Investigation of Focal Adhesions During Dental Pulp Stem Cell Differentiation

Objective: Focal adhesions are mediated by integrin clustering and they contain adaptor proteins such as vinculin.  Vinculin is a 117-kDa cytoskeletal protein containing binding sites for signaling and structural molecules such as actin.  Successful regeneration of the pulp-dentin tissue using biomimetic scaffolds will likely require cell-extracellular matrix (ECM) interactions. Our aim is to validate the hypothesis that, when exposed to osteogenic induction media, human dental pulp stem cells (hDPSC) physically and genetically modulate focal adhesions.

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).