The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young’s modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.

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.