Effects of Dexamethasone Dose and Timing on Tissue-Engineered Skeletal Muscle Units

Abstract

Our lab showed that administration of dexamethasone (DEX) stimulated myogenesis and resulted in advanced structure in our engineered skeletal muscle units (SMU). While administration of 25 nM DEX resulted in the most advanced structure, 10 nM dosing resulted in the greatest force production. We hypothesized that administration of 25 nM DEX during the entire fabrication process was toxic to the cells and that administration of DEX at precise time points during myogenesis would result in SMU with a more advanced structure and function. Thus, we fabricated SMU with 25 nM DEX administered at early proliferation (days 0–4), late proliferation (days 3–5), and early differentiation (days 5–7) stages of myogenesis and compared them to SMU treated with 10 nM DEX (days 0–16). Cell proliferation was measured with a BrdU assay (day 4) and myogenesis was examined by immunostaining for MyoD (day 4), myogenin (day 7), and α-actinin (day 11). Following SMU formation, isometric tetanic force production was measured. An analysis of cell proliferation indicated that 25 nM DEX administered at early proliferation (days 0–4) provided 21.5% greater myogenic proliferation than 10 nM DEX (days 0–4). In addition, 25 nM DEX administered at early differentiation (days 5–7) showed the highest density of myogenin-positive cells, demonstrating the greatest improvement in differentiation of myoblasts. However, the most advanced sarcomeric structure and the highest force production were exhibited with sustained administration of 10 nM DEX (days 0–16). In conclusion, alteration of the timing of 25 nM DEX administration did not enhance the structure or function of our SMU. SMU were optimally fabricated with sustained administration of 10 nM DEX.

Antenatal Steroid Exposure, Aerobic Fitness, and Physical Activity in Adolescents Born Preterm with Very Low Birth Weight

“…7,8 However, in vitro studies suggest that the effects are dependent on the dose and timing of exposure. 32,33 Muscle satellite cells are muscle-specific stem cells that play an important role in fiber plasticity, repair, and regenerative capacity. 34 Aerobic exercise training has been shown to activate satellite cells and promote proliferation and differentiation toward a more oxidative phenotype with greater mitochondrial protein content, oxidative capacity, and capillary density.…”

A tissue engineering approach for repairing craniofacial volumetric muscle loss in a sheep following a 2, 4, and 6-month recovery

“…Muscle progenitor cells including satellite cells were isolated as described previously [23,24,[29][30][31][32][33][34]. Briefly, muscle biopsies between 3g and 3.5g were sanitized in 70% ethanol and finely minced with a razor blade.…”

“…No. D4902)) [23,[29][30][31][32]35] and seeded at a density of 10,000 cells/cm 2 onto 150mm tissue culture dishes embedded with stainless steel pins positioned 9cm apart. The culture media was changed for the first time on day 4 followed by a Monday-Wednesday-Friday feeding schedule thereafter.…”

“…no. A8960)) to promote differentiation [23,[29][30][31][32]35]. Monolayers began to spontaneously delaminate on days 10-12 and were fully rolled up by day 15.…”

“…A subset of the SMUs, termed "sentinel SMUs", was reserved for in vitro characterization including biomechanical testing and histology. Approximately 24-48 hours after 3D formation, contractile properties of the single SMUs (not modularly fused) were measured as described previously [23,[29][30][31][32][33]35,36]. Briefly, contractions were elicited through field stimulation with a platinum electrode and measured by an optical force transducer (World Precision Instruments, cat.…”