Evaluation of the Effect of Casein Phosphopeptide – Amorphous Calcium Phosphate on
Fracture Resistance of Single Rooted EDTA Treated Teeth (An in Vitro Study).

doi:10.21203/rs.3.pex-2156/v1

Method Article

Evaluation of the Effect of Casein Phosphopeptide – Amorphous Calcium Phosphate on Fracture Resistance of Single Rooted EDTA Treated Teeth (An in Vitro Study).

https://doi.org/10.21203/rs.3.pex-2156/v1

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Aim: to evaluate the effect of CPP-ACP on fracture resistance of single rooted EDTA treated teeth when used as a final irrigant.

Methodology: A twenty one single rooted teeth will be randomly divided into three equal groups based upon the final irrigation protocol as follows: Group I (EDTA / CPP-ACP) – final irrigation with EDTA 17% for 1 min

followed by irrigation with CPP-ACP remineralizing agent for 10 min. Group II (EDTA alone) – final irrigation with EDTA 17% for 1 min. Group III (normal saline) – final irrigation with normal saline (0.9% NaCl) for 5 min. Then, Specimens from all groups will be prepared for test assembly. The apical 5 mm of roots will be embedded along the long axis in self-curing acrylic blocks, with 8 mm of each root exposed. The fracture resistance of the specimen will be tested using a Universal Testing Machine.

Endodontic treatment aims to remove all sources of infection within the complex root canal

system. This is obtained by multiple successive steps that end up with a clean, well-shaped canal,

free of necrotic tissue and suitable for receiving a three dimensional seal.

Subsequent to chemo-mechanical preparation, a residual smear layer is formed on the canal walls.

This amorphous layer contains organic and inorganic substances, microorganisms and toxins,

fragments of odontoblastic processes, and tissue debris¹. Its generation is inevitable during root

canal instrumentation, with the risk of influencing the sealing ability of the sealer-dentin interface,

and increasing the potential for microbial survival and reproduction. Moreover, it may limit the

penetration of irrigants and medicaments into the dentinal tubules. Therefore, removal of smear

layer is usually preferred².

Current methods of smear layer removal include the use of chelating agents in conjunction with

other irrigants to eliminate effectively such a layer. The use of sodium hypochlorite (NaOCl) has

a sufficient role in dissolving the organic component, but unfortunately it has a very limited action

against the inorganic part. Hereby, the critical role of chelating agents is evident, in which they

react with the calcium ions in dentin, converting them to soluble calcium chelates^2,3. Most

chelating solutions are based on Ethylenediaminetetraacetic (EDTA) in various concentrations.

Unfortunately, It has been reported that endodontic irrigation is capable of causing alterations in

the chemical composition of dentin, and this effect can increase by extended application time and

concentration of irrigation solutions. These changes in the chemical property of dentin arise

because of the changes in the inorganic and the organic phases of the dentin. In turn, the

microhardness, permeability, and solubility characteristics of dentin change, and this might also

adversely affect the fracture resistance of teeth⁴.There is no statistical difference in smear layer

removal when using 17% EDTA for 1 or 3 minutes⁵, but prolonged use of high concentrations of

17% EDTA may increase the risk of root fracture⁴

To overcome these alterations in the mechanical properties of dentin, the concept of

remineralization was introduced. It is a repair mechanism that aims to restore the mineral content

of the tooth structure in ionic forms to the hydroxyapatite crystal lattice⁶. Unfortunately, dentin

remineralization is more complicated and less effective than enamel remineralization because of

the fewer amounts of residual mineral crystals in dentin, and the presence of exposed collagen

fibrils on the dentin surface. This complex structure limits the classical ion-based crystallization

concept that is applicable for enamel. Herein, the concept of biomimetic remineralization appears,

which imitates the natural process of mineralization⁷.

Organic compartment of dentin is composed mainly of collagen fibrils, in addition to non- collagenous

proteins (NCPs) that comprise less than 10% of organic content. These NCPs have high affinity for

both calcium ions and collagen fibrils. They play a critical role in the regulation of mineralization in

which they control the apatite nucleation and growth in dentin during mineralization. Biomimetic

remineralization utilizes artificial NCP analogues to guide the calcium-phosphate recruitment to the

collagen matrix⁷.

