ABSTRACTObjectiveThe study evaluates three biologically active substances with known bone‐inductive potential on previously decontaminated titanium (Ti) discs.Material and MethodsRough and smooth Ti surfaces were contaminated with a multispecies biofilm and cleaned with a chitosan brush. Discs were treated either with injectable‐platelet‐rich fibrin (i‐PRF), advanced platelet‐rich fibrin (A‐PRF+), or enamel matrix derivatives (EMDs) before osteoblast seeding.ResultsBiocompatibility, adhesion, migration, and gene expression of runt‐related transcription factor 2 (RUNX2), collagen Type I Alpha 2 (COL1a2), alkaline phosphatase (ALP), osteocalcin (OC), and osteonectin (ON) were performed. All the tested biologic agents similarly increased cell viability. Specifically, osteoblasts seeded over i‐PRF and EMD‐treated surfaces showed improvement in adhesion and migration and significantly increased ALP, OC, ON, RUNX‐2, and COL1a2 mRNA levels up to 2.8 fold (p < 0.05) with no differences between Ti surfaces.Conclusionsi‐PRF and EMD possess beneficial bioactive properties that enhance tissue healing and promote regeneration on thoroughly sterilized surfaces. Biologically active materials may hold the potential to influence the process of implant re‐osseointegration, which warrants more research since sterilization of the affected surfaces under clinical conditions is still not reliably possible and remains one of the greatest challenges.