Generation of a Fibrin Based Three-Layered Skin Substitute

Abstract

A variety of skin substitutes that restore epidermal and dermal structures are currently available on the market. However, the main focus in research and clinical application lies on dermal and epidermal substitutes whereas the development of a subcutaneous replacement (hypodermis) is often disregarded. In this study we used fibrin sealant as hydrogel scaffold to generate a three-layered skin substitute. For the hypodermal layer adipose-derived stem cells (ASCs) and mature adipocytes were embedded in the fibrin hydrogel and were combined with another fibrin clot with fibroblasts for the construction of the dermal layer. Keratinocytes were added on top of the two-layered construct to form the epidermal layer. The three-layered construct was cultivated for up to 3 weeks. Our results show that ASCs and fibroblasts were viable, proliferated normally, and showed physiological morphology in the skin substitute. ASCs were able to differentiate into mature adipocytes during the course of four weeks and showed morphological resemblance to native adipose tissue. On the surface keratinocytes formed an epithelial-like layer. For the first time we were able to generate a three-layered skin substitute based on a fibrin hydrogel not only serving as a dermal and epidermal substitute but also including the hypodermis.

Integration of Mature Adipocytes to Build-Up a Functional Three-Layered Full-Skin Equivalent

“…Piccinno et al (2013) and Kober et al (2015) have shown that ASCs enhance adipocyte cell survival. 31 , 32 …”

Construction of a dermis–fat composite in vivo: Optimizing heterogeneous acellular dermal matrix with in vitro pretreatment

“…Previous studies have mainly used single hydrogel‐type materials as scaffolds such as a collagen–elastin matrix (Keck et al, ), fibrin hydrogel (Kober, Gugerell, Schmid, Kamolz, & Keck, ), human plasma hydrogel (Monfort, Soriano‐Navarro, García‐Verdugo, & Izeta, ), and collagen type I hydrogel (Birgit et al, ) to construct a dermis–fat composite. ADSCs cell sheets have been used to construct a three‐layer skin substitute containing subcutaneous fat in vitro without application of scaffold materials (Vidal et al, ); in another study, the dermis and hypodermis were constructed using a silk–collagen hydrogel and silk sponge respectively, and the two layers were then overlapped to form a full‐thickness skin equivalent (Bellas, Seiberg, Garlick, & Kaplan, ).…”

Biomaterials for Skin Substitutes

“…Vascularization seen in the scaffold is reported to be implemented by enhancing VEGF secretion and promoting vascular endothelial cell migration to the scaffold or differentiating residing stem cells to a vascular phenotype and developing a microvascular network . Fibrin scaffolds and gels have been seeded with both HDF and keratinocytes (capable of forming stratified epithelium with early expression of basement membrane proteins) as well as stem cells (while preserving their differentiation capacity) . Fibrin has also been utilized to prevascularize a dermo‐epidermal skin graft, forming blood and lymphatic capillaries in vitro …”

“…Fibrin may have the most beneficial role by acting as a keratinocyte cell delivery scaffold to develop the epidermal layer at the graft site, as fibrin has been shown to be important in epithelialization of the wound through facilitating keratinocyte migration . Its presence in the scaffold as an additive to other biomaterials can harness its specific capacity to promote vascularization in the scaffold.…”