Hollow Hydroxyapatite Microspheres Loaded with rhCXCL13 to Recruit BMSC for Osteogenesis and Synergetic Angiogenesis to Promote Bone Regeneration in Bone Defects

INTRODUCTION: Bone tissue engineering is a promising method to treat bone defects. However, the current methods of preparing composite materials that mimic the complex structure and biological activity of natural bone are challenging for recruitment of bone marrow mesenchymal stem cells (BMSCs), which affects the application of these materials in situ bone regeneration. Hollow hydroxyapatite microspheres (HHMs) possess a natural porous bone structure, good adsorption, and slow release of chemokines, but have low ability to recruit BMSCs and induce osteogenesis. In this study, The HHM/chitosan (CS) and recombinant human C-X-C motif chemokine ligand 13 (rhCXCL13)-HHM/CS biomimetic scaffolds that optimize bone regeneration and investigated their mechanism of BMSC recruitment and osteogenesis through cell and animal experiments and transcriptomic sequencing. METHODS: Evaluate the physical characteristics of the HHM/CS and rhCXCL13-HHM/CS biomimetic scaffolds through Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), and the cumulative release curve of rhCXCL13. Transwell migration experiments and co-culture with BMSCs were conducted to study the recruitment ability and osteogenic differentiation of the scaffolds. Transcriptomic sequencing was performed to analyze the osteogenic differentiation mechanism. The osteogenesis and bone healing performance were evaluated using a rabbit radial defect model. RESULTS: SEM demonstrated that the rhCXCL13-HHM/CS scaffold comprised hydroxyapatite microspheres in a porous three-dimensional network. The rhCXCL13 showed excellent sustained release capability. The rhCXCL13-HHM/CS scaffold could recruit BMSCs and induce bone regeneration. Transcriptome sequencing and experimental results showed that the osteogenesis mechanism of rhCXCL13-HHM/CS was through the PI3K-AKT pathway. In vivo, the rhCXCL13-HHM/CS scaffold significantly promoted osteogenesis and angiogenesis at 12 weeks after surgery. CONCLUSION: The rhCXCL13-HHM/CS scaffold demonstrates excellent potential for BMSC recruitment, osteogenesis, vascularized tissue-engineered bone reconstruction, and drug delivery, providing a theoretical basis for material osteogenesis mechanism study and promising clinical applications for treating large bone defects.