Development of a Novel Device for the Perfusion Driven Decellularization of Skeletal Muscle

Decellularization of skeletal muscle is a process that removes cellular components of skeletal muscle tissue while leaving behind the intact extracellular matrix (ECM). Skeletal muscle ECM is currently being studied as a biologic scaffold for repairing volumetric muscle loss (VML) because the removal of cells greatly reduces the antigenicity of the donor tissue. Decellularization usually relies on passive diffusion of detergents, surfactants and/or osmotic solutions to strip cells from the ECM. However, passive diffusion alone is usually not sufficient for complete removal of cells from the interior of large pieces of skeletal muscle using detergents, such as sodium dodecyl sulfate (SDS). The goal of this study was to develop a device that not only removes cells by perfusion from the interior of skeletal muscle, but also monitors the progress of decellularization in real-time. The device, based around a Raspberry Pi, is a standalone system that does not require a desktop computer or expensive software packages. Different flow rates (0.1, 1.0 and 10 mL/hr) along with different concentrations of SDS (0.2% and 1.0%) were tested. Decellularization progress was monitored and logged to an online spreadsheet. The device was found to be capable of decellularizing the medial gastrocnemius of a rat in under 10 hours. Complete decellularization was validated using fluorescent imaging. Perfusion decellularized muscle samples were found to have no significant differences in collagen or sulfated glycosaminoglycan (sGAG) content when compared to samples that were decellularized using current passive diffusion protocols. The ECM obtained through the use of this device is currently being used for the repair of VML in a rat model

Scaffold and Tissue Based Therapies to Improve Skeletal Muscle Regeneration After Volumetric Muscle Loss

Volumetric muscle loss (VML) is an injury to skeletal muscle characterized by a loss of more than 20% of a muscles volume. The combination of the bulk loss of tissue, transection and separation of myofibers proximal and distal to the injury, loss of innervation and blood supply, and the depletion of muscle progenitor cells results in permanent fibrosis and functional deficits due to loss of contractile tissue. Scaffolds, cells, and engineered constructs have been explored as potential therapeutic interventions to induce myogenesis at the site of a VML injury in animal models, in addition to limited clinical trials. This dissertation summarizes the current state of the field and explores possible strategies for repairing VML and understanding the mechanisms underlying the regenerative response of VML-damaged muscle. The challenges currently facing the skeletal muscle tissue engineering are presented along with potential approaches to further the field and deliver effective treatment options for patients and their physicians