Introduction: Over the past several years, the generation of a human plasma-derived fibrin bilayer (including dermis and epidermis) skin model by our group has been applied successfully to treat burns, traumatic and surgical wounds[1]. However, limitations of plasma-derived fibrin hydrogels such as low mechanical properties[2], high degradation rates and shrinking during in vitro culture or implantation necessitate the development of new approaches to address these needs. The combination of plasma-derived fibrin with other natural polymers can be considered an interesting possibility for improving the hydrogel mechanical properties without compromising biocompatibility. Hyaluronic acid (HA) is a natural polymer which is present in the extracellular matrix (ECM) of the skin (estimated 0.12-0.3% w/v) and it is also a contributor of dermal turgor. In the present work, we incorporated a thiolated form of HA crosslinked with poly(ethylene glycol) diacrylate (PEGDA) in a well-established protocol for producing plasma-derived fibrin dermal equivalents to improve their biological and mechanical properties.
Materials and Methods: Plasma-derived fibrin hydrogels were prepared following the protocol described previously by our group1. In this sense, 883 μl of human plasma (2.21 mg/ml of fibrinogen) was diluted in 600 μl of saline solution (0.9 % w/v). For crosslinking, 130 μl of a CaCl2 (1% w/v) solution was added. Then the protocol was modified to incorporate thiolated HA and PEGDA at concentrations of 0.1-0.2% and 0-0.05% (w/v) respectively, while concentrations of the rest of the reagents were kept constant in all hydrogels to comply with the above mentioned protocol. Formation of gels and gelation times were assessed using flip-flop method and rheology measurements. The swelling/deswelling studies of HA-fibrin hydrogels were performed. Human fibroblasts were embedded in the hydrogels following the same protocol and toxicity tests were performed. The capacity of the human fibroblasts to contract the hydrogels was also studied at 0, 3, 7 and 10 days.
Results and Discussion: Introduction of HA and PEGDA in fibrin hydrogels increased the gelation time to 16-35 minutes (with the exception of HA 0.2 % and PEGDA 0.05%) due to possible interactions between HA, PEGDA and fibrinogen respectively (Figure1). It was observed that HA-fibrin hydrogels retained shape and bulk integrity in contrast to fibrin hydrogels. Fibrin gels were confirmed to have strong contractile behaviour, while HA-fibrin hydrogels inhibited fibrin contraction (Figure2). Introduction of HA and PEGDA in fibrin gels did not affect the viability of the human fibroblasts. All hydrogels with human fibroblasts embedded contracted after 10 days, but HA-fibrin gels contracted significantly less than control fibrin gels. HA-fibrin gels with PEGDA enhanced very significantly fibroblast proliferation compared with fibrin hydrogels and HA-fibrin hydrogels without PEGDA at day 7 (Figure3).
Conclusion: In this work, it was demonstrated that the incorporation of HA and PEGDA to plasma-derived fibrin hydrogels increased the mechanical properties and inhibited the fibrin hydrogel contraction with and without cells. It was also confirmed a higher proliferation of human fibroblasts when using HA-fibrin hydrogels with PEGDA. Further in vitro and in vivo experiments are required to assess the quality of the engineered skin.
The authors acknowledge financial support of this work to the Universidad Carlos III de Madrid, and the plasma supply by the Centro Comunitario de Sangre y Tejidos in Asturias
References:
[1] Llames S.G., et al.Transplantation. (2004); 77:350-355.
[2] Haugh, M.G., et al. J. Mech. Behav. Biomed. Mater. (2012); 16:66-72.