Dynamic Stiffening Hydrogel with Instructive Stiffening Timing Modulates Stem Cell Fate in Vitro and Enhances Bone Remodeling in Vivo.

Biomechanical stimuli derived from the extracellular matrix (ECM) extremely tune stem cell fate through three-dimensional (3D) and spatiotemporal changes in vivo. The matrix stiffness as a crucial factor during bone tissue development is dynamic stiffening. However, most in vitro models to study the osteogenesis of mesenchymal stem cells (MSCs) are static or stiffening in a 2D environment. Here, we create a dynamic and controllable stiffening 3D biomimetic model to regulate the osteoblastic differentiation of MSCs with a dual-functional gelatin macromer that can generate a double-network hydrogel by sequential enzymatic and light-triggered crosslinking reactions. Our findings showed that these dynamic hydrogels allowed cells to spread and volume expansion prior to secondary crosslinking and to sense high stiffness after stiffening. The MSCs in the dynamic hydrogels, especially the hydrogel stiffened at the late period, presented the significantly elevated osteogenic ECM secretion, gene expression, and YAP/TAZ nuclear localization. In vivo evaluation of animal experiments further indicated that the enhancement of dynamic stiffening on osteogenesis of MSCs substantially promoted bone remodeling. Consequently, this work reveals that the 3D dynamic stiffening microenvironment as a critical biophysical cue not only mediates the stem cell fate in vitro, but also augments bone restoration in vivo. This article is protected by copyright. All rights reserved.