Application of cyclic strain for accelerated skeletal myogenic differentiation of mouse bone marrow-derived mesenchymal stromal cells with cell alignment.
The fabrication of biomimetic skeletal myocyte constructs continues to present a challenge to functional tissue engineering. The skeletal myogenesis of bone marrow-derived mesenchymal stromal cells (BMSCs) to mimic the native tissue architecture offers great therapeutic promise, but remains particularly difficult. The aim of this study was to examine the possibility of accelerating the skeletal myogenic differentiation of BMSCs with an aligned structure by applying cyclic strain. Mouse BMSCs (mBMSCs) were plated on silicone sheets that were coated with fibronectin and subjected to cyclic 10% uniaxial strain when they reached 80%-90% cell confluency. Cells cultured in a growth medium that were subjected to cyclic strain at a frequency of 0.17 Hz (10 times/min) demonstrated a shift of alignment within 48 h from a completely random orientation to a well-aligned morphology with well-organized actin stress fibers that were parallel to the strain vector. The cyclic strain restricted the motility and proliferation of the aligned mBMSCs in the growth medium, which resulted in tight cellular contact in the cell population. When mBMSCs were subjected to cyclic strain in a myogenic medium, reverse transcription-polymerase chain reaction analysis demonstrated the upregulation of skeletal myogenic marker genes (myogenic factor 5 [Myf5], myogenin, and myogenic regulatory factor 4 [MRF4]), but not smooth muscle marker genes (myocardin and α-smooth muscle actin). In addition, immunocytochemistry showed that the mBMSCs fused to form multinucleated myosin- and myogenin-positive myotubes in the direction of the applied tension within 5 days. These results demonstrate that our simple method of applying of cyclic strain to cells cultured in a myogenic medium greatly accelerates the skeletal myogenic differentiation of mBMSCs with an aligned structure, and they highlight the importance of cellular alignment for creating physiologically relevant environments to study the myogenesis of BMSCs and engineer skeletal muscle.
Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita City, Japan. egu@dent.osaka-u.ac.jp
Hiroshi
H
Egusa
Munemasa
M
Kobayashi
Takuya
T
Matsumoto
Jun-Ichi
J
Sasaki
Shinya
S
Uraguchi
Hirofumi
H
Yatani
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation / Q000235:genetics; D019070:Cell Lineage / Q000235:genetics; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002999:Clone Cells; D005786:Gene Expression Regulation; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D024510:Muscle Development / Q000235:genetics; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D013314:Stress, Mechanical