Introduction/Objectives Critical-size bone defects cause significant challenges to bone regeneration, which can affect the healing process and recovery. Bone implants are usually employed for treating these issues. Their biointegration, however, is challenged by large defect sizes and their effect on biological processes such as the transport of vital nutrients and cell growth.[1] This study focuses on developing bio-functionalized hydrogels from modified Human Platelet Lysate (PL) for enhancing the osseointegration of 3D-printed bone implants. By leveraging the cell-instructive and regenerative properties of PL and improving its mechanical strength through photopolymerizable derivatives, existing boundaries in scaffold-assisted bone regeneration can be overcome. Methods The PL was modified with photopolymerizable groups (i.e. allyl glycidyl ether (PLAGE)) and was subsequently crosslinked under UV irradiation. The hydrogel’s properties were compared to similar gelatin-based systems. Extensive swelling tests, in situ photorheology, and in vitro studies comprised a detailed characterization of the materials. Preliminary in vivo tests of the most promising PL-based hydrogels provided insight on the material’s performance in living organisms. Results A variety of photocrosslinkable PL alternatives were synthesized and crosslinked under UV light. The crosslinking of these photopolymerizable motifs in the modified PL’s backbone resulted in improvement of the mechanical properties compared to those of unmodified PL. Furthermore, successful hydrogel crosslinking was achieved both in presence and absence of thiols. Since PL is primarily based on serum albumin, cysteine is one of its most prevalent amino acid. Therefore, the thiol groups of the protein’s backbone further enhanced gel formation in PL. The applicability of the crosslinked modified PL in Tissue Engineering and Regenerative Medicine was investigated further. A pilot in vivo test on dorsal subcutaneous mouse model was conducted for the most promising PL-based hydrogels, in order to investigate the acute response, degradation and grade of inflammation. Conclusions A promising and versatile PL-based toolbox was developed within this study including a different photopolymerizable PL modifications. Different crosslinking approaches were tested and were followed by thorough mechanical characterization. By tweaking the parameters, a promising system for 3D biofabrication can be developed to provide a novel solution for bone tissue engineering. Acknowledgements Christian Doppler Research Association, Austrian Federal Ministry for Digital & Economic Affairs, National foundation for Research, Technology & Development References 1. Nauth A., Schemitsch E, Norris B., Nollin Z., Watson J.T., J. Orthop. Trauma, 2018. 32.