title: Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds.
abstract: 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, α10β1and 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 α1and α2subunits. 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.
  
  
  
  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.
authors.affiliation: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, United Kingdom; Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom. Electronic address: dvb24@cam.ac.uk.
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Bax
authors.affiliation: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, United Kingdom.
authors.forename: Natalia
authors.initials: N
authors.lastname: Davidenko
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Norway.
authors.forename: Donald
authors.initials: D
authors.lastname: Gullberg
authors.affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Samir W
authors.initials: SW
authors.lastname: Hamaia
authors.affiliation: Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, United Kingdom.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.affiliation: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, United Kingdom.
authors.forename: Serena M
authors.initials: SM
authors.lastname: Best
authors.affiliation: Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, United Kingdom.
authors.forename: Ruth E
authors.initials: RE
authors.lastname: Cameron
mesh_terms: D000818:Animals; D002412:Cations; D002417:Cattle; D002448:Cell Adhesion; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D003094:Collagen / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism; D051379:Mice; D000072417:Protein Domains; D012995:Solubility; D013388:Succinimides; D054457:Tissue Scaffolds / Q000737:chemistry*; D014162:Transfection
chemical_list: D002412:Cations; D003432:Cross-Linking Reagents; D038982:Integrin alpha2beta1; D013388:Succinimides; D003094:Collagen; C001426:N-hydroxysuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: Cell adhesion; Collagen; EDC crosslinking