PEG-Plasma Hydrogels Increase Epithelialization Using a Human Ex Vivo Skin Model

Abstract

In vitro cell culture methods are used extensively to study cellular migration, proliferation, and differentiation, which play major roles in wound healing but the results often do not translate to the in vivo environment. One alternative would be to establish an ex vivo model utilizing human discarded skin to evaluate therapies in a more natural setting. The purpose of this study was to institute such a model by creating ‘wounds’ in the center of a piece of discarded skin and treating them with three different biomaterials: collagen, polyethylene glycol (PEG)-fibrin, or PEG-platelet free plasma (PFP). Explants were cultured for 14 days with supernatant and microscopy images collected every 3 days to assess cytotoxicity and epithelialization. After 14 days, the explants were fixed, sectioned, and stained for cytokeratin-10 (CK-10), alpha-smooth muscle actin (α-SMA), and wheat germ (WG). Compared to controls, similar levels of cytotoxicity were detected for 12 days which decreased slightly at day 14. The PEG-PFP hydrogel-treated wounds epithelialized faster than other treatments at days 6 to 14. A 6-8 cell layer thick CK-10+ stratified epidermis had developed over the PEG-PFP hydrogel and cells co-stained by WG and α-SMA were observed within the hydrogel. An ex vivo model was established that can be used practically to screen different therapies exploring wound healing.

Effect of Fibrin Concentration on the In Vitro Production of Dermo-Epidermal Equivalents

“…Although PRP has shown its potency and capability in tissue regeneration, its experimental inconsistency and low reproducibility has meant that, in applications in which platelet-released growth factors are not crucial, PPP is generally used. Additionally, since PPP is cost-effective, suitable for bulk production, and easily translatable with minimal regulatory requirements for FDA approval, it is widely used in tissue regeneration [22,[40][41][42][43].…”

“…However, we found several persisting issues associated with the low final concentration of fibrin (1.2 mg/mL) used in the plasma derived-fibrin hydrogels when they are placed in transwell inserts for in vitro applications: (1) their height is reduced by 30% during the first 24 h and by 70% after 21 days in culture [17]; and (2) they suffer rapid degradation due to the skin cells present in the culture, limiting their lifespan (usually to 17 days) [15,44]. Several strategies have been proposed to overcome the limitations of plasma-derived fibrin-based hydrogels in skin tissue engineering, for example, combining fibrin (blood plasma-derived) with other molecules such as PEG or agarose polymers [40][41][42]50,51] or the use of highly concentrated commercial fibrinogen. In our experience, the use of commercial fibrinogen in organotypic skin cultures produced worse keratinocyte proliferative and differentiation behavior in comparison with PPP-hydrogel cultures.…”

Elastin-Plasma Hybrid Hydrogels for Skin Tissue Engineering

“…However, current drawbacks of these types of hydrogel lies not only in their moderate tensile strength, but they also suffer a rapid degradation and contraction over time, limiting their reproducibility and lifespan [ 18 , 19 , 20 ]. PEGylation of the fibrinogen contained in human plasma or the combination with natural polymeric networks, such as alginate or agarose [ 21 , 22 , 23 , 24 , 25 , 26 ], have been utilized to overcome the aforementioned limitations. Another promising strategy could be the preparation of interpenetrating polymer network (IPN) hydrogels based on plasma-derived fibrin and extracellular matrix (ECM) components.…”