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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 9  |  Issue : 3  |  Page : 72-77

Comparative efficacy of three deproteinizing agents on the shear bond strength of pit and fissure sealant: An in vitro study


1 Final Year PG Student, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
2 Former Professor and Head, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
3 Professor, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
4 Former Reader, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
5 Professor and Head, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
6 Reader, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India
7 Senior Lecturer, Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Ernakulam, Kerala, India

Date of Submission11-Aug-2022
Date of Acceptance25-Aug-2022
Date of Web Publication28-Sep-2022

Correspondence Address:
Dr. Saranya P Thankachan
Department of Pediatric and Preventive Dentistry, Annoor Dental College and Hospital, Perumattom, Muvattupuzha, Ernakulam - 686 673, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpcdr.ijpcdr_18_22

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  Abstract 


Objective: The objective of this study was to evaluate and compare the shear bond strength (SBS) of pit and fissure sealant with and without deproteinization with agents such as 5% NaOCl, papain gel, and bromelain gel.
Materials and Methods: In this in vitro study, 60 premolar teeth were divided into four groups of 15 teeth each. In control Group 1, enamel was etched for 60 s with 37% phosphoric acid and rinsed with water. In Group 2, deproteinizing agent 5% sodium hypochlorite was applied for 60 s before acid etching. In Group 3, deproteinizing agent papain gel was applied for 60 s before acid etching. In Group 4, deproteinizing agent bromelain gel was applied for 60 s before acid etching. Following this, Clinpro 3M ESPE pit and fissure sealant disc was built on the enamel buccal surface of each tooth. Samples were then tested for SBS using Universal Testing Machine after storage in distilled water for 24 h.
Results: The mean SBS was highest for Group 4 and lowest for Group 1. SBS was significantly higher in the bromelain gel group, followed by the papain gel group (P < 0.001), 5% NaOCl group (P < 0.001), and control group (P < 0.001).
Conclusion: Among deproteinizing agents, deproteinization when carried out with bromelain gel showed effective bond strength and lowest for the control group.

Keywords: Acid etching, bromelain gel, enamel deproteinization, papain gel, phosphoric acid, shear bond strength, sodium hypochlorite


How to cite this article:
Thankachan SP, Emmatty TB, Jose B, Krishna KK, Peter J, Methippara JJ, Sebastian R. Comparative efficacy of three deproteinizing agents on the shear bond strength of pit and fissure sealant: An in vitro study. Int J Prev Clin Dent Res 2022;9:72-7

How to cite this URL:
Thankachan SP, Emmatty TB, Jose B, Krishna KK, Peter J, Methippara JJ, Sebastian R. Comparative efficacy of three deproteinizing agents on the shear bond strength of pit and fissure sealant: An in vitro study. Int J Prev Clin Dent Res [serial online] 2022 [cited 2022 Dec 6];9:72-7. Available from: https://www.ijpcdr.org/text.asp?2022/9/3/72/357307




  Introduction Top


Dental caries among children is one of the greatest challenges faced by dentists globally. Modern dentistry requires preventive options to decrease the incidence of caries in both adults and children.

Pit and fissure sealants are one of the best preventive methods for deep pit and fissures and are very difficult to clean. A pit and fissure sealant is a resin material that is introduced into the pits and fissures of caries susceptible teeth, forming a micromechanically retained physically protective layer that acts to prevent demineralization of enamel by blocking the interaction of cariogenic bacteria and their nutrient substrates, thus eliminating the harmful acidic by-products.[1] They are effective in caries prevention and in preventing the progression of incipient lesions.

The success of pit and fissure sealants depends on factors such as an efficient marginal seal, retention, and resistance to microleakage related to the ability of adhesion between the fissure sealant and the enamel.[2],[3] The retention rate of a pit and fissure sealant is directly related to the micromechanical bond between the sealant and enamel. Shear bond strength (SBS) measures the ability of sealant to bond-to-tooth structure. Higher SBS enhances the performance of sealant. This makes it necessary to evaluate the bond strength of sealants.

