Lyophilized L-PRF enhances the bioactivity and rheological properties of 3D-printed and bioprinted scaffolds containing Dental pulp stem cells

Abstract

Background: Bone tissue engineering requires bioinks that combine suitable rheological properties, printability, and biological activity. Leukocyte and platelet-rich fibrin (L-PRF) is a platelet concentrate providing a sustained release of bioactive molecules involved in angiogenesis and osteogenesis. Objective: This study aimed to develop and characterize alginate/gelatin/hydroxyapatite (ALG5–GEL5–HAp10) composite bioinks supplemented with lyophilized L-PRF and to evaluate their rheological performance, cytotoxicity, and ability to promote dental pulp stem cell (DPSC) osteogenic differentiation. Methods: Bioinks were rheologically characterized. Extrusion-based 3D printing produced scaffolds evaluated for cell viability (MTS assay). Growth factor release (PDGF-BB, EGF, VEGF-C, FGF-2, BMP2) from L-PRF-loaded scaffolds was quantified by Luminex for up to 21 days in conditioned medium. Osteogenic differentiation was analyzed by qRT-PCR of key genes (RUNX2, SP7/OSX, ALPL, COL1A1, OCN, OPN, OPG, RANKL, BMP2, TGFB2, VEGF). Results: Inks exhibited shear-thinning and thixotropic recovery. L-PRF addition increased viscosity and storage modulus (G′) while reducing extrusion pressure, improving printability without compromising scaffold fidelity. L-PRF-loaded scaffolds provided sustained growth factor release, with early peaks in PDGF-BB and EGF and detectable BMP2 up to day 7. Conditioned media enhanced DPSC proliferation, peaking at day 3, indicating functional activity of released biomolecules. Bioprinted scaffolds with L-PRF significantly upregulated osteogenic gene expression compared to 3D-printed scaffolds with post-DPSC seeding. Conclusions: As a proof-of-concept, this study demonstrates that lyophilized L-PRF enhances the rheological, printability, and bioactive properties of ALG–GEL–HAp bioinks, supporting DPSC viability. Bioprinted scaffolds showed higher mRNA osteogenic gene expression. These findings support the potential of L-PRF-loaded bioprinted scaffolds for bone tissue regeneration applications, while highlighting the need for further mechanistic and in vivo validation.