Opn immobization on surface-graftedpoly(acrylic acid)brushes to promote osteoblast adhesion and proliferation

In this research, glass substrates were grafted with poly(acrylic acid) (PAA) brushes via surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. 1H NMR analysis of PAA formed in solution indicated that the polymerization was well-controlled. Osteopontin (OPN) synthesized from Tobacco plant was then immobilized on the surface-grafted PAA brushes (SiO2/Si-OPN) using EDC/NHS as coupling agents. Stepwise surface modification was verified by water contact angle measurements, Fourier transform-infrared spectroscopy (FT-IR), x-ray photoelectron spectroscopy (XPS) and atomic-force microscopy (AFM). Quantification of OPN immobilized on surface was determined by ELISA assay. The percentages of OPN immobilization efficiency were higher than 95% at all concentration tested. MC-3T3-E1 cells cultured on the SiO2/Si-OPN were better spreading than those on pristine glass substrate and gelatin-modified surface. Moreover, polymerase chain reaction (PCR) analysis indicated that expression levels of the following genes, namely collagen I (Col I), Osterix (Osx), Runt-related transcription factor 2 (Runx2) of MC-3T3-E1 cells on the SiO2/Si-OPN were higher as compared with those on pristine glass substrates and gelatin-modified surface. As determined by MTT assay, OPN could not promote cell proliferation. These results indicated that OPN immobilization on surface-grafted poly (acrylic acid) brushes can promote osteoblast adhesion and differentiation implying that OPN can potentially be used for bone tissue engineering applications.