect was used as a control, 30 mL of the primer solution was placed into matrix. Light-cured adhesive cylinders or 30 mL of each control group solutions (n = 3) were placed into well of 96-well cell culture plates (Corning Incorp., Corning, NY, USA), which contained 90 mL of bacterium Inoculum. After 5 min, 10 min, 30 min, 1 h and 24 h, 5 mL of microbial suspension from each well was taken and transferred to FAA 5% blood agar plates (anaerobics) or BHIA (facultatives). Agar plates were incubated for 48 h in the appropriate gaseous conditions and the time required for adhesive and control groups to kill the bacteria was recorded.

Table 2 – Mean bond strength (standard deviation) of adhesives systems after one week and one year storage times (in MPa). Adhesive systems

Storage time 1 week

Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond

43.1 51.1 34.8 47.3 51.1 51.7

(13.3) Aab (9.1) Aa (11.5) Ab (8.9) Aab (10.7) Aab (5.3) Aa

1 year 40.6 34.7 37.9 38.2 49.8 44.7

(10.8) Aa (8.9) Ba (12.2) Aa (8.2) Aa (10.5) Aa (8.0) Aa

Groups having similar letters (upper case in horizontal and lower case in vertical) are not significantly different ( p > 0.05).

3.

Results

Table 2 shows the bond strength means (standard deviation) for the adhesive systems after one week and one year of artificial saliva storage. The two-way ANOVA showed statistically significant differences for both factors (adhesive system, p = 0.0006 and storage time, p = 0.0070), but no significant interaction between them ( p = 0.0672). The one-year storage decreased the dentine bond strength only for Gluma Comfort Bond ( p < 0.05). However, this adhesive originally (at one week storage) did not show significant difference with most of the adhesives tested ( p > 0.05). When the adhesives were compared after the storage for one year no significant difference was observed among them ( p > 0.05).

Fig. 1 – Distribution of failure modes among experimental groups tested after 1 week and 1 year of storage in artificial saliva. Please cite this article in press as: Andre´ CB, et al. Dentine bond strength and antimicrobial activity evaluation of adhesive systems. Journal of Dentistry (2015), http://dx.doi.org/10.1016/j.jdent.2015.01.004


5

Fig. 2 – (A) Type 1 (cohesive failure within composite resin) fracture mode. It is possible to visualize the fillers particles of the composite resin (Peak Universal Bond tested after 24 h) (CR: composite resin); (B) Type 2 (adhesive failure between the adhesive system and the composite resin) fracture mode. The adhesive layer covers the dentine and the opening dentine tubules (Clearfil SE Protect tested after 24 h) (AL: adhesive layer); (C) Type 3 (adhesive failure between dentine and the adhesive system) fracture mode. The image shows dentine, opened dentine tubules (indicated by arrows) and the scratches of the SiC paper (600-grit) (Gluma Comfort Bond after 24 h) (D: dentine); (D) Type 4 (Mixed failure) fracture mode. The image shows type 1 and type 3 fracture modes together. This failure exposed the dentine surface (D) and cohesive fracture in the composite (CR) (Clearfil SE Bond after 24 h); (E) Type 5 (cohesive failure within the adhesive system) fracture mode. The adhesive layer (AL) appears fractured similarly to scales (Clearfil SE Protect after 1 year); (F) Type 6 (cohesive failure within the hybrid layer) fracture mode. There is no adhesive layer and the resin tags are occluding most of the dentine tubules. The arrows show some opened dentine tubules (Gluma 2Bond after 24 h) (RT: resin tag); (G) Type 7 (cohesive failure within dentine) fracture mode. The dentine (D) was fractured in different levels. It is also possible to visualize the opened dentine tubules (Peak LC Bond after 24 h).

Distribution of failure modes for the experimental groups is depicted in Fig. 1 and the representative images of each failure pattern are illustrated in Fig. 2. Adhesive failure along the dentine and mixed failure were observed in all groups (Fig. 2C and D, respectively). Cohesive failure within composite and within dentine also noted in all groups, except for Clearfil SE Protect at 24 h and Gluma 2Bond, respectively (Fig. 2A and G). Fig. 2B shows the adhesive failure between the adhesive system and the composite resin with the adhesive layer covering the dentine. The cohesive failure within adhesive layer was noted only for Gluma 2Bond at 24 h and Clearfil SE Protect (Fig. 2E). The cohesive failure within hybrid layer was observed basically for Gluma 2Bond at one week (Fig. 2F). The storage for one year did not change significantly the failure pattern for Clearfil SE Bond, Peak Universal Bond and Peak LC Bond. Three positive controls (5% glutaraldehyde, 0.2% and 2% chlorhexidine solutions) required only 5 min to eliminate all tested microorganisms; however, the primer of Clearfil SE Protect required 30 min to kill most of the oral pathogens, except for L. casei and F. nucleatum, which spent 1 h and 10 min, respectively. The negative controls (inoculums and physiological saline solution) did not kill any bacterium at all times tested. Gluma 2Bond and Gluma Comfort Bond required 24 h for killing microorganisms, except when the bacteria were P. gingivalis and P. intermedia, in which the time was reduced for 1 h. Clearfil SE Bond showed no ability to eliminate any facultative bacteria and F. nucleatum strict anaerobic microorganism. Clearfil SE Protect presented antibacterial activity

