Effects of Intracanal Cryotherapy on Biomarkers and Postoperative Pain in Apical Periodontitis

Apical periodontitis is an inflammatory condition of the periapical tissues associated with microbial infection of the root canal system. Inflammatory mediators released during the disease process contribute to periapical inflammation, tissue destruction, and bone resorption. Intracanal cryotherapy, involving the use of a cooled irrigation solution during root canal treatment, has been proposed as an approach to modulate the inflammatory response and reduce postoperative pain. However, the biological effects of different intracanal irrigation temperatures and the potential temperature-dependent response have not been fully characterized.

This prospective, randomized, controlled clinical study will investigate the biological and clinical effects of different irrigation temperatures in patients with asymptomatic apical periodontitis requiring root canal treatment. Participants will be randomly allocated to three groups according to the temperature of distilled water used for irrigation: 2-4°C, 8-10°C, or 22-24°C. The assigned temperature-controlled distilled-water irrigation protocol will be applied consistently during both the first and second treatment sessions. The temperature of the irrigation solution will be standardized and monitored according to the participant's allocated group throughout the irrigation procedure.

Root canal treatment will be performed using a standardized two-visit protocol. During the first visit, the tooth will be isolated with a rubber dam, working length will be determined, and chemomechanical root canal preparation will be performed according to a standardized protocol. The assigned temperature-controlled distilled-water irrigation method will be used during this session. A periapical tissue fluid (PTF) sample will be collected using sterile paper points and stored under appropriate conditions for subsequent biochemical analysis. Following completion of the first-session procedures, calcium hydroxide will be placed as an intracanal medicament.

During the second treatment session, the calcium hydroxide intracanal medicament will be removed and the same temperature-controlled distilled-water irrigation protocol assigned to the participant will again be applied. Thus, participants allocated to the 2-4°C, 8-10°C, or 22-24°C group will receive distilled-water irrigation at their respective assigned temperature during both treatment sessions. A second PTF sample will be collected using sterile paper points. Root canal treatment will then be completed with obturation using gutta-percha and an endodontic sealer.

The collected PTF samples will be analyzed using enzyme-linked immunosorbent assay (ELISA) to determine the levels of selected biomarkers, including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), receptor activator of nuclear factor kappa-B ligand/osteoprotegerin (RANKL/OPG), prostaglandin E2 (PGE2), transforming growth factor-beta (TGF-β), and matrix metalloproteinase-9 (MMP-9). These biomarkers will be used to assess changes related to inflammatory activity, bone metabolism, tissue destruction, and tissue response associated with the different irrigation temperatures.

In addition to the biochemical assessment, postoperative pain will be evaluated using a visual analog scale (VAS), and analgesic consumption will be recorded during the first 7 days following treatment. The clinical and biochemical findings will be evaluated together to determine whether temperature-controlled intracanal irrigation influences the periapical inflammatory response and postoperative pain and whether these effects vary according to irrigation temperature.

By comparing three different irrigation temperatures and evaluating both molecular biomarkers and patient-reported postoperative pain, the study aims to provide evidence regarding the biological and clinical effects of intracanal cryotherapy and to determine whether a temperature-dependent response exists.

• Temperature-Controlled Intracanal Irrigation (Procedure), also known as Intracanal Cryotherapy: Participants will receive temperature-controlled distilled-water intracanal irrigation during root canal treatment. According to computer-generated randomization, participants will be assigned to one of three irrigation-temperature groups: 2-4°C, 8-10°C, or 22-24°C. The assigned temperature-controlled distilled-water irrigation protocol will be applied during both treatment sessions. Irrigation conditions will be standardized across groups, with the irrigation temperature being the primary difference between the intervention groups.

Primary outcomes

• Change in Periapical Inflammatory Biomarker Levels [From the first treatment session to the second treatment session]: Changes in interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG), prostaglandin E2 (PGE2), transforming growth factor-beta (TGF-β), and matrix metalloproteinase-9 (MMP-9) levels in periapical tissue fluid (PTF) will be assessed using enzyme-linked immunosorbent assay (ELISA). Biomarker levels obtained during the first and second treatment sessions will be compared within and between the three irrigation-temperature groups to evaluate the biological response to temperature-controlled intracanal irrigation.

Secondary outcomes

• Postoperative Pain Intensity Assessed Using the Visual Analog Scale [Day 0, Day 1, Day 3, and Day 7 after the second treatment session.]: Postoperative pain intensity will be assessed using a 10-cm Visual Analog Scale (VAS), ranging from 0 to 10, where 0 represents no pain and 10 represents the worst imaginable pain. Higher scores indicate greater postoperative pain intensity. Participants will record their pain intensity after root canal treatment, and scores will be compared among the three irrigation-temperature groups.
• Postoperative Analgesic Consumption [From Day 0 through Day 7 after the second treatment session.]: Postoperative analgesic consumption will be assessed as the total number of analgesic tablets consumed by each participant during the postoperative follow-up period. Participants will record analgesic use on a daily patient record form. The type and dose of analgesic used will also be documented for descriptive purposes. The total number of tablets consumed will be compared among the three irrigation-temperature groups. Unit of measure: number of tablets.

Sex: All, ages: 18 Years to 65 Years

Inclusion Criteria:

* Age between 18 and 65 years.
* Presence of a posterior tooth indicated for primary endodontic treatment (excluding retreatment cases).
* Diagnosis of apical periodontitis, meeting the following criteria:
* Necrotic pulp based on pulp vitality testing (negative cold test and/or electrical pulp test \[EPT\]).
* Periapical Index (PAI) ≥3 on periapical radiography or presence of periapical radiolucency on cone-beam computed tomography (CBCT).
* Asymptomatic or mildly symptomatic apical periodontitis (excluding cases of severe acute apical periodontitis).
* Periodontal status meeting the following criteria:
* Probing depth ≤3 mm.
* Clinical attachment loss ≤2 mm.
* No periodontal pockets with bleeding on probing.
* The tooth to be treated must be suitable for isolation and sample collection.
* Participants must be able to understand the study procedures and provide informed consent.

Exclusion Criteria:

* Use of antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs), or -corticosteroids within the previous 3 months.
* Presence of a systemic inflammatory or autoimmune disease.
* Diabetes (glycated hemoglobin \[HbA1c\] ≥6.5%) or uncontrolled metabolic disease.
* History of immunodeficiency or immunosuppressive therapy.
* Pregnancy or breastfeeding.
* Active smoking.
* History of acute infection, febrile illness, or COVID-19 within the previous 3 -months.
* Retreatment cases.
* Presence of a fistula/sinus tract.
* Periodontal probing depth >3 mm.
* Tooth mobility ≥ Grade II.

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• Mourad A, Abdelsalam N, Fayyad D. Influence of intracanal cryotherapy on inflammatory mediators expression using different irrigation protocols: a randomized clinical trial. Saudi Endod J. 2021;11(2):246. doi:10.4103/sej.sej_203_20.
• Vera J, Ochoa J, Romero M, Vazquez-Carcano M, Ramos-Gregorio CO, Aguilar RR, Cruz A, Sleiman P, Arias A. Intracanal Cryotherapy Reduces Postoperative Pain in Teeth with Symptomatic Apical Periodontitis: A Randomized Multicenter Clinical Trial. J Endod. 2018 Jan;44(1):4-8. doi: 10.1016/j.joen.2017.08.038. Epub 2017 Nov 1. PMID 29079057

• Duygu Değirmencioğlu, Değirmencioğlu, Principal investigator, Medipol University