Extracellular Matrix Stiffness Regulates Osteogenic Differentiation through MAPK Activation
Fig 2
ECM stiffness stimulates osteoblast differentiation and represses adipocyte differentiation.
(A) A stiff hydrogel stimulates osteogenic differentiation. hMSCs were seeded on a 1.37 or 4.47 kPa hydrogel, and osteogenic differentiation was induced 24 h after seeding. Cells were stained for alkaline phosphatase at 9 days after differentiation and Von Kossa staining was assessed to see mineralization at 21 days after differentiation. (B) qRT-PCR analysis of the osteoblast marker genes DLX5, MSX2, osteocalcin, and RUNX2 of the cells used in panel (A). Expression of the marker genes was normalized to GAPDH expression. Data are presented as fold induction. (*p < 0.05, ***p < 0.005, t-test) (C) The luciferase reporter gene construct 6OSE2-luc was introduced into hMSCs along with a Runx2-expressing vector, which provides similar amounts of Runx2 protein in assay condition. After 16 h, the transfected cells were plated on a 1.37 or 4.47 kPa hydrogel. After 24 h, luciferase reporter gene activity was analyzed. The pGL3-basic luciferase reporter gene construct, which has no promoter for transcription, was used as a negative control. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity and is expressed as relative fold induction. (***p < 0.005, t-test) (D) hMSCs were seeded on a 1.37 or 4.47 kPa hydrogel, and adipogenic differentiation was induced 24 h after seeding. Oil Red O staining was assessed to see lipid droplet at 9 days after differentiation (E) The expression of the adipogenic marker genes adiponectin, aP2, and C/EBPα were analyzed by qRT-PCR in adipocyte-differentiated hMSCs. Adipogenic differentiation was assessed according to the protocol described in the Materials and Methods. Target gene expression was normalized to GAPDH. Data are expressed as relative fold induction. (*p < 0.05, ***p < 0.005, t-test)