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Address requests for reprints to Prof Ahmed Abdel Rahman Hashem, Endodontic Department, Faculty of Dentistry, Ain Shams University, Organization of African Unity Street, Al-Qubba Bridge, Abbasia, 79 Abou Bakr ElSedeek st., Heliopol, Cairo, Egypt.
The efficacy of trypsin-chymotrypsin in postoperative pain management following single-visit root canal treatment of teeth with symptomatic irreversible pulpitis was evaluated. Additionally, synergistic effects with nonsteroidal anti-inflammatory drugs and reported side effects were also investigated.
Methods
This prospective, parallel, triple-blinded phase IV randomized controlled trial included 60 patients with mandibular first molars exhibiting symptomatic irreversible pulpitis. The patients were randomly allocated using computer software to one of four treatment groups (n = 15 each), and either ibuprofen (600 mg), ambezim-G (trypsin 5mg-chymotrypsin 5 mg), a combination of both, or a placebo drug were administered postoperatively. The participants scored pain intensity at different time-intervals using a numerical scale, and passive surveillance of harm was used to detect clinical safety. Age was compared between groups using a one-way analysis of variance test. Pain scores were analyzed using the Kruskal-Wallis and Friedman's tests and, if significant, Dunn's test was used for pairwise comparisons. The chi-square test was used to compare qualitative data, and the significance level was set at P value ≤ .05.
Results
All interventions were found to be effective in reducing postoperative pain, and no statistically significant differences were observed between the ibuprofen, trypsin-chymotrypsin, and combination groups. However, all 3 groups differed significantly from the placebo group. The safety profile of the interventions did not differ significantly.
Conclusions
Trypsin-chymotrypsin exhibits comparable efficacy to nonsteroidal anti-inflammatory drugs. No synergistic effects occur when the 2 are used in combination. This is the first randomized controlled trial to assess the effects of proteolytic enzymes on postendodontic pain.
To equip dental clinicians with other decisions of new pain control strategies. Under the circumstances of this randomized controlled trial, Trypsin-Chymotrypsin can be used alternatively to nonsteroidal anti-inflammatory drugs to control postoperative pain after root canal treatment.
Postoperative pain is a common outcome encountered by clinicians. The International Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage”
. Evidence on the incidence of postoperative pain varies considerably, with some studies reporting high incidence rates and others reporting the contrary
, and this huge disparity can be attributed to variations in the sample sizes considered, methodologies used, and the cultural and ethnic characteristics of the patient populations
The etiology of postoperative pain is multifactorial and includes mechanical and chemical injuries to the pulpal and periapical tissues. Inflammatory mediators such as prostaglandins, leukotrienes, bradykinins, and other neuropeptides are released into the periapical tissues in response to injury, resulting in sensitization of the pain fibers. Furthermore, vasodilation, increased vascular permeability, and chemotaxis of the inflammatory cells can cause tissue edema and increased interstitial tissue pressure
Acute irreversible pulpitis is typically treated using endodontic measures such as primary root canal treatment. These procedures have high success rates when used in vital teeth
, but are often associated with complications such as postoperative pain. The use of various postoperative medicaments such as steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs), narcotics, anti-histamines, or flexible plan basis have been advocated previously in the literature
Post-operative endodontic pain management: an overview of systematic reviews on post-operatively administered oral medications and integrated evidence-based clinical recommendations.
Efficacy and safety of post-operative medications in reducing pain after nonsurgical endodontic treatment: a systematic review and network meta-analysis.
. Upon oral administration, this enzyme complex is absorbed into the bloodstream and exerts anti-inflammatory, analgesic, anti-edematous, fibrinolytic, and anti-infective effects
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
A randomized, double-blind, placebo-controlled study comparing the efficacy and safety of paracetamol, serratiopeptidase, ibuprofen and betamethasone using the dental impaction pain model.
