Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

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.

Bacterial cellulose membrane conjugated with plant-derived osteopontin: Preparation and its potential for bone tissue regeneration.

Bacterial cellulose membrane (BCM) has been recently recognized as a new generation of carbohydrate-based nanomaterial that possesses a great potential in tissue engineering applications. This research aims to develop an active non-resorbable guided tissue regeneration (GTR) membrane from BCM by conjugating with plant-derived recombinant human osteopontin (p-rhOPN), an economically produced and RGD-containing biomolecule. The BCM was initially grafted with poly(acrylic acid) (PAA) brushes to form poly(acrylic acid)-grafted BCM. Multiple carboxyl groups introduced to the BCM by PAA can serve as active anchoring points for p-rhOPN conjugation and yielded p-rhOPN-BCM. All chemically modified BCMs were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while their surface morphology was evaluated by field emission-scanning electron microscopy and atomic force microscopy analyses. The amount of p-rhOPN adhered on the membrane was quantified by enzyme-linked immunosorbent assay. The immunocytochemistry, two-stage quantitative real-time reverse transcriptase polymerase chain reaction and in vitro mineralization analyses strongly suggested that p-rhOPN-BCM could elicit biological functions leading to the enhancement of osteogenic differentiation of human periodontal ligament stem cells as effective as BCM conjugated with commercially available rhOPN from mammalian cells (rhOPN-BCM), suggesting its potential to be used as GTR membrane to promote bone tissue regeneration.

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

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 (SiO2/Si-OH) grafted with poly(acrylic acid) (SiO2/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 SiO2/Si-PAA substrate immobilized with various concentrations (0.6-30 ng/mL) of p-rhOPN (SiO2/Si-p-rhOPN) exhibited superior cell spreading compared to those cultured on SiO2/Si-OH or gelatin-modified glass substrate (SiO2/Si-gelatin). Polymerase chain reaction analysis indicated that the SiO2/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 SiO2/Si-OH or SiO2/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.