Optimisation of UV irradiation as a binding site conserving method for crosslinking collagen-based scaffolds

Abstract

Short wavelength (λ = 254 nm) UV irradiation was evaluated over a range of intensities (0.06 to 0.96 J/cm(2)) as a means of cross-linking collagen- and gelatin-based scaffolds, to tailor their material characteristics whilst retaining biological functionality. Zero-link carbodiimide treatments are commonly applied to collagen-based materials, forming cross-links from carboxylate anions (for example the acidic E of GFOGER) that are an essential part of integrin binding sites on collagen. Cross-linking these amino acids therefore disrupts the bioactivity of collagen. In contrast, UV irradiation forms bonds from less important aromatic tyrosine and phenylalanine residues. We therefore hypothesised that UV cross-linking would not compromise collagen cell reactivity. Here, highly porous (~99 %) isotropic, collagen-based scaffolds were produced via ice-templating. A series of scaffolds (pore diameters ranging from 130-260 μm) with ascending stability in water was made from gelatin, two different sources of collagen I, or blends of these materials. Glucose, known to aid UV crosslinking of collagen, was added to some lower-stability formulations. These scaffolds were exposed to different doses of UV irradiation, and the scaffold morphology, dissolution stability in water, resistance to compression and cell reactivity was assessed. Stabilisation in aqueous media varied with both the nature of the collagen-based material employed and the UV intensity. Scaffolds made from the most stable materials showed the greatest stability after irradiation, although the levels of cross-linking in all cases were relatively low. Scaffolds made from pure collagen from the two different sources showed different optimum levels of irradiation, suggesting altered balance between stabilisation from cross-linking and destabilisation from denaturation. The introduction of glucose into the scaffold enhanced the efficacy of UV cross-linking. Finally, as hypothesized, cell attachment, spreading and proliferation on collagen materials were unaffected by UV cross-linking. UV irradiation may therefore be used to provide relatively low level cross-linking of collagen without loss of biological functionality.

Fig. 1

SEM images of non-XL scaffolds of different compositions before and after UV irradiation with an intensity of 0.42 J/cm²

Fig. 2

Dependence of mass loss (%) over incubation time (days) on UV intensity for different scaffold compositions, a Col(S), b Col(D), c Col(S)–Gel and d Gel. Samples were crosslinked with increasing UV intensity (0—diamond, 0.12—square, 0.42/0.27—triangle, 0.96—circle in J/cm²

Fig. 3

Comparison of the dissolution profiles of Sigma and Devro collagen scaffolds. Mass loss (%) of Sigma and Devro Col UV treated scaffolds after 14 days in water

Fig. 4

Comparison of the influence of EDC treatment with different concentrations and UV irradiation at different intensities (J/cm²) on the free amine group content of Col(S), Col(S)–Gel and Gel scaffolds

Fig. 5

Influence of UV intensity on the compressive modulus (E*) of scaffolds composed of Col(S) (diamond), Col(S)–Gel (square) and Gel (triangle) (a). UV effect on successive compressions of Col(S) UV-irradiated scaffolds (b)

Fig. 6

Percentage of glucose release from scaffolds to water after incubation for 1 and 24 h

Fig. 7

Combined effect of glucose and UV light (0.42 J/cm²) on dissolution resistance of Col(D) (left), Col(D)–Gel (middle) or Gel (right) scaffolds

Fig. 8

Influence of the presence of glucose on the compressive modulus (E*) of mixed scaffolds composed of Col(S) and Col(D)

Fig. 9

Platelet adhesion to Col(S) (left), Col(S)–Gel (middle) or Gel (right) films irradiated with 0, 0.42 or 0.96 J/cm² UV. Platelet adhesion was measured in the presence of 5 mM MgCl₂ (dark grey) or 5 mM EDTA (light grey)

Fig. 10

HT1080 attachment (a) or spreading (b) to Col(S) films irradiated with 0, 0.42 or 0.96 J/cm² UV. Cell attachment was measured in the presence of 5 mM MgCl₂ (dark grey bars) or 5 mM EDTA (light grey bars). Cell spreading was measured in DMEM without additional MgCl₂

Fig. 11

HT1080 cell proliferation (a) and cell coverage (b, c) on Col(S) films irradiated with 0 or 0.42 J/cm² UV. Cells were cultured on the films in DMEM containing 10 % serum. For b and c cells were cultured for 4 days prior to analysis