Understanding Myoblast Differentiation Pathways When Cultured on Electroactive Scaffolds through Proteomic Analysis

Abstract

Electroactive materials allow the modulation of cell–materials interactions and cell fate, leading to advanced tissue regeneration strategies. Nevertheless, their effect at the cellular level is still poorly understood. In this context, the proteome analysis of C2C12 cell differentiation cultured on piezoelectric polymer films with null average surface charge (non-poled), net positive surface charge (poled +), and net negative surface charge (poled −) has been addressed. Protein/pathway alterations for skeletal muscle development were identified comparing proteomic profiles of C2C12 cells differentiated on poly­(vinylidene fluoride), with similar cells differentiated on a polystyrene plate (control), using label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS). Only significantly expressed proteins (P < 0.01, analysis of variance) were used for bioinformatic analyses. A total of 37 significantly expressed proteins were detected on the C2C12 proteome with PVDF “poled −” at 24 h, whereas on the PVDF “poled +”, a total of 105 significantly expressed proteins were considered. At 5 days of differentiation, the number of significantly expressed proteins decreased to 23 and 31 in cells grown on negative and positive surface charge, respectively, the influence of surface charge being more explicit in some proteins. In both cases, proteins such as Fbn1, Hspg2, Rcn3, Tgm2, Mylpf, Anxa2, and Anxa6, involved in calcium-related signaling, were highly expressed during myoblast differentiation. Furthermore, some proteins involved in muscle contraction (Acta2, Anxa2, and Anxa6) were detected in the PVDF “poled +” sample. Upregulation of several proteins that enhance skeletal muscle development was detected in the PVDF “poled −” sample, including Ckm (422%), Tmem14c (384%), Serpinb6a (460%), adh7 (199%), and Car3 (171%), while for the “poled +” samples, these proteins were also upregulated at a smaller magnitude (254, 317, 253, 123, and 72%, respectively). Other differentially expressed proteins such as Mylpf (189%), Mybph (168%), and Mbnl1 (168%) were upregulated only in PVDF “poled −” samples, while Hba-a1 levels (581%) were increased in the PVDF “poled +” sample. On the other hand, cells cultured on non-poled samples have no differences with respect to the ones cultured on the control, in contrary to the poled films, with overall surface charge, demonstrating the relevance of scaffold surface charge on cell behavior. This study demonstrates that both positive and negative overall surface charges promote the differentiation of C2C12 cells through involvement of proteins related with the contraction of the skeletal muscle cells, with a more pronounced effect with the negative charged surfaces.

High protein-containing new food by cell powder meat

“…However, because the CPM is not organized up to the level of muscle tissue, proteins that occur at the cellular level, such as those constituting the cell nuclei, cytoplasm, and ribosomes are upregulated. Also, proteins that are known to be expressed in the myoblasts and myotubes, that function in RNA binding, protein binding, and protein folding, are upregulated 18 , 19 . This suggests that CPM can be manufactured from myoblasts and myotubes, but not from muscle tissue.…”

Analysis of the impact of handling and culture on the expansion and functionality of NK cells

“…The impact of the physicochemical properties of the microenvironment on lymphocytes remains largely undescribed. However, properties such as the surface potential of the materials used in cell culture are determinant of cell response ( 37 ). It is hypothesized that surface charge may also affect non-adhering cells due to the long-range electrostatic interactions, via factor immobilization that might impact their biological activity or via short-term cell-substrate contacts.…”

Scaffold-Based Poly(Vinylidene Fluoride) and Its Copolymers: Materials, Fabrication Methods, Applications, and Perspectives

“…Highly aligned fibers can drive cell proliferation and migration along aligned directions, making them well-suited for the repair and regeneration of muscle and neural tissues. − Compared to random structures, aligned fibers have a higher piezoelectric output. The symmetric electrode collectors modify the electric field distribution through the geometry of the electrodes, resulting in opposite forces acting on the two ends of the fiber to obtain an aligned structure.…”

“…125 Although "poled+ " and "poled−" PVDF both can enhance the expression of skeletal muscle development proteins, "poled−" PVDF had higher ability. 78 In addition, skeletal muscle development proteins were only upregulated in "poled−" PVDF. To further evaluate polarization, Ribeiro et al used the maturity index and fusion index to evaluate the impact of surface polarization.…”

“…The polarization properties of piezoelectric scaffolds regulate and control the potential changes on the cell membrane surface and the opening and closing of ion channels, thereby affecting myogenic responses . Although “poled+ ” and “poled–” PVDF both can enhance the expression of skeletal muscle development proteins, “poled–” PVDF had higher ability . In addition, skeletal muscle development proteins were only upregulated in “poled–” PVDF.…”