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.Graphical
Highlights
The extracellular matrix (ECM) of tissues provides mechanical support for cells and supplies correct biological signals for cell activity [1,2,3,4]
Studies were first performed on monolayer coatings of the molecules of interest applied to a polystyrene tissue culture plastic surface
Cell lines selected in this work allow a comparison to be made between the interaction of Col and Gel-based compositions with cells that express Col-binding integrins (HT1080 and L3 expressing α2β1, and Rugli expressing α1β1) and a parent cell line C2C12, which only possess Gel-binding integrins, αvβ3 and α5β1
Summary
The extracellular matrix (ECM) of tissues provides mechanical support for cells and supplies correct biological signals for cell activity [1,2,3,4]. In particular fibrillar Type I, is the most abundant constituent of the ECM of many hard and soft tissues in the human body [2, 16,17,18,19] This protein provides both the structural support to resident cells and important cell surface receptor-recognition motifs that are essential for cell–substrate interaction [20,21,22]. The addition of Gel to Col and the variation in crosslinking status can tailor many important material properties of resultant matrices. These include the dissolution resistance in different biological environments, the swelling characteristics and the mechanical strength [15, 25]. In conjunction with this data, the main objective of this research is to evaluate cell interaction with Col and
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