Modulating Immune-Stem Cell Crosstalk Enables Robust Bone Regeneration via Tuning Compositions of Macroporous Scaffolds

Cellular crosstalk is critical for tissue healing. Following bone injury, macrophages (Mφ) initiate the immune response by secreting signals that recruit mesenchymal stem cells (MSC) and other niche cells to shape healing. Despite its importance, most studies focus on designing biomaterials that modulate individual cell type, leaving the potential of enhancing bone regeneration by modulating immune-stem cell crosstalk largely unexplored. Here, we report a macroporous microribbon (µRB) scaffold with tunable ratios of gelatin (Gel) and chondroitin sulfate (CS), achieving rapid endogenous bone regeneration in a critical-sized defect model without exogenous growth factors or cells. The 3D MSC/Mφ co-culture model, but not the mono-culture model, effectively identified Gel50_CS50 as the leading ratio for accelerating bone regeneration in vivo. Single-cell sequencing (scRNAseq) of cells infiltrated at day 7 showed Gel50_CS50 led to distinct cellular compositions changes compared to Gel100, and identified a unique M2-like 2 Mφ subpopulation associated with robust bone regeneration. CellChat analysis revealed that Gel50_CS50 significantly enhanced the frequency and intensity of cellular crosstalk among Mφ and other bone niche cell types, with signaling pathways linked to immunomodulation, angiogenesis, and osteogenesis. This study demonstrates Gel50_CS50 µRB as a promising biomaterials-based therapy for treating critical-sized bone defects by modulating cellular crosstalk. Cranial bone defect tissue samples were harvested at week 1 from mice treated with Gel100 and Gel50_CS50, with five mice per material composition. Single cell suspensions were prepared from harvested scaffolds and analyzed using scRNA-seq.