Hypoxia-Preconditioned Human Dental Pulp Mesenchymal Stem Cell Secretome Enhances Tissue Regeneration in a Medicinal Leech Wound Model

Background Chronic and non-healing wounds remain a major clinical challenge due to persistent inflammation, impaired angiogenesis, and defective extracellular matrix (ECM) remodelling. Human Mesenchymal Stem Cells have emerged as promising regenerative therapies, with their effects mainly attributed to the secretome. In particular, the secretome, derived from human Dental Pulp Stem Cells cultured under hypoxic conditions (hDPSC-HS), is enriched in pro-regenerative and immunomodulatory factors. Previous in vitro studies demonstrated that hDPSC-HS promotes endothelial cell migration and angiogenesis, enhances fibroblast proliferation and collagen deposition, and modulates macrophage polarization. However, its in vivo regenerative potential remains poorly defined. Here, we used the medicinal leech Hirudo verbana , an evolutionarily conserved model that reproduces key cellular and molecular mechanisms of tissue repair, to explore the biological effects of hDPSC-HS in vivo . Methods Standardized surgical wounds were created in adult leeches and then a locally injection was made with either hDPSC-HS or serum-free Dulbecco's Modified Eagle Medium (control). Tissues were collected at 48 h, 72 h, and 1 week post-treatment. Wound healing was evaluated by morphological, histological, immunofluorescence, and gene expression analyses focusing on ECM remodelling, angiogenic activation, and muscle regeneration. Results hDPSC-HS significantly accelerated wound closure and improved tissue restoration compared with controls. Treated animals showed enhanced muscle fibre regeneration and a more organized ECM architecture. Molecular and immunofluorescence analyses revealed modulation of key pathways involved in ECM remodelling and tissue regeneration, indicating the establishment of a pro-regenerative microenvironment that supports more efficient healing. Conclusions Although generated in an exploratory in vivo model, these findings demonstrate that hDPSC-HS promotes coordinated tissue repair by modulating evolutionarily conserved regenerative mechanisms. Because these biological pathways are shared with vertebrates and are critically involved in the pathophysiology of chronic human wounds, the Hirudo verbana model provides biologically relevant evidence supporting the translational potential of hDPSC-HS. These results provide a rationale for further preclinical validation and clinical investigation of hDPSC-HS as a therapeutic strategy for chronic wounds characterized by impaired healing.