Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED).

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.

Soft matrix supports osteogenic differentiation of human dental follicle cells.

The differentiation of stem cells can be directed by the grade of stiffness of the developed tissue cells. For example a rigid extracellular matrix supports the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs). However, less is known about the relation of extracellular matrix stiffness and cell differentiation of ectomesenchymal dental precursor cells. Our study examined for the first time the influence of the surface stiffness on the proliferation and osteogenic differentiation of human dental follicle cells (DFCs). Cell proliferation of DFCs was only slightly decreased on cell culture surfaces with a bone-like stiffness. The osteogenic differentiation in DFCs could only be initiated with a dexamethasone based differentiation medium after using varying stiffness. Here, the softest surface improved the induction of osteogenic differentiation in comparison to that with the highest stiffness. In conclusion, different to bone marrow derived MSCs, soft ECMs have a superior capacity to support the osteogenic differentiation of DFCs.

Tyrosine phosphorylation of vinculin at position 1065 modifies focal adhesion dynamics and cell tractions.

Focal adhesions (FAs) connect the cellular actin cytoskeleton via integrin with the extracellular matrix. They comprise of many structural and signaling proteins which are highly dynamic, well regulated, and responsible for the sensing of physical properties from the environment. Vinculin is a protein that incorporates all these functions. Here, we investigated the phosphorylation of Y1065 in the activation/regulation of vinculin. We used different vinculin mutants mimicking either a permanently activated or inhibited phosphorylation site at position 1065. Using these mutants, we determined their influence on the exchange dynamics and cell forces using fluorescence recovery after photobleaching and traction microscopy. The results indicate that phosphorylation at Y1065 significantly increases the amount of freely exchanging vinculin within FAs whereas inhibition of this phosphorylation site leads to an uncontrolled exchange of vinculin and reduced adhesive cell forces. In conclusion, we show that phosphorylation on position Y1065 is essential for accurate incorporation of vinculin into FAs and mechanical behavior of cells.

Becoming stable and strong: the interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation.

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.