Osteopontin Adsorption Research Articles

Controlling the adsorption of osteopontin for mediating cell behaviour by using self-assembled monolayers with varying surface chemistry.

Osteopontin (OPN) is an important protein for mediating cell behaviour on biomaterials. However, the interactions between the chemical groups on the biomaterial surface and OPN still need to be further clarified, which has restricted the application of OPN in biomaterial functionalization. In the present study, we developed different self-assembled monolayers (SAMs) with specific chemical groups, including SAMs-OH, SAMs-OEG, SAMs-COOH, SAMs-NH2, and SAMs-PO3H2, to study the behavior of OPN on these SAMs. The results showed that SAMs-NH2 could strongly adsorb OPN, and the amount of protein was highest on this material. Meanwhile, the lowest amount of OPN was present on SAMs-OEG. Interestingly, the unit-mass trend of bound OPN monoclonal antibodies (mAbs) on the SAMs was opposite to the OPN adsorption trend: lowest on SAMs-NH2 but highest on SAMs-OEG. In vitro cell assay results showed that mouse bone marrow mesenchymal stem cells (mBMSCs) on SAMs-COOH, SAMs-NH2, and SAMs-PO3H2 with pre-adsorbed OPN showed promoted behaviour, in terms of spreading, viability, and the expression levels of αv and β3 genes, compared with the other two SAMs, demonstrating the higher bioactivity of the adsorbed OPN. We believe that our findings will have great potential for developing OPN-activated biomaterials.

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates.

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019.

The correlation between osteopontin adsorption and cell adhesion to mixed self-assembled monolayers of varying charges and wettability.

Osteopontin (OPN) is a key mediator of cell interactions with biomaterials. However, few studies have been dedicated to studying cell adhesion on OPN-adsorbed substrates with controlled charge and wettability. Here, amino-carboxyl (NH2/COOH) and hydroxyl-methyl (OH/CH3) mixed self-assembled monolayers (SAMs) of varying charges and wettability, respectively, were used as controllable model surfaces to study OPN adsorption and subsequent mesenchymal stem cell (MSC) adhesion. The amount of OPN adsorbed onto the NH2/COOH mixed SAMs appeared to monotonically depend on the surface charge, whereas only a moderately hydrophilic surface was conducive to OPN adsorption on OH/CH3 mixed SAMs. The results correlated well with cell spreading on OPN-coated surfaces in a serum-free medium culture. In addition, the OH/CH3 mixed SAMs with moderate wettability tended to promote β1, β3, αv and α5 integrins, indicating that wettability may guide cell adhesion by mediating the integrins signaling pathway. This work will have reference value for designing biologically responsive substrate surfaces.

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow

ABSTRACTThe bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental caries or medical device–related infections. It further investigated if OPN’s effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN’s effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion in a dose-dependent manner for all investigated species (Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus paracasei subsp. paracasei, Staphylococcus epidermidis, Streptococcus mitis, and Streptococcus oralis). Likewise, adhesion forces decreased after OPN treatment. No effect of OPN on the lectin-accessibility to glycoconjugates was found. OPN reduces the adhesion and adhesion force/energy of a variety of bacteria and has a potential therapeutic use for biofilm control. OPN acts upon bacterial adhesion without blocking cell surface glycoconjugates.

Mediating Mesenchymal Stem Cells Responses and Osteopontin Adsorption via Oligo(ethylene glycol)-amino Mixed Self-assembled Monolayers

Oligo(ethylene glycol) (-OEG) and amino (-NH2) mixed self-assembled monolayers (SAMs) were employed as model substrates to investigate the effect of charge density on the fate of mesenchymal stem cells (MSCs) and osteopontin (OPN) adsorption. We found that all surfaces presenting -NH2 groups favored cell responses regardless of the surface charge. Meanwhile, OPN adsorption could remain stable on the mixed SAMs over a certain range of charge densities. Our work provides some insights into cell responses and protein adsorption to surface charge.

Role of Osteopontin in Modulation of Hydroxyapatite Formation

The presence of osteopontin (OPN) at high levels in both mineralized tissues such as bone and ectopic calcifications such as atherosclerotic plaque presents a conundrum: is OPN a promoter or inhibitor of hydroxyapatite (HA) formation? In vitro studies show that OPN adsorbs tightly to HA and is a potent inhibitor of crystal growth. Although the mechanism of the OPN-HA interaction is not fully understood, it is probably electrostatic in nature. Phosphorylation enhances OPN's ability to adsorb to and inhibit the growth of HA crystals, although other anionic groups also contribute to these properties. Recent findings suggest that OPN is an intrinsically unordered protein and that its lack of folded structure facilitates the protein's adsorption by allowing multiple binding geometries and the sequential formation of ionic bonds with Ca(2+) ions of the crystal surface. By analogy with other biominerals, it is likely that adsorption of OPN to HA results in "pinning" of growth steps. The abundance of OPN at sites of ectopic calcification reflects upregulation of the protein in response to crystal formation or even in response to elevated phosphate levels. Therefore, it appears that OPN is one of a group of proteins that function to prevent crystal formation in soft tissues. The role of OPN in bone mineralization, if any, is less clear. However, it is possible that it modulates HA formation, either by preventing crystal growth in "inappropriate" areas such as the osteoid seam or by regulating crystal growth habit (size and shape).

