Collagen-Fibrinogen Lyophilised Scaffolds for Soft Tissue Regeneration

Abstract

A significant body of research has considered collagen as a scaffold material for soft tissue regeneration. The main structural component of extra-cellular matrix (ECM), collagen's advantages over synthetic polymers are numerous. However, for applications where higher stiffness and stability are required, significant cross-linking may affect bioactivity. A carbodiimide (EDC) cross-linking route consumes carboxylate groups that are key to collagen's essential cell recognition motifs (GxOGER). Fibrinogen was considered as a promising additive as it plays a key role in the process of wound repair and contains RGD integrin binding sites which bind to a variety of cells, growth factors and cytokines. Fibrinogen's binding sites however, also contain the same carboxylate groups as collagen. We have successfully produced highly interconnected, porous collagen-fibrinogen scaffolds using a lyophilisation technique and micro-computed tomography demonstrated minimal influence of either fibrinogen content or cross-linking concentration on the scaffold structure. The specific biological effect of fibrinogen additions into cross-linked collagen are considered by using films as a model for the struts of bulk scaffolds. By considering various additions of fibrinogen to the collagen film with increasing degrees of cross-linking, this study demonstrates a significant biological advantage with fibrinogen addition across the cross-linking concentrations typically applied to collagen-based scaffolds.

Keywords: adhesion; collagen; fibrinogen; lyophilisation; micro-computed tomography.

Figure 1

Photographs demonstrating typical samples of the full range of freeze-dried scaffolds. Percentage cross-linking concentrations refer to proportion of standard cross-linking concentration as defined above. Gross structures were comparable and the variation in shrinkage associated with freeze-drying was insignificant.

Figure 2

Investigation of fibrinogen distribution in scaffolds using immunofluorescence. These representative confocal images are taken after incubation with primary antibodies for both collagen and fibrinogen and associated secondary antibodies.

Figure 3

Investigation of fibrinogen distribution in films using immunofluorescence. Again, representative images of the films are taken after incubation with primary antibodies for both collagen and fibrinogen and associated secondary antibodies.

Figure 4

Characteristic pore structures for lyophilized collagen-fibrinogen scaffolds as imaged directly using SEM ((a) and (c)) and generated from the reconstructed micro-CT data ((b) and (d)). Micro-CT images are at the same magnification as those from SEM. Images (a) and (b) are of a scaffold with 10% cross-linking and 10% fibrinogen, and images (c) and (d) of a scaffold with 100% cross-linking and 50% fibrinogen.

Figure 5

Percent adherence of seeded cells as a function of the percent of fibrinogen in film (a) at 10% cross-linking concentration; and (b) 100% cross-linking concentration; and (c) a comparison of the percent adhesion to bovine serum albumin (BSA) and tissue cultured plastic (TCP) controls at 100% cross-linking concentration. Adhesion is shown in the presence of 5 μM Mg²⁺ or EDTA.

Figure 6

Phase contrast images demonstrating cell spreading on (a) 0% fibrinogen; (b) 50% fibrinogen and controls; (c) tissue cultured plastic and (d) bovine serum albumin. (e) Percentage of spread cells on the various surfaces (***p < 0.0001). All films were cross-linked at 100%. Scale bar = 200 µm.

Figure 7

Spreading of CellTracker-stained C2C12 cells on 100% cross-linked films with increasing fibrinogen concentration compared to the negative control (BSA) and positive control (tissue-culture plastic). Scale bar = 200 μm.

Figure 7

Spreading of CellTracker-stained C2C12 cells on 100% cross-linked films with increasing fibrinogen concentration compared to the negative control (BSA) and positive control (tissue-culture plastic). Scale bar = 200 μm.

Figure 8

Specificity of the antibodies is demonstrated: 100% collagen films are imaged in the presence of both primary and secondary antibodies, and no fibrinogen-associated signal was detected.