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ORIGINAL ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 2  |  Page : 246-251

Influence of intracanal cryotherapy on postendodontic pain and interleukin-6 expression using different irrigation protocols: A randomized clinical trial


Department of Endodontics, Faculty of Dentistry, Suez Canal University, Egypt

Date of Submission06-Aug-2020
Date of Decision21-Aug-2020
Date of Acceptance23-Sep-2020
Date of Web Publication8-May-2021

Correspondence Address:
Dr. Ahmed Emad
Faculty of Dentistry, Suez Canal University, Port Said
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sej.sej_203_20

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  Abstract 

Introduction: Cryotherapy is a new form of treatment, in which the body is exposed to cold temperatures. Hence, this study was aimed to evaluate the effect of intracanal cryotherapy on postendodontic pain and interleukin-6 (IL-6) expression in teeth with symptomatic apical periodontitis.
Materials and Methods: Forty-eight single-rooted teeth in patients with symptomatic apical periodontitis were randomly divided into four groups based on the irrigation protocol. Group I (control): 5% sodium hypochlorite (NaOCl) at room temperature (RT) was used during instrumentation and final rinse. Group II: 5% cold NaOCl (2°C–5°C) was used during the root canal procedure. Group III: 5% NaOCl at RT during instrumentation, while the final rinse was 20 mL of 5% cold NaOCl (2°C–5°C). Group IV: 5% NaOCl at RT during instrumentation followed by 20 mL of cold saline (2°C–5°C). Postoperative pain score was recorded using a modified visual analog scale at 12, 24, 48, and 72 h and after 1 week. Paper point passing 2 mm beyond the apex was used to collect periapical fluid before, immediately after mechanical instrumentation, and after 1 week to characterize the mRNA expression of IL-6 using real-time polymerase chain reaction (PCR). Data were statistically analyzed. The significance level was set at P ≤ 0.05.
Results: The control group showed the significant highest mean pain score (7), whereas experimental Groups II, III, and IV showed significantly lower mean pain scores with no significant differences in between (P > 0.05). Group II showed the lowest levels of IL-6 expression with no statistical significance between the experimental groups (P > 0.05).
Conclusion: All irrigation protocols using intracanal cryotherapy resulted in lower postoperative pain levels and IL-6 expression than the control group.

Keywords: Cryotherapy, interleukins, postoperative pain, real-time polymerase chain reaction, sodium hypochlorite


How to cite this article:
Emad A, Abdelsalam N, Fayyad DM. Influence of intracanal cryotherapy on postendodontic pain and interleukin-6 expression using different irrigation protocols: A randomized clinical trial. Saudi Endod J 2021;11:246-51

How to cite this URL:
Emad A, Abdelsalam N, Fayyad DM. Influence of intracanal cryotherapy on postendodontic pain and interleukin-6 expression using different irrigation protocols: A randomized clinical trial. Saudi Endod J [serial online] 2021 [cited 2021 Jun 17];11:246-51. Available from: https://www.saudiendodj.com/text.asp?2021/11/2/246/315646


  Introduction Top


Apical periodontitis (AP) occurs initially as an inflammatory reaction to microorganisms colonizing the root canal system, then as an infectious disease when bacteria exit the root canal into the periapical tissue. It is similar to any other connective tissue inflammation with the same sequence of inflammatory events including the vasodilatation of blood vessels, the increase in vascular permeability, and the migration of the inflammatory cells such as lymphocytes, macrophages, and neutrophils to the inflamed site.[1]

The principal immunologically effector cells of AP were macrophages and T helper cells.[1],[2] Release of cell signaling molecules called cytokines helps in communication between cells in immune responses and enhance cell recruitment toward the infected and inflamed sites. Cytokines are present in many forms such as peptides, proteins, and glycoproteins. The development of AP is usually regulated by many cytokines such as interleukin-1 (IL-1) β, IL-6, and tumor necrosis factor.[3],[4],[5],[6]

ILs representing the functional cytokines are the main mediators of inflammatory reactions. Local effects include increased adherence of leukocytes to endothelial walls, lymphocytes stimulation, potentiation of neutrophil, increased production of prostaglandin and proteolytic enzymes, and mediate resorption of bone.[5]

