Botulinum toxin injection in masseter muscle evokes musculoskeletal impairment of the masticatory system.

The masticatory system is a finely coordinated machine, where minimal deregulation induces alterations of the whole system. Botulinum toxin type A (BoNTA) injection in masseter muscle is widely used as a clinical/aesthetic procedure. However, the non-desirable effects of masseter muscle paralysis in neighbor masticatory muscles or mandibular bone are unknown. In the current work, we aimed to evaluate musculoskeletal loss at the masticatory system after BoNTA-injection in masseter muscle of adult mice. Unilateral paralysis of masseter muscles was induced in male BalbC mice (8 weeks-old) by single-injection of BoNTA (0.2U/10 µL) in the right muscle and saline solution in the left muscle. Masticatory muscles (masseter, temporalis, lateral- and medial-pterygoid) and mandibles were dissected after 2-7-14d for molecular measurements (qPCR), histology, or morphology (microcomputed tomography). All procedures were approved by the IACUC-Universidad de Chile (# 20381-ODO-UCH). T-test or Wilcoxon-test were used for statistical comparisons between BoNTA- and Control-side (n=3-10, p<0,05). The expression of molecular markers of atrophy (Atrogin1, Murf1) and regeneration (myogenin) significantly increased in the BoNTA-side, as early as 2-7d, in the intervened masseter but also in the ipsilateral temporalis and medial pterygoid muscles. There was a time-dependent reduction in masseter fibers diameter from 2d to 14d. Masseter, temporalis, and medial pterygoid's volumes significantly reduced in the BoNTA-injected side at 14d. Interestingly, the volume of lateral pterygoid increased in the BoNTA side, with no change in atrophy/regeneration markers. Microstructural analysis of mandibular condyles demonstrated a progressive bone loss in the BoNTA-injected side, starting as early as 7d and worsening 14d after the intervention. In conclusion, BoNTA injection in the masseter muscle impairs the whole masticatory system, inducing atrophy of masseter but also the ipsilateral temporalis and medial pterygoid, and even early-bone loss of the mandibular condyle at the injected side. Then, clinical uses of BoNTA in masticatory muscles should be avoided or carried out under strict morpho-functional monitoring.