Development of a vascularized skin construct using adipose-derived stem cells from debrided burned skin

Abstract

Large body surface area burns pose significant therapeutic challenges. Clinically, the extent and depth of burn injury may mandate the use of allograft for temporary wound coverage while autografts are serially harvested from the same donor areas. The paucity of donor sites in patients with burns involving large surface areas highlights the need for better skin substitutes that can achieve early and complete coverage and retain normal skin durability with minimal donor requirements. We have isolated autologous stem cells from the adipose layer of surgically debrided burned skin (dsASCs), using a point-of-care stem cell isolation device. These cells, in a collagen-polyethylene glycol fibrin-based bilayer hydrogel, differentiate into an epithelial layer, a vascularized dermal layer, and a hypodermal layer. All-trans-retinoic acid and fenofibrate were used to differentiate dsASCs into epithelial-like cells. Immunocytochemical analysis showed a matrix- and time-dependent change in the expression of stromal, vascular, and epithelial cell markers. These results indicate that stem cells isolated from debrided skin can be used as a single autologous cell source to develop a vascularized skin construct without culture expansion or addition of exogenous growth factors. This technique may provide an alternative approach for cutaneous coverage after extensive burn injuries.

Figure 1

(a) Burn incidence superimposed onto the concurrent use of skin allograft in relation to TBSA. (b) Number of operations performed during the acute burn hospitalization as a function of TBSA. TBSA: total body surface area; CPA: cryo-preserved allograft.

Figure 2

(a) Total number of discarded skin samples collected per age group for the patient population included in this study. (b) Discarded burn tissue. (c) Hematoxylin and eosin staining of burn tissue showing the loss of epidermal and dermal layers of the discarded burn skin. The inset figure is the magnified field of the section showing presence of collapsed blood vessel within the hypodermal layer (asterisks). (d) Masson's trichrome stained adipose tissue sections of discarded burn tissue showing collapsed and viable hypodermis (arrows). The figure inset is the magnified field view of the viable hypodermis (asterisks). Original magnification: ×100 (c, d) and ×400 (figure insets).

Figure 3

(a) Discarded burn skin. (b–d) Immunohistochemical images of section from discarded skin biopsies: (b) stained with PDGFRβ (Alexa fluor 488), (c) Hoechst for nuclei, and (d) overlay of PDGFRβ and Hoechst. (e) Stem cell isolation process from the adipose tissue layer of discarded burn skin (dsASCs) using the Transpose RTtissue processing system. (f) Fluorescent-activated cell sorting (FACS) analysis of P1 dsASCs stained with phycoerythrin-labeled PDGFRβ. (g–i) Immunocytochemical images of P1 dsASCs: (g) stained with PDGFRβ (Alexafluor 594), (h) Hoechst for nuclei, and (i) overlay of PDGFRβ and Hoechst. Original magnification: ×200 (b–d and dsASCs in (e)) and ×600 (g–i).

Figure 4

Development of different layers of skin substitute using dsASCs and hydrogel-based matrices. Epithelial and hypodermal constructs are developed using collagen hydrogel and the vascularized dermal construct using collagen-PEGylated-fibrin-based bilayered hydrogel.

Figure 5

(a and b) Differentiation time course of dsASCs into epithelial-like cells on a collagen hydrogel showing (a) squamous cell-like morphology by day 4 and (b) epithelial-like cuboidal cell morphology by day 12. (c) Immunocytochemical image of section from dsASCs differentiated into epithelial-like cells on collagen gel (day 12) stained with pan cytokeratin. (d, e, g, and h) Differentiation time course of dsASCs in PEGylated-fibrin-collagen bilayer gels exhibiting bidirectional differentiation into fibroblast-like morphology in collagen layer (d; day 6) and tubular structures (g; day 6) in the PEGylated-fibrin layer. By day 12, collagen layers showed an increase in fibroblast-like cells (e) and complex networks in PEGylated-fibrin layers (h). Immunocytochemical image of section from bilayered hydrogel (day 12) depicting dsASCs to maintain stromal phenotype with collagen layer, stained with α-smooth muscle actin (f) and differentiated to vascular phenotype, stained with NG2 (i). (j and k) Differentiation time course of dsASCs into adipocytes on collagen showing appearance of oil vesicles by day 7 (j) and significant accumulation of oil droplets over time (k: day 14). (l) Oil Red O. staining of differentiated dsASCs (day 14) confirming formation of mature adipocytes within collagen gel. Bright field images original magnification: ×100 (a–h and l) and ×200 (j, k). Immunofluorescence original magnification ×400 (c, f, and i).