Compressive Loading Rescues the Effects of Botox Injection Into the Masseter in the Mandibular Condyle

Objectives: To evaluate whether the effects of botulinum neurotoxin (botox) injection into the masseter in the mandibular condylar cartilage (MCC) and subchondral bone are transient and/or if loading would rescue the osteopenia and cellular changes after injection.
Methods: Botox (0.3 U) was injected into the right masseter of 6-week-old female mice (C57BL/6; n = 21). Additionally, 13 mice were used as control and received no injections. Bone labels (3 days and 1 day before sacrifice) and the proliferation marker EdU (2 days and 1 day before sacrifice) were intraperitoneally injected into experimental and control mice. Experimental and matching control mice were sacrificed 4 or 8 weeks after botox injection. Another group of mice were subjected to temporomandibular joint (TMJ) compressive loading, by forced mouth opening, 4 weeks after botox injection. Mice were dissected and examined by x-ray, micro-CT and histology. Sagittal sections of condyles were stained for TRAP, EdU, alkaline phosphatase and toluidine blue. In addition, immunostaining for pSmad was performed.
Results: Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the subchondral bone of botox injected side when compared to control side and control mice, 4 and 8 weeks after injection. Furthermore, histological analysis revealed decrease in mineralization, matrix deposition, TRAP activity, EdU positive cells and pSmad expression in the MCC of the botox injected side, 4 and 8 weeks after injection. However, compressive loading reversed the reduced bone volume and density and the cellular changes in the MCC caused by Botox injection.
Conclusions:
The effects of Botox injection into the masseter muscle in the MCC and subchondral bone are not transient, but TMJ compressive loading rescues the negative effects of botox injection in the MCC and subchondral bone.