A new biofunctionalized and micropatterned PDMS is able to promote stretching induced human myotube maturation

Abstract

Inter-individual variability in muscle responses to mechanical stress during exercise is poorly understood. Therefore, new cell culture scaffolds are needed to gain deeper insights into the cellular mechanisms underlying the influence of mechanical stress on human myogenic progenitor cells behavior. To this end, we propose the first in vitro model involving uniaxial mechanical stress applied to aligned human primary muscle-derived cells, employing a biocompatible organic-inorganic photostructurable hybrid material (OIPHM) covalently attached to a stretchable PDMS support. Using a laser printing technique with an additive photolithographic process, we optimally micropatterned the PDMS support to create longitudinal microgrooves, achieving well-aligned muscle fibers without significantly affecting their diameter. This support was biofunctionalized with peptide sequences from the ECM, which interact with cellular adhesion receptors and prevent myotube detachment induced by stretching. X-ray photoelectron spectroscopy (XPS) of biofunctionalized PDMS with RGD-derived peptide deposition revealed a significant increase in nitrogen compared to silicon, associated with the presence of a 380 nm thick layer measured by atomic force microscopy (AFM). Upon cell culture, we observed that functionalization with an RGD peptide had a beneficial impact on cell fusion rate and myotube area compared to bare PDMS. At the initiation of the stretching protocol, we observed a three-fold rapid and transient increase in RNA expression for the mechanosensitive ion channel protein piezo and a decrease in the ratio of nuclei expressing myogenin relative to the total nuclei count (43 ± 16% vs. 6 ± 6%, p < 0.01). Compared to day 0 of differentiation, stretching the myotubes induced MHC and Titin colocalization (0.66 ± 0.13 vs. 0.93 ± 0.05, p < 0.01), favoring sarcomere organization and maturation. In this study, we propose and validate an optimized protocol for culturing human primary muscle-derived cells, allowing standardized uniaxial mechanical stress with a biocompatible OIPHM covalently linked to PDMS biofunctionalized with an ECM-derived peptide, to better characterize the behavior of myogenic progenitor cells under mechanical stress in future studies.