(F-204) Characterizing the effects of mechanical deformation on engineered skeletal muscle fiber morphology.

Introduction:: Skeletal muscle powers voluntary movement in our bodies. Disease or damage that disrupts intercellular communication in this multicellular tissue can thus severely impact health, mobility, and quality of life. There is a critical need for engineering in vitro models of skeletal muscle that capture the hierarchical organization of this tissue in vivo and enable studying how mechanical, biochemical, and electrical stimuli in the environment impact muscle’s assembly, maturation, and adaptation to pathological conditions. While there are several useful tools for monitoring electrical and biochemical changes within multicellular tissues, ranging from microelectrodes to transcriptomics, there is a significant lack of platform technologies that enable mapping and monitoring mechanical signaling within engineered tissues. We have developed an active magnetic extracellular matrix that enables precisely controlling mechanical deformation of engineered muscle tissues. Our investigations have revealed that dynamic mechanical stimulation guides assembly, maturation, and alignment of muscle fibers and their resultant contractile response. We anticipate that the technology we have developed will enable fabricating more physiologically relevant models of muscle tissue for applications ranging from high-throughput drug testing to regenerative medicine to biorobotics.


Materials and Methods:: We fabricated magnetic micropillars by mixing iron microparticles with poly (dimethyl siloxane) (PDMS, 1:10 monomer: crosslinker ratio). Micropillars were embedded within a fibrin hydrogel (4 mg/mL) within a 24-well plate (Fig. 1A). We 3D printed a custom-designed apparatus for controllable linear actuation of a permanent magnet under the well plate. The resultant mechanical motion was recorded with a stereoscope and custom automated tracking software (Matlab) was used to map deformation of the hydrogel in response to magnetic stimulation (Fig. 2A). C2C12 mouse myoblasts were seeded on the hydrogel and imaged daily to track muscle fiber formation, maturation, and alignment. Muscles were stimulated to contract after 1 week of culture and custom tracking software from our collaborators (Prof. Emma Lejeune, Boston University) was used to map the deformation of the gel in response to muscle contraction. Tissues were fixed and stained for cell nuclei and myosin expression to quantify muscle fusion index and other indicators of mature tissue formation.


Results, Conclusions, and Discussions:: There seems to be no significant difference in fiber length and width for the experimental (magnet stimulation) and control (no magnet stimulation) groups when compared to each other on the same day; however, there is a significant difference when the groups are compared across day 5 and day 8 (Fig. 2B). Dynamic mechanical stimulation has an effect on global fiber alignment, though this is not significant, and a significant impact on the dispersion of fiber alignment within an imaging area (Fig. 1D). Improved fiber alignment and reduced dispersion has a downstream effect by coordinating synchrony of response to optical stimulation (Fig. 2C). Qualitatively, the control group appears to beat more sporadically in response to optical stimulation compared to the experimental group, which has been corroborated quantitatively through motion tracking analysis. Additionally, it would appear that the muscle fibers take on a certain alignment early into the differentiation process which remains the same from that point onwards. Future work points to seeding the cells on the hydrogels at lower density to enable monitoring how magnetic stimulation impacts proliferation to confluency, and whether this improves our ability to control global alignment. Answering these questions on fiber formation and alignment will allow us to create sections of muscle fibers oriented in a specific direction within a larger 3D tissue engineered construct that could provide desirable morphological and functional properties such as pennation.




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