Efficacy of sub-mucosal injection of chymotrypsin, oral serratiopeptidase and oral corticosteroids for reducing postoperative complications following impacted mandibular third molars surgery: a randomized, three-arm, double-blind, controlled clinical trial.
. Therefore, the current study aimed to evaluate the efficacy of trypsin-chymotrypsin in postendodontic pain management following single-visit root canal treatment; investigate its synergistic effects with NSAIDs; and evaluate the incidence of reported side effects. The null hypothesis being tested was that ibuprofen, trypsin-chymotrypsin, and a combination of both would not differ in terms of postoperative pain management following single-visit root canal treatment of mandibular first molars with symptomatic irreversible pulpitis (SIP).
Materials and Methods
Ethical Considerations
This study was approved by the ethics committee of the Faculty of Dentistry, Ain Shams University, Cairo, Egypt (FDASU-Rec IM012110), and the study protocol was registered on a clinical trials website (http://www.clinicaltrials.gov Identifier: NCT05479747). The study was carried out in accordance with the World Medical Association Declaration of Helsinki (2008).
Trial Design
This prospective, parallel, triple-blinded phase IV randomized controlled trial (RCT) conformed to the consolidation standards of The Preferred Reporting Items for Randomized Trials in Endodontics 2020 guidelines
which proposes a checklist developed using the Consolidated Standards of Reporting Trials and Clinical and Laboratory Images in Publication guidelines. Figure 1 illustrates the study design.
Figure 1Preferred Reporting Items for Randomized Trials in Endodontics 2020 flowchart.
A power calculation was carried out using pain scores after 72 hours as the primary outcome. Five patients from each group participated in a pilot study, and the results were used to determine the final sample size
. A minimum sample size of 52 patients (ie, 13 patients per group) was estimated, and this was increased to 60 (ie, 15 patients per group) after taking a drop-out rate of 15% into consideration. The sample size calculation was carried out using the G∗Power software, Version 3.1.9.2. (Franz Faul, Universität Kiel, Germany).
Eligibility Criteria
The study participants were selected from the outpatient emergency clinic of the Endodontics Department, Faculty of Dentistry, Ain Shams University in Cairo, Egypt, and recruitment was carried out over a period of 6 months between 1st June and 31st December 2021. Informed consent was collected from all patients after provision of relevant information regarding the associated benefits and risks.
Inclusion Criteria
This study included Egyptian both gender patients that were healthy [defined as American Society of Anesthesiologists class I], aged 18–40 years, exhibited SIP of the mandibular first molars (characterized by 2 mesial canals and 1 distal canal), and showed no radiographic evidence of periapical or periodontal pathosis.
Exclusion Criteria
Pregnant females; patients experiencing difficulty reading, understanding, and completing the baseline patient questionnaire; those that had taken analgesics 24 hours prior to treatment; those subjected to over-instrumentation during the treatment procedure; and those with known sensitivity to pharmaceuticals used in this study were excluded.
Randomization and Blinding
Sixty patients who fulfilled the eligibility criteria were randomly allocated to 4 treatment groups in a ratio of 1:1:1:1. Computer-generated randomization was conducted using appropriate software (www.randome.org).
Allocation Concealment
Random sequence generation, allocation concealment, and preparation of the interventions were carried out by one independent person prior to commencement of the trial. The researchers were blinded to patient group by concealing the allocation sequence in a tightly sealed opaque envelope to minimize the risk of selection bias during the recruitment stage. Intervention preparation included the division of the postoperative medicaments into 60 tightly sealed opaque envelopes labeled using alphabetical symbols (X, Y, Z, R). Each envelope contained a treatment specification and the recommended dose.
Implementation
After completion of the root canal treatment, the postoperative medicaments were assigned by a telephonic conversation. This independent person informed the operator (A) which intervention should be prescribed. To ensure operator blinding, the appropriate interventions were informed using the assigned labels (X, Y, Z, or R) only. The investigators used computer-generated randomization to allocate eligible participants to one of the four intervention groups, and all pain assessment forms and data were assessed pre- and postoperatively by a trained clinician (H). This trained clinician was also blinded to minimize the risk of assessment bias
. Thus, ensuring triple-blinding (investigators, care providers, and outcome assessor). Additionally, the statistician responsible for data analysis was also blinded.