against some facultative bacteria and all strict anaerobic microorganisms, spending 30 min to kill P. gingivalis, P. intermedia and P. nigrescens, but needed only 10 min to eliminate F. nucleatum. This self-etching adhesive did not kill E. faecalis until 24 h, but was bactericidal for S. aureus and L. casei at 24 h and S. mutans at 30 min. Peak Universal Bond and Peak LC Bond killed only strict anaerobic microorganisms after 24 h by direct contact (Tables 3–10).

4.

Discussion

The first null hypothesis stating that the artificial saliva storage does not influence the bond strength of adhesive systems was rejected because one of adhesives had its bond strength reduced after artificial saliva-storage for one year. The differences in the composition between Gluma 2Bond and Gluma Comfort Bond are related to the incorporation of filler particle (amorphous silica) and glutaraldehyde. By analogy with restorative composites, studies have suggested that the addition of fillers may increase the strength the adhesive and hybrid layers.16,17 However, the main function of glutaraldehyde in this adhesive is the prevention of post-operative pain, as a result of collagen denaturation and the occlusion of dentinal tubules.4,18,19 Bond strength test was performed to estimate the composite retention on dentine structure immediately after composite placement and after storage for one year.20 It was compared adhesives with similar compositions and evaluated



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Table 3 – Bactericidal contact activity for Staphylococcus aureus. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

+ + +

10 min

30 min

1h

24 h + + + +

+

+

+ + +

+ + +

+ + +

30 min

1h

24 h

+

+

+ + +

+ + +

+ + +

+ + +

30 min

1h

24 h

+ + +

The positive signal indicates that the adhesive systems were bactericidal at that specific time.

Table 4 – Bactericidal contact activity for Enterococcus faecalis. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

+ + +

10 min

+ + +


Table 5 – Bactericidal contact activity for Lactobacillus casei. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

10 min

+

+ + +

+ + +

+ + +

+ + +

+ + + +

+ + +


if antibacterial agent addition could change the dentine bond strength. The investigation of failure pattern is an important tool to identify the weakest area of dentine–composite interface created by adhesives. The failure pattern analysis for specimens stored for one year showed possible changes occurred at bonded interface, which could be caused by long period immersed in artificial saliva. Glutaraldehyde is considered a cross-linking agent that can change the structure of collagen fibrils, improving degradation resistance and stabilization,21 which could explain the

maintenance of dentine bond strength for Gluma 2Bond after one year of storage and bond strength reduction for Gluma Comfort Bond, which does not contain glutaraldehyde. In addition, Gluma Comfort Bond increased the adhesive failure pattern after one year of storage, indicating some changes at dentine–composite interface over time. The 5% glutaraldehyde positive control group required 5 min to kill all types of microorganisms tested in this study, showing a strong antibacterial activity.4–6 Gluma 2Bond and Gluma Comfort Bond obtained the same results in the Direct Contact Method



7


Table 6 – Bactericidal contact activity for Streptococcus mutans. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

10 min

+ + +

30 min

1h

24 h

+ +

+ +

+ + + +

+ + +

+ + +

+ + +

30 min

1h

24 h

+ + +

+ + + + +

+ + + + + + +

+ + +

+ + +

+ + +

30 min

1h

24 h

+ + +

+ + + + +

+ + + + + + +

+ + +

+ + +

+ + +

+ + +


Table 7 – Bactericidal contact activity for Porphyromonas gingivalis. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

10 min

+ + +

+ + +


Table 8 – Bactericidal contact activity for Prevotella intermedia. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

+ + +

10 min

+ + +


and required 24 h for killing microorganisms, except when the bacteria were P. gingivalis and P. intermedia, in which the time was reduced for 1 h. Peak Universal Bond and Peak LC Bond differ basically by the presence of chlorhexidine and ethyl-4-dimethylamino benzoate. Both components did not seem to interfere with dentine bond strength, since they did not differ from each other, even after artificial saliva storage for one year. Chlorhexidine is a cationic polybiguanide, bisphenol component containing chlorine and have been used primarily as its

salts, such as dihydrochloride, diacetate and digluconate. It is a safe antiseptic with a broad spectrum of action, which is active against gram-positive and gram-negative organisms, facultative, anaerobes and aerobes. Clinically, it also reduces the microorganisms in plaque and saliva, decreasing the level of S. mutans.8,10,11,22 Both concentration of chlorhexidine solution (0.2 and 2%) used as control groups killed all bacteria tested after 5 min of direct contact, demonstrating its effectiveness bactericidal effect. However, the chlorhexidine-containing adhesive and its version without antibacterial



