Dual-functional β-TCP based injectable bone grafts functionalized with peptides for enhanced osteogenesis and...

Bone defects with irregular geometries and high infection risk remain a major clinical challenge. Injectable bone grafts (IBGs) offer minimally invasive and moldable solutions, yet conventional β-tricalcium phosphate (β-TCP)-based formulations often lack sufficient mechanical strength and antimicrobial activity. Here, a dual-functional β-TCP-based putty-form IBG was developed by combining powdered and sintered granules at optimized ratios to enhance mechanical stability, osteogenic potential, and handling properties. Antimicrobial peptides (AMPs), KR-12 and its anti-MRSA analog KR-12-a5, were covalently immobilized onto β-TCP surfaces via cold atmospheric plasma (CAP), which created reactive sites without compromising structural integrity to ensure stable peptide conjugation and sustained antimicrobial activity. The AMP-functionalized IBGs demonstrated potent anti-biofilm activity against Staphylococcus aureus, Escherichia coli, multidrug-resistant Pseudomonas aeruginosa, and MRSA with KR-12-a5, while KR-12 more effectively promoted human mesenchymal stem cell (hMSC) viability, osteogenic differentiation, and extracellular matrix deposition. Osteogenic markers were analyzed using alkaline phosphatase (ALP) activity and collagen deposition to assess protein levels, and the expression of OCN, OPN, COL1, ALP and RUNX2 genes was evaluated by quantitative PCR (qPCR). To our knowledge, this is the first injectable bone graft that simultaneously integrates osteogenic and broad-spectrum anti-biofilm functionalities for treating complex, infection-prone, and irregularly shaped bone defects.