Acceleration of Calvarial Bone Regeneration by Stem Cell Recruitment with a Multifunctional Hydrogel

Hydrogels have garnered increasing attention for critical-size bone regeneration. However, the limited functionality and tedious preparation procedure of current hydrogel often fall short of meeting clinical requirements. In this study, a simple and effective strategy for preparing a multifunctional physically crosslinked hydrogel (CHMgel) for bone regeneration, which integrates a carboxymethyl cellulose (CMC) framework with Mg²⁺ and hydroxyapatite (HAP), is presented. Through the formation of strong intermolecular hydrogen bonds, the hydrogel exhibits a range of desirable properties, including injectability, high adhesion, satisfactory self-healing capacity, moderate mechanical strength, good biodegradability, and excellent biocompatibility. In vivo testing further demonstrates that CHMgel significantly promotes stem cell recruitment and the formation of new lamellar bone. Single-cell RNA sequencing (scRNA-seq) and in vitro assays show that CHMgel enhances in situ stem cell proliferation, bone marrow mesenchymal stem cells (BMSCs) migration, and osteogenic potential, thereby accelerating bone regeneration. Notably, Cmss1(hi) stem cells are markedly upregulated, influencing endochondral ossification through the elevated expression of key proteins, including Filip1l, Celf2, and Cmss1. The cellular characteristics and interactions observed in this study deepen the understanding of skeletal stem cell subsets in early biomaterial-aided bone regeneration, providing a foundation for material strategies aimed at controlling osteogenesis.