8


Table 9 – Bactericidal contact activity for Prevotella nigrescens. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

+ + +

10 min

30 min

1h

24 h

+ + +

+ + +

+ + + + + + +

+ + +

+ + +

+ + +

30 min

1h

24 h

+ +

+ + + +

+ + +


Table 10 – Bactericidal contact activity for Fusobacterium nucleatum. 5 min Gluma 2Bond Gluma Comfort Bond Clearfil SE Protect Primer Clearfil SE Protect Clearfil SE Bond Peak Universal Bond Peak LC Bond 0.9% Physiological saline solution 2% Chlorhexidine 0.2% Chlorhexidine 5% Glutaraldehyde Inoculum

10 min

+ +

+ +

+ + + + +

+ + +

+ + +

+ + +

+ + +


component showed the same results, eliminating only strict anaerobic microorganisms after 24 h. These results might indicate that the chlorhexidine incorporated into the adhesive solution can be trapped in the polymer chain and, without contact with the bacteria, has no bactericidal effect. Also, this adhesive may not present antibacterial component release properties. The results found for anaerobic microorganisms can be from other compound present in the composition of both adhesives (Peak LC Bond and Peak Universal Bond). Studies showed that a self-etch primer containing 5% MDPB killed S. mutans within 30 s of contact before curing,23,24 and after light-curing, copolymerizing with other adhesive monomers, this self-etch primer presented only inhibitory effect on the growth and adherence of bacteria on adhesive surface.25,26 In this study, the primer of Clearfil SE Protect required 30 min to kill most of the oral pathogens. Regarding L. casei and F. nucleatum bacteria, this primer required 1 h and 10 min to kill these microorganisms, respectively. The commercial self-etching adhesive that contains this primer showed antibacterial activity against some facultative bacteria and all strict anaerobic microorganisms. This self-etching adhesive required 10 min to kill F. nucleatum and 30 min to kill P. gingivalis, P. intermedia and P. nigrescens. Regarding facultative bacteria, it was bactericidal for S. aureus and L. casei at 24 h and S. mutans at 30 min. Among adhesive systems tested in this study, including containing or not antibacterial agents, the Clearfil SE Protect and its primer showed higher potential bactericidal activity. No difference between Clearfil SE Protect

and Clearfil SE Bond was found for bond strength at both storage times. The second null hypothesis was also rejected because some antibacterial-containing adhesive systems showed antibacterial activity against strict anaerobic and facultative oral pathogens until 24 h. Clearfil SE Protect and Peak Universal Bond did not kill E. faecalis, which is a gram-positive bacterium and has been frequently found in contaminated root canal ranging from 30% to 90% of the cases.27 In root canal treatments sodium hypocloride and chlorhexidine have been indicated to eliminate E. faecalis, however a study showed that sodium hypocloride or chlorhexidine solutions presented low ability to kill E. faecalis.28 The results of Peak Universal Bond that contains 0.2% chlorhexidine di(acetate) showed that this adhesive did not kill E. faecalis until 24 h. L. casei and S. mutans were not killed by Peak Universal Bond too. L. casei is found in the mouth and intestines of human beings. It produces lactic acid, which decreases the pH levels in the mouth and is able to demineralize dental hard tissues. Studies have shown that lactobacilli are both acidogenic and acid tolerant, since it can grow and survive in acidic environment.29,30 S. mutans also produces lactic acid and is dominant in oral biofilms. This bacterium is considered primary causal agent of caries disease and able to survive at a low-pH environment during development stages.31,32 Studies have shown that MDPBcontaining self-adhesive adhesive system has a strong antibacterial activity against L. casei and S. mutans, having no adverse effect on dentine bond strength and monomeric conversion.14,23



