Biomedical Engineering - Theses

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Start date: 2014 × Subject: tissue engineering × Author: Maier IV, Michael Peter ×

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    Magnetic manipulation of cells to enhance tissue engineering
    Maier IV, Michael Peter ( 2023-03)
    One of the major detrimental effects of the aging process is the natural atrophy of skeletal muscle tissues, a process where individuals typically experience lower muscle mass, reduced muscle function and, as the problem worsens, compromised personal independence. This problem can become exacerbated due to injuries and diseases such as cancer, where patients may suffer from cachexia, a severe form of atrophy which leads to a 20-70% total volume loss in affected muscles. As it stands now, in vitro tissue engineered muscle fibres are often-times functionally immature, making it difficult to use these fibres as experimental models for drug testing. Part of the problem is that current tissue engineering methods use complex and expensive bioreactor systems to exercise skeletal muscle cells in vitro, systems that are inherently inflexible in terms of their potential applications. This technological shortcoming limits the insights that can be gained about muscle development and its disease states. Therefore, the aim of this thesis was to develop an in vitro magnetic stimulation method that provides a finer, more flexible alternative to typical bioreactors, and to investigate possible applications of this novel stimulation method for C2C12 myoblasts grown in a variety of 2D and 3D environments. To achieve this aim, magnetic chitosan microspheres (1-10 micron diameter) were produced and loaded with 30 nm, polyethylene glycol (PEG)-coated iron oxide nanoparticles (Mag30-CMs). The microspheres were produced at this size to limit cellular uptake prior to stimulation, and the microspheres were further functionalized with an RGD-containing peptide (Mag30-CM-RGDs) to enable external cellular stimulation through key integrin receptors. A magnetic stimulation chamber, optimized using finite element simulations, was 3D-printed and contained a sterile culture plate and two N45-grade neodymium magnets, allowing for the culture of magnetically-labelled C2C12 myoblasts inside a controlled and well-defined static magnetic field. After this system was developed, the same stimulation regime was applied to C2C12 myoblasts grown on a soft (8 kPa) 2D gelatin methacryloyl (GelMA) hydrogel scaffold, in order to determine the efficacy of this technique on softer substrates. Finally, the stimulation regime was then applied to C2C12 myoblasts grown in RGD-functionalized chitosan-gelatin cryogels, with macrostructures consisting of interconnected aligned pores roughly 50-250 micron wide. The maturity of the differentiated myotubes produced in the three environments was characterized based on the qPCR gene expression of key myogenic regulatory factors (MRFs), as well as morphological analysis (fusion index) done via confocal imaging. The magnetic stimulation regime method was modelled to produce piconewton-sized forces directly on the cell surface, resulting in a significantly higher fusion index as well as up-regulated gene expression of key MRFs for stimulated myotubes on hard tissue culture plastic (TCP). Stimulated myotubes had a 5-fold increase in multinucleated myotubes with 4+ nuclei over control, an important indicator of maturity. Significant increases from 2-6 fold were found in the gene expression of mature myosin heavy chain (MHC) genes MHC1, MHC2x, and MHC2a, in addition to a 2-2.3 fold increase in myogenin, a key MRF. These results indicate a promotion of myotube maturity in vitro in response to magnetic stimulation via the application of Mag30-CM-RGDs in a static magnetic field on hard TCP. In comparison, the soft GelMA hydrogels did not see the same benefits from the stimulation regime, producing no significant changes between the stimulated group and control. The 3D porous cryogels, functionalized with RGD-containing peptides, demonstrated some evidence of good cell attachment at early timepoints, but the attachment was not robust enough for the cryogels to serve as a scaffold for this kind of stimulation method, and cells did not survive long enough inside the gels to undergo differentiation. In this thesis, a robust and flexible magnetic stimulation method was produced and investigated in a novel skeletal muscle tissue engineering application. The Mag30-CM-RGDs showed evidence of maintaining cell attachment after 5 days of differentiation, and were able to promote muscle maturity in a 2D setting. This technique is not unique to skeletal muscle cells, and the methods utilized here can be adapted and applied to other types of tissues. As the materials are biocompatible, future experiments can be conducted to determine if these materials can be effective in vivo as well as in vitro.