The influence of collagen type I source and cross-linking on cell function and phenotype maintenance
Date
2023-09-13Author
Sorushanova, Anna
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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.