Intervention
Diagnosis of SIP was based on a positive history of spontaneous diffused lingering pulpal pain and observation of a moderate to severe exaggerated response to cold-sensitivity testing using Endo-Ice (1,1,1,2 Tetrafluoroethane; Hygenic Corp, Akron, OH). The vitality of the affected tooth was compared with that of the contralateral unaffected tooth (controls), and was confirmed clinically through direct observation of bleeding in the root canal. Only teeth with normal periapical conditions (no radiographic evidence of periapical or periodontal pathosis) and no sensitivity upon percussion or palpation during intraoral clinical examination were included in this study. Teeth were anesthetized by 3.6 ml of a local anesthetic solution [consisting of 4% articaine and 1:100,000 epinephrine (Scandonest, Barcelona, Spain)] using inferior alveolar nerve block technique with buccal infiltration and intraligament supplemental injections. A single clinician (Y) performed all diagnostic procedures and local anesthesia injections throughout the study.
Endodontic treatment was carried out by a postgraduate master's student (A) with 5 years of clinical experience in the relevant field. After caries removal, the access cavity was done under rubber dam isolation. Chemo-mechanical preparation was carried out using the rotary file system, ProTaper Next (Dentsply Sirona Endodontics, Ballaigues, Switzerland), up to size X3 (30#7) in the mesial canals and X5 (50#6) in the distal canal (speed: 300 rpm; torque: 2–3 N cm). The canals were individually irrigated using 15 ml of 5.25% sodium hypochlorite (NaOCl; Chlorox, Cairo, Egypt), and this was followed by the final flushing protocol consisting of 3 ml each of NaOCl, saline, ethylenediaminetetraacetic acid (EDTA; MD Cleanser, Meta biodent, Cheongju-si, Korea), and saline used sequentially. The canals were obturated using the cold lateral compaction technique with a root canal resin sealer (ADSEAL, Meta biodent, Cheongju-si, Korea) and restored with a temporary restorative material (Coltosol F, Coltene Whaledent, Altstatten, Switzerland; temporary restorative material). A trained clinician (H) assessed all patients pre- and postoperatively.
Of the 85 patients recruited initially, 25 were excluded as they did not meet the inclusion criteria (n = 21) or did not consent to participation (n = 4). The final trial included 60 patients (31 females and 29 males) who were randomly assigned to 4 intervention groups (n = 15 each) to allow evaluation of the primary (efficacy of trypsin-chymotrypsin in postoperative pain management after single-visit endodontic treatment of teeth with SIP), secondary (investigation of their synergistic effects with NSAIDs), and tertiary (observation of reported side effects) outcomes (Fig. 1).
Ibuprofen Group
Patients in this group received 600 mg of ibuprofen (Brufen; Abbott chemical laboratories, Advil, USA) 3 times a day (after meals) for 3 days after completion of treatment.
Trypsin-Chymotrypsin Group
Patients in this group received trypsin-chymotrypsin (5-mg each; Ambezim-G; Global Napi Pharmaceutical Company, Giza, Egypt) 3 times a day (1 h before meals) for 3 days after completion of treatment.
Ibuprofen + Trypsin-Chymotrypsin Group
Patients in this group received both protocols mentioned above [ie, 600-mg ibuprofen 3 times a day (after meals) for 3 days + trypsin 5 mg-chymotrypsin 5 mg 3 times a day (1 hour before meals) for 3 days].
Placebo Group
This group received placebo sugar pills (Department of Pharmaceutics & Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt) 3 times a day for 3 days.
