In vitro 3D full-thickness skin-equivalent tissue model using silk and collagen biomaterials

Abstract

Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.

Figure 1

Experimental outline for tri-layer culture. Skin and adipose tissues were cultured independently prior to being combined at ‘day 0’. At day -21, endothelial cells are seeded onto silk sponges, while adipose derived stem cells undergo adipogenic differentiation in 2D. At day 14, the adipocyte-like cells are added to the endothelial seeded silk sponges and the co-cultures were maintained in a 1:1 adipogenic:endothelial cell media. Also at day 14, fibroblasts are cast within a collagen gel. After the cell contracts, keratinocytes are seeded on top of the collagen gel. After epithelialization has occurred the construct was raised to the air-liquid interface for 3 days. On day 0, the 2 constructs were combined with a thin layer of collagen gel as a binder. At this point the tri-layer construct was cultured in a 1:1 mix of the co-culture media to skin media.

Figure 2

H&E images of positive controls for skin and adipose constructs after 2 and 8 days in culture. Skin constructs demonstrated a well-organized, multilayer epithelium by day 2 (a) that demonstrated all 4 morphological layers and a collagen lattice in which resident fibroblasts showed their characteristic spindled morphology. This epithelium was further developed by day 8 (c), where it showed a columnar basal cell layer and more complete development of the spinous cell layer. By day 8, cells populated the entire silk sponge in the adipose construct. Images are representative of n=2 per group.

Figure 3

Constructs cultured in 1:1 skin: adipose media had more physiologically relevant features. H&E images of tri-layer constructs in different media after 2 and 8 days in culture. Images are representative of n=2 per group.

Figure 4

Tri-layer constructs were responsive to various drugs. Rosiglitizone (Rosi), an activator of the adipogenic program, caused hyper-proliferation (bracket) at the basal layer of the epidermis at 9 days. TGF-β, also caused hyper-proliferation, to a lesser extent than the Rosi group, at the basal layer of the epidermis at 9 days. The adipose regions did not appear affected, morphologically, by the drug treatments. Images are representative of n=2 per group.