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 (TDSC).
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 with Botox to either induce mechanical stress deprivation of the patellar tendon or with normal saline (controls). At 2 weeks post-injection, animals were euthanized prior to harvesting of tissues for either evaluation of tendon morphology or in vitro studies. Tendon-derived stem cells were isolated by cell-sorting prior determination of viability, differentiation capacity and markers of senescence, 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 derived from either Botox-injected or vehicle-only control mice.
Results:
A single injection of Botox into the right vastus lateralis muscle of the quadriceps in mice caused loss of muscle contractility and reduction in loading on the patellar tendon. Two weeks after Botox injection, patellar tendons display atrophic features; including reductions in tissue volume and extracellular matrix and collagen fibre crimping and misalignment. The atrophic tendon tissues showed increased degradation of collagen fibres. The colony formation assay revealed that the numbers of TDSCs colony forming units in the Botox injected group were significantly reduced in comparison to vehicle-only controls. Multipotent differentiation capacity of patellar tendon TDSCs has also diminished after Botox injection. To examine if population of TDSC that has suffered from mechanical deprivation 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 fail 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.