Outcome Assessment
The efficacy of the intervention was assessed 6, 12, 24, 48, and 72 hours after endodontic treatment. Analgesic effects were measured by asking the patient to assign a numeric value to describe their pain intensity using an 11-point Numeric Rating Scale (NRS) with no graduation between the whole numbers. Scores ranged from 0–10 on a 10 cm line, where higher scores indicated greater pain intensity. The ranked value was recorded as the valuable answer, meaning we had only 11 possible answers
Test–retest reliability, validity, and minimum detectable change of visual analog, numerical rating, and verbal rating scales for measurement of osteoarthritic knee pain.
. Clinical safety was determined through passive surveillance of harm, defined as adverse events reported by the patient on their initiative during the study period
An intention-to-treat-analysis was carried out, and differences in age between groups were assessed using a one-way analysis of variance test. Pain scores were compared between groups using the Kruskal-Wallis test, and the Friedman's test was used to detect time interval changes within the same group. The Dunn's test was used for pairwise comparison when the Kruskal-Wallis or Friedman's test were found to be statistically significant. Qualitative data were compared using the chi-square test, and the significance level was set at P value ≤ .05. All data analyses were carried out using the IBM SPSS Statistics for Windows software, Version 23.0. (Armonk, NY: IBM Corp).
Results
The final study sample included 60 patients, and no patients were lost to follow-up. No adverse events were observed, and the safety profile of the various interventions did not differ significantly. The results of this study have been reported in 2 parts, with part I evaluating the overall effects of each medication on postoperative pain and part II assessing the effects over time within each group. No statistically significant differences in baseline characteristics (age and gender) were observed between the groups (Table 1)
Figure 2 and Table 2 compare the effects of each medication on pain (NRS) scores between the 4 groups. Although no differences were observed preoperatively, the groups were seen to significantly differ with regard to NRS scores 6, 12, 24, 48, and 72 hours after treatment. Pairwise comparisons between groups showed that the placebo group exhibited significantly higher median pain scores, while the ibuprofen, trypsin-chymotrypsin, and combination groups did not differ significantly. Figure 3 summarizes the effects of the medications on postoperative pain over time, and comparison of time-intervals within each group showed statistically significant differences after 6 hours, from 6 to 12 hours, and 12 to 24 hours. There was no statistically significant change in pain scores from 24 to 48 hours and 48 to 72 hours.
Figure 2Box plot showing median (and range) pain scores in the four groups (circles and stars represent outliers).
Previous studies have investigated the effects of various postoperative medicaments on postendodontic pain; however, to the best of our knowledge, this is the first RCT to assess the efficacy of proteolytic enzymes (trypsin-chymotrypsin) in postendodontic pain management.
The findings showed no statistically significant differences between the ibuprofen, trypsin-chymotrypsin, and combination groups and, as a result, the null hypothesis was accepted. The safety profile of the interventions also did not differ significantly, evidenced by the absence of any adverse effects. However, possible side effects of these medications have been reported previously, including allergic reactions, hypersensitivity, and gastrointestinal upset when using trypsin-chymotrypsin
Efficacy and safety of post-operative medications in reducing pain after nonsurgical endodontic treatment: a systematic review and network meta-analysis.
Previous evidence suggests that the intensity of postendodontic pain is higher in patients undergoing endodontic treatment of vital teeth when compared to those exhibiting a necrotic pulp
. This is further complicated by difficulty in achieving anesthesia in patients with SIP, with the success rates of standard inferior alveolar nerve blocks ranging between 19% and 55%
. Therefore, the current study used buccal infiltration and intraligament supplemental injections in patients diagnosed with SIP to improve the success rates of standard nerve blocks in a standardized manner.