Label-Free Mapping of Osteopontin Adsorption to Calcium Oxalate Monohydrate Crystals by Tip-Enhanced Raman Spectroscopy

In the ectopic biomineralization of calcium oxalate kidney stones, the competition between calcium oxalate monohydrate (COM) formation and its inhibition by the phosphoprotein osteopontin (OPN) plays a key role in COM stone-forming processes. To get more insights into these processes, tip-enhanced Raman spectroscopy (TERS) was used to provide surface-specific information about the adsorption of OPN to faces of COM crystals. In TERS, the surface plasmon resonance of a metallic AFM tip is locally excited when the tip is placed in the optical near-field of a laser focused on the crystal surface. Excitation of this localized surface plasmon resonance allows the enhancement of the Raman signal as well as the improvement of the spatial resolution beyond the diffraction limit of the light. As TERS works label free and noninvasively, it is an excellent technique to study the distribution of adsorbed proteins on crystal faces at the submicrometer scale. In the present work, we generated Raman intensity maps indicating high spatial resolution and a distinct variation in relative peak intensities. The collected TERS spectra show that the OPN preferentially adsorbs to edges and faces at the ends of COM crystals (order: {100}/{121} edge > {100} face > {100}/{010} edge ≈ {121}/{010} edge > {010} face) providing also relevant information on the inhibition of crystal growth. This study demonstrates that TERS is an excellent technique for detailed investigations of biomolecules adsorbed, layered, or assembled to a large variety of surfaces and interfaces.

COMPARISON OF ADSORPTION BEHAVIOR OF BOVINE SERUM ALBUMIN AND OSTEOPONTIN ON HYDROXYAPATITE AND ALUMINA

Adsorption behavior of bovine serum albumin (BSA) and osteopontin (OPN) on osteoconductive hydroxyapatite (HA) and non-osteoconductive alpha-type alumina (-Al2O3) was studied as function of protein concentration and pH, using Bradford dye binding assay. BSA showed a much larger binding capacity on -Al2O3 than that on HA irrespective of BSA solution concentration and pH. Furthermore, OPN is likely to show the same adsorption characteristic as BSA. Definite correlation was not observed between the albumin or OPN adsorption capacity and the osteoconductivity of materials, suggesting that other factors (e.g., orientation, arrangement, etc. of albumin and/or OPN) likely govern expression of the osteoconductivity. (Received February 2, 2012; Accepted March 30, 2012)

The adsorption characteristics of osteopontin on hydroxyapatite and gold

The adsorption of osteopontin on hydroxyapatite (HA) and reference gold (Au) surfaces was studied at different protein bulk concentrations over the temperature range 295–317K, using quartz crystal microbalance with dissipation (QCM-D) and X-ray photoelectron spectroscopy (XPS). The QCM-D protein adsorption studies were complemented with polyclonal antibodies to examine the availability of protein sequences on the resulting protein layer. The QCM-D and XPS results show that the osteopontin surface mass uptake is larger on Au as compared to HA surfaces within the range of experimental conditions examined (protein bulk concentrations and temperature range), in accordance with the formation of a more compact protein film on Au. The specific antibody binding to the resulting adsorbed osteopontin layer as measured by QCM-D further confirms that the protein packing and conformational/orientational changes occurring during OPN adsorption on Au and HA are different, since fewer antibodies are observed to bind per OPN molecule on Au as compared to HA. The adsorption process on the respective surfaces was modeled using both the Langmuir and Hill adsorption isotherms, and from these isotherm curves, the Gibbs free energy, ∆G, of the osteopontin adsorption was determined. The estimated ∆G values indicate that the osteopontin molecules have a high affinity towards Au, while a lower affinity is observed between osteopontin and HA. By examining the changes in ∆G as a function of temperature, we additionally find that the osteopontin adsorption on HA and Au is endothermic and driven by an increase in entropy.

Interaction of human mesenchymal stem cells with osteopontin coated hydroxyapatite surfaces

In vitro studies of the initial attachment, spreading and motility of human bone mesenchymal stem cells have been carried out on bovine osteopontin (OPN) coated hydroxyapatite (HA) and gold (Au) model surfaces. The adsorption of OPN extracted from bovine milk was monitored by the quartz crystal microbalance with dissipation (QCM-D) and the ellipsometry techniques, and the OPN coated surfaces were further investigated by antigen–antibody interaction. It is shown that the OPN surface mass density is significantly lower and that the number of antibodies binding to the resulting OPN layers is significantly higher on the HA as compared to the Au surfaces. The initial attachment, spreading and motility of human mesenchymal stem cells show a larger cell area, a faster arrangement of vinculin in the basal cell membrane and more motile cells on the OPN coated HA surfaces as compared to the OPN coated Au surfaces and to the uncoated Au and HA surfaces. These in vitro results indicate that there may be great potential for OPN coated biomaterials, for instance as functional protein coatings or drug delivery systems on orthopaedic implants or scaffolds for tissue-engineering.