IL-6 is a primary mediator in the induction and control of the acute phase of inflammation. After an injury, plasma concentrations of IL-6 can be measured in 60 min with a median rise of 4–6 h and can last for up to 10 days. It is considered the most important marker for the degree of tissue damage during surgical procedures. As excessive and prolonged preoperative production of IL 6 during acute phase of inflammation usually results in higher postoperative pain levels.[7],[8],[9]

Postendodontic pain most often occurs during the first 24–48 h following cleaning and shaping and generally recedes in a few hours although it occasionally persists for several days. Pain management during and after root canal treatment is one of the most important aspects of endodontic practice. To control preoperative and postoperative pain, various strategies have been suggested. One of the simplest and effective strategies for pain control in dentistry is “cryotherapy.”[10]

Employing cryotherapy during root canal treatment has recently gained popularity. Hence, this study was carried out to evaluate the effect of cold temperature of irrigants in reducing postendodontic pain and inflammation.


  Materials and Methods Top


The study was given approval by the Ethics Committee, Faculty of Dentistry, Suez Canal University (56/2017) and was registered on clinicaltrials.gov with ID: NCT04324398. Before starting the treatment procedures, all participants were discussed about the nature and objectives of the study, along with obtaining an authored consent.

A total sample size of 48 patients (12 for each group) suffering from symptomatic AP as defined by the American Association of Endodontists was considered to be sufficient to detect this high effect size of f = 0.5, a power of 80%, and a 5% significance level (calculation of sample size was done by IBMTM, Sample PowerTM Version 3.0.1 (IBM Corporation, U.S.A.), based on previous studies.[11],[12]

The patients were selected from the Department of Endodontics Outpatient Clinic, Faculty of Dentistry, Suez Canal University. Inclusion criteria were patients aging from 20 to 50 years suffering from symptomatic AP with a preoperative pain score > 7 on the visual analog scale (VAS) scoring.[13] The exclusion criteria were patients presenting with periapical abscess; patients under immunosuppressive chemotherapy, anti-inflammatory drugs, or antibiotic medications for the past 2 months; medically compromised patients; and pregnant or lactating females.

The selected patients were randomly classified into four groups (12 each) based on irrigation protocols. They were chosen from 48 opaque and tightly sealed envelopes containing the patients' coding. This study was double blinded by the observer and statistician. The only who knew which data belong to which group was the allocator.

Patients' grouping and evaluation methods

Group I (control): irrigation with 5% sodium hypochlorite (NaOCl) at room temperature (RT) from the beginning of cleaning and shaping, between each file. Final rinse was done by 20 mL of 5% NaOCl for 5 min. Group II: irrigation with 5% cold NaOCl (2°C–5°C) from the beginning of cleaning and shaping and between each file. Final rinse was done by 20 mL of 5% cold NaOCl (2°C–5°C) for 5 min. Group III: irrigation with 5% NaOCl at RT from the beginning of cleaning and shaping and between each file. Then, final rinse with 20 mL of 5% cold NaOCl (2°C–5°C) for 5 min. Group IV: irrigation with 5% NaOCl at RT from the beginning of cleaning and shaping and between each file, then final rinse with 20 mL of cold saline (2°C–5°C) for 5 min. Each root canal received a total of 30 mL irrigation using side-vented (30G) needle.

Clinical procedures of root canal treatment

Root canal therapy was conducted in two appointments. At the first appointment, all patients were anaesthetized using one carpule of articaine hydrochloride 40 mg/mL + 1/100,000 epinephrine bitartrate solution (Inibsa Dental, Barcelona, Spain). Cases in which additional anesthesia was needed during access cavity, another carpule of articaine hydrochloride 40 mg/ml +1/100,000 epinephrine bitartrate solution was administrated.

The access cavity was performed under absolute rubber dam isolation using sterile round carbide bur under copious water cooling; then, the initial glide path was obtained using #10 K-file (Micro Méga, Besançon, France).

Coronal flaring was done using SX rotary file (ProTaper, Dentsply Maillefer, Ballaigues, Switzerland), then root canals irrigation with 2 mL 5% NaOCl. The working length at 0.5 was determined using an electronic apex locator (Root ZX Mini; J. Morita Co, Tustin, CA, USA)[14] and confirmed by periapical radiograph. Biomechanical preparation was carried out by ProTaper Next rotary files (Dentsply Maillefer, Ballaigues, Switzerland) following the instructions of the manufacturer till X3 file.

