The influence of collagen isolation, cross-linking and sterilisation on macrophage response
Delgado, Luis Maria
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Collagen-based devices are frequently associated with foreign body response. Although several pre- (e.g. species, state of animal, tissue, isolation protocol) and post- (e.g. cross-linking, scaffold architecture, sterilisation) scaffold fabrication factors have a profound effect on foreign body response, little is known about how collagen-based devices fabrication mediates macrophage response. In this thesis, we assessed biophysically, biochemically and biologically three different treatments during the fabrication of a collagen type I film: collagen isolation, cross-linking and sterilisation. In the first phase of this thesis, we studied the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Subsequently, as extracted collagen forms are usually subjected to chemical cross-linking to enhance their stability and the traditional cross-linking approaches are associated with toxicity and inflammation, we investigated the stabilisation capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde, carbodiimide, 4-arm polyethylene glycol succinimidyl glutarate, genipin and oleuropein. Therefore, as a step forward and given that there is still no single cross-linking method / sterilisation treatment that can be used universally for collagen-based devices, we assessed the influence of ethylene oxide, ethanol, gamma irradiation and gas plasma sterilisation on the structural, biophysical, biochemical and biological properties of self-assembled collagen films cross-linked with the optimised cross-linking agents from the previous part, 4-arm polyethylene glycol succinimidyl glutarate and genipin. Acetic acid / pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the different extraction groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on collagen films extracted using acetic acid. Moreover, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1β and TNF-α release from macrophage with respect to acetic acid treatments. Salt precipitation did not influence the assessed parameters. Overall, our data suggest that collagen extraction variables affect the physicochemical and biological properties of collagen preparations. Regarding collagen cross-linking, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of glutaraldehyde, THP-1 cultures supplemented with conditioned media from the various groups showed no significant difference between the treatments. No significant difference in secretion of pro-inflammatory (e.g. IL-1β, IL-8, TNF-α) and anti-inflammatory (e.g. VEGF) cytokines was observed between the non-cross-linked and the 4SP and genipin cross-linked groups, suggesting the suitability of these agents as collagen cross-linkers. With regards to collagen sterilisation, our data clearly illustrate that gas plasma is not suitable for collagen-based devices. Ethylene oxide, after 4-arm polyethylene glycol succinimidyl glutarate cross-linking, resulted in significant reduction of the mechanical properties of the collagen films. Gamma irradiation and ethanol sterilisation did not significantly affect thermal, degradation, solubility and mechanical properties of the collagen films. Human skin fibroblast and human macrophage cultures did not reveal any considerable differences as a function of the cross-linking method / sterilisation treatment. Overall, our data illustrate that genipin cross-linking maintains collagen stability even against the most severe sterilisation treatments. Collectively, these data suggest that during collagen scaffold fabrication all processing variables should be monitored as, evidently, they affect collagen stability and biological response.
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