Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media

Abstract

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro-patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi-nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer-by-layer (LbL) polyelectrolyte multilayer deposition was combined with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self-assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4-styrene-sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly-L-arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)₅-coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro.

Biomimetic Extracellular Environment Based on Natural Origin Polyelectrolyte Multilayers

“…Depending on the structure assumed by the polypeptides the films will present different internal structures and mechanical properties. Among the polypeptides already used, the most common are poly ( l ‐lysine) (PLL), poly ( d ‐lysine) (PDL), Poly ( l ‐glutamic acid) (PGA), poly (aspartic acid) (Pasp), poly‐ l ‐arginine (Parg), and elastin‐like recombinamers (ELRs) . Among all, PLL can be consider as a model protein, since lysine together with arginine are the major positively charged amino acids .…”

Release Retardation of Model Protein on Polyelectrolyte-Coated PLGA Nano- and Microparticles

“…PLL and DES have been employed for PEM assembly with each other or other polyelectrolytes for cell-related applications, such as guiding cell differentiation [22], [23], changing cell behavior [24], or creating enzymatically degradable microcapsules [25]. The suitability of PLL/DES in this study extends to the fact that just as PAH/PSS, they are a weak and strong polycation and polyanion combination, respectively.…”

Surface Micro‐ and Nanoengineering: Applications of Layer‐by‐Layer Technology as a Versatile Tool to Control Cellular Behavior

“…The SMCs depends on the shape of the gelatin patterns: strip patterns have more power to limit the cells to only grow on gelatincoated surface, while square patterns do not. Fibronectin attract all the cells to grow on its surface no matter the shape of the patterns [94] Stripes 25/50/150 µm -0.03-0.2 µm C2C12 cell line Patterned substrates consisting of cell-repellant and cell-adhesive regions have been used for the spatial control of cell attachment in vitro; their parallel alignment was essential for their fusion into myotubes [140] Grooves 5/10/30/100 µm 5/10/30/100 µm 4 µm C2C12 cell line The substrate can serve as an inductive template for in vivo muscle tissue regeneration; it drives the development, alignment, and further fusion of cells after implantation [67] This originate changes in gene expression, opening space to modulate cell differentiation into a specific lineage.…”