Hyaluronic acid-fibrin hydrogels show improved mechanical stability in dermo-epidermal skin substitutes

Human plasma-derived bilayered skin substitutes have been successfully used by our group in different skin tissue engineering applications. However, several issues associated with their poor mechanical properties were observed, and they often resulted in rapid contraction and degradation. In this sense, hydrogels composed of plasma-derived fibrin and thiolated-hyaluronic acid (HA-SH, 0.05-0.2% w/v) crosslinked with poly(ethylene glycol) diacrylate (PEGDA, 2:1, 6:1, 10:1 and 14:1 mol of thiol to moles of acrylate) were developed to reduce the shrinking rates and enhance the mechanical properties of the plasma-derived matrices. Plasma/HA-SH-PEGDA hydrogels showed a decrease in the contraction behaviour ranging from 5% to 25% and an increase in Young's modulus. Furthermore, the results showed that a minimal amount of the added HA-SH was able to escape the plasma/HA-SH-PEGDA hydrogels after incubation in PBS. The results showed that the increase in rigidity of the matrices as well as the absence of adhesion cellular moieties in the second network of HA-SH/PEGDA, resulted in a decrease in contraction in the presence of the encapsulated primary human fibroblasts (hFBs), which may have been related to an overall decrease in proliferation of hFBs found for all hydrogels after 7 days with respect to the plasma control. The metabolic activity of hFB returned to the control levels at 14 days except for the 2:1 PEGDA crosslinking ratio. The metabolic activity of primary human keratinocytes (hKCs) seeded on the hydrogels showed a decrease when high amounts of HA-SH and PEGDA crosslinker were incorporated. Organotypic skins formed in vitro after 21 days with plasma/HA-SH-PEGDA hydrogels with an HA content of 0.05% w/v and a 2:1 crosslinking ratio were up to three times thicker than the plasma controls, evidencing a reduction in contraction, while they also showed better and more homogeneous keratin 10 (K10) expression in the supra-basal layer of the epidermis. Furthermore, filaggrin expression showed the formation of an enhanced stratum corneum for the constructs containing HA. These promising results indicate the potential of using these biomimetic hydrogels as in vitro skin models for pharmaceutical products and cosmetics and future work will elucidate their potential functionality for clinical treatment.