Casein phosphopeptide – amorphous calcium phosphate (CPP-ACP) is a protein nanotechnology

introduced by Eric Reynolds and co-workers, in which CPP is a milk protein derivative that acts as

NCP analogue⁶. GC Tooth Mousse⁽¹⁾ is a commercially available remineralizing agent that utilizes this

technology⁸. It can be used for dentin and enamel remineralization in cases of dentin hypersensitivity,

enamel demineralization and initial caries lesions.

Although there are many studies that discussed the effect of remineralization of coronal dentin on its

fracture resistance and mineral content^7,9,10,14,15, to our knowledge, there is lack of studies that

investigate the effect of remineralization of root dentin after using chelating agents on its fracture

resistance.

The aim of this in vitro study is to evaluate the effect of CPP-ACP on fracture resistance of

single rooted EDTA treated teeth when used as a final irrigant.

Teeth preparation:

The crown of each tooth will be sectioned perpendicular to the long axis of the root below

the cementoenamel junction using a high speed diamond bur under a water coolant so that

the length of the root will be adjusted to 13 mm. The working length of each root will be

determined by inserting #10 K-file until it just exits the apical foramen, and then 1 mm will

be subtracted from the obtained length.

Canal preparation technique:

The root canals will be prepared with the hyflex rotary system until file size 40 taper 4%

using a gentle in and out motion with a torque-controlled endodontic motor at a constant

speed of 350 rpm and torque of 1.5 N.cm. During biomechanical preparation, the canals will

be irrigated with 5 ml of 5.25% sodium hypochlorite solution prior to instrumentation, 5 ml

after each instrument change and 5ml as final flush after root canal instrumentation to reach

a total volume of 25 ml using 30-gauge, side-vented needle placed at 1 ml from the apex

The root samples will be randomly divided into three groups based upon the final

irrigation protocol as follows:

Group I (EDTA / CPP-ACP) – final irrigation with EDTA 17% for 1 min

followed by irrigation with CPP-ACP remineralizing agent for 10 min.

-Remineralizing solution will be prepared by diluting the GC Tooth Mousse to

10 times with distilled water17. Fracture resistance will be measured after the

final irrigation protocol.

Group II (EDTA alone) – final irrigation with EDTA 17% for 1 min.

Group III (normal saline) – final irrigation with normal saline (0.9% NaCl) for

5 min.

Treatment for intervention I group (EDTA / CPP-ACP):

After chemo-mechanical preparation, each sample will be irrigated with 5 ml

EDTA 17% for 1 minute, then washed thoroughly with normal saline, followed by

10 ml of the previously prepared remineralizing solution for 10 minutes, then

washed adequately with normal saline.

Treatment for intervention II group (EDTA alone):

After chemo-mechanical preparation, each sample will be irrigated with 5 ml

EDTA 17% for 1 minute, then washed thoroughly with normal saline.

Treatment for control group (normal saline):

After chemo-mechanical preparation, final irrigation with 10 ml normal saline for

5 minutes.

Preparation for Fracture resistance testing: Specimens from all groups will be

prepared for test assembly. The apical 5 mm of roots will be embedded along the

long axis in self-curing acrylic blocks, with 8 mm of each root exposed

Fracture resistance measurement:

The fracture resistance of the specimen will be tested using a Universal Testing Machine (Lloyd, LR50K, UK). A custommade stainless steel loading indenter with a round tip (r = 2 mm) will be used to deliver force directed along the long axis of the tooth. The load will be increased at a rate of 1 mm/min until fracture occurs. The fracture will be evidenced by an audible crack and/or a sudden drop in load. The force necessary to fracture the root will be recorded in Newtons (N).

1. Violich, D. R., Chandler N. P. The smear layer in endodontics - A review. Int. Endod. J.

2010;51:605-621.

2. Tsenova--ilieva I. and Karova E. Effect of Endodontic Irrigants on Root Dentin

Microhardness : A Systematic Review. Int. J. Sci. Res. 2020;9: 491–496.

