Introduction: Volumetric skeletal muscle lost through trauma or disease has proven difficult to regenerate due to the challenge of differentiating human myoblasts into aligned, contractile tissue in vitro. Specifically, we are interested in how the structure and composition of the basal lamina influences differentiation and alignment of skeletal muscle myoblasts. Previously we demonstrated that the geometric patterning of extracellular matrix (ECM) protein cues, specifically fibronectin (FN), can guide aligned myotube formation and increase twitch stress[1]. We hypothesized that ECM proteins found in the basal lamina, including laminin (LAM) and collagen IV (Col IV), would further increase differentiation. To test this we investigated how ECM protein type in combination with micropatterned line geometry impacts differentiation and alignment of murine C2C12 and human skeletal muscle myoblasts in our 2D system.
Materials and Methods: Glass coverslips were spincoated with polydimethylsiloxane (PDMS) and microcontact printed (μCP) with lines of Col IV, Collagen I (Col I), FN, or LAM[1]. Line widths were 20, 50, 100, and 200 μm and line spacings were 10, 15, 20, and 30 μm, as well as an isotropic control. C2C12 or human myoblasts were seeded on μCP coverslips, grown to confluence and differentiated for 6 days until myotubes formed. Samples were fixed and stained for nuclei and myosin heavy chain (MHC) and imaged using confocal microscopy. Images were post-processed using ImageJ to quantify percent area myotubes, myotube length, myotube fusion index, and myotube alignment.
Results and Discussion: For both C2C12 and human myoblasts, LAM significantly increased myotube formation compared to FN, Col IV, and Col I (Figure 1). C2C12 myotubes were uniaxially aligned on 20, 50, and 100 μm widths with 20 and 30 μm spacing, but were off-axis on the other patterns. C2C12 myotube density was higher on 50, 100, and 200 mm line widths and 10 and 15 mm line spacings. Human myoblasts differentiated into myotubes on 50, 100, and 200 mm line widths with 10, 15, and 20 mm spacings. Uniquely, human myotubes were uniaxially aligned with comparable density on all line geometries, in contrast to the C2C12, which showed variability. Thus, while human myotube differentiation increased on the LAM, they were less sensitive to variation in line geometry than the C2C12 cells.
Conclusion: We found that LAM increased C2C12 and human myotube formation, and human myotube density and alignment was not affected by line geometry. For future work, we will assess the contractility of human engineered muscle using the muscular thin film (MTF) contractility assay[1], which can be used to assess muscle function in response to pharmaceutical agents and for in vitro diagnostic applications.
NIH Director's New Innovator Award; John and Claire Bertucci Fellowship
References:
[1] Sun Y, Duffy R, Lee A, Feinberg AW. Optimizing the structure and contractility of engineered skeletal muscle thin films. Acta Bio. 2013 Aug; 9(8): 7885-94.