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Background Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN‐induced osteogenic differentiation of human PDL cells. Methods Full length rhOPN (FL‐OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium‐binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL‐OPN, N142, C122, or C122δ‐coated surfaces. Real‐time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein‐protein interaction. In vivo analysis was performed using a rat calvaria defect model. Results Human PDL cells seeded on FL‐OPN and C122‐coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL‐OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor‐like kinase (ALK‐1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK‐1 receptor. C122, but not C122δ coated surfaces, induced the expression of p‐Smad‐1 and this induction was inhibited by an ALK‐1 inhibitor and RNAi against ALK‐1. In vivo data showed that 3D porous scaffold containing C‐122 enhanced new bone formation as compared to scaffold alone. Conclusion The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK‐1 receptor.

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