Abstract
Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.
Highlights
Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; the success of these strategies is dependent on faithful reproduction of native cellular organization
Material cues can be used to generate aligned structures at single cell resolution, each different pattern requires a new mask, mold, or scaffold.[3,4,5]. Many of these limitations can be addressed by acoustic manipulation, whereby cells are translated toward the static pressure nodes of ultrasound standing waves.[6]
We tested the compatibility of acoustic patterning for muscle engineering by exposing myoblasts suspended in cell medium to a 2.0–2.1 MHz field for 30 min. This exposure produced no significant detrimental effects upon cell metabolic activity, cell proliferation (PicoGreen DNA assay; 1–2 d), myogenic gene expression (MYOG, MRF4; 2–8 d), or muscle protein expression (α-myosin skeletal fast; 7 d) (Figure 1E and Figures S5 and S6, Supporting Information). We showed that these field parameters could be used to pattern myoblasts within a range of hydrogels, including agarose, Matrigel, and poly(ethylene glycol) (PEG) norbornene (Figure S7, Supporting Information)
Summary
Acoustic patterning enables remote and dynamic manipulation of cell populations within biomaterials, while complex, nonlinear architectures can be generated using hologram-based acoustics.[7] To date, 2D and 3D acoustic cell patterning has been used to study biological processes such as neurite guidance,[8] angiogenesis,[9] neural differentiation,[10] and cardiomyocyte beating.[11,12] Here, we use ultrasound standing waves to direct the assembly of myoblasts in collagen-based hydrogels, and stimulate these patterned materials to undergo in situ myogenesis and engineer bundles of aligned myotubes (Figure 1A) Future work will focus on macroscale organization, potentially by integrating a vertical standing wave to levitate myoblasts for the assembly of 3D, close-packed fibers
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