Gli1⁺ Cells Residing in Bone Sutures Respond to Mechanical Force via IP₃R to Mediate Osteogenesis

Figure 3

Mechanical stretching induces Gli1⁺ cell increases and regulates JBMMSC osteogenesis via IP₃R-mediated intracellular calcium increases. (a) Experimental procedure: Gli1-mT/mG mice were treated with tamoxifen for 4 consecutive days and were sacrificed after 7 days to isolate JBMMSCs. (b) Flow cytometry analysis of the proportion of Gli1⁺ cells in JBMMSCs; the stretch-treated group showed an increase in Gli1⁺ cells; . (c) The proportion of Gli1⁺ cells was higher in the stretch group. ; . (d) Alkaline phosphatase (ALP) activity of JBMMSCs subjected to mechanical stretching was higher after osteogenic induction for seven days. Mineralized nodules formed by JBMSCs were detected using alizarin red staining after osteogenic induction for 14 days. (e) The abundance of mineralized nodules in the stretch group was higher. ; . (f) Western blot analysis of ALP, RUNX2, Col1, and IP₃R protein expression levels in JBMSCs which after mechanical stretching were upregulated, compared with control cells; . (g) RT-qPCR showed upregulation of ALP, RUNX2, and Col1 RNA expression in JBMSCs after mechanical stretching, compared with control cells. ; ^ns; . (h) RT-qPCR showed upregulation of IP₃R1, IP₃R2, and IP₃R3 RNA expression in JBMSCs after mechanical stretching, compared with control cells. ; ^ns; . (i) Intracellular calcium concentration visualized at mean fluorescence intensity was increased in the stretch group. ; .

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