Injectable carboxymethyl chitosan hydrogel loaded with platelet-rich plasma enhances bone regeneration in diabetic mice

Diabetes impairs bone repair via oxidative stress, chronic inflammation, and reduced osteogenesis. We engineered an injectable carboxymethyl chitosan (CMC) hydrogel incorporating platelet-rich plasma (PRP) to counter these barriers. The CMC–PRP hydrogel was systematically characterized for its rheological properties (steady-shear viscosity, gelation kinetics), porosity, and mechanical strength. In vitro evaluations using murine bone-marrow mesenchymal stem cells (BMSCs) assessed cell viability, intracellular reactive oxygen species (ROS), total antioxidant capacity, and antibacterial activity against Staphylococcus aureus and Escherichia coli. Osteogenic differentiation was quantified via Alizarin Red S and Sirius Red staining. In vivo, critical-size femoral defects were created in streptozotocin (STZ)-induced diabetic mice. Bone regeneration was evaluated using micro-CT and comprehensive histomorphometry (including osteoblast, osteoid, and osteoclast indices). The involvement of the PI3K/Akt signaling pathway was investigated via Western blot and pharmacological inhibition using LY294002. The hydrogel exhibited excellent injectability with shear-thinning behavior and rapid in situ gelation at 37 °C (G’–G’’ crossover = 8 min). It featured an interconnected porous network (median ECD = 14.3 µm) and a compressive modulus of 9.8 ± 1.1 kPa, significantly higher than individual components. Sustained release of PDGF-BB reached approximately 95% over 120 h. In vitro, the hydrogel demonstrated superior biocompatibility, potent antioxidant activity by reducing ROS, and broad-spectrum antibacterial effects. Histomorphometric analysis of diabetic defects revealed that CMC–PRP treatment significantly increased bone mineral density (BMD) and bone volume fraction (BV/TV). Notably, it enhanced osteoblast surfaces and osteoid thickness while maintaining physiologic osteoclast activity. Mechanistically, the hydrogel upregulated key osteogenic markers (RUNX2, Osterix, OPN, and Collagen I) and activated the PI3K/Akt pathway, effects that were significantly attenuated by LY294002. The CMC–PRP hydrogel is a promising bioactive scaffold that facilitates bone regeneration in diabetic environments by mitigating oxidative stress and activating the PI3K/Akt signaling axis, demonstrating strong potential for clinical translation in diabetic bone tissue engineering.