Evaluation of the Effect of Different Deproteinizing Agents on the Clinical Success of Fissure Sealants in Primary Teeth: Splint Mouth Clinical Study
Phase: NA
Conditions: Fissure Sealant
Sponsor: Tokat Gaziosmanpasa University

 
 
Evaluation of the Effect of Different 
Deproteinizing Agents on the Clinical 
Success of Fissure Sealants in 
Primary Teeth: Splint Mouth Clinical 
Study 
 
 
18/12/2023 
 

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RESEARCH PROTOCOL 
 
Evaluation of the Effect of Different Deproteinizing Agents on the Clinical Success of Fissure 
Sealants in Primary Teeth: Splint Mouth Clinical Study 
 
 
1. PURPOSE and HYPOTHESIS OF THE RESEARCH 
Dental caries is a chronic infectious, preventable disease in which prolonged exposure 
to fermentable carbohydrate causes an ecological shift in the composition and activity of the 
bacterial biofilm, resulting in the physical and chemical destruction of dental hard tissues and 
disrupting the balance between demineralization and remineralization of tooth surfaces.1 The 
teeth with the highest caries risk are primary molars, respectively. 2 The retantive areas formed 
by pits and fissures on the occlusal surfaces of molars are more favorable areas for caries 
initiation from the eruption of teeth.3 Pit and fissure sealants are the most effective method used 
to prevent caries formation in these areas. Pit and fissure sealants are a non-invasive method 
that has been used for many years to prevent caries formation or to stop the progression of initial 
caries lesions limited to enamel on tooth surfaces considered to be at high risk of caries.4 There 
are many clinical studies in the literature reporting that fissure sealants applied with non- 
invasive techniques are highly effective in preventing pit and fissure caries.5,6 Various methods 
such as pumice prophylaxis and deproteinization have been recommended as surface 
preparation methods to increase the success of fissure sealants.7,8 During the application of pit 
and fissure sealants, acid roughening of the enamel as a pre- application is an inevitable step.9 
Removal of organic materials from the enamel surface before acid roughening provides better 
acid roughening on the enamel, increases bond strength, improves adhesion and retention of the 
adhesive material. There are many studies in the literature showing that the use of 5.25% NaOCl 
as an enamel deproteinization agent is successful in positively affecting adhesion by removing 
the organic elements of the pellicle both in the enamel structure and on the enamel surface 
before acid application.10-13 Hypochlorous acid is produced as a result of oxidative reactions by 
cells of the body immune system, neutrophilic polymorphonuclear leukocytes, to overcome 
pathogenic microorganisms and fight infection.14,15 Recently, slightly acidic HOCl solutions 
developed by electrolyzing NaOCl have been widely used as disinfectants.16 Hypochlorous acid 
has been shown to significantly reduce the lipopolysaccharide concentration of Porphyromonas 
gingivalis compared to NaOCl and chlorhexidine, and is a well-tolerated agent by oral tissues.17 
Furthermore, HOCl used as a mouthwash has been reported to produce no systemic side 

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effects.18 In clinical dentistry, HOCl has been shown to be highly effective as a result of its 
antimicrobial and deproteinizing properties. With its low acidity, HOCl is an antiseptic irrigant 
with exceptional properties, exhibiting not only biocompatibility and low cytotoxicity, but also 
rapid and highly effective antimicrobial activity in physiological concentration ranges.19 The 
aim of this study was to compare the effects of sodium hypochlorite and hypoclous acid, 
deproteinizing agents used to prepare the tooth surface for fissure sealant application in primary 
teeth, on the clinical success of fissure sealants. 
 
 
The null hypotheses of our study; 
 
I. There is no significant difference in terms of retention loss and new caries formation between 
the fissure sealants in the group treated with sodium hypochlorite as deproteinizing agent and 
the fissure sealants in the group treated with hypoclurous acid. 
II. There is no significant difference in edge integrity between the fissure sealants in the group 
treated with sodium hypochlorite as deproteinizing agent and the fissure sealants in the group 
treated with hypoclurous acid. 
III. There is no significant difference in edge coloration between the fissure sealants in the group 
treated with sodium hypochlorite as deproteinizing agent and the fissure sealants in the group 
treated with hypoclurous acid. 
IV. There is no significant difference in anatomical form between the fissure sealants in the 
group treated with sodium hypochlorite as deproteinizing agent and the fissure sealants in the 
group treated with hypoclurous acid. 
 