The anesthetic solution consisted of 4% articaine hydrochloride and 1:100,000 adrenaline. Articaine not only provides rapid onset anesthesia for prolonged durations but also exhibits greater efficacy when compared to lidocaine in patients diagnosed with SIP
Neutrophils are the first leukocytic cells to reach the affected site following acute tissue injury. They typically secrete elastase, responsible for the degradation and removal of cellular debris, and reactive oxygen species with anti-infective properties. However, elastase activity, unless controlled, can transform acute inflammations into chronic ones. Therefore, the body's defense mechanism regulates this through a sharp increase in the secretion of acute-phase proteins
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
(eg, α1-antitrypsin and α2-macroglobulin) that inhibit several proteolytic enzymes that can lead to unregulated inflammation and healing impairment. These proteins also demonstrate various affinities, with α1-antitrypsin exhibiting the highest affinity for elastase, followed by chymotrypsin, cathepsin G, trypsin, and plasmin. Similarly, α2-macroglobulin exhibits the highest affinity for cathepsin G.
Plasmin, another proteolytic enzyme, plays an essential role in the fibrinolytic process and its inhibition can lead to a period of fibrinolytic shutdown and delayed repair. The oral supplemental form of trypsin-chymotrypsin preserves the role of plasmin by competing with it for protease inhibitor binding sites in the early inflammatory stages. The higher affinity of the oral enzyme complex favors its binding to the protease inhibitors, thereby increasing the availability of plasmin for fibrinolysis, resolution of inflammation, and enhancement of tissue repair
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
. Trypsin and chymotrypsin further enhance healing and potentiate anti-inflammatory actions by blocking the release of pain-inducing amines from inflamed tissues
A randomized, double-blind, placebo-controlled study comparing the efficacy and safety of paracetamol, serratiopeptidase, ibuprofen and betamethasone using the dental impaction pain model.
and increasing enzymatic and non-enzymatic antioxidant levels.
The anti-edematous effects of trypsin-chymotrypsin can be attributed to the breakage of large polypeptide chains into smaller chains that return to circulation and contribute to the resolution of edema and attenuation of pain
. Although previous studies suggest that the analgesic effects of this enzyme complex occur secondary to its anti-inflammatory and anti-edematous actions
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
, a primary analgesic effect through direct interaction between serine proteases (trypsin and chymotrypsin) and nociceptors and consequent reduction of pain mediators such as bradykinin has also been reported
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
Ibuprofen is effective when inflammation has sensitized the pain receptors (hyperalgesia), and its action can be attributed to the blockage of cyclo-oxygenase-1 and cyclooxygenase-2 enzymes that play a key role in synthesizing prostaglandins from arachidonic acid
Efficacy and safety of post-operative medications in reducing pain after nonsurgical endodontic treatment: a systematic review and network meta-analysis.
The findings of this trial are in agreement with Chandanwale et al, who compared the efficacy and tolerability of three oral supplemental drugs, trypsin-chymotrypsin; serratiopeptidase; and a combination of trypsin, bromelain, and rutoside
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
. They found that trypsin-chymotrypsin enhanced the resolution of inflammatory symptoms following orthopedic surgery to a greater extent when compared to the other drugs, thus leading to better healing and pain attenuation
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
. Additionally, Al-sandook et al investigated the effects of proteolytic enzymes (trypsin and chymotrypsin) on the postoperative sequelae of impacted lower third molar extraction
Clinical evaluation of the efficacy of orthal-forte (prolytic enzymes, trypsin and chymotrypsin) on postoperative sequel following the removal of lower impacted third molar.
Clinical evaluation of the efficacy of orthal-forte (prolytic enzymes, trypsin and chymotrypsin) on postoperative sequel following the removal of lower impacted third molar.
. Al-Moraissi et al compared the efficacy of preoperative injections of chymotrypsin, oral serratiopeptidase, and oral dexamethasone in managing postsurgical complications following impacted lower wisdom tooth extraction
Efficacy of sub-mucosal injection of chymotrypsin, oral serratiopeptidase and oral corticosteroids for reducing postoperative complications following impacted mandibular third molars surgery: a randomized, three-arm, double-blind, controlled clinical trial.
Efficacy of sub-mucosal injection of chymotrypsin, oral serratiopeptidase and oral corticosteroids for reducing postoperative complications following impacted mandibular third molars surgery: a randomized, three-arm, double-blind, controlled clinical trial.
