Matrix dimensionality and stiffness cooperatively regulate osteogenesis of mesenchymal stromal cells.

Osteogenic potential of mesenchymal stromal cells (MSCs) is mechanosensitive. It's affected by the mechanical properties of the cellular microenvironment, particularly its mechanical modulus. To explore the effect of mechanical modulus on osteogenesis in the third dimension (3D), this study used a novel polyacrylamide (PA) scaffold whose pores are monodisperse and spherical, the mechanical moduli of which can be tuned across a wide range. It was found that MSCs have similar proliferation rates in PA scaffolds independent of the matrix stiffness. The contractile force exerted by MSCs inside PA scaffolds was strong enough to deform the pores of scaffolds made of more compliant PAs (whose shear modulus, G'scaffold<4 kPa). Only scaffolds of the highest stiffness (G'scaffold=12 kPa) can withhold the contraction from MSCs. After osteogenic induction for 21 days, the expression profiles of marker genes showed that PA scaffolds of G'scaffold=12 kPa promoted osteogenesis of MSCs. Confocal image analysis demonstrated that there are more F-actin cytoskeletons and bundled stress fibers at higher matrix moduli in 2D and 3D. Moreover, the 3D porous structure promotes osteogenesis of MSCs more than 2D flat substrates. Together, the differences of cellular behaviors when cultured in 2D and 3D systems are evident. The PA scaffolds developed in the present study can be used for further investigation into the mechanism of MSC mechanosensing in the 3D context. STATEMENT OF SIGNIFICANCE: Mechanical properties of the microenvironment affect cellular behaviors, such as matrix stiffness. Traditionally, cell biological investigations have mostly employed cells growing on 2D substrates. The 3D porous PA scaffolds with the same topological conformation and pore sizes but different stiffness generated in this study showed that the differences of cellular behaviors in 2D and 3D systems are evident. Our 3D scaffolds provide insights into tissue engineering when stem cells incorporated with 3D scaffolds and support the future studies of cellular mechanobiology as well as the elucidation the role mechanical factor plays on the physiology and fate determination of MSCs in the 3D context.