A Comparative Evaluation of fibrin density with Chitosan, Papain and 17% EDTA-Normal saline combination as irrigants in teeth with open apices: An ex vivo SEM study.

Context: In regenerative endodontic techniques, a blood clot acts as a natural scaffold for revitalizing dental pulp tissue. Although 17% ethylene diamine tetra acetic acid (EDTA) releases growth factors from the dentin matrix and induces odontoblast differentiation, it has anticoagulant property. Aims: The aim of the study is to evaluate the ability of alternatives (0.2% chitosan and papain) in fibrin formation when used as root canal irrigants during regenerative endodontic procedures in teeth with open apices. Also investigated whether 0.2% chitosan and papain solutions can be used as alternatives to 17% EDTA-normal saline solution (EDTA-NSS) combination, as root canal irrigants during regenerative endodontic procedures in teeth with open apices. Subjects and
Methods: Twenty-five human freshly extracted single-rooted mandibular premolars were taken. They were then divided into five groups and irrigated accordingly. Human donor blood samples were collected and placed in the specimens and fibrin density was evaluated using scanning electron microscopy. Comparison of fibers per 10 μm among 5 experimental groups at each level was performed using one-way analysis of variance test.
Results: Results revealed that the specimens irrigated with EDTA-NSS followed by papain had an increased fibrin density when compared with chitosan. Conclusions: All the root canal irrigants that were used in this study as part of treatment protocol for open apex exhibited positive results. Copyright:

INTRODUCTION

The preparation of root canal surfaces is a major problem for modern endodontic instruments. More than 35% of the root canal surfaces are left uninstrumented by the techniques utilizing nickel titanium files. The goal of regenerative endodontic therapy (RET) is to restore the pulp-dentin complex that has been impaired by infection.

Ethylene diamine tetra acetic acid (EDTA) is a biocompatible artificial amino acid and also an anticoagulant. The ability of EDTA solution to chelate metallic ions is one of its major characteristics.[12]

Therefore, an ex vivo study was conducted with the aim of evaluating fibrin density when Chitosan, Papain, and 17% EDTA-Normal saline combination are used as irrigants in teeth with open apices.

SUBJECTS AND METHODS

This ex vivo study was conducted to evaluate fibrin density when Chitosan, Papain (Alpha Omega Research Foundation, Salem, Tamilnadu), and 17% EDTA (Avue Prep, + Dental avenue (l) Pvt Ltd, Thane, Mumbai)-Normal saline combination (Aculife Healthcare Pvt. Ltd., Ahmedabad, Gujarat, India) are used as irrigants in teeth with open apices.

The study protocol was approved by the Scientific Advisory committee and Institutional Ethics Committee IEC/24270919/S/6 and the study methodology followed the checklist for reporting in vitro studies guidelines.

This study was carried out by obtaining twenty-five freshly extracted human single-rooted mandibular premolars. Then, using a slow-speed diamond saw bur (Isomet; Buehler Limited, Lake Bluff, IL), the teeth were sliced perpendicularly to the long axis of the root at the cementoenamel junction.

Sample preparation

Trimming was done in the apical end of a sectioned root until a uniform length of 9 mm was obtained. The root segment was prepared using no. 1-4 Gates Glidden drills (Kerr, Kerr Corporation, Orange, CA) through each canal to achieve an open apex of 1 mm in diameter under 20 ml 1.5% sodium hypochlorite (NaOCl) (Prime dental products private Ltd.) irrigation.[3] Without striking the root canal, two longitudinal grooves were made on the external surface along the specimen at the buccal and lingual aspects using a high-speed cylindrical diamond bur (Meisinger) under copious irrigation. Then, two parallel horizontal grooves were made beneath the cementoenamel junction for 3 and 6 mm (at the longitudinal grooves), to separate the specimen into coronal middle and apical portions, respectively.

The specimens were then divided equally into five experimental groups and then settled in plastic droppers with impression material before irrigation. A 20-mL disposable plastic syringe with a 25-G irrigation needle was used. It was positioned approximately 1 mm from the root end to reproduce the clinical protocol.[4] The specimens were then irrigated with the corresponding irrigating solutions in consonance with the groups.

Irrigation process

For irrigation in teeth with open apices, we divided into five groups (one control group and four experimental group).

