Clinical and early biochemical responses to botulinum toxin type-A in refractory masticatory myofascial pain: a pilot single-arm study

BACKGROUND: Masticatory myofascial pain (MMP) is a common subtype of temporomandibular disorders (TMD) characterized by muscle tenderness and pain during jaw function. Botulinum toxin type-A (BoNT-A) is often used for refractory cases unresponsive to conservative therapies; however, its biological mechanisms remain unclear. This exploratory pilot study aimed to evaluate both clinical outcomes and biochemical changes following BoNT-A injections in patients with refractory MMP. METHODS: Twenty-seven patients diagnosed with refractory MMP according to DC/TMD criteria received intramuscular BoNT-A injections into the masseter and temporalis muscles. Pain intensity (VAS), pressure pain threshold (PPT), maximum mouth opening (MMO), and oral health-related quality of life assessed using the OHIP-14 were recorded at baseline (T0) and 28 days post-treatment (T1). Serum and saliva samples were collected to measure inflammatory cytokines (IL-1β, IL-6, TNF-α) and neuropeptides (CGRP, NGF) using ELISA. Data were analyzed using the paired t-test or Wilcoxon signed-rank test, as appropriate. RESULTS: At 28 days, improvements were observed in pain (VAS, p < 0.001), PPT for the masseter and temporalis muscles (p < 0.001), and OHIP-14 scores (p < 0.001), while MMO remained unchanged. Changes in cytokine levels were smaller and more variable. In contrast, CGRP and NGF concentrations increased in both serum and saliva and showed the strongest evidence of change among the evaluated biomarkers. CONCLUSION: BoNT-A produced measurable clinical improvements and modulated biochemical profiles in refractory MMP. Similar trends observed in serum and saliva suggest that salivary biomarkers may provide a noninvasive approach for monitoring biochemical responses. Given the exploratory design and limited sample size, these findings should be interpreted with caution. Larger, controlled trials with extended follow-up are needed to clarify BoNT-A's mechanisms and long-term effects. TRIAL REGISTRATION: ClinicalTrials.gov (NCT06840730), retrospectively registered (2025-02-21).