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  • br Conclusion Our objective is to

    2020-05-22


    Conclusion Our objective is to promote cell activity on biomaterials, especially for use in regenerative medicine, by grafting THPs containing collagen-binding sites onto inert substrates. We have previously reported a methodology to derivatize EDC/NHS crosslinked films with photoreactive THPs. This resulted in increased cell reactivity towards the collagen substrate, illustrated by cell binding and spreading, after attaching a GFOGER-containing THP that supports the attachment of collagen-binding integrins. Here, we diversified our range of photoreactive ligands to address different collagen-binding receptors for a wider range of applications. Of particular interest is Diaz-ES-VWFIIINle, containing the GPRGQOGVNleGFO active sequence, which targets both DDR1 and DDR2, and VWF. DDRs have been implicated in wound healing and cell migration, and DDR2 has been reported in epicardium-derived cardiac fibroblasts [29] and cardiac mesenchymal stem R115777 [28]. The proper regulation of these receptors is likely to be important to direct cell behavior and regeneration in several tissue engineering settings. VWF can equally present multiple interests in vascular repair, as it is a key factor in platelet thrombus formation in flowing blood. We have demonstrated that Diaz-ES-VWFIIINle-derivatised films had an increased affinity for recombinant DDR2 and VWF A3. Beyond simple binding activity, we have shown that receptors could be activated by our THPs covalently bound to collagen films, which was highlighted by tyrosine phosphorylation of DDR2 in transfected cells, and platelet deposition in human blood. Finally, we have demonstrated that derivatised collagen films could support HUVECs activity, a key step towards the production of collagen-based structures promoting angiogenesis. In this way, we have overcome the loss of reactivity that occurs when collagen R115777 is crosslinked to enhance its mechanical properties, paving the way for the construction of collagen scaffolds and other biomaterials for diverse applications.
    Data availability
    Acknowledgments The work was supported in Department of Biochemistry by grants from British Heart Foundation (SP/15/7/31561, FS/15/20/31335 and RG/15/4/31268). At the Department of Materials Science, Cambridge, funding was from the People Programme of the EU 7th Framework Programme (RAE no: PIIF-GA-2013-624904, to DVB), a Proof of Concept grant from the EPSRC Medical Technologies IKC, and an ERC Advanced Grant 320598 3D-E (to REC). At the National Heart and Lung Institute, London, funding was from MRC Doctoral training partnership PhD studentship, and Biomedicine and Bioengineering in Osteoarthritis (BBOA) studentship. We are grateful for help and advice from Dr Daniel Bax, Department of Materials Science, Cambridge, and Mr Douglas Sammon, National Heart and Lung Institute, London.
    Introduction During endochondral ossification (EO), chondrocytes within the cartilage template undergo a highly co-ordinated sequence of proliferation, maturation and hypertrophy with the concomitant changes in the synthesis and deposition of extracellular matrix (ECM) components at each stage (Ortega et al., 2004). Many paracrine and endocrine factors are now known to be regulators of EO (Hering, 1999, de Crombrugghe et al., 2000, van der Eerden et al., 2003). Indian Hedgehog and parathyroid hormone related protein have been shown to regulate chondrocyte hypertrophy in a negative feedback control loop (Vortkamp et al., 1996), possibly acting in conjunction with several other regulatory factors including bone morphogenetic proteins and fibroblast growth factors (Minina et al., 2001, Minina et al., 2002). The ECM also regulates EO by mediating cell migration and shape changes, cell proliferation and differentiation either through direct cell–matrix interactions or by facilitating growth factor binding to cells (Shimazu et al., 1996, Bass and Humphries, 2002). It has been generally accepted that the hypertrophic ECM serves as a permissive matrix to vascularisation and mineralisation. It is therefore conceivable that collagen X, which exhibits unique temporal and spatial expression patterns in the hypertrophic zone, would have specific regulatory roles in EO besides the maintenance of tissue structure and integrity.