Group A (Control Group) -20 Ml 17% EDTA for 5 min followed by 20 Ml normal saline solution (NSS) for 5 min, Group B - 20 Ml 0.2% chitosan solution for 5 min, Group C - 20 Ml papain solution for 5 min, Group D - 20 Ml 0.2% chitosan solution for 5 min followed by 20 Ml papain solution for 5 min, Group E - 20 Ml 0.2% chitosan for 5 min, 20 Ml papain solution for 5 min and 20 Ml NSS for 5 min.

Collection of blood sample

Blood samples were collected from a healthy volunteer (SELF) who did not take any anticoagulant medication. The samples were then spread on the specimens that have been irrigated according to the irrigating protocol and fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for 1 h. The sample was washed 3 times in distilled water for 10 min. Subsequently, they were dehydrated in a series of ethanol dilutions (30%, 50%, 70%, 90%, and 95% and 3 times at 100% with an equilibration step of 5 min each).

Specimen preparation for scanning electron microscopy

After fixation, samples were dried using hexamethyldisilazane (Sigma-Aldrich, St Louis, MO) for 2 h under an analog steel fume hood. All processes were conducted at room temperature. The samples were sputter coated with 12.5 nm platinum-palladium at 2.2 kV and 20 mA for 3 min using an E-102 Ion Sputter. The areas at the coronal, middle, and apical portions of the root were visualized with a JSM 6610 LV Scanning electron microscope (SEM) (JEOL, Tokyo, Japan) at ×500 and ×2500 magnifications.

Characterization of blood clot

The characteristics of blood clots were observed by direct visual examination at ×2500 magnification in 3 random areas at each level of the sample. Low-magnification images were taken at ×500 magnification in the center of all levels to validate and evaluate the homogeneity of the specimen in a larger area.[5]

Fiber density of blood clot

Micrographs at ×2500 magnification were evaluated using Image software. A 5 × 6 line grid was placed on individual images, and the number of fibers intersecting the lines was counted using ImageJ software. The total number of fibers crossing the line per 10 mm at each level of the sample from the 3 micrographs was averaged.

Comparison of fibers per 10 μm among 5 experimental groups at each level was performed using one-way analysis of variance test.

RESULTS

Characteristic of blood clot

The clot surface in Group 1 (EDTA-NSS) was composed of a dense meshwork of fibrins with abundant biconcave erythrocytes at all levels of the root canal surfaces. The most commonly found elements that were trapped inside the fibrin network were biconcave erythrocytes [Figure 1a]. The surfaces of fibrin clots in the Group 3 (Papain) followed by Group 2 (Chitosan) groups were also composed of a dense meshwork of fibrins with abundant biconcave erythrocytes at all levels of the root canals [Figure 1b and c].

Figure 1

A root canal treated with various irrigation protocols at the apical portion of the root at a magnification of ×2500 (a) Ethylene diamine tetra acetic acid-normal saline solution group (b) Chitosan group (c) Papain group (d) Chitosan-papain group (e) Chitosan-papain-normal saline solution group

The number of fibrin networks in Group 4 (Chitosan-papain) and Group 5 (Chitosan-papain-NSS) specimens at all levels of root canal surfaces were lower and shorter than that observed in the above groups [Figure 1d and e].

Fiber density

Fiber density in the EDTA-NSS group exhibited the greatest values of 8.94, 9.13, 9.57 fibers per 10 μm at the coronal, middle, and apical portions, respectively [Table 1]. Group 4 (Chitosan-papain) and Group 5 (Chitosan-papain-NSS) specimens showed statistically significantly lower fiber density values than Group 1 (EDTA-NSS), Group 2 (Chitosan) and Group 3 (Papain) specimens at coronal, middle, and apical portions of the root (P < 0.001) [Table 1].

