Nanostructured Graphene Surfaces Promote Different Stages of Bone Cell Differentiation.

Nanostructured graphene films were used as platforms for the differentiation of Saos-2 cells into bone-like cells. The films were grown using the plasma-enhanced chemical vapor deposition method, which allowed the production of both vertically and horizontally aligned carbon nanowalls (CNWs). Modifications of the technique allowed control of the density of the CNWs and their orientation after the transfer process. The influence of two different topographies on cell attachment, proliferation, and differentiation was investigated. First, the transferred graphene surfaces were shown to be noncytotoxic and were able to support cell adhesion and growth for over 7 days. Second, early cell differentiation (identified by cellular alkaline phosphatase release) was found to be enhanced on the horizontally aligned CNW surfaces, whereas mineralization (identified by cellular calcium production), a later stage of bone cell differentiation, was stimulated by the presence of the vertical CNWs on the surfaces. These results show that the graphene coatings, grown using the presented method, are biocompatible. And their topographies have an impact on cell behavior, which can be useful in tissue engineering applications.

Human endothelial cells grow poorly on vitronectin: role of PAI-1.

The cell adhesive protein vitronectin is a common component of interstitial extracellular matrix and circulates in plasma. It competes effectively with other plasma proteins to adsorb to certain biomaterial surfaces, and is likely to represent an important cell adhesion mediator on the luminal surface of vascular grafts. It is also found associated with certain vascular pathologies. We have shown previously that human endothelial cells grow poorly on a vitronectin surface compared with other extracellular matrix molecules. In this paper we show that endothelial cells seeded on vitronectin and fibronectin produced substantially different profiles of extracellular matrix molecules. The most outstanding difference was in the amount of matrix-localised plasminogen activator-inhibitor-1 which was high on vitronectin and negligible on fibronectin. This was correlated with a small but significant inhibition of cell adhesion to vitronectin compared with fibronectin, and very significant interference with dissociation of cell: extracellular matrix contacts, resulting either from direct inhibition of the proteolytic activity of urokinase, or from interference with urokinase-receptor signaling and consequent focal adhesion turnover. Such interference would inhibit cell proliferation by disabling the cells from loosening their matrix contacts in order to proceed through mitosis. This would seriously compromise endothelial recovery in cases of damage to the vascular wall and placement of stents or grafts, where the presence of surface-adsorbed vitronectin is likely to modulate the tissue response.

Specific affinity depletion of cell adhesion molecules and growth factors from serum.

Serum is a common component of most in vitro cell culture media, particularly of primary cells. Studies of cellular responses to particular adhesion molecules or growth factors are often confounded by the presence of these molecules in the serum supplement. We describe a combined affinity protocol for removing vitronectin and fibronectin from serum. This protocol can also be used to purify these molecules. We also describe the removal of growth-promoting elements using heparin-Sepharose. As vitronectin and fibronectin each bind to heparin, these molecules are removed first and the heparin-Sepharose depletion occurs last in the sequence. This protocol provides a detailed step-by-step guide to achieve quantitative depletion of serum in an optimised format, with additional information on pitfalls and problems. It should be of use to people who wish to accurately determine the relationship between cells, extracellular matrix molecules and growth factors.

Inhibition of endothelial cell adhesion and proliferation by extracellular matrix from vascular smooth muscle cells: role of type V collagen.

Endothelial cells recovering from damage due to disease or surgical procedures come into close contact with extracellular matrix (ECM) secreted by intimal vascular smooth muscle cells (VSMCs). We have investigated these relationships using human umbilical artery endothelial cells (HUAECs) and human mammary artery VSMC in vitro. HUAEC adhesion and proliferation were significantly lower on ECM secreted by VSMC compared with HUAEC ECM or surface-coated fibronectin. Characterisation of the ECM of both cell types with monoclonal antibodies showed that the ECM secreted by VSMC contained significantly more elastin, chondroitin sulphate and collagen types I, III and V than that from HUAECs. HUAECs adhered poorly to collagen type V coated on plastic and not at all to elastin. When these proteins were co-coated with fibronectin, elastin did not inhibit migration or proliferation compared to the response on fibronectin but collagen type V significantly inhibited both. Treatment of VSMC ECM with enzymes which selectively depleted the matrix of collagen types I, III and IV, or chondroitin sulphate, had no effect on HUAEC responses to the ECM, suggesting that these molecules did not contribute to the inhibition of HUAECs. Treatment of VSMC ECM with a mixture of collagenases, selectively depleted the matrix of collagen type V, as well as types I, III and IV. Such depleted ECMs supported increased proliferation of HUAECs compared to buffer controls. Overall these results suggest that collagen V secreted into the ECM of VSMC may inhibit the recovery of adjacent endothelium.

