Dynamic stiffening hydrogels orchestrate macrophage- mesenchymal stem cells interactions for bone regeneration

Slow stiffening hydrogel was achieved via gradual low-dose Ca 2+ crosslinking. • The hydrogel replicates early bone repair's dynamic mechanical niche. • Dynamic stiffness fosters a pro-osteogenic immune microenvironment. • Dynamic stiffness-driven macrophage-MSCs interaction promotes osteogenesis. During bone defect repair, the phenotypes, differentiation, and paracrine functions of both macrophages (Mφ) and mesenchymal stem cells (MSCs) are critically regulated by matrix stiffness. These two cell types interact via paracrine signaling, forming a bidirectional “immune-osteogenic” regulatory axis that jointly drives bone regeneration. Based on this, the present study employed a dynamically stiffening hydrogel system based on calcium ion-crosslinked gelatin/sodium alginate (Gel/SA), combined with a conditioned medium (CM)-based coculture model, to investigate the interaction between Mφ and MSCs under conditions of dynamic matrix stiffness evolution. Experimental results demonstrated that dynamic stiffness, together with inflammatory factors secreted by stiffness-modulated Mφ, synergistically enhanced the osteogenic differentiation of MSCs, leading to significantly higher differentiation levels in the dynamic stiffness group compared to other groups. Meanwhile, dynamic stiffness, along with prostaglandin E2 (PGE2) secreted by stiffness-modulated MSCs, collectively influenced Mφ, resulting in an inflammatory factor expression profile intermediate between those in the high- and low-stiffness groups. The data demonstrated that dynamic rigidity steers Mφ polarization and MSCs immunomodulation, and that dynamic stiffness-mediated Mφ-MSCs crosstalk tailors the injury microenvironment to accelerate early osteogenic commitment. These findings reveal the mechanism by which dynamic matrix mechanics guide bone regeneration through modulation of the early inflammatory microenvironment. Low-stiffness static hydrogels (Low-stiffness group) were prepared using gelatin (Gel) and sodium alginate (SA), while high-stiffness static hydrogels (High-stiffness group) were fabricated by adding a high concentration of CaCl₂ to the Gel/SA mixture. Hydrogels with dynamically increasing stiffness (Dynamic group) were achieved through daily supplementation of a low concentration of CaCl₂. All hydrogels were crosslinked via ethyldimethylaminopropyl carbodiimide/N-hydroxysuccinimide (EDC/NHS) for 24 h. Furthermore, by integrating the dynamic-stiffness hydrogel system with a conditioned medium-based coculture model, this work systematically investigated the effects of the dynamic mechanical microenvironment on macrophage phenotype polarization and the intercellular crosstalk between macrophages and mesenchymal stem cells (MSCs), thereby elucidating its role in modulating the immune microenvironment and directing osteogenic differentiation. Download: Download high-res image (302KB) Download: Download full-size image