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

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 Abstract

PLA-Based Hybrid and Composite Electrospun Fibrous Scaffolds as Potential Materials for Tissue Engineering

“…GEL is also known to effectively reduce the hydrophobicity of the substrate surface and can be the source of the amino acid sequence (RGD) enhancing cellular adhesion in in vitro studies [30].…”

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“…Aside from their mechanical role in attachment, integrins play a prominent role in cell signalling that provides information regarding the local environment, adhesive properties and surrounding matrix characteristics [41]. Integrins are important in governing cell attachment to the biological scaffolds for tissue engineering, as a poor attachment will cause cells to grow abnormally and increase the chances of losing homeostasis [43]. One study conducted on β 1 integrins illustrated how IL-β 1 is important in binding fibronectin matrices and can even facilitate adhesion when the matrix is damaged (Figure 4) [44].…”

“…IL-β 1 is a cytokine that plays an important role in cell adhesion to damaged ECM by activating matrix metalloproteinases to reconstruct the matrix [44]. However, the specific mechanisms behind cell attachment may also be dependent on the structure of the biological scaffold; 3D scaffolds that are highly porous may be able to entrap cells via a non-integrin based attachment [43]. Nevertheless, proper cell attachment to tissue scaffolds continues to be a major consideration in tissue engineering design and research and integrins remain the central player in this regard.…”

Home-Grown Kidneys: Challenges and Mechanisms Behind Tissue Engineering for the Treatment of Chronic Kidney Disease

“…41 Integrins are important in governing cell attachment to the biological scaffolds for tissue engineering, as a poor attachment will cause cells to grow abnormally and increase the chances of losing homeostasis. 43 One study conducted on β 1 integrins illustrated how IL-β 1 is important in binding fibronectin matrices and can even facilitate adhesion when the matrix is damaged ( Figure 4). 44 IL-β 1 is a cytokine that plays an important role in cell adhesion to damaged ECM by activating matrix metalloproteinases to reconstruct the matrix.…”

“…44 However, the specific mechanisms behind cell attachment may also be dependent on the structure of the biological scaffold; 3D scaffolds that are highly porous may be able to entrap cells via a non-integrin based attachment. 43 Nevertheless, proper cell attachment to tissue scaffolds continues to be a major consideration in tissue engineering design and research and integrins remain the central player in this regard. Integrin signalling: The binding of integrins to the ECM facilitates cell attachment via a signaling cascade that drives fibrillogenesis.…”