Background <p>Bone regenerative medicine focuses on restoring damaged tissue, with bone augmentation materials commonly used to fill defects, support recovery and addressing issues related to aging, bone diseases or trauma in dental and orthopedic procedures. To avoid complications associated with harvesting autogenous tissue grafts, novel applications focus on alloplastic materials to support regenerative and healing processes. However, current synthetic materials demonstrate shortcomings specifically pertaining to mimicking bone regenerative properties of autogenous bone. Whether bioactive fractions enriched for human platelet lysate derived extracellular vesicles (hPLEV-Fs) could biologize alloplastic materials with their non-immunogenic tissue-restorative potential, stimulate intercellular communication between bone-forming osteoblasts and bone-resorbing osteoclasts and transform alloplastic materials in potent regenerative grafts needs to be determined.</p> Methods <p>This study investigated hPLEV-Fs impact on bone regenerative pathways and evaluated whether combination with a collagen-embedded β-tricalcium phosphate (β-TCP) three-dimensional matrix enhances bone regeneration.</p> Results <p>Treatment with hPLEV-F improved osteoblasts’ proliferation, differentiation and mineralization in both murine and human primary osteoblasts while reducing inflammatory responses, which was further supported by systems-wide phosphoproteome-screening of bone-remodeling pathways. Although initial pre-osteoclastic differentiation was enhanced under hPLEV-F treatment, cells remained in a non-resorbing state, indicating potential for increased net bone formation. Furthermore, hPLEV-F stimulated osteoblasts to increase osteoprotegerin secretion, limiting osteoclast differentiation, especially in combination with β-TCP biomaterial.</p> Conclusions <p>Our data demonstrate the potential of hPLEV-F to stimulate bone cell interaction and support bone regenerative pathways, thereby suggesting it as a biologizing agent in combination with synthetic biomaterial. This creates innovative possibilities in biointerface engineering thereby advancing patient care in clinical applications.</p> Graphical Abstract <p>①This study examined human platelet lysate-derived extracellular vesicles (hPLEV-F) in bone metabolism. ②hPLEV-F modulated osteoblastic secretion and ③improved differentiation and mineralization in murine and human primary osteoblasts accompanied by enhanced secretion of osteoclastic inhibitor osteoprotegerin (OPG). Moreover, osteoclasts remained in an undifferentiated and non-resorbing state when challenged with hPLEV-F. ④Combination of hPLEV-F and bone augmentation material β-TCPCM enhanced osteoblastic bone regenerative potential and OPG-mediated bone protection, ⑤indicating a role of hPLEV-F in biologizing alloplastic materials for augmentation procedures&#xa0;Created in BioRender. Döding, A. (2025)&#xa0;(<a href="https://BioRender.com/xncb1y0">https://BioRender.com/xncb1y0</a>).</p> <p></p>