Evidence for the location of a binding sequence for the alpha 2 beta 1 integrin of endothelial cells, in the beta 1 subunit of laminin.

To date no specific location on laminin 1 for the binding of alpha 2 beta 1 integrin has been described, although recent evidence supports a location in the E1XNd fragment of the cross region. We have identified a peptide sequence from this region, in the beta 1 chain of laminin 1, YGYYGDALR, which inhibits the adhesion of endothelial cells to laminin 1 and type-IV collagen. A structurally related sequence from the CNBr-cleaved fragment CB3 of the alpha 1 chain of collagen type IV, FYFDLR, inhibits endothelial cell adhesion to both collagen types I and IV and laminin 1. The CB3 fragment containing the FYFDLR sequence has been shown to contain binding sites for both alpha 1 beta 1 and alpha 2 beta 1 integrins. Present experiments with anti-integrin antibodies indicate that the alpha 2 beta 1 integrin on endothelial cells can account for all the cell binding to collagen types I and IV, and that this integrin makes a major contribution towards the adhesion of these cells to laminin 1. We therefore propose that the peptide FYFDLR participates in alpha 2 beta 1 binding to collagen type IV and that the putatively structurally similar peptide, YGYYGDALR, participates in alpha 2 beta 1 binding to laminin 1. This is the first account of structurally related peptide sequences from laminin 1 and type-IV collagen which show reciprocal inhibition of cell adhesion to either ligand and which might form part of a common integrin-binding site, as well as the first suggestion of a precise location contributing to the alpha 2 beta 1 integrin binding site on laminin 1.

Relative topography of biologically active domains of human vitronectin. Evidence from monoclonal antibody epitope and denaturation studies.

A panel of monoclonal antibodies to human vitronectin was used to define some epitopes for the multifunctions of this protein. Separate antibodies were identified which strongly inhibited cell spreading activity and which prevented binding to collagen. A third group interfered with the ability of vitronectin to inhibit complement-mediated guinea pig erythrocyte reactive lysis. None of the antibodies from these three groups prevented heparin binding, providing evidence that this reaction occurs at a fourth location; a different monoclonal antibody partially inhibited the binding of heparin. The relative accessibility of each biologically active epitope was assessed by the differential binding of the monoclonals to native and denatured vitronectin. Reactivity of the antibody which inhibited heparin binding greatly increased upon denaturation of vitronectin, implying that this region is normally inaccessible in the native form of the molecule. By contrast, epitopes for cell spreading, collagen binding, and inhibition of terminal complement complex lysis were destroyed by denaturation. On the basis of denaturation data and epitope mapping by competitive exclusion of monoclonal antibodies, a Venn diagram was constructed to represent overlap of monoclonal antibody epitopes in the tertiary structure. Linear epitopes for the antibodies were identified using cyanogen bromide and plasmin-derived peptides from vitronectin. The antibody which strongly inhibited cell binding reacted with a region containing the RGD site, whereas linear epitopes for collagen binding and complement inhibition appeared to reside in a 43-kDa peptide representing the internal region of the amino acid sequence, excluding the heparin-binding site. The latter two epitopes were differentiated from each other by reactivity of the antibody which inhibited collagen binding toward a smaller 20-kDa plasmin-derived peptide.

Hazards of the limiting-dilution method of cloning hybridomas.

Three hybridoma clones, which were shown to change the characteristics of their antibody specificities when grown under different culturing conditions, are described in detail. This phenomenon was shown to be due to the persistence of mixed clones, even under conditions where standard statistical treatment indicated a high probability of monoclonality. Such mixed clones persisted, sometimes undetected, through repeated cycles of re-cloning. It was shown that the assumption that every viable clone has the same random chance of monoclonality, is invalid, and can lead to misleadingly high estimates for the probability of monoclonality. Verification of seeding of individual wells with single cells is recommended and the relative merits of this versus repeated limiting-dilution cloning are discussed.

The influence of methods of production, purification and storage of monoclonal antibodies upon their observed specificities.

A panel of 38 monoclonal antibodies was exposed to a variety of physical conditions commonly used in their purification and storage. Reactivity with the homologous (immunising) antigen was reduced in a significant proportion (42%) of antibodies following these physical treatments. Sensitivity to particular physical treatments correlated with antibody subclass. In 3 cases the relative specificity of the antibody (as measured by cross-reactivity with 16 closely related antigens) was also altered. Following passage of the cloned hybridomas as ascites, 3 antibodies exhibited marked differences in specificity, which suggested the selection of different genotypes. The importance of documenting the methods of production, purification and storage of particular monoclonal antibodies used in comparative assay is stressed.