Replicating and modulating skin fibrosis in vitro: Multi-compartment collagen devices as dual drug delivery vehicles

Complex pathologies, such as fibrosis, are often not the result of a singular event, but rather the consequence of multiple disruptions in the normal balance between intricately connected organs, tissues and signalling pathways. This imposes a burden on global healthcare systems, affecting millions of patients every year, with estimates indicating that, in the USA alone, approximately 45 % of non-accident-related deaths are attributable to chronic fibro-proliferative diseases. In the case of skin fibrosis, wounding or pathological triggers can lead to alterations in the normal wound healing cycle, which in turn can result in chronic inflammation, excessive fibroblast activation, formation of excessive fibrous connective tissue and aberrant scarring. The alteration of the dermis’ architecture then takes place, potentially compromising the skin’s function and aesthetic, causing a lasting impact on the quality of life of the patient. Given its complexity, conventional single treatments often fail to achieve reliable therapeutic efficacy, and as such, a synergistic approach aiming to modulate multiple targets appears to be the most promising form of successful treatment for skin fibrosis. As such, it is paramount to develop multi-compartment systems capable of delivering multiple bioactive agents in a controlled, safe and cost-effective way. Some technologies already succeed in doing this, having shown superiority over traditional administration approaches with respect to pharmacokinetics and distribution profiles, accumulation ratios, circulation times and target selectivity. However, they often involve the use of more than one type of material, which can raise concerns regarding potential cytotoxicity and host immune response, controlled release and increase the cost of production. Single material multi-compartment systems from widely used biomaterials (e.g. collagen) seem to be a promising alternative, as the risk for immune reactions from the patient should be smaller, while production costs can be reduced, and regulatory approval can be streamlined. Herein, an in vitro model of skin fibrosis using macromolecular crowding and transforming growth factor β1 (TGFβ1) supplementation was developed for drug screening, which was then used to assess the therapeutic efficacy of a multi-compartment, crosslinked collagen type I hydrogel system loaded with two potential anti-fibrotics. Initially, isoelectric focusing was assessed as a potential fabrication method of anisotropic multi-compartment collagen type I fibres. Despite initial promising results, where it was possible to obtain crosslinked collagen type I fibres with a high degree of alignment, several problems with reproducibility were encountered, which compromised its use. The in vitro model of fibrosis was developed by promoting the activation of primary dermal fibroblasts into myofibroblasts and inducing a fibrotic phenotype for drug screening of potential anti-fibrotics through supplementation with macromolecular crowding and TGFβ1. This resulted in an increase of the expression of α-smooth muscle actin and deposition of collagen type I (hallmarks of fibrosis), thus validating the model. A wide range of anti-fibrotic compounds (triamcinolone acetonide: corticosteroid; trichostatin A: inhibitor of histone deacetylases; serelaxin, pirfenidone: pleiotropic inhibitors of fibrotic activation; activin IIB receptor inhibitor and soluble TGFβ receptor traps: inhibitors of TGFβ signalling; and β-aminopropionitrile: inhibitor of lysyl oxidase activity/collagen crosslinking) were then assessed via quantification of collagen type I deposition inhibition. Data obtained demonstrated that trichostatin A, serelaxin, pirfenidone and soluble TGFβ receptor trap T122bt resulted in decreased collagen type I deposition, illustrating their anti-fibrotic potential. Afterwards, this model would then be used to test the multi-compartment system, obtained by fabricating a core-shell collagen type I hydrogel with two compartments through differential crosslinking with 4 arm polyethylene glycol succinimidyl glutarate and loaded with two of the best performing anti-fibrotics, trichostatin A and a TGFβ trap, T122bt. It was observed that the produced hydrogel systems possessed adequate biophysical and biological properties and were effective at delivering the encapsulated biomolecules to the in vitro model. This resulted in a decrease of collagen I deposition and α-smooth muscle actin expression, while mitigating the negative effect on cell proliferation and cell viability observed after treatment with the free forms of the used therapeutic agents, illustrating the potential of this technology in the treatment of skin fibrosis. Summarily, these results indicate that the produced in vitro model represents a robust screening tool for future drug testing and development as it successfully replicates the main hallmarks of a fibrotic condition. Furthermore, treatment with the core-shell hydrogel system dual loaded with two anti-fibrotics proved to have a therapeutic effect in the in vitro fibrotic model, paired with reduced negative side effects when compared to the free administration of the anti-fibrotic drugs. Altogether, these results illustrate the therapeutic potential of these systems in the treatment and prevention of abnormal scarring.2024-01-1