Specific Adsorption of Osteopontin and Synthetic Polypeptides to Calcium Oxalate Monohydrate Crystals

Protein-crystal interactions are known to be important in biomineralization. To study the physicochemical basis of such interactions, we have developed a technique that combines confocal microscopy of crystals with fluorescence imaging of proteins. In this study, osteopontin (OPN), a protein abundant in urine, was labeled with the fluorescent dye AlexaFluor-488 and added to crystals of calcium oxalate monohydrate (COM), the major constituent of kidney stones. In five to seven optical sections along the z axis, scanning confocal microscopy was used to visualize COM crystals and fluorescence imaging to map OPN adsorbed to the crystals. To quantify the relative adsorption to different crystal faces, fluorescence intensity was measured around the perimeter of the crystal in several sections. Using this method, it was shown that OPN adsorbs with high specificity to the edges between {100} and {121} faces of COM and much less so to {100}, {121}, or {010} faces. By contrast, poly-L-aspartic acid adsorbs preferentially to {121} faces, whereas poly-L-glutamic acid adsorbs to all faces approximately equally. Growth of COM in the presence of rat bone OPN results in dumbbell-shaped crystals. We hypothesize that the edge-specific adsorption of OPN may be responsible for the dumbbell morphology of COM crystals found in human urine.

Controlling the orientation of bone osteopontin via its specific binding with collagen I to modulate osteoblast adhesion

Osteopontin (OPN) is an important matricellular protein that modulates cell functions. It is potentially an excellent surface-coating component for engineered biomaterials. It is believed that in its preferred orientation and conformation on a surface, the functional domains of OPN such as the arginine-glycine-aspartic acid (RGD) motif will be presented to cells to the greatest extent. Previously, the authors demonstrated that OPN orientation could be modulated by surface charge. In this work, the authors attempt to control the orientation/conformation of bone OPN via its specific interactions with type I collagen. Surface plasmon resonance was used to confirm the specific binding between bone OPN and collagen I. A radiolabeled OPN adsorption assay was used to determine the amount of adsorbed OPN on tissue culture polystyrene (TCPS) surfaces with or without collagen I as an interlayer. An in vitro cell adhesion assay using osteoblast MC3T3-E1 was performed to compare the functionality of collagen-bound OPN and adsorbed OPN on TCPS. With the same amount of OPN on the surfaces, the number of cells adhered to collagen-bound OPN is significantly higher than to OPN alone on TCPS. A cell inhibition assay using soluble GRGDSP peptides showed that a higher GRGDSP concentration was needed to completely block osteoblast adhesion to collagen-bound OPN than to OPN directly on TCPS. Enhanced cell adhesion and higher blocking peptide concentration suggest that collagen-bound bone OPN has a preferable orientation/conformation for cell adhesion compared with OPN alone on TCPS. Thus, the specific binding of OPN to collagen I may naturally orient OPN, thus influencing osteoblast adhesion.

Controlling osteopontin orientation on surfaces to modulate endothelial cell adhesion

Osteopontin (OPN) is an important extracellular matrix protein that has been shown to impact wound healing, inflammation, and the foreign body reaction, and has been identified as a potential surface component for engineered biomaterials. OPN contains the arginine-glycine-aspartic acid (RGD) moiety that has been shown to mediate cell adhesion through interactions with integrins. In its preferred orientation and conformation on a surface, the functional domains of OPN will be presented to cells to the greatest extent. However, control of protein orientation and conformation is still challenging. In this work, we investigated OPN adsorption and cell adhesion to the OPN layer on self-assembled monolayers (SAMs) of alkanethiols terminated with various functional groups and on a gold surface. The four SAM terminal groups studied were --CH3, --OH, --NH2, and --COOH, representing hydrophobic, hydrophilic but neutral, positively charged, and negatively charged surfaces, respectively. Surface plasmon resonance biosensor and atomic force microscopy were used to characterize the adsorption of OPN on these surfaces. An in vitro cell adhesion assay of bovine aortic endothelial cells was performed to test the functionality of OPN on various SAMs. Surface plasmon resonance results showed that the amount of protein adsorbed on the --NH2 surface is close to a monolayer and similar to that on the --COOH surface, consistent with the atomic force microscopy results. However, based on cell adhesion experiments, both cell count and average cell spreading area on the --NH2 surface are much higher than those on the --COOH surface. From these results, it is suggested that the orientation and conformation of OPN on a positively charged --NH2 surface is more favorable for cell adhesion and spreading than on a negatively charged --COOH surface. The surface coverage of bovine aortic endothelial cells on the surfaces studied decreased in the following order: --NH2 > Au > --CH3 > --COOH > --OH whereas the mean cell spreading area decreased in the following order: --NH2 > Au > --CH3 > --COOH. Our studies show that surface properties will alter OPN behavior on surfaces, thus influencing cell interactions.