RCTs are known to provide the strongest level of scientific evidence, and the key strength of the current study lay in its design. Random sequence generation and allocation concealment were carried out to avoid selection bias, while blinding of the investigators, care providers, and outcome assessors ensured minimization of performance and detection bias. The risk of attrition bias was also eliminated as none of the patients included in this study were lost to follow-up. This trial also assessed both favorable (efficacy of postoperative pain relief) and unfavorable (reported side effects) outcomes to avoid reporting bias
. To the best of our knowledge, this is the first RCT to assess the efficacy of trypsin-chymotrypsin in postendodontic pain management.
The main limitation of this study was the unidirectional scale, NRS, used to assess pain. All scales are valid, reliable, and suitable for practical clinical work, and the NRS benefits from having good sensitivity and producing numerical scores that can be analyzed statistically
Test–retest reliability, validity, and minimum detectable change of visual analog, numerical rating, and verbal rating scales for measurement of osteoarthritic knee pain.
. However, pain is a subjective measure, making precise description challenging. Therefore, future studies should utilize novel objective pain measures such as heart rate variability, functional magnetic resonance imaging, electroencephalography, and electromyography along with conventional pain scales
. Another limitation of the current study was that medication intake was found to be unnecessary in patients with SIP after the first 24 hours, and reduction of the dosage is recommended for future research.
Future studies using larger sample sizes, different dosage protocols, and routes of administration are necessary. Moreover, the efficacy of trypsin-chymotrypsin in postendodontic pain management should be compared to that of other drugs such as acetaminophen, narcotics, corticosteroids, other proteolytic enzymes, and cyclo-oxygenase–inhibiting nitric oxide donators in the future. The anti-infective effects of trypsin-chymotrypsin in patients exhibiting necrotic teeth and periapical pathosis should also be explored.
This study found that trypsin-chymotrypsin is a suitable alternative for NSAIDs used to control postoperative pain after root canal treatment. These findings can help inform dental clinicians about newer pain control strategies, thereby assisting in the development of treatment plans.
Conclusion
The current study found that the efficacy of trypsin-chymotrypsin in postendodontic pain management after single-visit treatments in patients diagnosed with mandibular lower first molar SIP was comparable to that of NSAIDs. Furthermore, no adverse events were detected when using trypsin-chymotrypsin. Lastly, no synergistic effects were observed when the enzyme complex was used in combination with NSAIDs.
Acknowledgments
The authors contributed equally to this work.
The authors thank Dr Khaled Kerra for his help in the statistical analysis.
This research was self-fund by the authors.
The authors deny any conflicts of interest related to this study.
References
Merskey H.
Pain terms: a list with definitions and notes on usage. Recommended by the IASP subcommittee on taxonomy.
Post-operative endodontic pain management: an overview of systematic reviews on post-operatively administered oral medications and integrated evidence-based clinical recommendations.
Efficacy and safety of post-operative medications in reducing pain after nonsurgical endodontic treatment: a systematic review and network meta-analysis.
A randomized, clinical trial to evaluate efficacy and tolerability of trypsin: chymotrypsin as compared to serratiopeptidase and trypsin: bromelain: rutoside in wound management.
A randomized, double-blind, placebo-controlled study comparing the efficacy and safety of paracetamol, serratiopeptidase, ibuprofen and betamethasone using the dental impaction pain model.
Efficacy of sub-mucosal injection of chymotrypsin, oral serratiopeptidase and oral corticosteroids for reducing postoperative complications following impacted mandibular third molars surgery: a randomized, three-arm, double-blind, controlled clinical trial.
Test–retest reliability, validity, and minimum detectable change of visual analog, numerical rating, and verbal rating scales for measurement of osteoarthritic knee pain.
Clinical evaluation of the efficacy of orthal-forte (prolytic enzymes, trypsin and chymotrypsin) on postoperative sequel following the removal of lower impacted third molar.
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