Enhanced Osteoinduction and Osteogenesis Promotion by Bovine Freeze-Dried Bone Combined with <i>Rehmannia glutinosa</i> Libosch Polysaccharide on Bone Defect and Fracture Repair in Rats

Freeze-dried bone (FDB) scaffolds present several advantages, including mechanical strength, osteoconductive properties, and abundant availability. This study aimed to investigate the osteogenic effects and underlying mechanisms of bovine FDB combined with <i>Rehmannia glutinosa</i> Libosch polysaccharide (RGP) on bone defects and fractures in rats. The characteristics of the scaffolds were analyzed through observational studies, scanning electron microscopy imaging, mechanical strength testing, cytotoxicity assessments, and in vitro RGP release. Subsequently, femoral condyle and radial defect models mimicking bone defect and fracture were used to evaluate the scaffolds' repair capabilities for cortical and cancellous bone defects. Results obtained at the eighth week showed that FDB&RGP combination outperformed FDB alone in various parameters, indicating superior repair potential for both bone types, as well as for bone defects and fractures. The combination scaffold exhibited enhanced osteoinductive properties, rendering it more suitable for bone defect repair. The osteogenic mechanisms of FDB&RGP were investigated using alkaline phosphatase (ALP) and Alizarin Red S staining, as well as assessments of ALP, OCN, and COL1 activities, and the activation of the BMP-9/GSK3β signaling pathway. The results indicate that FDB&RGP enhances osteo-differentiation, with the BMP-9/GSK3β pathway potentially serving a pivotal role. This study establishes a foundation for the future clinical application of xenograft FDB&RGP.