Method: Osteogenic Induction: At passage 4 DPSCs were incubated with the osteogenic media containing 10nM dexamethasone, 10mM β-glycerophosphate and 0.05 mM ascorbic acid 2-phosphate. Samples were subcultured onto 22x22 mm coverslips and changes in the formation of focal adhesions and actin reorganization were fluorescently visualized. Immunofluorescence: Cultured DPSCs were fixed with 4% formalin, permeabilized, and incubated with mouse anti-human primary vinculin antibody overnight at 4°C, and further treated with fluorescently conjugated goat-anti-mouse secondary antibody for 1 hour at room temperature. F-actins were visualized using 0.5 uM phalloidin.
Results: Both the number and physical size of focal adhesions in hDPSCs increased in response to the osteogenic media. Up-regulation of vinculin expression was also evident. Increase in the formation of focal adhesions was consistent with actin remodeling to stress fibers.
Conclusions: Our preliminary results suggest that the hDPSCs become strongly attached to the substrate in response to the osteogenic factors. Comparison with our previous results using bone marrow-derived stem cells, the hDPSC adhesion and actin remodeling are differentially regulated. The surprising findings suggest that engineering an appropriate microenvironment is critically important for manipulating tissue-specific stem cells. Future work includes quantification of the gene expression for odontoblast differentiation in 3D scaffolds.
This work was supported by a NIH/NIDCR grant (DE019514-SBA).
Keywords: Adhesion, Bioengineering, Dentin, Regeneration and Tissue engineering
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