Effects of electrical stimulation in C2C12 muscle constructs

Abstract

Electrical stimulation affects the deposition of extracellular matrices and cellular differentiation. Type I collagen is one of the most abundant extracellular matrix proteins; however, not much is known about the effects of electrical stimulation on collagen type I deposition in C2C12 cells. Thus, we studied the effects of electrical voltage and stimulation frequency in 3D cultured C2C12 muscle cells in terms of metabolic activity, type I collagen deposition and cell morphology. Electrically excitable C2C12 muscle cells were seeded in collagen scaffolds and stimulated with rectangular signals of voltage (2, 5, 7 V) and frequency (1, 2 Hz), using parallel carbon electrodes spaced 1 cm apart. Metabolic activity was quantified by the glucose: lactate concentration ratio in the medium. Apoptotic activity was assessed by TUNEL staining and changes in collagen deposition were identified by immunohistology. The ultrastructure of the tissue was examined by TEM. Glucose and lactate analysis indicated that all groups had similar metabolic activity. TUNEL stain showed no significant difference in apoptotic damage induced by electrical stimulation compared to the control. Samples stimulated at 2 Hz exhibited reduced collagen deposition compared to the control and 1 Hz stimulated samples. Muscle-protein marker desmin was highly expressed in constructs stimulated with 1 Hz/5 V sample. TEM revealed that the stimulated samples developed highly organized sarcomeres, which coincided with improved contractile properties in the 1 Hz/5 V-and 2 Hz/5 Vstimulated groups. Our data implicate that a specific electrical frequency may modulate type I collagen accumulation and a specific voltage may affect the differentiation of muscle sarcomeres in excitable cells.

Acceleration of Myofiber Formation in Culture by a Digitized Synaptic Signal

“…Such studies typically use high-voltage artificial stimulation. 10,[13][14][15][16][17][18] We did not examine the impact of the synaptic signal on inducing twitching. Of interest would be to examine the efficacy of this and/or a more extensive synaptic stimulation regimen on twitching on cultured myofibers.…”

“…[10][11][12][13][14] These approaches attempt to mimic developmental cues using a multi-volt electrical stimulation to induce differentiation of myoblast cells in vitro. A variety of electrical stimulation protocols have been developed to induce muscle cell differentiation.…”

“…A variety of electrical stimulation protocols have been developed to induce muscle cell differentiation. Application of signals from 1 to 50 V induced differentiation, 10,[13][14][15][16][17][18] while those of 100 mV were ineffective, 11 suggesting the existence of a voltage threshold to foster differentiation.…”

A contactless electrical stimulator: application to fabricate functional skeletal muscle tissue

“…It is well known that muscle cells are able to differentiate under stimulation with an electrical field, which leads to an increase in myosin production and the formation of myofibers and contractile proteins (Park et al 2008). Advances in microscale technologies used in biology and regenerative medicine can provide reliable methods and devices to enable the use of electrical stimulation (ES) for tissue engineering applications in a precise and controllable manner.…”

A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering

“…Doping with anionic or cationic dopants imparts conductivity to these polymers [11]. In the presence of an electrical stimulation, these conductive polymers can modulate cell adhesion, migration, protein secretion and DNA synthesis of electrically responsive cells, such as nerve, bone, muscle and cardiac cells [12][13][14][15][16][17][18][19][20][21].…”