Surface-immobilized plant-derived osteopontin as an effective platform to promote osteoblast adhesion and differentiation

Abstract In this report, recombinant human osteopontin synthesized in tobacco plants (p-rhOPN) is introduced as a potential bioactive molecule that can promote osteoblast adhesion and differentiation. A glass substrate (SiO 2 /Si-OH) grafted with poly(acrylic acid) (SiO 2 /Si-PAA) was prepared by surface-initiated reversible addition-fragmentation chain transfer polymerization and used as a carboxyl-rich platform for the chemical conjugation of p-rhOPN. The PAA grafting and subsequent p-rhOPN immobilization were confirmed by water contact angle, Fourier transform-infrared spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy analyses. Indirect ELISA quantification revealed that the p-rhOPN immobilization efficiency was above 95% and the surface coverage was a function of the p-rhOPN concentration. MC-3T3-E1 cells cultured on the SiO 2 /Si-PAA substrate immobilized with various concentrations (0.6–30 ng/mL) of p-rhOPN (SiO 2 /Si-p-rhOPN) exhibited superior cell spreading compared to those cultured on SiO 2 /Si-OH or gelatin-modified glass substrate (SiO 2 /Si-gelatin). Polymerase chain reaction analysis indicated that the SiO 2 /Si-p-rhOPN substrates with high level of immobilized p-rhOPN promoted MC-3T3-E1 cell differentiation, as demonstrated by the higher transcript expression levels of the osteogenic differentiation regulatory gene, Runt-related transcription factor 2, compared to cells cultured on SiO 2 /Si-OH or SiO 2 /Si-gelatin. Given that p-rhOPN can be more economically produced than the commercially available OPN derived from human or mammalian sources, then, together with its well-preserved biological function in spite of being chemically conjugated to the substrates, it is likely that p-rhOPN could be more broadly applied for the development of materials for bone tissue engineering with a promising medical and commercial value.