End-tail soaking strategy toward robust and biomimetic sandwich-layered hydrogels for full-thickness bone regeneration

Despite an increasing number of tissue-engineered scaffolds have been developing for bone regeneration, simple and universal fabrication of biomimetic bone microstructure to repair full-thickness bone defects remains a challenge and an acute clinical demand due to the negligence of microstructural differences within the cortex of cancellous bone. In this work, a biomimetic sandwich-layered PACG-CS@Mn(III) hydrogel (SL hydrogel) was facilely fabricated in an end-tail soaking strategy by simply post-crosslinking of poly(acryloyl 2-glycine)-chitosan (PACG-CS) composite hydrogel using trivalent manganese solutions. Taking the merits of <i>in-situ</i> formation and flexible adjustment of chain entanglements, hydrogen bonds and metal chelate interactions, SL hydrogel with sandwich-like three-layered structures and anisotropic mechanical performance was easily customized through control of the manganese concentration and soaking time in fore-and-aft sides, simulating the structurally and mechanically biomimetic characteristics of cortical and cancellous bone. Furthermore, the produced SL hydrogel also demonstrated favorable biocompatibility and enhanced MnSOD activity via a peroxidase-like reaction, which enabled the excellent radical scavenging efficiency and anti-inflammatory regulation for facilitating the activity, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). <i>In vivo</i> studies further revealed that these SL hydrogels achieved restrictive pro-vascular regeneration through their stratified structure, thereby promoting the differentiation of osteoblasts. Simultaneously, the mechanical cues of stratified structure could mediate macrophage phenotype transitions in accordance with stem cell-osteoblast differentiation process via the PI3K-AKT pathway, resulting in robust osteogenesis and high-quality bone reconstruction. This facile yet efficient strategy of turning anisotropic hydrogel offers a promising alternative for full-thickness repair of bone defects, which is also significantly imperative to achieve high-performance scaffolds with specific usage requirements and expand their clinic applicability in more complex anisotropic tissues.