An Ju-Hyeon, Kim Chun-Choo, Lee Junil, Kim Junhyeok, Lee Jeong-Chae, Kook Sung-Ho
Cluster for Craniofacial Development and Regeneration Research, Institute of Oral Biosciences, School of Dentistry, Jeonbuk National University, Jeonju 54896, Republic of Korea.
Department of Bioactive Material Sciences, Research Center of Bioactive Materials, Jeonbuk National University, Jeonju 54896, Republic of Korea.
Cells. 2025 Jun 28;14(13):991. doi: 10.3390/cells14130991.
Mechanical stresses affect a variety of cellular events in relation to the frequency, magnitude, and duration of the stimuli applied. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) are pluripotent stem cell aggregates and comprise all somatic cells. Numerous studies have highlighted the effects of mechanosignals on stem cells, whereas their impact on EBs has been barely investigated. Here, we examined how cyclic tensile stress affects the behavior of EBs to differentiate into mineralized osteocytes by applying 2% elongation at 0.5 Hz frequency for 1 h once or 1 h every other day for 5 or 14 days in osteogenic medium. EBs that expressed undifferentiated markers, Oct4 and Sox2, were differentiated into mineralized cells, along with the accumulation of runt-related transcription factor 2 (RUNX2) and β-catenin in osteogenic medium. The application of tensile force inhibited EB' mineralization via the downregulation of bone sialoprotein, osteocalcin, osterix, and RUNX2. While the transfection with si-β-catenin did not affect the osteogenic potency of EBs at a significant level, treatment with 10 μM of PD98059, but not of SP600125 or SB203580, diminished the mineralization of EBs and the expression of RUNX2 and RUNX2-regulated osteoblastic genes. The level of phosphorylated extracellular signal-regulated kinase-1 (p-ERK1) rather than p-ERK2 was more apparently diminished in tension-applied EBs. The transfection with si-ERK1, but not with si-ERK2, suppressed the mineralization of osteogenic medium-supplied EBs and the expression of osteoblast-specific genes. Collectively, this study demonstrates that tensile stress inhibits osteogenic potency of EBs by downregulating ERK1-mediated signaling and osteogenic gene expression.
机械应力会影响与所施加刺激的频率、大小和持续时间相关的各种细胞事件。胚胎干细胞（ESC）来源的胚状体（EBs）是多能干细胞聚集体，包含所有体细胞。众多研究强调了机械信号对干细胞的影响，而其对EBs的影响却鲜有研究。在此，我们通过在成骨培养基中以0.5Hz频率施加2%伸长，每次1小时或每隔一天1小时，持续5天或14天，研究了循环拉伸应力如何影响EBs分化为矿化骨细胞的行为。在成骨培养基中，表达未分化标志物Oct4和Sox2的EBs分化为矿化细胞，同时 runt相关转录因子2（RUNX2）和β-连环蛋白积累。拉伸力的施加通过下调骨唾液蛋白、骨钙素、osterix和RUNX2抑制了EBs的矿化。虽然用si-β-连环蛋白转染在显著水平上不影响EBs的成骨能力，但用10μM的PD98059处理，而不是用SP600125或SB203580处理，减少了EBs的矿化以及RUNX2和RUNX2调节的成骨细胞基因的表达。在施加张力的EBs中，磷酸化细胞外信号调节激酶-1（p-ERK1）的水平比p-ERK2更明显降低。用si-ERK1转染，而不是用si-ERK2转染，抑制了成骨培养基供应的EBs的矿化和成骨细胞特异性基因的表达。总的来说，这项研究表明拉伸应力通过下调ERK1介导的信号传导和成骨基因表达来抑制EBs的成骨能力。