3. Baldasso F. E. R., Roleto L., Silva V. D., da Morgental R. D. and Kopper P. M. P. Effect of

final irrigation protocols on microhardness reduction and erosion of root canal dentin. Braz.

Oral Res. 2017;31:e40.

4. Uzunoglu E., Aktemur S., Uyanik M. O., Durmaz V. and Nagas E. Effect of

ethylenediaminetetraacetic acid on root fracture with respect to concentration at different time

exposures. J. Endod. 2012;38:1110-13.

5. Safwat, H., Nour El Deen, M., Bastawy, H. Evaluation of Smear Layer Removal and Calcium

Ions Concentration in Intraradicular Dentin Treated with Apple Vinegar (SEM Study). Al-Azhar

Dental Journal for Girls, 2017; 4(3): 279-287.

6. Arifa M. K., Ephraim R. and Rajamani T. Recent Advances in Dental Hard Tissue

Remineralization: A Review of Literature. Int. J. Clin. Pediatr. Dent. 2019;12:139-144.

7. Cao C. Y., Mei M. L., Li Q. L., Lo E. C. M. and Chu C. H. Methods for biomimetic

remineralization of human dentine: A systematic review. Int. J. Mol. Sci. 2015;16:4615-

4627.

8. Garg P., Singh U., Sinha D. and Tyagi S. An update on remineralizing agents. J. Interdiscip.

Dent. 2013;3:151-158.

9. Gandolfi M. G., Taddei P., Pondrelli A., Zamparini F., Prati C, Spagnuolo G.

Demineralization, collagen modification and remineralization degree of human dentin after

EDTA and citric acid treatments. Materials (Basel). 2019;12(1):25.

10. Bertassoni L. E., Habelitz S., Marshall S. J. and Marshall G. W. Mechanical recovery of

dentin following remineralization in vitro - An indentation study. J. Biomech.

2011;44(1):176-81.

11. Schneider SW. A comparison of canal preparations in straight and curved root canals.

Oral Surg Oral Med Oral Pathol 1971;32:271-5.

12. Uzunoglu E, Yilmaz Z, Erdogan O, Görduysus M. Final Irrigation Regimens Affect

Fracture Resistance Values of Root-filled Teeth. J Endod. 2016 Mar;42(3):493-5.

13. Uzunoglu E, Aktemur S, Uyanik MO, Durmaz V, Nagas E. Effect of

ethylenediaminetetraacetic acid on root fracture with respect to concentration at different

time exposures. J Endod. 2012 Aug;38(8):1110-3..

14. Firouzmandi M, Afzali N, Parsaie Z, Mohammadi N. Effect of Casein Phosphopeptide-

Amorphous Calcium Phosphate on Fracture Resistance of Reattached Tooth Fragments

Using Conventional and Self-Adhesive Bioactive Flowable Composite. Eur J Dent. 2020

Mar;14(2):288-293.

15. Surekha GL, Vinay C, Baliga S, Uloopi KS, RojaRamya KS, Penmatsa C. Effect of

preconditioning the fractured coronal fragments with remineralizing agents on fracture

resistance of re-attached teeth. J Indian Soc Pedod Prev Dent. 2021 Oct-Dec;39(4):384-

387.

16. Bhandary S, Kakamari S, Srinivasan R, Chandrappa MM, Nasreen F, Junjanna P. A

comparative evaluation of the effect of 8% and 17% ethylenediaminetetraacetic acid

exposure for 1 min and 10 min on the fracture resistance of endodontically treated roots:

An in vitro study. J Conserv Dent. 2017 Jan-Feb;20(1):21-24.

17. CPP-ACP as a final irrigant in improving the micro-hardness of erosive root dentin and its

influence on the bond strength of self etch resin sealer - An in-vitro study. J. Clin. Diagnostic

Res. 2016;10:53–56.

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Evaluation of the Effect of Casein Phosphopeptide – Amorphous Calcium Phosphate on Fracture Resistance of Single Rooted EDTA Treated Teeth (An in Vitro Study).

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Evaluation of the Effect of Casein Phosphopeptide – Amorphous Calcium Phosphate on Fracture Resistance of Single Rooted EDTA Treated Teeth (An in Vitro Study).

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Introduction

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