Effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide concentrations on the mechanical and biological characteristics of cross-linked collagen fibres for tendon repair

Abstract

Reconstituted type I collagen fibres have received considerable interest as tendon implant materials due to their chemical and structural similarity to the native tissue. Fibres produced through a semi-continuous extrusion process were cross-linked with different concentrations of the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS). Tensile properties of the fibres were considered, along with imaging of both surface structure and fibrillar alignment. Resistance of the fibres to bacterial collagenase was investigated and fibre sections seeded with human tendon cells for biological characterization, including cell adhesion and proliferation. The work clearly demonstrated that whilst the concentration of EDC and NHS had no significant effect on the mechanics, a higher concentration was associated with higher collagenase resistance, but also provided a less attractive surface for cell adhesion and proliferation. A lower cross-linking concentration offered a more biocompatible material without reduction in mechanics and with a potentially more optimal degradability.

Keywords: biopolymer; scaffolds; soft tissue.

Figure 1

Extrusion of collagen fibres: collagen slurry drawn into two syringes and using a vertically oriented syringe pump (a), the slurry extruded through a three channel manifold system (b), to form six channels flowing along the length of the FFB bath (c). The six strands are then collected together at the end of the bath and wound onto a spool (d). Fibre bundles are allowed to dry overnight on the spools (e) before cross-linking and/or washing.

Figure 2

Effect of cross-linking on single fibre mechanics: (a) ultimate tensile strength, (b) strain to failure, (c) tensile modulus defined at 3-5% strain (n = 5-7; mean ± SD). No statistically significant difference was observed with cross-linking concentration on any of the three parameters considered.

Figure 3

SEM images of sections of extruded collagen fibre: (a, b) without any cross-linking and (c, d) with the standard 25 mM EDC and 12.5 mM NHS cross-linking.

Figure 4

AFM images of collagen fibre sections: (a) without cross-linking, (b) 1/100 cross-linking concentration, (c) standard cross-linking concentration.

Figure 5

(a) number of human cells adherent to fibres with the different cross-linking concentrations after 24 h (n = 3; mean ± SD); (b, c) SEM images of tenocytes adherent to fibres with standard cross-linking and 1/100 cross-linking, respectively; (d) higher magnification image of tenocytes on fibre with the lowest cross-linking demonstrating the flattened morphology and cellular processes.

Figure 6

Amount of DNA extracted from human tenocytes seeded on fibres of the various cross-linking concentrations (n = 3; mean ± SD). Data for fibres without cross-linking are also included on the chart.