3D Osteoimmune Stem Cell Spheroids with Osteoinduction and Immunomodulation Dual Functionality for In Vivo Bone Tissue Engineering

Effective bone regeneration requires not only robust osteoinduction but also precise immunomodulation to orchestrate the complex healing process. In this study, we present a strategy for engineering multifunctional three-dimensional (3D) stem cell spheroids (Sphe-BP-IL4-BMP2) by integrating black phosphorus (BP) nanosheets coloaded with interleukin-4 (IL-4) together with recombinant human bone morphogenetic protein-2 (rhBMP-2). BP nanosheets served as a biodegradable scaffold and a delivery vehicle, enabling sustained release of rhBMP-2 and IL-4 to enhance osteogenic differentiation and to promote anti-inflammatory M2 macrophage polarization, respectively. The resulting spheroids exhibited a well-defined morphology, enhanced cell viability, and uniform BP nanosheet distribution. The in vitro studies demonstrated Sphe-BP-IL4-BMP2 has significantly upregulated osteogenic markers and ALP activity alongside potent immunomodulatory effects on macrophages. Further in vivo implantation into a rat calvarial defect model led to increased angiogenesis and accelerated bone regeneration without adverse effects. The results highlight the therapeutic synergy between osteoinductive and immunomodulatory cues within a 3D spheroid platform, offering a promising avenue for treating critical-sized bone defects.