The process of EDC-NHS Cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

Abstract

We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was considered. Atomic Force Microscopy (AFM) and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clear fibrillar structure comparable to native collagen was observed which was not observed in the non-cross-linked fibre. The amide III doublet in the Raman spectra provided additional evidence of alignment in the cross-linked fibres. Raman spectroscopy also indicated no residual polyethylene glycol (from the fibre forming buffer) or water in any of the fibres.

Figure 1. SEM imaging of extruded collagen fibres: (a,b) with no cross-linking, (c,d) with EDC-NHS cross-linking

Figure 2. AFM image of: (a, b) fibres without cross-linking, (c, d) with the EDC-NHS treatment

Figure 3. Raman spectra of the collagen fibres.

The amide doublet is clearly marked. The doublet appears more prominent where cross-linking has been applied and the peak at approximately 1270cm⁻¹ absent where the fibre axis is parallel to the laser polaristion direction.

Figure 4. Investigation of residual PEG (a) and significant water content using Raman spectroscopy ((b) and (c)).

None of the fibres demonstrated the characteristic PEG peak at a wavenumber of around 2900 cm⁻¹ [31]. There were also not significant peaks present attributable to the δ or ν modes of water absorption at 1640 or 3280 cm⁻¹ [32].

Figure 5. SEM image of a section of Kew et al’s synthetic collagen fascicle cross-linked with EDC/NHS.

This single fibre length was produced by a multiple fibre overlayer process where the individual strands are clearly differentiated. Reproduced from [11]