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  • br Declaration of interest statement br Introduction

    2020-07-30


    Declaration of interest statement
    Introduction Collagen is widely used in tissue engineering [[1], [2], [3]]. It is the most abundant protein in the human body and is a major constituent transcription factor of the extracellular matrix (ECM), providing natural structural and biological support for transcription factor [4]. The mechanical properties of collagen-based biomaterials can be tailored to match the strength, stiffness and architecture of the host tissue. Chemical crosslinking of a collagen-based scaffold using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), is widely used to increase its stiffness and to resist swelling in biological media [[5], [6], [7]]. However, we have previously shown that such crosslinking ablated the recognition of the collagen-binding integrins (α1β1, α2β1, α10β1 and α11β1) and led to a dramatic loss of cell adhesion compared to native type I collagen [[8], [9], [10], [11], [12]], as a result of consumption of the glutamate residues in integrin binding Gxx\'GEx\'\' motifs [13,14] in forming isopeptide bonds with lysines. Other collagen receptors might also be affected if their binding sequences contain aspartate, glutamate or lysine residues, or if they are adjacent to such EDC/NHS-sensitive residues. We show in this study that DDR2 and the A3 domain of VWF are affected by EDC/NHS crosslinking due to the presence of adjacent glutamate and lysine, even though their immediate binding site in collagen lacks such residues. For use in regenerative medicine, these materials must support cell attachment, subsequent differentiation and proliferation to fulfil their physiological function in the target tissue. Previously, we reported a methodology to derivatise collagen films with photoreactive triple-helical peptides (THPs) containing the GFOGER integrin binding motif [15]. THPs mimic the natural structure of collagen by adopting a triple helix conformation, essential for recognition by collagen binding receptors. THPs, however, remain rarely used in tissue engineering and, although examples can be found on various surfaces [16,17], their incorporation into collagen-based materials has only been investigated in this laboratory, to our knowledge. In our hands, this led to complete restoration of integrin mediated cell binding and spreading to EDC/NHS crosslinked collagen films [15]. We aim to apply the same technology to target other collagen-binding proteins using motifs from collagens that are specific for other receptors or matrix components. Previously, we synthesized THP libraries, called Toolkits, composed of an active guest sequence flanked by five GPP host triplets that form a triple helix [18]. Screening of collagen binding proteins against Toolkits II and III (encompassing the sequences of collagen II and III respectively), led to the identification of a THP ligand for both Discoidin Domain Receptors (DDRs) [19,20] and von Willebrand Factor (VWF) [18,[21], [22], [23]]. The shared binding motif GPRGQOGVMGFO, referred to as VWFIII in the literature, is derived from the active sequence of Toolkit III-23, GPOGPSGPRGQOGVMGFOGPKGNDGAO. A similar motif, GARGQOGVMGFO, is present in collagen II, and an equivalent sequence, GARGQAGVMGFO, occurs in the α1 chain of collagen I (bold text represents differences in sequence).