Figure 1.
Surgical procedures of co-transplantation.
The autologous epidermal sheet and ADM are co-transplanted to the full-thickness skin defect.
Figure 2.
Histological characterization of epidermal sheet.
(A) Gross appearance of the split-thickness skin. (B) Gross appearance of the epidermal sheet. It appears milk white, soft and elastic, and can be lifted and stretched by forceps. (C) H&E staining of the split-thickness skin. It is composed of an epidermis (solid arrows) and an underlying partial dermis, including some sebaceous glands (dotted arrow) and hair follicles (asterisks). Scale bar = 200 µm. (D) H&E staining of the epidermal sheet. It contains only an intact epidermis. Scale bar = 100 µm. (E–H) Immunohistochemical staining reveals that the split-thickness skin contains continuous distributions of collagen IV (arrows in E) and laminin (arrows in G) at the epidermal-dermal junction, and neither of them is detected in the epidermal sheet (F and H). Dotted lines indicate the location of basal keratinocytes. Scale bars = 100 µm.
Figure 3.
(A) Viability index of the epidermal sheet determined by CCK-8 assay. There is no significant difference in the viability index between the 8- and 10-h digestion groups, while the value of the 12-h digestion group is significantly lower than that of the 8-h digestion group. n = 5. (B) The percentage of viable cells detected by Hoe/PI staining. With the duration of Dispase II digestion prolonging, the percentage of viable cells in the epidermal sheet decreases gradually. n = 5.
Figure 4.
Cell proliferating ability analysis.
(A–B) Hoechst staining shows the cellular structure of the split-thickness skin (A) and epidermal sheet (B). (C–D) Immunohistochemical staining reveals that most basal keratinocytes (single arrows) in the split-thickness skin (C) and epidermal sheet (D) are positive for P63. Cells around the follicular structures (double arrows) are also positive for P63. (E) The percentage of P63 positive keratinocytes in the epidermal sheet is not significantly different from that in the split-thickness skin (p = 0.086, n = 5). Scale bars = 100 µm.
Figure 5.
(A) and (B) show gross appearance of the wound 2 and 3 weeks after co-transplantation of the epidermal sheet and ADM, respectively. The epidermal sheet survives and forms a new epidermis (asterisks in A) by week 2. The arrow indicates un-epithelialized area. By week 3, the wound is completely re-epithelialized. The newly generated epidermis becomes apparently thicker, and the healed wound surface is smooth with a mild degree of contraction (dotted lines in B).
Figure 6.
Histological analysis of the wound.
(A) H&E staining of the wound 3 weeks after co-transplantation. The newly formed epidermis lies directly on the ADM surface. Scale bar = 200 µm. (B) H&E staining of the ADM 3 weeks after subcutaneous implantation. The ADM is laid between the host dermis and deep fascia. Scale bar = 500 µm. (C–D) H&E staining of the ADM-fascia junction area in the co-transplantation and subcutaneous implantation groups, respectively, indicating no apparent inflammatory response around the ADM, and fibroblasts and new blood vessels (arrows) have infiltrated into the ADM. Scale bars = 100 µm. (E–F) Masson’s trichrome staining of the ADM 3 weeks after co-transplantation (E) and subcutaneous implantation (F) show the broad presence of neo-capillaries (arrows) in both groups. Scale bars = 50 µm.
Figure 7.
BM formation detected by immunohistochemistry.
(A–C) Collagen IV and (D–F) laminin distribute intermittently at the epidermis-ADM junction of the wound 2 weeks after co-transplantation (arrows in A and D), and by week 4 their distributions have become continuous (arrows in B and E), similar to that in normal skin (arrows in C and F). Scale bars = 100 µm.
Figure 8.
(A) Discontinuous lamina densa (arrows) exists at the epidermis-ADM junction of the wound 2 weeks after co-transplantation. (B) The laminin densa has become continuous and thick by week 4, and hemidesmosomes can also be observed between the basal keratinocytes and laminin densa. (C) The normal skin serves as control. Scale bars = 1 µm.