AVS 50th International Symposium
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuP

Paper BI-TuP23
Control of Osteopontin Behavior on Surfaces for Cell Adhesion

Tuesday, November 4, 2003, 5:30 pm, Room Hall A-C

Session: Poster Session
Presenter: L. Liu, University of Washington
Authors: L. Liu, University of Washington
S. Chen, University of Washington
B.D. Ratner, University of Washington
S. Jiang, University of Washington
Correspondent: Click to Email
Osteopontin (OPN) is an important extracellular matrix protein shown to function in wound healing, inflammation and foreign body reaction and has been identified as a potential target for engineered biomaterials. It contains RGD moiety that has been shown to mediate cell adhesion through interaction with integrins. In preferred orientation and conformation, the RGD tripeptide of OPN will be presented to the cells to the greatest extent. However, control of OPN orientation/conformation is seldom investigated so far. In this work, we investigate OPN adsorption and cell adhesion on self-assembled monolayers (SAMs) of alkanethiols terminated with different functional groups to tailor surface properties. Four different alkanethiols terminated with -CH3, -OH, -NH2 and -COOH were used to form surfaces representing hydrophobic, hydrophilic, positive and negative surfaces. Atomic force microscopy (AFM) is used to characterize the adsorption of OPN on various SAM surfaces. Our AFM results show that the amount of adsorbed OPN on -COOH surface is slightly less than that on -NH2 surface. In vitro cell adhesion assay of bovine aortic endothelial cells (BAEC) was performed to test OPN function on various SAMs. Our results show that on -NH2 surface BAEC adhesion is the most and cell appears most spread. Both cell counts and average cell spreading area on -COOH and -CH3 surfaces are much less than those on -NH2 surface. The -OH surface is resistant to both OPN adsorption and cell adhesion. By comparing results from AFM and cell adhesion experiments, it is suggested that the orientation/conformation of OPN on -NH2 positively charged surface is more favorable for cell interaction than on -COOH negatively charged surface. This is consistent with our molecular simulation prediction. Our studies clearly show that surface properties will alter OPN behavior on surfaces, thus cell interactions with OPN. In addition, we use atomic force microscopy (AFM) to image the binding of OPN onto type I collagen monomer. Interactions among extracellular matrix (ECM) proteins are important in many aspects such as orientating ligand proteins and correctly delivering signals into a cell. Recent experiments show that osteopontin (OPN)-incubated collagen I chemically immobilized on poly(HEMA) promotes cell adhesion in vitro. It is speculated that bound OPN on a collagen matrix may have better orientation/ conformation and thus influence its cell-binding ability. Although it has been reported that OPN is able to bind with type I collagen, direct visualization of OPN-collagen binding complexes has not been reported and OPN binding sites on collagen I are still unknown. In our work, AFM is used to image the binding of OPN onto individual triple-helical collagen I monomer on freshly cleaved mica for the first time. Analysis of AFM results clearly shows binding patterns of OPN to collagen I. This work provides a direct means to identify binding among ECM proteins and a better understanding of ECM proteins on cell adhesion.