 
Keywords: Deproteinization, Fissure sealant, Hypoclous acid, Sodium hypochlorite 
 
 
REFERENCES 
1. Young DA, Novy BB, Zeller GG, Hale R, Hart TC, Truelove EL, et al. The American Dental 
Association Caries Classification System for clinical practice: a report of the American Dental 
Association Council on Scientific Affairs. J Am Dent Assoc. 2015;146(2):79-86. 
2. Batchelor PA, Sheiham A. Grouping of tooth surfaces by susceptibility to caries: a study in 
5-16 year-old children. BMC Oral Health. 2004;4(1):2. 
3. Horst JA, Tanzer JM, Milgrom PM. Fluorides and Other Preventive Strategies for Tooth 
Decay. Dental Clinics of North America. 2018;62(2):207-34. 

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4. Salama, F. S., and Al-Hammad, N. S. (2002). Marginal seal of sealant and compomer 
materials with and without enameloplasty. International Journal of Paediatric Dentistry, 12(1), 
39-46. 
5. Mertz-Fairhurst EJ, Adair SM, Sams DR, Curtis JW, Jr, Ergle JW, Hawkins KI, et al. 
Cariostatic and ultraconservative sealed restorations: nine-year results among children and 
adults. ASDC J Dent Child 62: 97-107, 1995. 
6. Morphis TL, Toumba KJ, Lygidakis NA. Fluoride pit and fissure sealants: a review. Int J 
Paediatr Dent 10: 90-98, 2000. 
7. Garrocho-Rangel A, Lozano-Vázquez C, Butrón-Tellez-Girón C, Escobar-García D, Ruíz- 
Rodriguez S, Pozos-Guillén AJEjopd. In vitro assessment of retention and microleakage in pit 
and fissure sealants following enamel pre-etching with sodium hypochlorite deproteinisation. 
2015;16(3):212-6. 
8. Hegde RJ, Coutinho RCJJJoISoP, Dentistry P. Comparison of different methods of cleaning 
and preparing occlusal fissure surface before placement of pit and fissure sealants: An in vivo 
study. 2016;34(2):111. 
9. Buonocore, M. G. (1955). A simple method of increasing the adhesion of acrylic filling 
materials to enamel surfaces. Journal of Dental Research, 34(6), 849-853. 
10. Espinosa, R., Valencia, R., Uribe, M., Ceja, I., and Saadia, M. (2008). Enamel 
deproteinization and its effect on acid etching: an in vitro study. Journal of Clinical Pediatric 
Dentistry, 33(1), 13-19. 
11. Valencia, R., Espinosa, R., Borovoy, N., Perez, S., Ceja, I., and Saadia, M. (2018). 
Deproteinization Effectiveness on Occlusal Enamel Surfaces and Resultant Acid Etching 
Patterns: An in vitro Study. Journal of Clinical Pediatric Dentistry, 42(6), 434-441. 
12. Justus, R., Cubero, T., Ondarza, R., &amp; Morales, F. (2010, March). A new technique 
with sodium hypochlorite to increase bracket shear bond strength of fluoride-releasing resin- 
modified glass ionomer cements: comparing shear bond strength of two adhesive systems with 
enamel surface deproteinization before etching. Seminars in Orthodontics, 16(1), 66-75. 
13. Harleen N, Ramakrishna Y, Munshi AJJoCPD. Enamel deproteinization before acid etching 
and its effect on the shear bond strength-An in vitro study. 2011;36(1):19-24. 
14. Mainnemare, A., Megarbane, B., Soueidan, A., Daniel, A., and Chapple, I. L. (2004). 
Hypochlorous acid and taurine-N-monochloramine in periodontal diseases. Journal of Dental 
Research, 83(11), 823-831. 
15. Fukuzaki, S. (2006). Mechanisms of actions of sodium hypochlorite in cleaning and 
disinfection processes. Biocontrol Science, 11(4), 147-157 
16. Wang, L., Bassiri, M., Najafi, R., Najafi, K., Yang, J., Khosrovi, B., Hwong, W., Barati, E., 
Belisle, B., Celeri, C., and Robson, M. C. (2007). Hypochlorous acid as a potential wound care 
agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of 
innate immunity. Journal of Burns and Wounds, 6(e5). 
17. Block, M. S., and Rowan, B. G. (2020). Hypochlorous Acid: A Review. Journal of Oral 
Maxillofacial Surgery, 78(9), 1461-1466. 

