Objectives: For many years, a three-dimensional (3D) collagen hydrogel has been used for embedding the fibroblasts to provide them a physiological 3D environment. However, the stiffness of tissue-engineered skin analogues based on collagen hydrogels is insufficient. In order to improve the mechanical properties of a two-layer skin construct, we stabilized the collagen hydrogel by fibrin-modified nanofibrous membrane.
Methods: Nanofibrous polylactid acid (PLA) membranes were modified with fibrin homogenous mesh. Fibrin-modified membranes were seeded with human dermal fibroblasts or adipose-derived stem cells (ASCs). After few days, collagen hydrogel was applied on the surface of fibrin-modified membranes preseeded with fibroblasts or stem cells and then primary keratinocytes were seeded on the surface of collagen hydrogel. Cell proliferation, migration and morphology were evaluated by MTS assay and by microscopy.
Results: We observed that the fibrin homogenous mesh enhanced the attachment and growth of fibroblasts and ASCs and additionally it supported their migration upwards into the collagen hydrogel. On top of that, the contraction and shrinkage of the collagen hydrogels by the traction forces of fibroblasts were minimalized by their gradual migration from membrane into the hydrogel. The keratinocytes on the top of the collagen construct formed a basal layer of highly dividing cells, and a suprabasal layer.
Conclusions: The construction of full-thickness skin substitutes that are similar to natural tissue is a promising approach to deep skin wound repairs. We can conclude that the collagen hydrogel reinforced by synthetic membrane provides a suitable microenvironment for the skin cells with sufficient mechanical properties for possible clinical applications.

1 - A full-thickness skin construct made of a collagen hydrogel strengthened by a fibrin-modified nanofibrous membrane