title: Short periods of cyclic mechanical strain enhance triple-supplement directed osteogenesis and bone nodule formation by human embryonic stem cells in vitro.
abstract: 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.
authors.affiliation: Faculty of Dentistry, National University of Singapore, 11 Lower Kent Ridge Road, Singapore.
authors.forename: Mingming
authors.initials: M
authors.lastname: Li
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Li
authors.forename: Murray C
authors.initials: MC
authors.lastname: Meikle
authors.forename: Intekhab
authors.initials: I
authors.lastname: Islam
authors.forename: Tong
authors.initials: T
authors.lastname: Cao
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D003094:Collagen / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D010012:Osteogenesis / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical
chemical_list: D003094:Collagen