Proteomic analysis of endothelial progenitor cells secretome identifies Serpine 1 as a potent regulator of osteogenesis

Endothelial progenitor cells (EPCs) exert potent paracrine effect on bone regeneration, yet the specific protein mediators remain incompletely characterized. Through proteomic profiling of EPC-conditioned medium, we identified eight highly secreted proteins with angiogenic and osteogenic potential and were evaluated in vitro using bone marrow human mesenchymal stromal cells (MSCs) and microvascular endothelial cells (HMECs). Proliferation (XTT assay), migration (scratch assay), and osteogenic differentiation (ALP and Alizarin Red staining) were assessed. Serpine-1 significantly increased MSC and HMEC proliferation in a dose-dependent manner and enhanced osteogenic differentiation. Migration was primarily driven by platelet-derived growth factor subunit B (PDGFB) and BMP2. Based on its dual bioactivity, Serpine-1 was encapsulated in PLGA microspheres embedded in a collagen scaffold (Col-Micro-Serpine) and applied to 4-mm calvarial defects in mice (C57BL/6JOIaHsd, Female, 6 weeks old, 20-25 g). Defects filled with collagen and empty microspheres (Col-Micro) served as control. Micro-CT analysis revealed significantly enhanced bone volume (0.036 ± 0.033, 0.003 ± 0.003 mm3, p < 0.01), BV/TV ratio (2.71 ± 2.91, 0.24 ± 0.27%, p < 0.05), bone density (0.78 ± 0.68, 0.13 ± 0.11 1/mm, p < 0.01 ), and trabecular thickness (0.01 ± 0.05, 0.04 ± 0.04 mm, p < 0.05) in Col-Micro-Serpine versus Col- Micro groups respectively. De novo bone formation within the defect center was observed exclusively in the Col-Micro-Serpine group. This study establishes Serpine-1 as a previously unrecognized osteogenic cue and presents a novel sustained-delivery platform for its application in bone regeneration. These findings offer new insights into cell-free regenerative strategies and highlight Serpine-1 as a promising therapeutic target in skeletal tissue engineering.