Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry

Studies of cell attachment to collagen-based materials often ignore details of the binding mechanisms—be they integrin-mediated or non-specific. In this work, we have used collagen and gelatin-based substrates with different dimensional characteristics (monolayers, thin films and porous scaffolds) in order to establish the influence of composition, crosslinking (using carbodiimide) treatment and 2D or 3D architecture on integrin-mediated cell adhesion. By varying receptor expression, using cells with collagen-binding integrins (HT1080 and C2C12 L3 cell lines, expressing α2β1, and Rugli expressing α1β1) and a parent cell line C2C12 with gelatin-binding receptors (αvβ3 and α5β1), the nature of integrin binding sites was studied in order to explain the bioactivity of different protein formulations. We have shown that alteration of the chemical identity, conformation and availability of free binding motifs (GxOGER and RGD), resulting from addition of gelatin to collagen and crosslinking, have a profound effect on the ability of cells to adhere to these formulations. Carbodiimide crosslinking ablates integrin-dependent cell activity on both two-dimensional and three-dimensional architectures while the three-dimensional scaffold structure also leads to a high level of non-specific interactions remaining on three-dimensional samples even after a rigorous washing regime. This phenomenon, promoted by crosslinking, and attributed to cell entrapment, should be considered in any assessment of the biological activity of three-dimensional substrates. Spreading data confirm the importance of integrin-mediated cell engagement for further cell activity on collagen-based compositions. In this work, we provide a simple, but effective, means of deconvoluting the effects of chemistry and dimensional characteristics of a substrate, on the cell activity of protein-derived materials, which should assist in tailoring their biological properties for specific tissue engineering applications.The authors would like to thank the British Heart Foundation (Grants NH/11/1/28922, RG/15/4/31268 and SP/15/7/31561), The Welcome Trust (Grant 094470/Z/10/Z), the ERC Advanced Grant 320598 3D-E and EPSRC Doctoral Training Account for providing financial support for this project. D. V. Bax is funded by the Peoples Programme of the EU 7th Framework Programme (RAE no: PIIF-GA-2013-624904) and was also supported by an EPSRC IKC Proof of Concept Award

Human cell types important for Hepatitis C Virus replication in vivo and in vitro. Old assertions and current evidence

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro

Research data supporting "Selecting the Correct Cellular Model for Assessing of the Biological Response of Collagen-based Biomaterials"

The zipped folder called “Raw data for Acta Biomaterilaia article” contains 8 files as described below: Article title: “Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterilas”, accepted in Acta Biomaterilalia. Authors: Natalia Davidenko, Samir Hamaia, Daniel V. Bax, Jean-Daniel Malcor, Carlos F. Schuster, Donald Gullberg, Richard W. Farndale, , Serena M. Best, Ruth E. Cameron. File cold “Fig 2” contains raw data of the adhesion values and profiles on collagen and peptide coatings for C2C12 cell lines transfected with all collagen-binding integrins (showed in Fig 2). Files “Fig 3” contain raw data of magnesium-dependent, non-specific adhesion profiles of different C2C12-α1+, C2C12-α2+, Rugli and Ht1080 cells on coatings (showed in Fig.3). File “Fig 4” contains results of adhesion dependence on initial cell concentration for collagen I, collected in Fig 4. File “Fig 5” contains results of cell spreading on collagen surfaces showed in Fig.5. File “Fig 6”contains results cell adhesion on films with different composition and crosslinked status (showed in Fig.6). File “Fig 7” contains results of the e of adhesion profiles of HT1080 and Rugli cells differently crosslinked collagen films (collected in Fig. 7). File “Fig 8” contains results of the attachment of α1 I domine on different collagens and selected synthetic and Toolkit peptides (showed in Fig8). File “Supplementary data” shows data of collagen detection on coated surfaces (results collected in Supplementary materilas”

The effects of inhibition and siRNA knockdown of collagen-binding integrins on human umbilical vein endothelial cell migration and tube formation.

Blood vessels in the body are lined with endothelial cells which have vital roles in numerous physiological and pathological processes. Collagens are major constituents of the extracellular matrix, and many adherent cells express several collagen-binding adhesion receptors. Here, we study the endothelium-collagen interactions mediated by the collagen-binding integrins, α1β1, α2β1, α10β1 and α11β1 expressed in human umbilical vein endothelial cells (HUVECs). Using qPCR, we found expression of the α10 transcript of the chondrocyte integrin, α10β1, along with the more abundant α2, and low-level expression of α1. The α11 transcript was not detected. Inhibition or siRNA knockdown of the α2-subunit resulted in impaired HUVEC adhesion, spreading and migration on collagen-coated surfaces, whereas inhibition or siRNA knockdown of α1 had no effect on these processes. In tube formation assays, inhibition of either α1 or α2 subunits impaired the network complexity, whereas siRNA knockdown of these integrins had no such effect. Knockdown of α10 had no effect on cell spreading, migration or tube formation in these conditions. Overall, our results indicate that the collagen-binding integrins, α1β1 and α2β1 play a central role in endothelial cell motility and self-organisation

Chain alignment of collagen I deciphered using computationally designed heterotrimers

The most abundant member of the collagen protein family, collagen I (COL1), is composed of two similar (chain A) and one unique (chain B) polypeptides that self-assemble with one amino acid offset into a heterotrimeric triple helix. Given the offset, chain B can occupy either the leading (BAA), middle (ABA) or trailing (AAB) position of the triple helix, yielding three isomeric biomacromolecules with different protein recognition properties. Despite five decades of intensive research, there is no consensus on the position of chain B in COL1. Here, three triple-helical heterotrimers that each contained a putative Von Willebrand Factor (VWF) and discoidin domain receptor (DDR) recognition sequence from COL1 were designed with chain B permutated in all three positions. AAB demonstrated a strong preference for both VWF and DDR and also induced higher levels of cellular DDR phosphorylation. Thus, we resolve this long-standing mystery and show that COL1 adopts an AAB register

SARS-CoV-2 Omicron subvariant spike N405 unlikely to rapidly deamidate.

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVβ3 and α5β1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVβ3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions