Larson Alexie A, Syverud Brian C, Florida Shelby E, Rodriguez Brittany L, Pantelic Molly N, Larkin Lisa M
Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA.
Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA.
Cells Tissues Organs. 2018;205(4):197-207. doi: 10.1159/000490884. Epub 2018 Aug 17.
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.
我们实验室的研究表明，给予地塞米松（DEX）可刺激肌生成，并使我们构建的骨骼肌单元（SMU）结构更先进。虽然给予25 nM DEX会产生最先进的结构，但给予10 nM DEX时产生的力量最大。我们推测，在整个构建过程中给予25 nM DEX对细胞有毒性，而在肌生成的精确时间点给予DEX会使SMU具有更先进的结构和功能。因此，我们在肌生成的早期增殖阶段（第0 - 4天）、晚期增殖阶段（第3 - 5天）和早期分化阶段（第5 - 7天）给予25 nM DEX构建SMU，并将其与用10 nM DEX处理（第0 - 16天）的SMU进行比较。通过BrdU检测法（第4天）测量细胞增殖，并通过对MyoD（第4天）、生肌调节因子（第7天）和α - 辅肌动蛋白（第11天）进行免疫染色来检测肌生成。在SMU形成后，测量等长强直收缩力的产生。细胞增殖分析表明，在早期增殖阶段（第0 - 4天）给予25 nM DEX比给予10 nM DEX（第0 - 4天）能使肌源性增殖提高21.5%。此外，在早期分化阶段（第5 - 7天）给予25 nM DEX显示生肌调节因子阳性细胞密度最高，表明成肌细胞分化改善最大。然而，持续给予10 nM DEX（第0 - 16天）表现出最先进的肌节结构和最高的力量产生。总之，改变25 nM DEX给药时间并未增强我们构建的SMU的结构或功能。持续给予10 nM DEX能最佳地构建SMU。
