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Human embryonic stem cells (hESCs) are uniquely endowed with a capacity for both self-renewal and multilineage differentiation. The aim of this investigation was to determine if short periods of cyclic mechanical strain enhanced dexamethasone, ascorbic acid, and β-glycerophosphate (triple-supplement)-induced osteogenesis and bone nodule formation by hESCs. Colonies were cultured for 21 days and divided into control (no stretch) and three treatment groups; these were subjected to in-plane deformation of 2% for 5 s (0.2 Hertz) every 60 s for 1 h on alternate days in BioFlex plates linked to a Flexercell strain unit over the following periods (day 7-13), (day 15-21), and (day 7-21). Numerous bone nodules were formed, which stained positively for osteocalcin and type I collagen; in addition, MTS assays for cell number as well as total collagen assays showed a significant increase in the day 7-13 group compared to controls and other treatment groups. Alizarin Red staining further showed that cyclic mechanical stretching significantly increased the nodule size and mineral density between days 7-13 compared to control cultures and the other two experimental groups. We then performed a real-time polymerase chain reaction (PCR) microarray on the day 7-13 treatment group to identify mechanoresponsive osteogenic genes. Upregulated genes included the transcription factors RUNX2 and SOX9, bone morphogenetic proteins BMP1, BMP4, BMP5, and BMP6, transforming growth factor-β family members TGFB1, TGFB2, and TGFB3, and three genes involved in mineralization-ALPL, BGLAP, and VDR. In conclusion, this investigation has demonstrated that four 1-h episodes of cyclic mechanical strain acted synergistically with triple supplement to enhance osteogenesis and bone nodule formation by cultured hESCs. This suggests the development of methods to engineer three-dimensional constructs of mineralized bone in vitro, could offer an alternative approach to osseous regeneration by producing a biomaterial capable of providing stable surfaces for osteoblasts to synthesize new bone, while at the same time able to be resorbed by an osteoclastic activity-in other words, one that can recapitulate the remodeling dynamics of a naturally occurring bone matrix.

tissue engineering. part a

Short Periods of Cyclic Mechanical Strain Enhance Triple-Supplement Directed Osteogenesis and Bone Nodule Formation by Human Embryonic Stem Cells <i>In Vitro</i>

Short Periods of Cyclic Mechanical Strain Enhance Triple-Supplement Directed Osteogenesis and Bone Nodule Formation by Human Embryonic Stem Cells In Vitro