Heparin-Mimicking Polymer-Based In Vitro Platform Recapitulates In Vivo Muscle Atrophy Phenotypes

Abstract

The cell–cell/cell–matrix interactions between myoblasts and their extracellular microenvironment have been shown to play a crucial role in the regulation of in vitro myogenic differentiation and in vivo skeletal muscle regeneration. In this study, by harnessing the heparin-mimicking polymer, poly(sodium-4-styrenesulfonate) (PSS), which has a negatively charged surface, we engineered an in vitro cell culture platform for the purpose of recapitulating in vivo muscle atrophy-like phenotypes. Our initial findings showed that heparin-mimicking moieties inhibited the fusion of mononucleated myoblasts into multinucleated myotubes, as indicated by the decreased gene and protein expression levels of myogenic factors, myotube fusion-related markers, and focal adhesion kinase (FAK). We further elucidated the underlying molecular mechanism via transcriptome analyses, observing that the insulin/PI3K/mTOR and Wnt signaling pathways were significantly downregulated by heparin-mimicking moieties through the inhibition of FAK/Cav3. Taken together, the easy-to-adapt heparin-mimicking polymer-based in vitro cell culture platform could be an attractive platform for potential applications in drug screening, providing clear readouts of changes in insulin/PI3K/mTOR and Wnt signaling pathways.

Mechanosensation mediates volume adaptation of cardiac cells and spheroids in 3D

“…GelMA hydrogels were prepared based on a previously described method [ 21 , 23 , 24 ]. In brief, a mould was constructed by 3D-printing a resin structure with a 12 ​× ​12 ​× ​0.5 ​mm 3 empty space in the centre.…”

Volume adaptation of neonatal cardiomyocyte spheroids in 3D stiffness gradient GelMA

“…A precursor gelatin methacryloyl (GelMA) solution was prepared as per our previously described method. 14 , 24 , 25 Lyophilized GelMA was dissolved in PBS to and combined with the photoinitiator Irgacure 2959 (Sigma Aldrich) in 70% ethanol to achieve final concentrations 0.09 g/ml of GelMA and 1 μg/ml of Irgacure. The mixture was warmed to 37°C in a water bath for 1 h to allow the GelMA to hydrate, before being stored at 4°C for use the next day.…”

“…In our study, we used a versatile GelMA hydrogel platform to mimic biophysical elements, healthy and diseased cardiac tissues, on a single linear stiffness gradient. GelMA is a well characterized, collagen derived material 14 , 22 , 23 , 24 , 25 , 26 that has been used for 3D mechanobiology, stem cell differentiation and tissue engineering. 6 , 14 , 23 , 24 , 26 , 27 The biophysical properties of GelMA are highly tuneable 14 , 23 , 24 , 28 , 29 and GelMA has an abundance of the arginine‐glycine‐aspartic acid (RGD) motif; one of several ECM binding motifs known to facilitate integrin‐mediate mechanosensation in cardiomyocytes.…”

Versatile Potential of Photo-Cross-Linkable Silk Fibroin: Roadmap from Chemical Processing Toward Regenerative Medicine and Biofabrication Applications

“…With this reaction, the methacryloyl groups are introduced on the reactive amines of the residues of amino acids present in SF. Noting that the total amount of amine in SF is approximately 0.2%, this is a maximum limit of the degree of methacrylation, overall being low compared to many other biopolymers. , Nevertheless, we expect the possibility to modulate it within this range by other factors, such as controlling temperature or the total time of the reaction. So far only two studies focused on the methacrylation by means of MA, however, no information has been provided on the final achieved degree of methacrylation. , Consequently, it is possible the minor variation in the degree of methacrylation in this range may result in SFMA products with lower number of cross-links and thus variable mechanical properties when subsequently photo-cross-linked.…”