D Bax

Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics.

Graphical abstract

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.Graphical Abstract

Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α1β1, α2β1, α10β1 and α11β1, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α1 and α2 subunits. Integrin α2β1-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance. STATEMENT OF SIGNIFICANCE Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines.

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

The article “Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry”, written by Natalia Davidenko, Carlos F. Schuster, Daniel V. Bax, Richard W. Farndale, Samir Hamaia, Serena M. Best and Ruth E. Cameron, was originally published Online First without open access. After publication in volume 27, issue 10, page 148 it was noticed that the copyright was wrong in the PDF version of the article. The copyright of the article should read as “© The Author(s) 2016”. The Open Access license terms were also missing.

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”.

Research data supporting the article 'Collagen-Fibrinogen Lyophilised Scaffolds for Soft Tissue Regeneration'

Data originating from a broad spectrum of work considering the lyophilisation of collagen based scaffolds for tissue engineering applications. A paper focusing on the feasiblity of the production of collagen-fibrinogen scaffolds and an investigation of the specific biological effect of the fibrinogen additions. The raw data is included for the quantitative pore analysis using micro-CT, cell adhesion and cell spreading. For access to any of the images from which these data were collected please contact the author.

Collagen-Fibrinogen Lyophilised Scaffolds for Soft Tissue Regeneration

A significant body of research has considered collagen as a scaffold material for soft tissue regeneration. The main structural component of extra-cellular matrix (ECM), collagen’s advantages over synthetic polymers are numerous. However, for applications where higher stiffness and stability are required, significant cross-linking may affect bioactivity. A carbodiimide (EDC) cross-linking route consumes carboxylate groups that are key to collagen’s essential cell recognition motifs (GxOGER). Fibrinogen was considered as a promising additive as it plays a key role in the process of wound repair and contains RGD integrin binding sites which bind to a variety of cells, growth factors and cytokines. Fibrinogen’s binding sites however, also contain the same carboxylate groups as collagen. We have successfully produced highly interconnected, porous collagen-fibrinogen scaffolds using a lyophilisation technique and micro-computed tomography demonstrated minimal influence of either fibrinogen content or cross-linking concentration on the scaffold structure. The specific biological effect of fibrinogen additions into cross-linked collagen are considered by using films as a model for the struts of bulk scaffolds. By considering various additions of fibrinogen to the collagen film with increasing degrees of cross-linking, this study demonstrates a significant biological advantage with fibrinogen addition across the cross-linking concentrations typically applied to collagen-based scaffolds.

Research Data Supporting "Evaluation of Cell Binding to Collagen and Gelatin: A Study of the Effect of 2D and 3D Architecture and Surface Chemistry"

The zipped folder called “Raw data for cell article” contains 7 files as described below: Article title: “Evaluation of Cell Binding to Collagen and Gelatin: A Study of the Effect of 2D and 3D Architecture and Surface Chemistry”, accepted in Journal of Materials Science: Materials in Medicine. Authors: Natalia Davidenko, Carlos F. Schuster, Daniel V. Bax, Richard W. Farndale, Samir W. Hamaia, Serena M. Best, Ruth E. Cameron. File cold “Fig3n” contains raw data of percentage of adhesion values of HT1080 and Rugli cells on surfaces of different compositions as a function of initial cell concentration (showed in Fig 3). Files “Fig4n” and “Fig4s” contain raw data of magnesium-dependent, non-specific and integrin mediated adhesion profiles of different cell lines on treated surfaces (showed in Fig.4). File “Fig5n” contains results of spreading studies collected in Fig 5. File “Fig6n”contains results cell adhesion on films with different composition and crosslinked status (showed in Fig.6). File “Fig7n” contains results of the percentage of adhesion of HT1080, Rugli, C2C12 and L3 cells on 100% EDC-XL scaffolds of different compositions (collected in Fig. 7). Files “Fig8n” contains results of the effect of crosslinking on the adhesion of cells expressing Col-binding integrins (HT1080 and Rugli) on Col scaffold (Fig8).