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

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 zerolength 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.

Targeted protein delivery:carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds

Tissue engineering response may be tailored via controlled,sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional(3D)ice-templated collagen scaffolds.However,the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored.Here,we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide)microparticles.We probe the effects of subsequent N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride crosslinking on protein release,using microparticles with different internal protein distributions.Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug.The scaffolds display a homogeneous microparticle distribution,and a reduction in pore size and percolation diameter with increased microparticle addition,although these values did not fall below those reported as necessary for cell invasion.The protein distribution within the microparticles,near the surface or more deeply located within the microparticles,was important in determining the release profile and effect of crosslinking,as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold.Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release.Protein located within the bulk of the microparticles,was protected from the crosslinking reaction and no delay in the overall release profile was seen.

Avoiding artefacts in MicroCT imaging of collagen scaffolds:Effect of phosphotungstic acid(PTA)-staining and crosslink density

X-ray micro-computed tomography(μ-CT)can be used to provide both qualitative and quantitative information on the structure of three-dimensional(3D)bioactive scaffolds.When performed in a dry state,μ-CT accurately reflects the structure of collagen-based scaffolds,but imaging in a wet state offers challenges with radiolucency.Here we have used phosphotungstic acid(PTA)as a contrast agent to visualise fully hydrated collagen scaffolds in a physiologically relevant environment.A systematic investigation was performed to understand the effects of PTA on the results of μ-CT imaging by varying sample processing variables such as crosslinking density,hydration medium and staining duration.Immersing samples in 0.3% PTA solution overnight completely stained the samples and the treatment provided a successful route forμ-CT analysis of crosslinked samples.However,significant structural artefacts were observed for samples which were either non-crosslinked or had low levels of crosslinking,which had a heterogeneous interior architecture with collapsed pores at the scaffold periphery.This work highlights the importance of optimising the choice of processing and staining conditions to ensure accurate visualisation for hydrated 3D collagen scaffolds in an aqueous medium.