Many studies have shown that conventional etching with 37% phosphoric acid is affected negatively by the higher amount of organic structure. Deproteinization involves the removal of organic content, i.e., the removal of proteins from the enamel. Various deproteinizing agents such as papain gel from papaya plant, bromelain enzyme from the stem of pineapple plant, and sodium hypochlorite are recommended in the literature.[4]

Although there are various studies on bond strength, microleakage, and retention of pit and fissure sealant, none of the studies compare the effect of deproteinizing agent bromelain gel, papain gel, and sodium hypochlorite before etching on the SBS of sealant.

This in vitro study is therefore being done to

  1. To evaluate the SBS of pit and fissure sealant ClinproTM 3MTM ESPETM with and without deproteinization with agents 5% NaOCl, papain gel, and bromelain gel
  2. To compare the SBS of pit and fissure sealant ClinproTM 3MTM ESPETM with and without deproteinizing agents 5% NaOCl, papain gel, and bromelain gel.



  Materials and Methods Top


This comparative experimental in vitro study was undertaken in the department of pediatric and preventive dentistry. The institutional ethical committee approval was obtained before the start of the study.

Morphologically intact caries-free premolar teeth freshly extracted for orthodontic purposes were collected. The extracted teeth were thoroughly washed in running water to remove blood and adherent tissue removed using hand scalers. All teeth were stored in sterile water until used for the study. Materials used for this study were conventional pit and fissure sealant (ClinproTM 3MTM ESPETM), Ivoclar Vivadent) and deproteinizing agents, 5% sodium hypochlorite solution (Asian acrylates – Mumbai), papain gel (Carie-careTM), and indigenously prepared bromelain gel [Figure 1].
Figure 1: Deproteinizing agents used

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Bromelain solution was prepared by dissolving 10 mg of bromelain powder (HerbaDiet) in 10 ml of saline as vehicle. Freshly prepared concentration of this solution was again diluted with saline in the ratio of 1:2, 2:2, and 3:2. Thickening agent methylcellulose was added to the bromelain solution. In the present study, we used 60% concentration (3:2) of bromelain gel.

After decoronation, teeth were embedded in self-curing acrylic resins in a Teflon mold such a way that its buccal surface is exposed [Figure 2]. Once the acrylic sets, the Teflon molds were removed. The enamel buccal surfaces were divided into three parts, and middle segment was used to standardize the bonding of specimens. A total of 60 premolar teeth were divided into four groups of 15 teeth each.
Figure 2: Prepared specimens in acrylic block

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Group 1 (control group) – There was no deproteinization done. Enamel surfaces were etched with 37% phosphoric acid gel (Eco-Etch Etching Gel, Ivoclar Vivadent) for 20 s, washed with water, and air dried. Pit and fissure sealant ClinproTM 3MTM ESPETM was placed in a hollow plastic tube of 3 mm height with internal diameter of 4 mm in an incremental fashion to form a button on the buccal surface of the teeth. Light curing was done using LED optical fiber instruments (high power blue light led 420–480 nm) according to manufacturers' instructions for 20 s.

Group 2 – Deproteinizing agent, 5% sodium hypochlorite (Novo Dental Products Pvt. Ltd., Mumbai) was applied with sterile cotton pellet for 60 s, rinsed with water and dried. Thirty-seven percent phosphoric acid etchant (Eco-Etch Etching Gel, Ivoclar Vivadent) was applied to the enamel for 20 s, rinsed and dried with air spray for 20 s. Pit and fissure sealant ClinproTM 3MTM ESPETM was placed in a hollow plastic tube of 3 mm height with internal diameter of 4 mm in an incremental fashion to form a button on the buccal surface of the teeth. Light curing was done using LED optical fiber instruments (high power blue light led 420–480 nm) according to manufacturers' instructions for 20 s.

Group 3 – Deproteinizing agent papain gel (86.9% in Carie-Care, Eco-works Solutions Pvt. Ltd.,) was applied with sterile cotton pellet for 60 s, rinsed with water and dried. Thirty-seven percent of phosphoric acid etchant was applied to the enamel for 20 s, rinsed and dried with air spray for 20 s. Pit and fissure sealant ClinproTM 3MTM ESPETM was placed in a hollow plastic tube of 3 mm height with internal diameter of 4 mm in an incremental fashion to form a button on the buccal surface of teeth. Light curing was done using LED optical fiber instruments (high power blue light led 420–480 nm) according to manufacturers' instructions for 20 s.

