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    • Films treated with Diaz ES VWFIIINle but not

    2020-10-15

    Films treated with Diaz-ES-VWFIIINle but not exposed to UV light supported only slight platelet deposition, suggesting that passively-bound peptides were removed under shear. However, treatment with Diaz-ES-VWFIIINle and UV restored platelet surface coverage to about 50% of the level on native collagen films, although under these conditions thrombi were smaller. Integrin α2β1-dependent binding will remain ablated by EDC/NHS treatment, with the loss of Gxx\'GEx\'\' motifs. Our findings are consistent with a synergic effect between GPVI, VWF and integrins being required for full thrombus formation [21]. In our current study, only GPVI (through GPO repeats in the collagen) and VWF (through GPRGQOGVNleGFO) binding occurs, but not integrin binding, leading to only partial thrombus formation. We observed a similar effect previously, where platelets were perfused on slides coated with GFOGER-, GPO- or GPRGQOGVNleGFO-containing peptides [21]. We expect that full restoration of platelet aggregation would require derivatisation of crosslinked films with both GPRGQOGVNleGFO- and GFOGER- containing THPs, an experiment for the future. Such a material might be valuable in stents used to treat aortic aneurysm, for example, where promotion of blood clotting stabilises the distension of the aorta. Use of an insoluble biomaterial would be a means of constraining clotting to the desired location. The last step of this study was to evaluate the feasibility of transposing THP-derivatised collagen substrates to tissue engineering applications. Endothelial AH 7614 have the ability to undergo angiogenesis, the process of formatting new blood vessels from pre-existing vasculature, which is essential for tissue repair at the site of injury. These cells are therefore prominent candidates for seeding on biomaterials. As a consequence, it was important to ensure that endothelial cells could fulfil their physiological function on our derivatised crosslinked collagen films. We selected HUVECs for this purpose for their convenience and because they are widely used in experimental settings. The uptake and regulation of Ac-LDL is a widely used marker to assess the activity of endothelial cells. We demonstrated that covalently linked Diaz-ES-VWFIIINle THPs were able to stimulate Ac-LDL internalization in HUVECs on 100% crosslinked films. The patterns observed in the presence of peptides is similar to what is observed on empty tissue culture treated plastic dishes, with Ac-LDL accumulation in the cytoplasm, indicating normal functioning of the cells. Again, some notable difference could be observed between results on derivatised crosslinked films and non-crosslinked films. Diaz-ES-VWFIIINle restores LDL uptake only partially, and additional ligands for other collagen binding receptors might be required for full HUVEC activity. At the moment, we can only speculate on how the THP ligands can beneficially stimulate HUVECs and we are currently investigating the repertoire of collagen receptors expressed on their surface to provide insight on these mechanisms. Nevertheless, our data highlights the potential of derivatised films for use as a biomaterial promoting angiogenesis and tissue regeneration. The present work, together with our previous study, offers a means of conferring collagen-binding integrin-, VWF- and DDR-reactivity upon an inert substrate. GPRGQOGVNleGFO is known to bind not only VWF and DDR2, but also DDR1 and SPARC (Secreted Protein Acidic and Rich in Cysteine) [20,52]. We therefore expect this peptide to be able to promote DDR1 and SPARC recruitment to crosslinked films, increasing the restoration of physiological function to cross-linked collagens in tissue engineering. The scope of the method developed here can also be extended to include three-dimensional matrices [10] and to other inert substrates, not necessarily collagen-based [53], due to the broad reactivity of the diazirine group. Other collagen motifs for other receptors, for example for GPVI or OSCAR, are currently being studied in order to broaden the methodology further to allow enhancement of reactivity beyond that of native collagen. A wider range of THP ligands will allow us to adjust cell activity for different cell types to particular applications in tissue engineering.