Proteomic profiling links increased mast cell activity in regenerating callus to impaired scaffold-guided bone regeneration in diabetic rats

Type 2 diabetes mellitus significantly impairs bone healing, with delayed regeneration observed in over 60% of patients with diabetes. Using time-resolved proteome profiling of critical-sized femoral defects in diabetic and non-diabetic rats treated with polycaprolactone scaffolds, we investigated the molecular mechanisms underlying diabetes-associated compromised regeneration. Explanted regenerated callus and contralateral bone tissue were analyzed at 21 and 42 days post-surgery using mass spectrometry-based quantitative proteomics and functional enrichment analysis, complemented by histological analysis and histamine quantification. Diabetic defects were characterized by disorganized fibrous and adipose tissue without mineralized bone after 42 days, whereas non-diabetic defects exhibited new bone formation. Deep proteome coverage identified 4384 proteins. At 42 days post-surgery, diabetic defects showed significantly reduced extracellular matrix proteins, while inflammatory proteins were higher abundant. Neutrophil degranulation signatures persisted at 42 days, suggesting sustained neutrophil extracellular trap (NET) formation. Notably, a distinct mast cell protease cluster (tryptase, chymase, carboxypeptidase A3, mast cell protease-1) was enriched in diabetic tissue at both timepoints, linking molecular to cellular dysregulation. Immunofluorescence staining revealed 2.5-fold more mast cells with peripheral, halo-like histamine structures typical for degranulation. Additionally, tissue histamine was elevated in diabetic defects. Conclusively, this comprehensive proteomic study points to a previously uncharacterized mast cell-neutrophil crosstalk in diabetic bone regeneration, converting physiologically transient inflammatory processes into pathologically sustained networks. This creates a self-reinforcing catabolic niche that prevents inflammatory resolution and matrix maturation. These findings identify mast cell stabilization and NET modulation as promising therapeutic targets for improving scaffold-guided bone regeneration in diabetic rats.