Updated project metadata. Type 2 diabetes mellitus significantly impairs bone healing, with delayed regeneration observed in over 60% of diabetic patients. This study investigated the molecular mechanisms underlying diabetes-associated compromised regeneration using time-resolved proteome profiling of critical-sized femoral defects in diabetic (Lepr-/-) and non-diabetic LundMetS rats treated with polycaprolactone scaffolds. Explanted regenerated callus and contralateral bone tissue were analyzed at 21- and 42-days post-surgery using TMT-based quantitative proteomics and functional enrichment analysis. Deep proteome coverage identified 4,384 proteins, revealing significantly reduced extracellular matrix proteins and elevated inflammatory proteins in diabetic defects at 42 days. A distinct mast cell protease cluster emerged as a novel mechanism, with diabetic callus containing 2.5-fold more mast cells and elevated tissue histamine. The findings demonstrate that diabetes converts physiological transient inflammatory processes into pathologically sustained networks through mast cell-neutrophil crosstalk, creating a self-reinforcing catabolic niche that prevents inflammatory resolution and matrix maturation.