Materials 3D printed via fused deposition modelling elicit myofibrillar alignment and enhanced differentiation of skeletal muscle cells in-vitro

Materials 3D printed via fused deposition modelling elicit myofibrillar alignment and enhanced differentiation of skeletal muscle cells in-vitr

Physiological and pathophysiological concentrations of fatty acids induce lipid droplet accumulation and impair functional performance of tissue engineered skeletal muscle

Fatty acids (FA) exert physiological and pathophysiological effects leading to changes in skeletal muscle metabolism and function, however, in vitro models to investigate these changes are limited. These experiments sought to establish the effects of physiological and pathophysiological concentrations of exogenous FA upon the function of tissue engineered skeletal muscle (TESkM). Cultured initially for 14 days, C2C12 TESkM was exposed to FA‐free bovine serum albumin alone or conjugated to a FA mixture (oleic, palmitic, linoleic, and α‐linoleic acids [OPLA] [ratio 45:30:24:1%]) at different concentrations (200 or 800 µM) for an additional 4 days. Subsequently, TESkM morphology, functional capacity, gene expression and insulin signaling were analyzed. There was a dose response increase in the number and size of lipid droplets within the TESkM (p < .05). Exposure to exogenous FA increased the messenger RNA expression of genes involved in lipid storage (perilipin 2 [p < .05]) and metabolism (pyruvate dehydrogenase lipoamide kinase isozyme 4 [p < .01]) in a dose dependent manner. TESkM force production was reduced (tetanic and single twitch) (p < .05) and increases in transcription of type I slow twitch fiber isoform, myosin heavy chain 7, were observed when cultured with 200 µM OPLA compared to control (p < .01). Four days of OPLA exposure results in lipid accumulation in TESkM which in turn results in changes in muscle function and metabolism; thus, providing insight ito the functional and mechanistic changes of TESkM in response to exogenous FA.</div

Scalable 3D Printed Molds for Human Tissue Engineered Skeletal Muscle

Tissue engineered skeletal muscle allows investigation of the cellular and molecular mechanisms that regulate skeletal muscle pathology. The fabricated model must resemble characteristics of in vivo tissue and incorporate cost-effective and high content primary human tissue. Current models are limited by low throughput due to the complexities associated with recruiting tissue donors, donor specific variations, as well as cellular senescence associated with passaging. This research presents a method using fused deposition modeling (FDM) and laser sintering (LS) 3D printing to generate reproducible and scalable tissue engineered primary human muscle, possessing aligned mature myotubes reminiscent of in vivo tissue. Many existing models are bespoke causing variability when translated between laboratories. To this end, a scalable model has been developed (25–500 μL construct volumes) allowing fabrication of mature primary human skeletal muscle. This research provides a strategy to overcome limited biopsy cell numbers, enabling high throughput screening of functional human tissue

Design and additive manufacture of microphysiological perfusion systems for pharmaceutical screening of tissue engineered skeletal muscle

The methodologies utilised by pharmaceutical companies for the toxicity screening of developmental drugs are currently based on outdated two-dimensional (2D) plate-based assay systems. Although such methods provide high-throughput analysis, limitations surrounding the biomimicry of the culture environment reduces the accuracy of testing, making the process cost and time inefficient. To significantly enhance the current methods, a screening platform that is both flexible in its design and is amenable toward physiologically representative engineered tissue is required. Incorporating a flow environment within the system elicits a variety of advantages over standard static cultures, pertinently the ability to couple the flow path with automated analytical systems via the use of intuitive software. Musculoskeletal pathological conditions account for £4.76 billion of NHS spending as of 2011 (Department of Health), affecting one in four of the UK adult population. Skeletal muscle, a highly metabolic and regenerative tissue, is involved in a wide variety of functional, genetic, metabolic and degenerative pathological conditions such as muscular dystrophy, diabetes, osteoarthritis, motor neuron disease and pertinently muscular weakness associated with aging populations. Skeletal muscle tissue engineering is centred on the in vitro creation of in vivo-like tissue within laboratory environments and seeks to aid the development of future therapies, by reliably elucidating the molecular mechanisms that regulate such conditions. However, the translation of such models toward systems amenable to pharmaceutical companies has to date been limited. Microphysiological perfusion bioreactors for in vitro cell culture are a rapidly developing research niche, although state of the art systems are currently limited due to the biologically non-representative 2D culture environment, lack of adaptability toward different experimental requirements and confinement to offline analytical methods. Advancements in additive manufacture (AM), commonly known as three-dimensional (3D) printing has provided a method of production that enables researchers to hold complete design freedom and facilitate customisation of required parts. The low cost, rapid prototyping nature of AM further lends itself toward the development of such technology, with design iterations quickly and easily printed, tested and re-designed where appropriate. Issues do however, currently persist regarding the biological compatibility of printed polymers and functional material properties of parts created. As such, this thesis investigated the use of AM as a rapid and functional prototyping technique to design and develop microphysiological perfusion bioreactors. Here, biocompatibility of candidate polymers derived from commercially available 3D printing processes; fused deposition modelling (FDM), stereolithography (SL), selective laser sintering (LS) and PolyJet modelling (PJM) were elucidated. Following the biological evaluation of these polymers, their suitability, and the applicability of each process in function and manufacture of perfusion bioreactors were assessed alongside the research and development process of system designs. Specifically, attention was afforded to the homeostatic environment within bio-perfusion systems. Once finalised, the biological optimisation of designs; biocompatibility and rates of proliferation in response to the perfusion environment, was undertaken. Protocols were then established for the automated perfusion of skeletal muscle cells in both monolayer and tissue engineered 3D hydrogels. This research outlined significant contributions to the scientific literature in 3D printed polymer biocompatibility, in addition to creating bio-perfusion systems that are adaptable, analytical and facilitate the in situ phenotypic development of physiologically representative skeletal muscle tissue. Polymer biocompatibility elucidated in this work will help to facilitate the wide-ranging use of AM in biological settings. However, advancements in the chemical properties of liquid resins for advanced photo-curable processes remain necessitated for AM to be considered as a primary manufacturing technique in the biological sciences. Furthermore, although systems developed in this work have provided a base technology from which to develop and build upon, significant challenges remain in the integration of tissue engineered perfusion devices within pharmaceutical settings. Although it is plausible that the technology created in its current guise would facilitate the automated generation of skeletal muscle tissue, systems require further development to aid their usability and scale. Furthermore, work is also required to optimise the biological environment prior to mass manufacture. As such, to truly influence the pharmaceutical industry, which has invested so heavily in more traditional screening technology, a system that is all-encompassing in biology, technology and automated analytics is required