Evaluation of postendodontic pain

Patients were told about the pain they might experience in the days following treatment and were told to fill a modified VAS with the following criteria: 0 indicated no pain, 1–3 indicated mild pain, 4–6 indicated moderate pain, 7–9 indicated severe tolerable pain, and 10 indicated severe intolerable pain to assess their postoperative pain scores at 12, 24, 48, and 72 h and after 1 week to be submitted at the next appointment, scheduled after 1 week.[15]

Sample collection for evaluation of gene expression of cytokines

In each group, after obtaining a glide path using #10 K-file (Micro Méga, Besançon, France), sample collection was done before and immediately after root canal cleaning and shaping and then after 1 week for characterization of the messenger RNA (mRNA) expression profile of IL-6. Root canals were dried, followed by inserting three paper points into the root canal to pass 2 mm beyond the apical foramen into the periapical tissues and was hold in place for 1 min to be soaked by the periapical interstitial fluid. 4 mm from the tip of each paper point was cut and dropped into a microcentrifuge tube then stored at −70°C.

The extraction of pure RNA was done using a total RNA purification kit following the protocol of the manufacturer (Thermo Scientific, Fermentas, #K0731). Complementary DNA (cDNA) synthesis was carried out using RevertAid H minus Reverse Transcriptase to convert RNA into cDNA.

The absorption of ultraviolet (UV) light by the ring structure of purines and pyrimidines for very pure samples (without significant contamination from protein, phenol, free nucleic acids, organic solvents, carbohydrates, etc.) was used to measure the amount of nucleic acids to quantify the concentration of RNA and cDNA to ensure that the purity of the concentrations is adequate to conduct real-time polymerase chain reaction (PCR). The spectrophotometer Q5000 (UV-Vis/USA) was used to ensure accuracy for all the measurements and calculations.[16]

Real-time polymerase chain reaction

The expression of mRNAs of target genes in periapical interstitial fluid was measured using real-time PCR with SYBR® Green (a trade name for cyanine dye produced by Synergy brands Inc. [SYBR]) and an internal reference using β-actin. Amplification of the isolated cDNA was done using 2X Maxima SYBR Green/ROX quantitative PCR Master Mix according to the instructions of the manufacturer (Thermo Scientific, USA, # K0221) and gene-specific primers [Table 1].
Table 1: Forward and reverse primers used in quantitative-polymerase chain reaction

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The mixture of the final reaction was inserted into a step one plus real-time thermal cycler (Applied Biosystems, Life Technology, USA), and the program of PCR was carried out in 45 cycles under the conditions of PCR: initial denaturation at 95°C for10 min, denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The temperature was increased from 60°C to 95°C at the end of the last cycle to produce a melting curve. The housekeeping gene (β-actin) represented as normalized used to calculate the relative gene expression or the fold change in the target gene. Therefore, the quantities critical threshold (Ct) of the target gene was normalized with quantities Ct of housekeeping gene using the 2−ΔΔCt.[17]

Statistical analysis

Data of VAS recording postendodontic pain and PCR analysis for gene expression of cytokines were collected, tabulated, and statistically analyzed.

Exploring the normality of the numerical data was done by checking the distribution of data and using normality tests (Kolmogorov–Smirnov and Shapiro–Wilk tests). The presentation of the data was in the form of mean, standard deviation, median, and range values. One-way ANOVA was used to compare between all groups. Fisher's exact test was compared between the groups regarding the severity of pain. The level of significance was set at P < 0.05. Statistical analysis was carried out using IBM® SPSS® Statistics software version 20 for Windows. (IBM Corporation, U.S.A.).


