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 α$_{1}$β$_{1}$, α$_{2}$β$_{1}$, α$_{10}$β$_{1}$ and α$_{11}$β$_{1}$, 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 α$_{1}$ and α$_{2}$ subunits. Integrin α$_{2}$β$_{1}$-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.This work was supported by the British Heart Foundation (Grant NH/11/1/28922, RG/15/4/31268, SP/15/7/31561 and RG/09/003/27122) and the ERC Advanced Grant 320598 3D-E. D. V. Bax is funded by the Peoples Programme of the EU 7th Framework Programme (RAE no: PIIF-GA-2013-624904) and was supported by an EPSRC IKC Proof of Concept Award

Measurement of the Interaction Between Recombinant I-domain from Integrin alpha 2 beta 1 and a Triple Helical Collagen Peptide with the GFOGER Binding Motif Using Molecular Force Spectroscopy.

The role of the collagen-platelet interaction is of crucial importance to the haemostatic response during both injury and pathogenesis of the blood vessel wall. Of particular interest is the high affinity interaction of the platelet transmembrane receptor, alpha 2 beta 1, responsible for firm attachment of platelets to collagen at and around injury sites. We employ single molecule force spectroscopy (SMFS) using the atomic force microscope (AFM) to study the interaction of the I-domain from integrin alpha 2 beta 1 with a synthetic collagen related triple-helical peptide containing the high-affinity integrin-binding GFOGER motif, and a control peptide lacking this sequence, referred to as GPP. By utilising synthetic peptides in this manner we are able to study at the molecular level subtleties that would otherwise be lost when considering cell-to-collagen matrix interactions using ensemble techniques. We demonstrate for the first time the complexity of this interaction as illustrated by the complex multi-peaked force spectra and confirm specificity using control blocking experiments. In addition we observe specific interaction of the GPP peptide sequence with the I-domain. We propose a model to explain these observations

Research data supporting "Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds"

Excel work book containing 11 spread sheets labeled figure 1, figure 2, figure 3, figure 4, figure 5, figure 6, figure 7, figure 8, figure 9, figure 10 and figure 11. Spread sheet 'figure 1' contains the absorbance values obtained on a plate reader from a platelet adhesion assay to collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 2' contains 3 data sets. A) absorbance values obtained on a plate reader from a HT1080 cell adhesion assay to collagen films crosslinked with increasing % of NHS/EDC. B) cell counts for a HT1080 cell spreading assay to collagen films crosslinked with increasing % of NHS/EDC. C) representative phase contrast micrographs of HT1080 cells spreading onto collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 3' contains 2 data sets. A) absorbance values obtained on a plate reader from a HT1080 cell adhesion assay conducted in PBS in the absence of cations or with added 2mM MgCl2 or CaCl2 B) absorbance values obtained on a plate reader from a HT1080 cell adhesion assay conducted in PBS with added MgCl2 or CaCl2 Spread sheet 'figure 4' contains 3 data sets. A) absorbance values obtained on a plate reader from a Rugli cell adhesion assay to collagen films crosslinked with increasing % of NHS/EDC. B) cell counts for a Rugli cell spreading assay to collagen films crosslinked with increasing % of NHS/EDC. C) representative phase contrast micrographs of Rugli cells spreading onto collagen films crosslinked with increasing % of NHS/EDC Spread sheet 'figure 5' contains 4 data sets. A) absorbance values obtained on a plate reader from a purified alpha 1 I domain adhesion assay to collagen films crosslinked with increasing % of NHS/EDC. B) calculations of the Mg dependent adhesion of purified alpha 1 I domain binding to collagen films crosslinked with increasing % of NHS/EDC. C) absorbance values obtained on a plate reader from a purified alpha 2 I domain adhesion assay to collagen films crosslinked with increasing % of NHS/EDC. D) calculations of the Mg dependent adhesion of purified alpha 2 I domain binding to collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 6' contains 4 data sets of the absorbance values obtained on a plate reader from A) non-transfected C2C12 cell, B) alpha 2 transfected C2C12 cell, C) alpha 10 transfected C2C12 cell and D) alpha 11 transfected C2C12 cell adhesion assays to collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 7' contains 3 data sets of the cell counts obtained by manually scoring phase contrast micrographs of A) non-transfected C2C12 cell, B) alpha 2 transfected C2C12 cell and C) alpha 10 transfected C2C12 cell spreading on to collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 8' contains 4 data sets of the correlations between the number of free amine groups and the Mg dependent A) HT1080 cell, B) alpha 2 transfected C2C12 cell, C) alpha 10 transfected C2C12 cell and D) alpha 11 transfected C2C12 cell adhesion to collagen films. Spread sheet 'figure 9' contains 2 data sets. A) representative fluorescent and bright field images of AnnexinV-FITC stained HT1080 cells on collagen films crosslinked with increasing % of NHS/EDC. B) counts of AnnexinV-FITC positive and total cell numbers of HT1080 cells on collagen films crosslinked with increasing % of NHS/EDC. Spread sheet 'figure 10' contains 2 data sets. A) Cell number manually counted from phase contrast images of HT1080 cells cultured on collagen films crosslinked with increasing % of NHS/EDC for 1,2,3 or 5 days. Representative micrographs are shown. B) measurement of the surface area coved by cells using Image J. The measurement is converted into microns square. The HT1080 cells were cultured on collagen films crosslinked with increasing % of NHS/EDC for 5 days prior to measurement. Spread sheet 'figure 11' contains a confocal micrograph of rhodmaine-phalloidin stained HT1080 cells on a collagen film with restricted 200% NHS/EDC crosslinking. The image was generated by compressing a Z stack into a single image

A simple bioconjugate attachment protocol for use in single molecule force spectroscopy experiments based on mixed self-assembled monolayers.

Single molecule force spectroscopy is a technique that can be used to probe the interaction force between individual biomolecular species. We focus our attention on the tip and sample coupling chemistry, which is crucial to these experiments. We utilised a novel approach of mixed self-assembled monolayers of alkanethiols in conjunction with a heterobifunctional crosslinker. The effectiveness of the protocol is demonstrated by probing the biotin-avidin interaction. We measured unbinding forces comparable to previously reported values measured at similar loading rates. Specificity tests also demonstrated a significant decrease in recognition after blocking with free avidin