Stem cells sense and respond to the mechanical properties of the extracellular matrix. However, both the extent to which extracellular-matrix mechanics affect stem-cell fate in three-dimensional microenvironments and the underlying biophysical mechanisms are unclear. We demonstrate that the commitment of mesenchymal stem-cell populations changes in response to the rigidity of three-dimensional microenvironments, with osteogenesis occurring predominantly at 11–30 kPa. In contrast to previous two-dimensional work, however, cell fate was not correlated with morphology. Instead, matrix stiffness regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem-cell populations. These findings suggest that cells interpret changes in the physical properties of adhesion substrates as changes in adhesion-ligand presentation, and that cells themselves can be harnessed as tools to mechanically process materials into structures that feed back to manipulate their fate.

Significance: Cell volume is thought to be a well-controlled cellular characteristic, increasing as a cell grows, while macromolecular density is maintained. We report that cell volume can also change in response to external physical cues, leading to water influx/efflux, which causes significant changes in subcellular macromolecular density. This is observed when cells spread out on a substrate: Cells reduce their volume and increase their molecular crowding due to an accompanying water efflux. Exploring this phenomenon further, we removed water from mesenchymal stem cells through osmotic pressure and found this was sufficient to alter their differentiation pathway. Based on these results, we suggest cells chart different differentiation and behavioral pathways by sensing/altering their cytoplasmic volume and density through changes in water influx/efflux.

Injectable biomaterials are increasingly being explored to minimize risks and complications associated with surgical implantation. We describe a strategy for delivery via conventional needle–syringe injection of large preformed macroporous scaffolds with well-defined properties. Injectable 3D scaffolds, in the form of elastic sponge-like matrices, were prepared by environmentally friendly cryotropic gelation of a naturally sourced polymer. Cryogels with shape-memory properties may be molded to a variety of shapes and sizes, and may be optionally loaded with therapeutic agents or cells. These scaffolds have the capability to withstand reversible deformations at over 90% strain level, and a rapid volumetric recovery allows the structurally defined scaffolds to be injected through a small-bore needle with nearly complete geometric restoration once delivered. These gels demonstrated long-term release of biomolecules in vivo. Furthermore, cryogels impregnated with bioluminescent reporter cells provided enhanced survival, higher local retention, and extended engraftment of transplanted cells at the injection site compared with a standard injection technique. These injectable scaffolds show great promise for various biomedical applications, including cell therapies.

Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm² and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.