  Results Top


Comparison regarding postoperative pain

After 12 h, the four groups showed a statistically significant difference in between (P < 0.001). Groups III and IV presented the highest prevalence of no pain, Group II showed the highest prevalence of mild pain, Groups I and III presented the highest prevalence of moderate pain, while Group I (control group) was the only group that showed severe tolerable pain. All four groups showed a statistically significant difference in between (P < 0.001) after 24 and 48 h. Group III presented the highest prevalence of no pain; Group II showed the highest prevalence of mild pain, while control group was the only group that showed moderate pain. After 72 h, the four groups showed a statistically significant difference in between (P < 0.001). Group III presented the highest prevalence of no pain, while Group I showed the highest prevalence of mild pain. After 1 week, the four groups showed a statistically significant difference in between (P < 0.001). Groups II, III, and IV showed the highest prevalence of no pain, while Group I was the only group that showed mild pain [Table 2].
Table 2: Descriptive statistics and results of Fisher's exact test for comparison between severity of pain in the four groups

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Comparison regarding time effect on IL-6 expression

All groups showed downregulation of IL-6 expression after 1 week without significant differences (P > 0.05). Group II recorded the lowest levels of IL-6 expression, followed by Group III then Group IV, while Group I showed the highest levels of IL-6 expression [Figure 1].
Figure 1: Bar chart representing relative expression of interleukin-6/β-actin gene in the four groups

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  Discussion Top


The mechanism of action of cold application (cryotherapy) can be divided into three basic actions: decrease in metabolic activity, blood flow, and inhibition in the skin and subcutaneous tissues neural receptors. This renders it efficient in reducing inflammation, pain, edema, and recovery time in the short-term application.[18]

The optimum application time of cryotherapy has not been determined. However, different application time according to the depth of tissues was recommended; 3–5 min of cryotherapy was advised when there is minimum fat and muscle, whereas approximately 20 min was recommended for deeper tissues such as the hip.[19],[20]

The transmission of cold to the periodontal ligament in the apical and coronal portions of the root is not the same because of differences in the thickness of dentin and the numbers and direction of dentinal tubules between the two levels. Apical dentin has fewer and more mineralized tubules, which encourage more efficient cold transmission than the cervical dentin, which exhibits a larger and increased number of dentinal tubules.[21] Furthermore, the apical third of teeth with a single root usually exhibits 1–7 pulp ramifications, which increase the rate of cold transmission compared to multirooted teeth.[22]

In the study, patients presenting with symptomatic AP and pain score exceeding 7 on the VAS were selected, because postoperative pain is mostly anticipated when preoperative pain is present.[23],[24],[25],[26],[27],[28]

In the present study, all the cryotherapy groups resulted in lower postoperative pain values than the control group without a statistically significant difference between the three experimental groups. These results were congruent with Vera et al.,[13] although they conducted the study on patients with pulp necrosis and symptomatic AP, they only used cold saline as a final rinse similar to Group IV in the present study.

They were also consistency in the study results with Gundogdu et al.[29] who investigated the effect of intracanal, intraoral, and extraoral cryotherapy on postoperative pain in molar teeth in 100 patients with symptomatic AP. All cryotherapy groups showed less pain with percussions and lower postoperative pain in comparison with the control group after 1, 3, 5, and 7 days. The results could be attributed to cold irrigation, in which decreasing temperature causes vasoconstriction restricting edema development, decreasing tissue pressure, and relieving pain.[30]

In the current study, all experimental groups (cryotherapy) showed a reduction in the expression of IL-6 in comparison to the control group with no statistically significant difference.

This might be inferred to cryotherapy reduced the count of leukocytes adhering to the capillary endothelial walls, thus reducing their migration into affected tissues and reducing endothelial dysfunction. Furthermore, decreasing cell metabolism, which results in a reduction of the oxygen demand of cells and limitation of the production of free radicals in tissues and minimizing the recruitment of pro-inflammatory mediators to the site of inflammation.[31] Cryotherapy also results in blocking nociception within the spinal cord by deactivation of thermoreceptors, which are pain receptors activated by cytokines.[32],[33],[34]

The lowest nonsignificant levels of IL-6 expression in Group II might be attributed to longer application time and larger volume of cold irrigation, which allowed for a slightly more pronounced physiological action of cryotherapy on the periapical tissue expressed by the lower level IL-6.

The only limitation of the present study was the relatively moderate number of samples.

Cryotherapy was proved to be an effective, practical, and inexpensive method to control postoperative pain. However, further studies regarding antimicrobial efficacy of cold NaOCl should be conducted.


  Conclusion Top


The cryotherapy irrigation protocols yielded lower levels of postoperative pain and periapical IL-6 expression when compared to the control group.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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