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Intramuscular Injection of Botox Causes Tendon Atrophy by Induction

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Intramuscular Injection of Botox Causes Tendon Atrophy by Induction Chen et al. Stem Cell Research & Therapy (2021) 12:38 https://doi.org/10.1186/s13287-020-02084-w RESEARCH Open Access Intramuscular injection of Botox causes tendon atrophy by induction of senescence of tendon-derived stem cells Peilin Chen1 , Ziming Chen1, Christopher Mitchell1, Junjie Gao2, Lianzhi Chen1, Allan Wang1,3, Toby Leys4, Euphemie Landao-Bassonga1,2, Qiujian Zheng5*, Tao Wang1,5* and Minghao Zheng1,2* 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 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. (Continued on next page) * Correspondence: [email protected]; [email protected]; [email protected] 5Division of Orthopaedic Surgery, Department of Surgery, Guangdong General Hospital, Guangdong Academy of Medical Science, Guangzhou, Guangdong, China 1Center for Orthopaedic Translational Research, Medical School, University of Western Australia, Nedlands, Australia Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. Stem Cell Research & Therapy (2021) 12:38 Page 2 of 13 (Continued from previous page) 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. Keywords: Tendon-derived stem cell, Tendon homeostasis, Mechanical loading, Mechanobiology, Bioreactor Introduction mechanical force on the associated tendons. Insufficient Botulinum toxin (Botox) is a potent neurotoxin in wide- mechanical loading on tendon has been shown to have ad- spread clinical use for the treatment of a variety of mus- verse effects on tendon morphology and causing increased culoskeletal disorders [23, 30]. Botox acts by blocking stiffness, collagen turn-over and tissue atrophy [2, 24, 26]. acetylcholine release from cholinergic nerves and end- Tendon-derived stem cells (TDSC), a population of resi- plates into the neuromuscular junction, resulting in a re- dent tendon stem cells, are known to be responsive to duction of muscular activity [29]. Botox injections have mechanical signal-induced stimuli within tendon tissue been shown to effectively reduce muscle tone, alleviating [20, 25, 31, 40]. As Botox injection impairs mechanical the symptoms caused by muscular spasm and providing stimulation of tendon tissue in vivo, we hypothesised that an alternative therapy to invasive surgery when conven- intramuscular Botox injection leads to cellular dysfunction tional therapies have failed. One of the common uses of of TDSCs. In this study, we injected Botox into the quad- Botox in musculoskeletal disorders is for the symptom- riceps muscles in mice, examined the patellar tendon tis- atic treatment of cerebral palsy. Site-specific injection of sue structure, isolated TDSCs and generated an ex vivo Botox reduces tibialis posterior and gastrocnemius-soleus tendon construct in the bioreactor. We showed that Botox contracture, resulting in the restoration of foot position injection into quadriceps muscle induced atrophy, colla- [1, 21, 30]. In addition, Botox injection is also used for gen fibre misalignment and degeneration of the patellar restoration of the range of head and neck motions in pa- tendon. Botox injection of this muscle also causes irrevers- tients with congenital/acquired muscular torticollis who ible senescence of TDSCs through modulation of the have failed in response to conventional physiotherapy PTEN/AKT signalling pathway. [12, 27] Recently, there has been an increase in clinical use of Botox injections for the treatment of tendinopathy Materials and methods or tendon injuries where excessive muscle contraction Animals and Botox-induced patellar tendon unloading may contribute to inadequate healing [13]. Several stud- protocol ies have reported pain relief following Botox injection All animal experiments were approved by the Animal after achieving sufficient suppression of muscular func- Ethics Committee, University of Western Australia. A tion in lateral epicondylitis [14, 22]. A potential mechan- total of 36 male C57BL/6J mice (5 weeks old) were ran- ism of improved tendon function was reported in an domly assigned into two groups: a Botox (18 mice) or animal study where Botox was injected into the supras- vehicle-only control group (18 mice). The Botox group pinatus muscle for repair of rotator cuff injury showing were injected with 3 unit/kg of Botox (10 μL per injec- an improvement of collagen fibres alignment at the tion with a Hamilton syringe) into the right vastus later- tendon-bone interface [7]. alis muscle of the quadriceps group. The mice in the Tendon structure and function are closely regulated by control group were injected with an equal volume of the mechanical stimulation of passive and dynamic mech- phosphate-buffered saline (PBS) at the same site. Two anical loading that results from daily activity [15]. Regard- weeks after injection, the mice were sacrificed, and hind less of its’ specific use in muscle or tendon-related limb samples were processed for either histological ana- disorders, intramuscular injection of Botox causes either lysis (6 animals in each group) or TDSC isolation (12 partial or full relaxation of the muscle and a reduced mice in each group). Chen et al. Stem Cell Research & Therapy (2021) 12:38 Page 3 of 13 Histological analysis of the patellar tendons triple helical collagen molecule where collagen denatur- Two weeks after Botox injection into the quadriceps, ation occurs was detected with 5-FAM conjugated colla- mice were euthanized and the hind-limbs were harvested gen mimic peptide (CMP; 3Helix, Inc.). The CMP and macroscopically observed. The maximal patellar ten- solution (10 μM, in PBS) was heated to 80 °C for 5 min don width was recorded before fixation (Fig. 1a). Then in a heat block and immersed in an ice-water bath for whole hind-limbs were immerse-fixed in 4% paraformal- 60 s to return the solution to room temperature and pre- dehyde in PBS (PFA) for 48 h. The tendons were then vent damage to the samples. Cooled CMP solution was rinsed thoroughly with PBS, dehydrated in increasing added, and the samples incubated overnight at 4 °C. gradients of ethyl alcohol, embedded in paraffin and sec- After staining,
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