Strategies for engineering skeletal muscle: an important link in the neuroprosthetic interface of bionic limbs
AuthorNgan, Catherine G. Y.
AffiliationSurgery (St Vincent's)
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-05-27.
© 2019 Dr. Catherine G.Y. Ngan
Limb amputation is a major cause of disability in our community, for which motorised prosthetic devices offer a return to function and independence. Advanced robotic limb technology utilises a range of mind-prosthetic interfacing strategies to intuitively drive the limb. These approaches include direct recording of peripheral nerves, brain-recording interfaces, or the transposition of transected nerves to remaining muscle groups for myoelectric recording. All of the current methods are hampered by delicate neural biology, either leading to premature device failure or introducing unnecessary surgical risk. As an alternative, this thesis proposes a new solution: to develop a bioelectrode using tissue engineered skeletal muscle as a signal amplifier of activity from residual nerves for intuitive prosthetic control. Conceptually, the fabrication of such a device would begin with a tissue biopsy from the patient from which a pool of myogenic stem cells would be derived and expanded. These autologous cells would be used for the tissue engineering of skeletal muscle fibres, primed with neurotrophic biofactors to optimise the tissue for innervation. Flexible recording wires could be incorporated into this fabrication step, thus eliminating the trauma of electrode insertion and also optimising its biocompatibility. The bioelectrode device could also be designed to patient or prosthetic anatomy as required. This thesis developed key elements of the above proposal. Firstly, a bioprinting technique was established to tissue engineer functional skeletal muscle using a gelatin methacryloyl (GelMA) bioink. Bioprinting enabled the rapid deposition of muscle progenitor cells (primary mouse myoblasts) in layered fibres, reminiscent of native muscle architecture. Fabrication parameters were optimised to produce fibres with high cell viability and print resolution. These bioprinted constructs were then able to support advanced maturation of cells into multinucleated muscle fibres, as evidenced by molecular analysis and functional testing. There was a significantly greater upregulation of genetic markers of myogenesis when compared to monolayer myotube cultures, and this result was complemented with functional testing that demonstrated mature patterns of calcium handling and electrical activity. The bioprinted muscle was then implanted in the nude rat to assess its capacity for innervation and vascularisation. The tissue construct was implanted in an in vivo chamber, which was supplied by a surgically formed arteriovenous loop and transected nerve. After only two weeks, independent bundles of mature muscle fibres had developed, with histological evidence of neural integration and vascularisation. In vivo electrophysiological studies confirmed the presence of innervation by demonstrating muscle activity in response to neural stimulation. Lastly, the triad of bioprinted muscle, vasculature and nerve was housed in a customised 3D printed chamber as a prototype for a bioelectrode that could be surgically grafted onto transected peripheral nerves after limb amputation. To conclude, this thesis developed the principle elements of designing a bioelectrode for neuroprosthetic interfacing and provides the foundation for further tissue optimisation and integration of electrodes. Although the work presented is in the frontier stages of development, it offers an exciting glimpse into the future of modern medicine and brings the dream of mind-controlled motorised prosthetic limbs closer to everyday reality.
Keywordstissue engineering; bioprinting; skeletal muscle; neuroprosthetic; bionic limbs; 3D printing; neuromuscular junctions; bioelectrode
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