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18. Morita, C., Nishida, T., and Ito, K. (2011). Biological toxicity of acid electrolyzed functional 
water: effect of oral administration on mouse digestive tract and changes in body weight. 
Archives of Oral Biology, 56(4), 359-366. 
19. Pashley, D. H., Tao, L., Boyd, L., King, G. E., and Horner, J. A. (1988). Scanning electron 
microscopy of the substructure of smear layers in human dentine. Archives of Oral Biology, 
33(4), 265-270. 
 
 
2. MATERIALS AND METHOD 
 
Case Selection 
 
This study will include patients between the ages of 4 and 6 years old who are admitted to Tokat 
Gaziosmanpaşa University, Faculty of Dentistry, Department of Pedodontics for routine check- 
ups, have no systemic problems, have a score of 3 and 4 according to the Frankl Behavior Scale 
20 (Table 1), have no parafunctional habits such as clenching and/or grinding, have erupted 
caries-free primary lower second molars, and have an indication for fissure sealant application. 
In our single-center, split mouth clinical study, sodium hypochlorite (Wizard, Turkey) will be 
applied to one of the lower second molars and hypochlorous acid (Superox, Anolit Hijyen ve 
Kimya Sanayi Anonim Şirketi, Ankara, Turkey) will be applied to the other as deproteinizing 
agent before non-invasive (without any abrasion on the tooth) fissure sealant application under 
rubber-dam isolation. Then 37% phosphoric acid (K-ETCHANT Syringe, Kuraray Noritake 
Dental Inc., Japan) will be applied. ClinproTM Sealant (3M ESPE, St. Paul, MN, United States 
of America), a resin-based sealant that polymerizes with light, changes color during 
polymerization, contains fluorine and has a low filler content, will be used as fissure sealant in 
both groups. 
Table 1. Frankl Behavior Scale 
 
 
1 
 
- 
 
Definitely negative 
Refusal of treatment, 
crying forcefully, 
fearful or any other 
overt evidence of 
extreme negativism. 
 
 
2 
 
 
-- 
 
 
Negative 
 
 
Reluctant to accept 
treatment; 
uncooperative, some 
evidence of negative 
attitude but not 

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pronounced, i.e. 
/sullen, withdrawn. 
3 
+ 
Positive 
Acceptance of 
treatment; at times 
cautious, willingness 
to comply with the 
dentist, at times with 
reservation but patient 
follows the dentist's 
directions 
cooperatively. 
4 
++ 
Definitely positive 
Good rapport with the 
dentist, interested in 
the dental procedures, 
laughing and enjoying 
the situation. 
 
 
 
 
Power Analysis of the Study 
 
As a result of the power analysis of the study with 95% confidence (1-α), 95% test power (1-β) 
and Chi-square test with an effect size of w=0.576, the number of samples to be included in the 
study was found to be 47. When the dropout rate was taken as 20%, the study was planned to 
be conducted with 60 samples.21 
Clinical Assessments 
 
The patients included in the study will be followed up by two observers at 3,6, 12 and 24 
months. At the control sessions, loss of retention and new caries formation (Table 2) according 
to the Modified Simonsen Criteria (1991) (22), edge integrity (Table 3) and edge coloration 
(Table 4) according to the criteria of Feigal et al. (2000) and anatomical form (Table 5) will be 
evaluated. 
Table 2. Modified Simonsen Criteria 
 
Score 0 Fissure sealant completely in the mouth and no new caries formation. 
Score 1 Partial loss of fissure sealant, no new caries formation. 
Score 2 Partial loss of fissure sealant and new caries formation. 

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Score 3 Complete loss of fissure sealant, no new caries formation. 
Score 4 Complete loss of fissure sealant and new caries formation. 
 
