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bioRxiv · 10.1101/2025.10.03.680271

Hypertrophic bone proliferation at enthesis induced by unilateral injection of botulinum toxin in masticatory muscles in adult rats is characterized by chondrocyte proliferation without inflammatory process at enthesis

Mermet, M.·Massiquot, Q.·Gaborit, N.·Lemiere, S.·Kun-Darbois, J.-D.·Libouban, H.
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Abstract

A single injection of botulinum toxin injected in masticatory muscles induces alveolar bone loss associated with muscle enthesis hypertrophic bone proliferation. The tissular and cellular mechanisms involved in this phenomenon are unknown. Because new bone formation due to inflammation at enthesis is observed in articular disorder like spondyloarthritis, we hypothesize that an inflammatory process could explain the development of hypertrophic bone proliferation in the BTX model. The aims of the study were to test this hypothesis and to determine whether IL-17A was involved. Mature Sprague-Dawley male rats (n=36) were randomized into 4 groups. Three groups received BTX injections in right masseter and temporalis muscle. One BTX group was treated with an anti-IL17A antibody for 31 days (BTX31+antiIL17A). The two other groups were respectively sacrificed at 21 (BTX21) and 31 (BTX31) days post BTX injection. The remaining non-BTX group received anti-IL17A treatment for 31 days (control+anti-IL-17A). Alveolar bone loss and hypertrophic bone proliferation at enthesis were studied using microcomputed tomography. The presence of inflammatory process and the implication of IL17A was assessed by histology and immunohistochemistry on decalcified right side hemimandibles. Quantitative measurement of the hypertrophic bone (bone volume and thickness) showed no significant differences between the 3 BTX groups. Histology revealed the presence of chondrocytes in large hypertrophic area but no inflammatory infiltrated cells. Immunochemistry confirmed the absence of inflammatory process but positive reaction for Ki67 which is in favor of actively growing and proliferative chondrocytes. BTX-related muscle atrophy of masticatory muscle induced chondrocyte proliferation at enthesis without inflammation process.

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Mermet, M., Massiquot, Q., Gaborit, N., Lemiere, S., Kun-Darbois, J.-D., Libouban, H.. 2025-10-06. Hypertrophic bone proliferation at enthesis induced by unilateral injection of botulinum toxin in masticatory muscles in adult rats is characterized by chondrocyte proliferation without inflammatory process at enthesis. https://doi.org/10.1101/2025.10.03.680271

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