Abstract: Covalent binding between bioactive substances and materials in different ways can significantly improve the bone inductivity and biological activity of bone repair materials. However, there is a lack of systematic understanding of how these binding modes affect biological activities of the active substances. In this study, four kinds of functionalized Multi-walled carbon nanotubes (MWCNTs) were prepared, ensuring the same grafting rate of different functional groups. Subsequently, two kinds of osteogenic-related peptides, bone morphogenetic protein-2 mimicking peptides and osteogenic growth mimicking peptides, were covalently bound to functionalized MWCNTs, ensuring the same molar mass of peptides bound to different functionalized MWCNTs in this process. Then the same amount of functionalized MWCNTs/Peptides composites were introduced into the scaffolds, and through the ectopic osteogenesis model in rats and calvarial defect model in rabbits, ectopic osteogenesis and bone repair ability of the composites were analyzed. Furthermore, the effects of different covalent binding modes on peptide-induced osteogenesis and bone repair were studied. The results showed that the negative influencing trend of different covalent binding modes of osteogenic-related peptides with artificial carriers on their biological activities was in the order as follows: amide binding (carboxyl) > silane coupling > dopamine binding > amide binding (amino), whose mechanism might be mainly that the covalent binding of peptides with different functional groups resulted in different charges. We believe that the results of this study have important guiding significance for the research and development of bone repair materials covalently bound with bioactive substances.

Key words: Osteogenic-related peptides, Covalent binding, Ectopic osteogenesis, Calvarial defect