Comparative Regenerative Mechanisms of Adipose-Derived Mesenchymal Stem Cell- and Conditioned Medium-Loaded Three-Dimensional Bioprinted Hydrogels in Chronic Diabetic Wounds

Abstract Chronic diabetic wounds remain a major clinical challenge owing to impaired angiogenesis, persistent inflammation, and cellular dysfunction. In this study, we developed a bioadhesive composite hydrogel scaffold (gelatin methacryloyl/alginate methacrylate [GA]) fabricated using digital light processing three-dimensional (3D) bioprinting, in which human adipose-derived mesenchymal stem cells (ADSCs) or their conditioned medium (CM) were incorporated to generate 3D skin constructs, and compared the paracrine effects on skin regeneration. We characterized the microstructures of GA-ADSC and GA-CM scaffolds; profiled CM proteins; and systematically compared the effects of GA-ADSCs and GA-CM on fibroblast proliferation, migration, angiogenesis, and macrophage polarization in vitro. Their therapeutic efficacy was further evaluated in diabetic mouse wound models, including analyses of collagen deposition, angiogenesis, and fibrosis markers. Furthermore, proteomic analysis was performed to understand the underlying mechanisms. In vitro, both GA-ADSCs and GA-CM promoted fibroblast proliferation, migration, angiogenesis, and macrophage M2 polarization. Moreover, they accelerated wound closure in diabetic mice by enhancing collagen deposition and neovascularization (CD31) and suppressing α-smooth muscle actin and transforming growth factor-β1 expressions. Notably, GA-ADSCs showed prolonged cell viability and exhibited stronger immunomodulatory and antifibrotic effects than GA-CM. Proteomic analysis revealed distinct mechanistic differences: GA-ADSCs markedly activated Toll-like receptor signaling, necroptosis, and extracellular matrix remodeling pathways, suggesting their key roles in immune regulation and metabolic reprogramming. In contrast, GA-CM primarily activated the Hippo signaling pathway, consistent with its role in growth factor-mediated tissue repair. Furthermore, we compared the differentially regulated proteins between ADSCs and CM in diabetic wounds and showed that ADSCs significantly modulated RGL2, RSPH9, and CRTC3, which are associated with enhanced cellular function and energy metabolism. These findings provide mechanistic insights into ADSC- and CM-mediated effects and support the development of bioinspired stem cell-based therapeutic strategies for chronic diabetic wound repair.