An Assessment of Myotube Morphology, Matrix Deformation, and Myogenic mRNA Expression in Custom-Built and Commercially Available Engineered Muscle Chamber Configurations

Abstract

There are several three-dimensional (3D) skeletal muscle (SkM) tissue engineered models reported in the literature. 3D SkM tissue engineering (TE) aims to recapitulate the structure and function of native (in vivo) tissue, within an in vitro environment. This requires the differentiation of myoblasts into aligned multinucleated myotubes surrounded by a biologically representative extracellular matrix (ECM). In the present work, a new commercially available 3D SkM TE culture chamber manufactured from polyether ether ketone (PEEK) that facilitates suitable development of these myotubes is presented. To assess the outcomes of the myotubes within these constructs, morphological, gene expression, and ECM remodeling parameters were compared against a previously published custom-built model. No significant differences were observed in the morphological and gene expression measures between the newly introduced and the established construct configuration, suggesting biological reproducibility irrespective of manufacturing process. However, TE SkM fabricated using the commercially available PEEK chambers displayed reduced variability in both construct attachment and matrix deformation, likely due to increased reproducibility within the manufacturing process. The mechanical differences between systems may also have contributed to such differences, however, investigation of these variables was beyond the scope of the investigation. Though more expensive than the custom-built models, these PEEK chambers are also suitable for multiple use after autoclaving. As such this would support its use over the previously published handmade culture chamber system, particularly when seeking to develop higher-throughput systems or when experimental cost is not a factor.

ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

“…Conversely, the addition of anti-NGF/proNGF impaired myogenic differentiation, as observable by the significant decrease in myotube number and by the reduction in myotube length and diameter ( Figure 3 B). Importantly, the fusion index, a widely employed parameter to quantify myogenic differentiation in cell culture [ 32 ], was deeply affected upon neutralizing antibody incubation ( Figure 3 C). Taken together, these results suggest that the addition of exogenous proNGF does not affect SCDM differentiation, since endogenous proNGF is already sufficient to induce a maximal effect.…”

Scalable 3D Printed Molds for Human Tissue Engineered Skeletal Muscle

“…Advancements in additive manufacture (AM) have prompted a trend toward TE platforms (Truskey et al, 2013) that are manufactured by automated technology (Costa et al, 2015; Zhang and Zhang, 2015), due to the associated design flexibility of processes, such as 3D printing. The model presented here, demonstrates the use of biocompatible PLA and PA-12 (Rimington et al, 2017, 2018) molds building on previously published molds that feature a twin or single based post design at decreasing construct volumes, utilizing user-friendly accessible FDM and LS 3D printing techniques (Jones et al, 2018). Such designs ensure there is minimal friction between the side surfaces of the constructs; eliciting cell-mediated mechanical matrix contraction in the X axis that facilitates myoblast alignment and myogenic differentiation for the formation of 3D multinucleated myotubes.…”

Engineered Whole Cut Meats Assembled of Cell Fibers Constructed by Tendon-Gel Integrated Bioprinting

“…3c). Studies related to muscle tissue engineering have implied that an 7 anchor structure enables 3D muscle tissue to not only maintain its initial shape but also improve 8 the cell alignment, fusion, and differentiation against the muscle fiber's contraction 15, [34][35][36][37][38] . We 9 placed a printed cell fiber onto a silicone rubber and anchored it with needles to fasten both 10 ends to withstand cell contractions (Fig.…”