Substrate Compliance Directs the Osteogenic Lineages of Stem Cells from the Human Apical Papilla via the Processes of Mechanosensing and Mechanotransduction.

It is well recognized that the interaction between stem cells and their physical microenvironment plays a fundamental role in controlling cell behaviors and directing lineage commitment, which eventually determines cell fate. Any change in the physical characteristics of the extracellular matrix in terms of topography, geometry, and stiffness has a strong effect on this interaction. Nevertheless, the precise biomechanism that regulates the responses of stem cells to the biophysical properties of substrates is not fully understood. In this study, we generated a series of polydimethylsiloxane (PDMS) substrates with different stiffness properties and explored the whole process involved in the determination of osteogenic lineage in stem cells from the human apical papilla (hSCAPs) in response to substrate stiffness. We first found that the hSCAPs responded to different substrate stiffnesses by changing their cell morphologies and cytoskeletons (via changes in α-tubulin and β-tubulin in microtubules and F-actin in microfilaments). We then found that the hSCAPs secreted more fibronectin in response to the stiffer substrates. We next found that fibronectin interacted with focal adhesion kinase (FAK) and paxillin in the FA plaques, and moreover, the expressions of FAK and paxillin were enhanced as the substrate stiffness increased. We further found that FAK and paxillin directly interacted with β-catenin. Furthermore, the accumulation of β-catenin in the nuclear region was strengthened as the substrate stiffness increased. We finally detected the changes of Lef-1 and TCF-1 in osteogenic-induced hSCAPs and found that their expressions were enhanced as the substrate stiffness increased. Lef-1 and TCF-1, as the transcriptional factors in the nucleus, potentially bound to the promoter region of Runx2 and might ultimately determine the osteogenic lineage in hSCAPs. These results indicate the important effect of stiffness in the microenvironment on the osteogenic lineage of hSCAPs and increase the understanding of the biomechanisms involved in the molecular signal cascade during mechanosensing, mechanotransduction, and stem cell differentiation, which will be useful in the biological fields of cell-matrix/cell-cell interactions and tissue engineering/regenerative medicine.