Intramuscular Injection of Botox Induces Tendon Atrophy and Senescence of Tendon-Derived Stem Cells

Abstract

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 in 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into to right vastus lateralis of quadriceps muscles either with Botox to induce mechanical stress deprivation of the patellar tendon or with normal saline as control. At 2 weeks post-injection, animals were euthanized prior to tissues harvest for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity and senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, key proteins in the PTEN/AKT pathway were evaluated in TDSCs in both groups. Results: Two weeks after Botox injection, patellar tendons displayed atrophic features including tissue volume reduction and collagen fibre misalignment and increased degradation. The colony formation assay revealed the significantly reduced colony units of TDSCs in Botox injected group compared to controls. Multipotent differentiation capacity of TDSCs has also diminished after Botox injection. To examine if mechanical deprived TDSC is capable of forming tendon tissue, we used an isolated bioreactor system to culture 3D TDSCs constructs. The result showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of PTEN/AKT signalling pathway revealed that injection of Botox into quadriceps muscle 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 and tendon injury could cause adverse effects in human tendons and re-evaluation of its long-term efficacy is warranted.