Investigating the response of skeletal muscle to prosthesis-related loading conditions : an ex vivo animal model

Graser, Marisa and Wark, Alastair and Day, Sarah and Sandison, Mairi and McConnell, Gail and Buis, Arjan (2021) Investigating the response of skeletal muscle to prosthesis-related loading conditions : an ex vivo animal model. In: International Society for Prosthetics and Orthotics 18th World Congress, 2021-11-01 - 2021-11-04, Virtual.

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BACKGROUND The use of lower-limb prosthetics puts the soft tissue of the residuum, including the muscle envelop, under constant physical stress. To adapt to this unphysiological mechanical loading, the muscles need to maintain the balance between tissue damage and regenerative processes. However, in extreme cases of overload or with repeated impact, this balance may be disturbed [1], potentially leading to residual limb pain and Deep Tissue Injuries [2]. AIM A new ex vivo model of rat skeletal muscle tissue was developed to quantify cellular damage from prosthesis-related loading protocols (Fig. 1a). Preliminary exploration of different imaging procedures and the relevance of results for prosthetic research and practice are discussed. METHOD Freshly isolated soleus and extensor digitorum longus muscles dissected from male Sprague Dawley rats were subjected to transverse compressive loading (9-32kPa through a 2mm indenter). Control tissues were held in the same conditions for the same time without loading. Tissues were subsequently processed for imaging by standard histological procedures, using H&E staining for visualising cell and tissue morphology and Procion Yellow for fluorescent dead cell staining [3]. In addition, tissue clearing methods were investigated to enable full tissue depth imaging by confocal microscopy. Furthermore, biochemical changes caused by cellular damage were visualised via multiphoton Raman microscopy of unstained samples. RESULTS During the maximum experimental time frame of 3h, the control samples showed only minor loss in cell viability. By comparison, the extent of mechanical damage in loaded tissues was readily distinguishable by imaging (Fig. 1b), with partial loss of striations, disrupted muscle fibres, increased interstitial space, and loss of cell viability. With careful control of the experimental setup, detailed imaging of local cellular damage in response to loading conditions could be obtained. DISCUSSION AND CONCLUSION Our preliminary studies present an ex vivo model and experimental procedures that are suitable for quantifying cellular damage from prosthesis-related loading conditions on skeletal muscle. Looking at this microscale will provide important insights into the adaptive capabilities of skeletal muscle. This can provide the basis for further research into the role of soft tissue deformation in limb pain and ulcer formation and could inform future directions for socket design and fit.