Biomechanical Stimulation of Mesenchymal Stem Cells in 3D Peptide Nanofibers for Bone Differentiation

Mechanical stimulation critically regulates mesenchymal stem cell (MSC) differentiation, yet its effects in three-dimensional (3D) environments remain poorly defined. Here, we developed a custom dynamic stretcher integrating poly(dimethylsiloxane) (PDMS) chambers to apply cyclic strain to human MSCs encapsulated in Fmoc-diphenylalanine (Fmoc-FF) peptide hydrogels-a fully synthetic, tunable extracellular matrix mimic. Finite element modeling verified uniform strain transmission across the hydrogel. Dynamic stretching at 0.5 Hz and 10% strain induced pronounced cytoskeletal alignment, enhanced actin stress fiber formation (coherency index ≈ 0.85), and significantly increased proliferation compared to static or high-frequency (2.5 Hz, 1%) conditions (coherency index ≈ 0.6). Quantitative image analysis confirmed strain-dependent increases in coherency index and F-actin intensity, indicating enhanced mechanotransductive remodeling. Biochemical assays and qRT-PCR revealed 2-3-fold upregulation of osteogenic markers-<i>RUNX2</i>, <i>ALP</i>, <i>COL1A1</i>, <i>OSX</i>, <i>BMP</i>, <i>ON</i>, and <i>IBSP</i>-under optimal strain. These results demonstrate that low-frequency, high-strain mechanical loading in 3D peptide hydrogels activates RhoA/ROCK and YAP/TAZ pathways, driving osteogenic differentiation. The integrated experimental-computational approach provides a robust platform for studying mechanobiological regulation and advancing mechanically tunable biomaterials for bone tissue engineering.