Group 4 – Indigenously prepared bromelain gel applied on enamel buccal surface of decoronated premolar teeth with sterile cotton pellet for 60 s, rinsed with water and dried. Thirty-seven percent of phosphoric acid etchant was applied to the enamel for 20 s, rinsed and dried with air spray for 20 s. Pit and fissure sealant ClinproTM 3MTM ESPETM was placed in a hollow plastic tube of 3 mm height with internal diameter of 4 mm in an incremental fashion to form a button on the buccal surface of the teeth. Light curing was done using LED optical fiber instruments (high power blue light led 420–480 nm) according to manufacturers' instructions for 20 s.

After setting, the plastic tube was removed and all the prepared samples [Figure 3] were stored in distilled water at 37°C for 24 h to avoid dehydration.
Figure 3: Prepared samples for the study

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The specimens were later tested for SBS by mounting them on Universal Testing Machine (UTM, INSTRON) [Figure 4]. Custom-made metallic jig of 4 mm thickness with a perforation of 5 mm diameter at one end was used for measuring the SBS. Shearing force was applied at a crosshead speed of 1 mm/min until a fracture occurred. This procedure was repeated for all the samples.
Figure 4: Universal testing machine

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Results obtained were analyzed in SPSS (Statistical Package for the Social Sciences) version 24 SPSS Inc., Chicago, Illinois, USA. Analysis of variance (ANOVA) was done to find statistically significant difference between the four groups, and post hoc analysis was done to compare the individual groups with the control. P < 0.05 was considered statistically significant.


  Results Top


The SBSs of pit and fissure sealant without the use of deproteinizing agent and after the use of three different deproteinizing agents were tabulated and summarized as mean ± standard deviation [Table 1].
Table 1: Comparison of shear bond strengths (MPa) of pit and fissure sealant before and after deproteinization

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The mean value of SBS in MPa for the 15 samples of Group 1 samples without any deproteinization was 3.26 samples after deproteinization with 5% NaOCl was 7.75. The mean value of SBS in MPa for the 15 samples of Group 3, samples after deproteinization with papain gel was 10.25, and samples after deproteinization with bromelain gel was 15.

Group 4 (bromelain gel + acid etching) shows better bond strength as compared to other groups and the difference is statistically significant with other groups [Figure 5].
Figure 5: Bar diagram showing mean shear bond strength of all samples in each group

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The comparisons of SBS s between different groups were done using One-way ANOVA and concluded that there was a statistically significant difference in the mean value of SBS among all the groups.

The bar diagram shows that the SBS of Group 4, i.e., samples treated after deproteinization with bromelain shows the maximum values, and Group 1, i.e., the control group shows the minimum value [Figure 5].

As there was a significant difference in mean SBS of pit and fissure sealant among the four groups, Tukey's post hoc analysis was done to observe which two pairs of groups had a significant difference. Among deproteinizing agents, deproteinization when carried out with bromelain gel showed effective bond strength followed by the papain gel group and 5% NaOCl group.


  Discussion Top


Pit and fissure sealants are commonly used to prevent dental caries in permanent molars, especially in individuals with a higher risk of dental caries.

Sealants act as a physically protective layer which prevents enamel demineralization by preventing the interaction of fissure microorganisms and their nutrient substrates. The first clinical sealant trial was reported in 1965.[5] Since then, many clinical and laboratory reports have documented sealant safety and effectiveness.[6]

If the sealant remains in place completely covering the pits and fissures it will affect its effectiveness and is directly related to its retention. The consequences of sealant loss can be diminished with regular maintenance. Adequate marginal seal, retention, and resistance to microleakage are the factors essential for the adhesion between the fissure sealant and the enamel. The longest clinical study done on sealant retention, the retention rates recorded with 3M™ ESPE™ Concise™ Sealant were 82% at 5 years, 57% at 10 years, and 28% at 15 years.[7]

3M™ Clinpro™ Sealant is a light-cure, fluoride-releasing pit and fissure sealant with a unique color-change feature. Clinpro™ Sealant is pink when applied to the tooth surface, and changes to an opaque off-white color when exposed to light. Rose bengal dye provides a pink color, which disappears when exposed to a dental curing light. Clinpro™ sealant contains a patented soluble organic fluoride source. The fluoride is released from the sealant in a diffusion-limited process by the exchange of hydroxide for the fluoride ion.

