Crosslinking and composition influence the surface properties, mechanical stiffness and cell reactivity of collagen-based films

Abstract

This study focuses on determining the effect of varying the composition and crosslinking of collagen-based films on their physical properties and interaction with myoblasts. Films composed of collagen or gelatin and crosslinked with a carbodiimide were assessed for their surface roughness and stiffness. These samples are significant because they allow variation of physical properties as well as offering different recognition motifs for cell binding. Cell reactivity was determined by the ability of myoblastic C2C12 and C2C12-α2+ cell lines (with different integrin expression) to adhere to and spread on the films. Significantly, crosslinking reduced the cell reactivity of all films, irrespective of their initial composition, stiffness or roughness. Crosslinking resulted in a dramatic increase in the stiffness of the collagen film and also tended to reduce the roughness of the films (R(q) = 0.417 ± 0.035 μm, E = 31 ± 4.4 MPa). Gelatin films were generally smoother and more compliant than comparable collagen films (R(q) = 7.9 ± 1.5 nm, E = 15 ± 3.1 MPa). The adhesion of α2-positive cells was enhanced relative to the parental C2C12 cells on collagen compared with gelatin films. These results indicate that the detrimental effect of crosslinking on cell response may be due to the altered physical properties of the films as well as a reduction in the number of available cell binding sites. Hence, although crosslinking can be used to enhance the mechanical stiffness and reduce the roughness of films, it reduces their capacity to support cell activity and could potentially limit the effectiveness of the collagen-based films and scaffolds.

Supplementary Figure 1

Supplementary Figure 2

Fig. 1

Tensile testing setup: the load cell is connected to the grips via a pulley system. Both grips and film are fully submerged. Scale bar is 25 mm.

Fig. 2

Effect of composition on film microstructure. FEGSEM images of non-crosslinked collagen and gelatin films.

Fig. 3

Effect of composition and crosslinking on root mean squared roughness (R, μm) of Coll, Coll-Gel and Gel films (nX non-crosslinked, XL crosslinked). Note: ^∗ indicates statistically significant difference in comparison to non-crosslinked sample of same composition, ■ indicates statistically significant difference between compositions for statistical significance of P ⩽ 0.05 in Student’s t-test. The error bars indicate the standard error of the mean.

Fig. 4

Effect of film composition and crosslinking on surface roughness and height as measured by AFM topography images of Coll, Coll-Gel and Gel films (nX non-crosslinked, XL crosslinked).

Fig. 5

Periodic banding structure of collagen fibres within the film as measured from AFM amplitude image: alternating dark and light bands indicate regions of variation of topography and the periodic repeat was measured along the straight line between the two cross-points as 64.4 ± 2.91 nm. The graph shows the change in topography between the two cross-points, and acts as a graphical representation of the banding pattern.

Fig. 6

Effect of composition and crosslinking on the Young’s modulus of films at different strains (5%, 20% and high strain: in the linear region of the stress–strain curve, nX non-crosslinked, XL crosslinked). Non-crosslinked films were weak when hydrated and only Coll nX was able to be tested. Note: ^∗ indicates statistically significant difference in comparison to non-crosslinked sample of same composition, ■ indicates statistically significant difference between compositions for statistical significance of P ⩽ 0.05 in Student’s t-test. The error bars indicate the standard error of the mean.

Fig. 7

Phase contrast images (a, d) and immunofluorescence images showing staining for live (Hoechst, b, e) and dead (Propidium Iodide, c, f) C2C12-α2+ cells after culture for 72 h on gelatin films of different crosslink status. (a–c) Gel nX, (d–f) Gel XL. Scale bar is 100 μm.

Fig. 8

Effect of film composition and crosslinking on live cell count (a), surface coverage (c), cell area (μm², b) and aspect ratio (d), for C2C12-α2+ cells after 72 h culture. Note: ^∗ indicates statistically significant difference in comparison to non-crosslinked sample of same composition, ■ indicates statistically significant difference between collagen-based and gelatin-based films for statistical significance of P ⩽ 0.05 in two-way ANOVA. The error bars indicate the standard error of the mean.

Fig. 9

Effect of film composition and crosslinking on cell count of (a) C2C12-α2+ and (b) C2C12 cells after 72 h culture. Note: ^∗ indicates statistically significant difference in comparison to non-crosslinked sample of same composition, ■ indicates statistically significant difference between cell lines (C2C12 and C2C12-α2+) for statistical significance of P ⩽ 0.05 in two-way ANOVA. The error bars indicate the standard error of the mean.

Fig. 10

Real-time adhesion and spreading of (a) C2C12 and C2C12-α2+ cells to GFOGER peptide (10 μg ml^–1) and (b) C2C12 cells or (c) C2C12-α2+ cells to gelatin (100 μg ml^–1). In (b) and (c) cells were blocked by pre-incubation with cyclic-RGD (c-RGD) or anti-β3 integrin (anti-β3). Incubations were carried out in the presence of 1 mM Mg²⁺, to enhance, or EDTA, to inhibit, the integrin-mediated cell binding. The xCELLigence system measured impedance every 30 s, reported as cell index value. Lines represent mean (standard error measurements not significant) for n = 6. Different conditions are denoted with shaped markers.