S. aureus is also a gram-positive coccal bacterium and frequently found in the human respiratory tract, oral cavity and on the skin. This bacteria is not always pathogenic and might be found in the saliva and supragingival biofilm of periodontally healthy adults.33,34 Clearfil SE Bond, Peak Universal Bond and Peak LC Bond didn’t kill this bacterium until 24 h, while other adhesives eliminated S. aureus at 24 h. S. mutans initiate biofilm formation colonizing tooth surface by the ability to synthesize extracellular polysaccharides from sucrose.35 Because further accumulation of biofilm, the number of capnophilic and obligatory anaerobic bacteria increase, changing the antimicrobial biofilm composition from streptococcus-dominated to Actinomyces spp. and P. gingivalis,35 which are involved in root caries and periodontal disease, respectively.36,37 Although anaerobic bacteria were more related to periodontal disease they can be found in cariogenic biofilm around the gingival margin.35 The adhesives systems containing antimicrobial can be used in all types of restoration, as class V, that is located close to gingival margin. Despite the thin layer of adhesives systems that is in contact with the oral environment, they can bring antimicrobial effects. In general, the strict anaerobes were more susceptible than the facultative microorganisms in this study. P. gingivalis belongs to the phylum bacteroidetes and is gram-negative, rod-shaped, anaerobic, pathogenic bacterium. It is found in the oral cavity, as well as the upper gastrointestinal tract, respiratory tract, and in the colon. This bacterium is involved in periodontitis and peri-implantitis, being present in most subjects with these diseases and rarely detected in subjects with good periodontal health.38,39 P. intermedia are anaerobic, non-spore forming, gram-negative rods and also periodontal pathogen. This microorganism colonizes in the periodontal pockets, where they co-exist with other microorganisms. It is found in patients with early periodontitis, advanced periodontitis, and acute necrotizing ulcerative gingivitis.40,41 P. nigrescens is part of the human oral cavity. It also plays a role in the pathogenesis of periodontal disease, gingivitis and some odontogenic infections.42,43 In 2002, a finding indicated that P. intermedia would be a periodontal pathogen, whereas P. nigrescens seemed a marker of relative periodontal health.44 F. nucleatum is an anaerobic gram-negative non-spore, fusiform rod bacterium of variable length. It is frequently associated with periodontal diseases and finds commonly in human dental plaque with a wide range of other plaque microbes, in which plays a crucial role in plaque development.45–47 The bactericidal effects of adhesives tested in this study against P. gingivalis, P. intermedia and P. nigrescens was similar, but the time to kill each bacterium depended on the type of adhesive. For the F. nucleatum, the self-etching system containing MDPB kill it in 10 min, however, the same adhesive without MDPB did not kill it until 24 h. Further long-term clinical studies involving the antibacterial activity of dentine bonding agents in adhesive interface are necessary to confirm and understand better the potential of antimicrobial effects of these adhesive systems without compromising their biological sealing and adhesion properties.

5.

9

Conclusion

The MDPB-containing bonding agent showed the strongest antibacterial activity among the adhesive tested and presented stable dentine bond strength for one year.

Acknowledgements This work was supported by Sa˜o Paulo Research Foundation (FAPESP) (2010/13599-0 and 2011/17841-2) and WDS Foundation.

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Bond strength of adhesive resin cement with different adhesive systems.

Analysis of micro-shear bond strength of self-etch adhesive systems with dentine: An in vitro study.

Evaluation of Shear Bond Strength of Giomer in Comparison with Two Dentin Adhesive Systems.

Longitudinal Evaluation of Bond Strength to Enamel of Dental Adhesive Systems Associated with Nd:YAG Laser.

The Effect of Temperature on Shear Bond Strength of Clearfil SE Bond and Adper Single Bond Adhesive Systems to Dentin.

Comparative evaluation in vitro of caries inhibition potential and microtensile bond strength of two fluoride releasing adhesive systems.

Adhesive interface and bond strength of endodontic sealers to root canal dentine after immersion in phosphate-buffered saline.

Microtensile Bond Strength and Micromorphology of Bur-cut Enamel Using Five Adhesive Systems.

Bond strength tests of dental adhesive systems and their correlation with clinical results - A meta-analysis.

Early marginal leakage and shear bond strength of adhesive restorative systems.

Effect of remaining dentin thickness on microtensile bond strength of current adhesive systems.

Repair Bond Strength of Aged Silorane-based Composite Using Intermediate Adhesive Systems Based on Different Monomers.

Long-Term Bond Strength of Two Benzalkonium Chloride-Modified Adhesive Systems to Eroded Dentin.

Dentine bonding agents--characteristic bond strength as a function of dentine depth.

Halloysite nanotube incorporation into adhesive systems—effect on bond strength to human dentin.

Shear bond strength of a 4-META adhesive system.

Effect of cariogenic challenge on bond strength of adhesive systems to sound and demineralized primary and permanent enamel.

Shear bond strengths of different adhesive systems to biodentine.

Shear bond strength of calcium enriched mixture cement and mineral trioxide aggregate to composite resin with two different adhesive systems.

Shear bond strengths of ten dentin adhesive systems.

Evaluation of Shear Bond Strength of Newer Bonding Systems on Superficial and Deep Dentin.

Effect of Saliva on the Tensile Bond Strength of Different Generation Adhesive Systems: An In-Vitro Study.

Evaluation of Shear Bond Strength of Total- and Self-etching Adhesive Systems after Application of Chlorhexidine to Dentin Contaminated with a Hemostatic Agent.

Shear Bond Strength of Orthodontic Brackets Fixed with Remineralizing Adhesive Systems after Simulating One Year of Orthodontic Treatment.