The use of phosphoric acid as a conventional etching used to create microporosities removes organic matter from the tooth surface. The presence of organic remnants as well as fissure morphology and aprismatic enamel structure can decrease etching ability and thus prevent adequate adhesion. Various methods for preparing fissures, such as pumice prophylaxis, bonding agents, lasers, air abrasion, and sodium hypochlorite deproteinization, have been recommended to improve sealant retention.[7],[8]

Various studies suggested that deproteinization removes the surface organic matter layer before acid etching, which aids in creating the desired type I or type II etching pattern.[9],[10]

In the present study compared the effect of three deproteinizing agents, namely, 5% NaOCl (Asian Acrylates – Mumbai), papain Gel (Carie-Care), and bromelain gel (freshly prepared) on the SBS of Clinpro™ 3M™ ESPE™ pit and fissure sealant.

Sodium hypochlorite is an excellent protein denaturant.[11] Removal of excess proteins may enhance adhesion of material to the tooth surface. Studies carried out by Espinosa et al. and Ahuja et al. with 5% NaOCl as a deproteinizing agent after acid etching in permanent teeth have shown effective bond strength as compared to conventional acid etching.[12],[13]

Carie-care™ (Unibiotech Pharmaceuticals, India) is a chemomechanical caries removal agent consisting of papaya extract, clove oil, colored gel, chloramines, sodium chloride, and sodium methylparaben. Papain, a papaya extract, is a proteolytic enzyme similar to human pepsin and has antibacterial and anti-inflammatory properties. The absence of alpha-1-antitrypsin in infected tissues allows papain to break the partially degraded collagen and facilitates caries removal along with increased microbial properties.

In the present study, 10% papain gel (Carie-Care) was used as a deproteinizing agent in Group 3. This was in accordance with the study by Pithon et al. where enamel pretreatment with 10% papain gel increased the bond strength of orthodontic brackets bonded with resin-modified glass ionomer cement on bovine incisors.[14]

Bromelain is a deproteinization agent obtained from pineapple fruit stem. It catalyzes the hydrolysis of protein into amino acids. In the present study, freshly prepared bromelain gel was used as deproteinizing agent in Group 4. Bromelain gel was prepared in accordance with the study by Hasija et al.[15] Group 4 (Bromelain gel + acid etching) shows slightly better bond strength as compared to other groups and the difference is statistically significant.

The results showed that incorporating a step of deproteinization after acid etching has definitely affected the mean SBS. In the present study, sealant that is deproteinized with bromelain gel had the highest SBS. The result is comparable with the study by Hasija et al., where they compared the effect of different deproteinizing agents on SBS of composite to primary teeth enamel and deproteinization was done after etching and their study concluded that deproteinization when carried out with bromelain gel and NaOCl showed effective bond strength as compared to papain.[15]

The result showed that incorporating a step of deproteinization before acid etching has definitely improved the SBS of pit and fissure sealant. An addition of deproteinization step before acid etching in sealant would increase the chairside time and require cooperation from pediatric patients.

Deproteinizing agents such as NaOCl, papain gel, and bromelain gel improved the SBS of pit and fissure sealant. However, NaOCl is a strong oxidizing agent and has some disadvantages such as poor taste, smell, and adverse soft-tissue reaction.[13],[16] Natural agents such as papain and bromelain are more advantageous in pediatric patients. According to this study, bromelain gel had the highest SBS than papain gel and NaOCl and that was statistically significant (P < 0.001). Bromelain gel can be considered an alternative to NaOCl and papain as deproteinizing agent in pediatric patients.

To achieve good bond strength of enamel surface in pediatric patients the addition of a deproteinizing step before acid etching is justified as it increases the bond strength. Higher the bond strength, the stronger the resistance to curing stress and masticatory function loading. Furthermore, rubber dam isolation is necessary for pit and fissure sealant application, especially while deproteinizing using sodium hypochlorite.

Limitations of this study are small sample size, only one physical property was tested in vitro and thermocycling was not done. Further clinical studies on large samples are recommended to confirm the role of new agent bromelain gel in enamel deproteinization.


  Conclusion Top


The conclusions that can be drawn from the study include:

  • An addition of deproteinization step before acid etching in sealant placement significantly increases the SBS of the conventional pit and fissure sealant
  • SBS was significantly higher in the bromelain gel group, followed by papain gel group (P < 0.001), 5% NaOCl group (P < 0.001), and control group (P < 0.001).