Table 1

Number of fibers per 10 μm in different groups after irrigating with each protocol

	Coronal	Middle	Apical
Group 1 (EDTA-NSS)	8.94	9.13	9.57
Group 2 (chitosan)	6.45	6.73	6.94
Group 3 (papain)	6.58	6.74	6.94
Group 4 (chitosan-papain)	5.73	5.85	5.93
Group 5 (chitosan-papain-NSS)	4.56	4.73	4.84
P (ANOVA)	0.001	0.001	0.001

EDTA: Ethylene diamine tetra acetic acid, NSS: Normal saline solution, ANOVA: Analysis of variance

DISCUSSION

Trauma or dental caries exposure at the time of root development results in an immature tooth with open apex. Techniques to deal with open apex include custom-made roll cone technique, short fill technique, obturation done with guttapercha and sealer with periapical surgery, apical closure technique, and induction of periapical bleeding with instrumentations.

The conventional method for treating necrotic teeth with open apex is apexification, a method to induce calcific barrier in a tooth with open apex or the continued apical development of an incompletely formed root in teeth with necrotic pulp tissue. The most commonly advocated medicaments to induce calcific barrier are calcium hydroxide, mineral trioxide aggregate (MTA), and other biomimetic materials.[6]

Most material used for apexification has got some limitations. For example, Calcium hydroxide is used to stimulate hard tissue deposition at the apex forming osteodentin. However, it involves a long treatment time and the prognosis is always uncertain. Long-term exposure of the tissue to calcium hydroxide may weaken the root structure, resulting in cervical fractures, as well as inducing periapical bone necrosis when there is overfilling of the material.[6]

MTA has certain drawbacks such as long setting time and discoloration of the teeth. Above all, after apexification with any of the biomimetic material, root canal treatment has to be done once the root formation gets completed, thus compromising the vitality of tooth.[6]

Regenerative endodontics is the use of biologically based procedures that are designed to replace the damaged tooth structures such as pulp, dentin, root structures, cells of pulp-dentin complex with live viable tissues, preferably of the same origin, that restore the normal physiologic functions of the pulp-dentin complex.[7]

REP is aimed to regenerate the pulp-dentin complex damaged by infection, trauma or developmental anamoly of immature permanent teeth with necrotic pulp.

For regenerating dental pulp tissue, a blood clot acts as a natural scaffold. Revascularisation is achieved by inducing bleeding into the pulp canal space from periapical tissues. After tissue injury, a clot occurs by the activation of thrombin and fibrinogen to form a cross-linked fibrin network scaffold.[7]

Blood clotting also known as coagulation is the process by which blood changes from a liquid to gel, forming blood clot. This process involves clotting factors and many blood cells. Blood clot has many advantages over other alternative scaffolds in regenerative endodontics such as no allergic reaction, reduced cost, increased prognosis, convenience, and comfort for patients.[8] Calcium ions play an important role as a cofactor in clotting process. They help in activation of II, VII, IX, X factors, and platelets.[9]

Various irrigating solutions such as EDTA, NaOCl, and chlorhexidine have been used in regenerative endodontics. 17% EDTA, a commonly used root canal irrigant, is an irreversible chelating agent. It is highly recommended for liberating growth factors from root dentin. EDTA induces odontoblast differentiation by releasing growth factors from the dentin matrix. However, due to its anticoagulant property, it affects the clot formation and fibrin deposition by chelating the calcium ions in the blood.[1]

Seventeen percent EDTA is recommended in regenerative endodontics because it liberates bioactive molecules from dentin and these molecules modulate the biological activities of cells recruited from the periapical tissues.[10]

The calcium ions that are present in the blood are chelated by residual EDTA and result in interruption of clotting process.

Thus, the aim of this study was to find alternative sources for fibrin-forming agents in regenerative endodontic procedures in teeth with open apices. The present study investigated the clotting ability of EDTA, proteolytic enzyme papain, and a linear polysaccharide chitosan on blood-dentin contacted surface.

In this study, twenty-five freshly extracted human single-rooted mandibular premolars were obtained. They were divided into five groups and irrigated accordingly. Blood samples were collected and placed on the irrigated specimens to evaluate the fibrin density using scanning electron microscopy. Scanning electron microscopy was used to analyze the blood clots because it can visualize the microscopic structure of fibrin network density.

In this study, the result values revealed that the fibrin density achieved for specimens irrigated with Group 1 (EDTA-NSS) was (9.57 ± 0.02). This was followed by Group 3 (papain) (6.94 ± 0.02) which had an increased fibrin density when compared with Group 2 (Chitosan) (6.94 ± 0.02), Group 4 (Chitosan-Papain) (5.93 ± 0.02), and Group 5 (Chitosan-Papain-NSS) (4.84 ± 0.03) [Table 1].

