Full Skin Equivalent Models for Simulation of Burn Wound Healing, Exploring Skin Regeneration and Cytokine Response

Abstract

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm^® and Mucomaix^®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Keywords: burn injury; cytokine response; fibroblasts; in vitro model; keratinocytes; skin morphogenesis; tissue engineering; wound repair.

Figure 1

Experiment scheme showing the timing of the performed steps.

Figure 2

Evaluation of skin development of cultured skin models. Images of ex vivo human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm, and Mucomaix (right) at T0 or T + 2 weeks. (A) Macroscopy and H&E staining; (B) Immunohistochemical pan-cytokeratin staining. Models were produced from 3 different skin donors in duplicate. For the full skin equivalent models, T0 was after the initial 3 weeks of culture. Black scale bar = 100 µm.

Figure 3

Evaluation of cytokeratin 15, collagen IV, laminin α 5, and vimentin expression in cultured skin models. Images of ex vivo human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm, and Mucomaix (right). Immunohistochemical (A) cytokeratin 15, (B) collagen IV, (C) laminin α 5, and (D) vimentin DAB staining. Models were produced from 3 different skin donors in duplicate. For the full skin equivalent models, T0 was after the initial 3 weeks of culture. Black scale bar = 100 µm.

Figure 4

Effect of burn injury on cultured skin models. Images of ex vivo human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm, and Mucomaix (right). Models were produced from 3 different skin donors in duplicate. For the full skin equivalent models, T0 was after the initial 3 weeks of culture. Black scale bar = 100 µm.

Figure 5

Evaluation of proliferation and re-epithelization in burn-injured skin models. Images of ex vivo human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm, and Mucomaix (right) at T0 and T + 2 weeks after burn. Immunohistochemical (A) Ki67; (B) BrdU DAB staining. Because the culture of ex vivo human skin models started at T0 and BrdU needed to be added 24 h prior to termination of the models, no BrdU was present in these models at T0. (C) Length of re-epithelization after burn injury at T + 1 week and T + 2 weeks after burn (diamonds represent the mean per model, and squares represent the mean of all models). Models were produced from 3 different skin donors in duplicate. For the full skin equivalent models, T0 was after the initial 3 weeks of culture. Black scale bar = 100 µm. Statistically significant differences are indicated by asterisks: *: p < 0.05.

Figure 6

Cytokines detected in medium of burn-injured and uninjured skin models. Ex vivo human skin (left) and full skin equivalents generated from de-epidermalized dermis, MatriDerm, and Mucomaix (right). Level of (A) IL-6; (B) IL-8; (C) MCP-1; (D) IL-1β; (E) IL-10; (F) IP-10 in the culture medium at T0, T + 1–4 days, T + 5–7 days, and T + 8–11 days (after burn injury). Samples from biological duplicates were pooled per donor (n = 3 donors) and re-calculated into pg/mL per day of culture to compensate for differences in intervals of medium changes. Striped line indicates the highest or lowest level of quantification. Because ex vivo human skin models were started on the first day, no levels are shown for day 0.