The addition of deproteinization would increase the chairside time and require cooperation from pediatric patients. However, it is justified as it increases the bond strength, retention, and longevity of the sealant.

Long-term clinical trials as well as in vitro studies on larger samples are need to be undertaken in permanent and primary teeth to prove the role of new agent bromelain gel in enamel deproteinization before drawing any definite conclusion.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Simonsen RJ. Retention and effectiveness of dental sealant after 15 years. J Am Dent Assoc 1991;122:34-42.  Back to cited text no. 1
    
2.
Garcia-Godoy F, Gwinnett AJ. Penetration of acid solution and gel in occlusal fissures. J Am Dent Assoc 1987;114:809-10.  Back to cited text no. 2
    
3.
Feigal RJ. The use of pit and fissure sealants. Pediatr Dent 2002;24:415-22.  Back to cited text no. 3
    
4.
Ferreira MB, Carlini Júnior B, Galafassi D, Gobbi DL. Calcium hypochlorite as a dentin deproteinization agent: Microleakage, scanning electron microscopy and elemental analysis. Microsc Res Tech 2015;78:676-81.  Back to cited text no. 4
    
5.
Gwinnett AJ, Buonocore MG. Adhesives and caries prevention; a preliminary report. Br Dent J 1965;119:77-80.  Back to cited text no. 5
    
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Hatibovic-Kofman S, Butler SA, Sadek H. Microleakage of three sealants following conventional, bur, and air-abrasion preparation of pits and fissures. Int J Paediatr Dent 2001;11:409-16.  Back to cited text no. 6
    
7.
Beauchamp J, Caufield PW, Crall JJ, Donly K, Feigal R, Gooch B, et al. Evidence-based clinical recommendations for the use of pit-and-fissure sealants: A report of the American Dental Association Council on Scientific Affairs. J Am Dent Assoc 2008;139:257-68.  Back to cited text no. 7
    
8.
Duffy MB, Bernet JK, Chovanec GK, Majerus GJ, Frazier PJ, Newell KJ. Dental hygienists' knowledge, opinions, and use of pit and fissure sealants: A comparison of two states. J Public Health Dent 1987;47:121-33.  Back to cited text no. 8
    
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Erickson RL, De Gee AJ, Feilzer AJ. Effect of pre-etching enamel on fatigue of self-etch adhesive bonds. Dent Mater 2008;24:117-23.  Back to cited text no. 9
    
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Sönmez IS, Aras S, Tunç ES, Küçükeşmen C. Clinical success of deproteinization in hypocalcified amelogenesis imperfecta. Quintessence Int 2009;40:113-8.  Back to cited text no. 10
    
11.
Ayman E, Amera A, Khursheed AM. Sodium hypochlorite as a deproteinizing agent optimize orthodontic brackets adhesion using resin modified glass ionomer cement. Austin J Dent 2016;3:1-7.  Back to cited text no. 11
    
12.
Espinosa R, Valencia R, Uribe M, Ceja I, Saadia M. Enamel deproteinization and its effect on acid etching: An in vitro study. J Clin Pediatr Dent 2008;33:13-9.  Back to cited text no. 12
    
13.
Ahuja B, Yeluri R, Baliga S, Munshi AK. Enamel deproteinization before acid etching – A scanning electron microscopic observation. J Clin Pediatr Dent 2010;35:169-72.  Back to cited text no. 13
    
14.
Pithon MM, Ferraz Cde S, de Oliveira Gdo C, Pereira TB, Oliveira DD, de Souza RA, et al. Effect of 10% papain gel on enamel deproteinization before bonding procedure. Angle Orthod 2012;82:541-5.  Back to cited text no. 14
    
15.
Hasija P, Sachdev V, Mathur S, Rath R. Deproteinizing agents as an effective enamel bond enhancer – An in vitro study. J Clin Pediatr Dent 2017;41:280-3.  Back to cited text no. 15
    
16.
Harleen N, Ramakrishna Y, Munshi AK. Enamel deproteinization before acid etching and its effect on the shear bond strength – An in vitro study. J Clin Pediatr Dent 2011;36:19-23.  Back to cited text no. 16
    


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