The results exhibited platelet clumping in the EDTA groups. This may be due to the fact that EDTA affected a receptor in platelet membranes and caused the loss of fibrinogen binding function, resulting in the clumping of platelets.[1112]

The use of EDTA followed by NSS did not affect fiber density but may possibly affect the amount of growth factor released from root dentin. The results of this study relate with the study by Taweewattanapaisan P et al.,[2] who stated that EDTA followed by NSS irrigation will aid in improved blood clot formation as NSS flushes out the residual EDTA in the root canal, thus reducing the chelation of calcium ions in the blood by EDTA, aiding in hemostasis.

Papain, also known as papaya proteinase I, is an acysteine protease enzyme extracted from the raw fruit of papaya plant. It is a true thrombin and acts directly on fibrinogen to form an insoluble modification resembling fibrin.[1] The fibrin density formed when irrigated with papain solution is slightly lower (6.94 ± 0.02) than EDTA-NSS (9.57 ± 0.02) [Table 1]. It may be due to the fact that it acts directly on fibrinogen to form a fibrillar gel resembling fibrin. Papain converts prothrombin to thrombin only because of its calcium content but the enzyme as such coagulates fibrinogen.[13]

Chitosan, a linear polysaccharide, is a deacetylated derivative of arthropod chitin that is made by treating the chitin shells of shrimp and other crustaceans with an alkaline substance, like sodium hydroxide. It is a hemostatic agent with chelating and antimicrobial properties. It belongs to polysaccharide family and its molecular structure contains N-acetyl-glucosamine and D-glucosamine that are connected by random β (1–40) linkages.[14]

Chitosan has the capacity to remove smear layer as effectively as done by 17% EDTA. It is also an effective chelating agent with less physical and chemical changes in radicular dentin and can be considered as a less invasive alternative or replacement to 17% EDTA.[15]

Higher degree of deacetylation of chitosan improves erythrocyte and platelets aggregation that is required for initiating hemostasis. The negative charges on platelets and erythrocytes cause electrostatic repulsion between them thus hindering the aggregation process causing hemostasis.[14]

Erythrocyte aggregation through electrostatic repulsion is facilitated by the amino groups on chitosan (poly-N-acetyl glucosamine) with its surface charges and hemostasis is induced once it activates platelets. Chitosan should not be deacetylated completely. Another important property of chitosan is its bactericidal action which is one of the important requirements for an ideal root canal irrigant. Free amine groups present on the chitosan give antimicrobial property by binding with bacterial cell wall causing cell lysis.[14]

EDTA enhances the antibacterial activity of chitosan by facilitating its entry into the bacterial cell. Both EDTA and chitosan restrain the growth of microorganisms by inhibiting various enzymes. Furthermore, binding of chitosan causes potential extraction of lipoteichoic acids that eventually lead to a cascade of events resulting in cell death. Chitosan-EDTA (1:1) proves to be a potential endodontic irrigant with significant antimicrobial activity against Enterococcus faecalis.[16]

Chitosan insolubility in aqueous medium limits its antimicrobial efficacy to acidic environment and it fails to produce effective antibacterial activity at alkaline pH. In this study, chitosan, when used as a fibrin-forming agent exhibited fibrin density of about (6.94 ± 0.02 fibers/10 μm).

CONCLUSIONS

Thus, from the results of the study, it was observed that all the root canal irrigants that were used in this study as part of treatment protocol for open apex exhibited positive results. That is, all the groups had an increase in fibrin density formation.

However, EDTA-NSS combination exhibited the maximum fibrin density formation followed by the experimental alternatives (Papain and Chitosan).

In this study, the other combinations that were experimented (Chitosan-Papain), (Chitosan-Papain-NSS) also did exhibit fibrin density. But not to the extent of (EDTA-NSS) (Papain) and (Chitosan) groups.

However, further studies are suggested, especially regarding the release of growth factors by the irrigants from the root canal dentin, exact mechanism of papain and chitosan in fibrin formation, alteration in fibrinogen molecule, dentin-pulp regeneration, etc., such that it can be proposed to be used in clinical RET, as alternative materials for treatment of blunderbuss canals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.