Intramuscular injection of Botox causes tendon atrophy by induction of senescence of tendon-derived stem cells

BACKGROUND: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. HYPOTHESIS: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). STUDY DESIGN: Controlled laboratory study METHODS: A total of 36 mice were randomly divided into 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into the right vastus lateralis of quadriceps muscles either with Botox (to induce mechanical stress deprivation of the patellar tendon) or with normal saline as a control. At 2 weeks post-injection, animals were euthanized prior to tissues being harvested for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity or the presence of senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, the PTEN/AKT-mediated cell senescence pathway was evaluated in TDSCs from both groups. RESULTS: Two weeks after Botox injection, patellar tendons displayed several atrophic features including tissue volume reduction, collagen fibre misalignment and increased degradation. A colony formation assay revealed a significantly reduced number of colony forming units of TDSCs in the Botox-injected group compared to controls. Multipotent differentiation capacities of TDSCs were also diminished after Botox injection. To examine if mechanically deprived TDSC are capable of forming tendon tissue, we used an isolated bioreactor system to culture tendon constructs using TDSC. These results showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of the signalling pathway revealed that injection of Botox into quadriceps muscles causes PTEN/AKT-mediated cell senescence of TDSCs. CONCLUSION: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. CLINICAL RELEVANCE: Intramuscular Botox injection for tendinopathy or tendon injury could result in adverse effects in human tendons and evaluation of its long-term efficacy is warranted.