Background: The management of temporomandibular disorders (TMD) remains challenging, with its underlying pathological mechanisms requiring further investigation. This study aimed to explore the effects of Botulinum Toxin A (Botox A) on the lateral pterygoid (LP) muscle and its impact on condylar subchondral bone mass.

Methods: Rats were randomly assigned to either an experimental group (injected with Botox A) or a control group (injected with an equal volume of 0.9% normal saline). All rats were sacrificed at 2-, 4-, 8-, or 12-weeks post-injection. The right condyles were harvested and analyzed using Micro-computed tomography (CT) scanning and hematoxylin-eosin staining to evaluate changes in the condylar subchondral bone. Osteoclast activity in the subchondral bone was assessed via tartrate-resistant acid phosphatase (TRAP) staining. The activities of osteoclasts and osteoblasts in subchondral bone were detected by western blotting.

Results: At 2-weeks post-Botox A injection, the trabecular number (Tb.N) was significantly lower (p < 0.01). At 2- and 4-weeks, bone volume fraction (BV/TV) and trabecular thickness (Tb.Th) were significantly lower (p < 0.05), while trabecular space (Tb.SP) and the ratio of bone surface area to bone volume (BS/BV) were significantly increased in the experimental group compared to control group (p < 0.05). At 8-weeks, BS/BV remained significantly elevated (p < 0.05), but no significant differences were observed in Tb.N, Tb.Th, BV/TV, or Tb.Sp at 8- and 12- weeks. Osteoclast numbers in the condylar subchondral bone were significantly higher in the Botox A group at 2-, 4-, and 8-weeks compared to the control group (p < 0.05). Additionally, protein expression levels of osteocalcin (OCN) and type I collagen (COL1A1) were markedly increased in the experimental group at 8- and 12-weeks (p < 0.05).

Conclusions: Botox A induces a significant reduction in condylar subchondral bone mass in rats during the early post-injection period, with subsequent time-dependent recovery.