The influence of collagen type I source and cross-linking on cell function and phenotype maintenance
Sorushanova, Anna
Sorushanova, Anna
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Publication Date
2023-09-13
Type
Thesis
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Abstract
Collagen is a complex supramolecular structure that occurs in highly diverse morphologies across different tissues, lending them a range of physical and biological functions. Collagens have a long history in both evolution and biotechnology and continue to offer both challenges and exciting opportunities in biomedicine as nature’s biomaterial of choice. Despite the significant advancement in the development of collagen-based devices, clinical data clearly demonstrate an inconsistent therapeutic efficiency, even when collagen devices are used that were produced from collagen extracted from the same species, using the same extraction protocol. These observations clearly illustrate that there are other factors at play, when one considers collagen as a raw material for medical device development. To this end, herein the properties of collagen-derived biomaterials and their effect on the behaviour and phenotype of permanently differentiated cells (human adult dermal fibroblasts and human tenocytes) as a function of collagen origin (e.g. species, tissue, gender) and cross-linking type [e.g. 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate, glutaraldehyde, carbodiimide] were investigated. Although collagen type I is extensively used in biomedicine, no study to-date has assessed how the properties of the produced scaffolds are affected as a function of species, gender and tissue from which the collagen was extracted. Herein, collagen from porcine and bovine, male and female and skin and tendon tissues was extracted and characterised and subsequently collagen sponges were fabricated and their structural, biophysical, biochemical and biological properties were assessed. All collagen preparations were of similar purity and free amine content. In general, the porcine groups yielded more collagen; had higher denaturation temperature and resistance to enzymatic degradation; and lower swelling ratio and compression stress and modulus than the bovine groups of the same gender and tissue. All collagen preparations supported growth of human dermal fibroblasts and exhibited similar biological response to human THP-1 monocytes. These results further illustrate the need for standardisation of collagen preparations for the development of reproducible collagen-based devices. Recent data suggest that collagen retains memory from the tissue that derives from and therefore affecting the properties of the produced devices. With this in mind, collagen (from bovine skin and tendon tissues) sponges were fabricated with different crosslinking densities of 4-arm polyethylene glycol succinimidyl glutarate and their physicochemical and biological properties were assessed. Structural analysis revealed that crosslinking significantly reduced % porosity of both skin- and tendon- derived collagen sponges. In general, as the crosslinking density was increased, the resistance to enzymatic degradation, denaturation temperature, compressive stress and compressive modulus were significantly increased and the free amine content, % swelling and cytocompatibility (using human dermal fibroblasts) were significantly reduced. The tendon-derived collagen scaffolds exhibited significantly higher compressive stress and compressive modulus values and induced significantly higher human tenocyte DNA concentration and metabolic activity than the skin-derived collagen scaffolds. In human tenocyte cultures at day 14, the 1 mM 4-arm polyethylene glycol succinimidyl glutarate tendon-derived collagen sponges induced significantly higher collagen type III synthesis (as expected at early stages of physiological tendon healing) and downregulated actin alpha 2 (associated with myofibroblast differentiation) and the skin-derived collagen sponges induced significantly higher collagen type IV synthesis (found primarily at the dermal-epidermal junction) and upregulated prolyl 4-hydroxylase subunit alpha-1 (associated with collagen biosynthesis and constitutes a target for antifibrotic compounds). Data obtained indicate that the tissue from which collagen is extracted should be considered in the development of medical devices. Various chemical, natural, or synthetic in origin, crosslinking methods have been proposed over the years to stabilise collagen fibres. However, an optimal method has yet to be identified. Herein, the potential of 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate, as opposed to glutaraldehyde and carbodiimide, on the structural, physical and biological properties of collagen fibres was assessed. The 0.0475 mM 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate induced an intermedium surface smoothness, denaturation temperature and swelling. The 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate fibres had significantly higher stress at break values than the carbodiimide fibres, but significantly lower than the glutaraldehyde fibres. With respect to strain at break, no significant difference was observed among the crosslinking treatments. 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate of 1 mM significantly reduced the amount of free amines and significantly increased resistance to degradation and denaturation temperature. Moreover, mechanical properties of 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate collagen fibres were significantly higher compared to glutaraldehyde, independent of concentration. The 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate fibres exhibited significantly higher cell metabolic activity and DNA concentration that all other crosslinking treatments, promoted consistently cellular elongation along the longitudinal fibre axis and by day 7 they were completely covered by cells. This work clearly demonstrates the potential of 4-star poly(ethylene glycol) ether tetrasuccinimidyl glutarate as collagen crosslinker. Collectively, this work provides further knowledge on the importance of collagen source and cross-linker type and concentration for the development and use of collagen-derived biomaterials.
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Publisher
NUI Galway