Hydrostatic Pressure Induces Osteogenic Differentiation of Single Stem Cells in 3D Viscoelastic Microgels

Sustained mechanical stimulation represents a powerful strategy for directing stem cell fate, yet its application within microscale injectable carriers remains limited. This study presents a dynamic microgel platform enabling osteogenic differentiation of single mesenchymal stem cells (MSCs) solely through hydrostatic pressure, without biochemical induction. Individual MSCs are encapsulated in ionically crosslinked, cell-adhesive alginate microgels and stabilized using an alginate–poly-l-lysine–alginate and calcium coating. Application of cyclic hydrostatic pressure at 200 kPa and 0.5 Hz frequency for 30 min per day leads to upregulation of early osteogenic markers RUNX2 and alkaline phosphatase, enhanced collagen I synthesis, and mineralization over 21 days. Results demonstrate that mechanical cues alone are sufficient to orchestrate osteogenic commitment in soft, confined microenvironments, offering a scalable approach to stem cell programming. This work establishes a versatile, high-resolution platform for engineering lineage specification at the single-cell level and highlights the potential of force-driven strategies for scalable production of therapeutic stem cells.