No abstract

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit ␤1 (Y783 and Y795) to phenylalanines markedly reduces the capability of ␤1A integrins to mediate directed cell migration. In this study, ␤1-dependent cell spreading was found to be delayed in GD25 cells expressing ␤1A Y783/795F compared to that in wild-type GD25-␤1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to ␤1-dependent adhesion in GD25-␤1A Y783/795F cells compared to that in wild-type GD25-␤1A or mutants in which only a single tyrosine was altered (␤1A Y783F or ␤1A Y795F ). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via ␤1A Y783/795F lies at the level of the initial autophosphorylation step. Indeed, ␤1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the ␤1A Y783/795F cells, consistent with the impairment in FAK activation. In contrast, p130 CAS overall tyrosine phosphorylation was unaffected by the ␤1 mutations. Despite the defect in ␤1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-␤1A Y783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit ␤1A are critical mediators of FAK activation and cell spreading in GD25 cells.Integrins are a family of adhesion receptors, each consisting of one ␣ and one ␤ subunit. Among the known integrins, 12 contain the ␤1 subunit, and ligands for this subfamily include collagens, laminins (LNs), and fibronectin (FN).Integrin subunit ␤1, as well as all other integrin subunits except for ␤4, consists of a large extracellular domain, a single transmembrane stretch, and a short intracellular cytoplasmic domain. Although devoid of an intrinsic enzymatic activity, the cytoplasmic domain regulates the conformation and ligand binding activity of the extracellular domain (so-called "insideout signaling"), as well as mediates interactions with the cytoskeleton and the transduction of intracellular signals ("outside-in" signaling). Specificity for this range of dynamic signaling events has been validated in many systems by using truncated and mutated integrin receptors (15,16,23,26,27,29,32,33,51).Ligand binding to integrins triggers the assembly of a large number of cytoplasmic and transmembrane molecules into discrete regions referred to as focal adhesions (FAs) (5, 10). These complexes serve both as structural anchorage sites that connect the extracellular matrix with the intracellular actin cytoskeleton and as signaling complexes, which initiate signaling pathways in the cell cytoplasm and nucleus. The signaling molecules found in FAs include tyrosine kinases, serine/threonine kinases, phospholipid kinases, phosphatases, Ras superfamily proteins, and various adapter proteins (e.g., those containing SH2 and SH3 domains) (...

Thin, nano-porous, highly adherent layers of anodised aluminium formed on the surface of titanium alloys are being developed as coatings for metallic surgical implants. The layers are formed by anodisation of a 1-5 microm thick layer of aluminium which has been deposited on substrate material by electron beam evaporation. The surface ceramic layer so produced is alumina with 6-8 wt % phosphate ions and contains approximately 5 x 10(8) cm(-2) pores with a approximately 160 nm average diameter, running perpendicular to the surface. Mechanical testing showed the coatings' shear and tensile strength to be at least 20 and 10 MPa, respectively. Initial cell/material studies show promising cellular response to the nano-porous alumina. A normal osteoblastic growth pattern with cell number increasing from day 1 to 21 was shown, with slightly higher proliferative activity on the nano-porous alumina compared to the Thermanox control. Scanning electron microscopy (SEM) examination of the cells on the porous alumina membrane showed normal osteoblast morphology. Flattened cells with filopodia attaching to the pores and good coverage were also observed. In addition, the pore structure produced in these ceramic coatings is expected to be suitable for loading with bioactive material to enhance further their biological properties.

A new method is proposed for coating implants that produces a metal implant covered in a layer of nano-porous alumina ceramic. These layers are produced by first depositing a layer of aluminium on the implant surface and then anodising it in phosphoric acid to produce the nano-porous structure. This process results in the conversion of the aluminium to alumina containing 6-8wt% phosphate ions. The surface alumina layer is bonded to the substrate via an interfacial layer of fully dense anodised titanium oxide. Mechanical measurements have shown that the shear and tensile strength of this coating are in excess of 20MPa and 10MPa, respectively.The biological performance of nano-porous alumina material has been assessed and shown to be highly favourable, supporting normal osteoblastic activity and maintaining the osteoblastic phenotype. The filling of the nano-porous coating with bioactive material to achieve enhanced biological performance has been investigated using colloidal silica as an analogue for a Bioglass sol. The coating has been loaded with silica by dipping in colloidal silica with a pH of 5.6. Pore filling equivalent to 1.3 wt% SiO 2 in the coating as a whole has been achieved in this way.

This study evaluates neutrophil responses on aluminum oxide membranes. Using an in vitro cell culture system, we have found that the pore size (20 and 200 nm in diameter) of alumina membranes have a significant effect on leukocyte morphology and activation. Specifically, our results show that 20-nm pore-size membranes were more potent in triggering PMN spreading and extending of pseudopodia than 200-nm pore-size membranes. The morphological changes are also associated with cell activation. In fact, adherent neutrophils on 20-nm pore-size membranes elicit much stronger initial oxygen free radical production. Overall, our results point out that membrane pore size significantly affects the extent of cellular responses of adherent neutrophils.

This study evaluates human neutrophil responses to aluminum oxide membranes with different pore sizes (20 nm and 200 nm in diameter) uncoated and pre-coated with serum, collagen I, or fibrinogen. The effect of released neutrophil granule components on the survival of osteoblastic cells (MG63) bound to the alumina membranes has also been evaluated. Without protein coatings the 20 nm pore-size membranes prompt higher reactive oxygen species (ROS) production as assessed by luminol-amplified chemiluminescence than the 200 nm pore-size membranes. Such pore-size depending responses were also found on membranes pre-coated with fibrinogen, but not with collagen or serum were in fact a much lower ROS production was observed. In addition, uncoated and fibrinogen-coated membranes prompt stronger release of the granule enzymes, myeloperoxidase and elastase, than collagen or serum-coated alumina. Equally important, we found that surface-mediated phagocyte activation and the subsequent release of granule components had a significant affect on the adhesion, viability and proliferation of osteoblasts. This stresses the importance of studying not only cell/surface interactions but also cell/cell interactions in wound healing and tissue regeneration processes.