Other ; This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s10856-016-5763-9 ; Abstract ; 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. ; Other ; 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.