 
Table 3. Edge Integrity Evaluation Criteria 
 
0 Fissure sealant and tooth surface are intact and indistinguishable with the probe. 
 
1 The edge of the fissure sealant can be distinguished with a probe. 
 
2 There is spacing along the edge of the fissure seal and deep cracks reaching the central 
fossa. 
Table 4. Edge Coloration Evaluation Criteria 
 
0 No discoloration between fissure sealant and tooth 
 
1 Discoloration in only one area 
 
2 Discoloration in many areas 
 
3 Severe discoloration indicating the presence of leakage 
 
Table 5. Anatomical Form Evaluation Criteria 
 
0 Consistent and continuous and structure in harmony with occlusal form 
 
1 Change in anatomical form but all pits and fissures covered 
 
2a Partial loss of one or two pits or fissures, but no need to repair or replace fissure sealant 
2b Partial loss of pits and fissures, replacement or repair of fissure sealant required 
3 Loss in all pits and fissures 
7 Partial loss due to occlusion 
9 Bubble not connected to margins 
 
 
Statistical Analysis 
 
All statistical analyses will be analyzed using IBM SPSS V23. Chi-square test will be used to 
compare categorical data by groups. 

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REFERENCES 
20. Frankl S, Shiere F, Fogels H. Should the parent remain with the child in the dental operatory. 
J Dent Child 1962; 29(1): 150-163. 
21. Rishika, Garg, N., Mayall, S. S., Pathivada, L., &amp; Yeluri, R. (2018). Combined Effect 
of Enamel Deproteinization and Intermediate Bonding in the Retention of Pit and Fissure 
Sealants in Children: A Randomized Clinical Trial. The Journal of clinical pediatric dentistry, 
42(6), 427-433. 
22. Simonsen RJ. Retention and effectiveness of dental sealant after 15 years. J Am Dent Assoc 
1991; 122(10): 34-42. 
23. Feigal RJ, Musherure P, Gillespie B, Levy-Polack M, Quelhas I, Hebling J.Improved sealant 
retention with bonding agents: A clinical study of two-bottle and single-bottle systems. J Dent 
Res 2000; 79(11): 1850-1856. 
 
 
PLACE(S) OF IMPLEMENTATION OF THE RESEARCH 
 Single Center 
 Multicenter 
 
 
Name of the center 
 
Name of the principal investigator 
 
phone 
 
Center 1 
 
Tokat 
Gaziosmanpasa 
University, Faculty 
of Dentistry 
 
Dr. Öğr. Üyesi Alem COŞGUN 
 
+90 552 413 32 22 
 
 
 
 
 
 
INFORMATION ABOUT RESEARCHERS 
 
Name-Surname 
Title 
Mission 
phone 
Merve İNAN 
GÜL 
Research assistant 
Obtaining and evaluating 
data, writing the research 
+90 545 615 99 01 
Alem COŞGUN 
Assistant professor Planning the research, 
Evaluation of the data 
+90 552 413 32 22 

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INFORMATION ABOUT VOLUNTEERS 
 
 
Qualification of volunteers 
Patients aged 4-6 years with no systemic problems, 
a score of 3 or 4 on the Frankl Behavior Scale, no 
habit of clenching and/or grinding, erupted caries- 
free primary lower second molars, and indication 
for fissure sealant application 
 
Working group 
 
60 
 
Number of control groups 
 
60 
Inclusion criteria 
He has no systemic problems and scores 3 and 4 
on the Frankl Behavior Scale, 
No habit of clenching and/or grinding teeth, 
He has erupted primary lower second molars 
without decay, 
Patients aged 4-6 years with an indication for 
fissure sealant application. 
 
Exclusion criteria 
Teeth with fissure sealant applied, 
Teeth with developmental defects such as 
hypoplasia, 
Teeth with aproximal or initial carious lesions on 
one or both lower primary second molars, 
Children who are not systemically healthy, 
Non-cooperative children with scores of 1 and 2 
on the Frankl Behavior Scale, 
Children and parents who refuse to attend follow- 
up appointments, 
Children with parafunctional habits such as teeth 
clenching. 
 
 
 
I will comply with the World Medical Association (WMA) HELSINKI Declaration 
(and/or World Psychiatric Association HAWAI Declaration), Good Clinical Practices and Good 
Laboratory Practices Rules during this research, and I will immediately notify the Clinical 
Research Ethics Committee in case of any unexpected adverse effect or event, I promise to 
notify the committee in writing in case of any changes in the study protocol during the research, 

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to submit a report on the progress of the research to the Clinical Research Ethics Committee 
every 6 months according to the duration of the research, and to notify the Clinical Research 
Ethics Committee immediately if the research is stopped. 
 
 
RESEARCH RESPONSIBLE COORDINATOR 
 
Asst. Prof.