Full Thickness Skin Expansion ex vivo in a Newly Developed Reactor and Evaluation of Auto-Grafting Efficiency of the Expanded Skin Using Yucatan Pig Model

Abstract

Background: Skin grafts are required in numerous clinical procedures, such as reconstruction after skin removal and correction of contracture or scarring after severe skin loss caused by burns, accidents, and trauma. The current standard for skin defect replacement procedures is the use of autologous skin grafts. However, donor-site tissue availability remains a major obstacle for the successful replacement of skin defects and often limits this option. The aim of this study is to effectively expand full thickness skin to clinically useful size using an automated skin reactor and evaluate auto grafting efficiency of the expanded skin using Yucatan female pigs.

Methods: We developed an automated bioreactor system with the functions of real-time monitoring and remote-control, optimization of grip, and induction of skin porosity for effective tissue expansion. We evaluated the morphological, ultra-structural, and mechanical properties of the expanded skin before and after expansion using histology, immunohistochemistry, and tensile testing. We further carried out in vivo grafting study using Yucatan pigs to investigate the feasibility of this method in clinical application.

Results: The results showed an average expansion rate of 180%. The histological findings indicated that external expansion stimulated cellular activity in the isolated skin and resulted in successful grafting to the transplanted site. Specifically, hyperplasia did not appear at the auto-grafted site, and grafted skin appeared similar to normal skin. Furthermore, mechanical stimuli resulted in an increase in COL1A2 expression in a suitable environment.

Conclusions: These findings provided insight on the potential of this expansion system in promoting dermal extracellular matrix synthesis in vitro. Conclusively, this newly developed smart skin bioreactor enabled effective skin expansion ex vivo and successful grafting in vivo in a pig model.

Keywords: Animal model; Skin bioreactor; Skin transplantation; Tissue expansion devices.

Fig. 1

Skin bioreactor and grafting procedure. A Skin was expanded on the bioreactor. B–D Full-thickness porcine inguinal skin with an area of 4 cm × 4 cm (B) was removed and measured (C), and then expanded and incubated in the bioreactor for 48 h (D). E Recipient dorsal skin with an area of 6 cm × 6 cm was removed. F The expanded skin was grafted on the dorsal skin

Fig. 2

Skin expansion rate. Full-thickness porcine inguinal skin was expanded on the bioreactor for 48 h. A The expanded area was calculated by ImageJ. B The mean expansion rate was 182.68 ± 29.72%. The measurement was replicated four times. Statistical significance: *p < 0.05 versus pre-expanded skin

Fig. 3

Physical properties. A Maximum load, B tensile strength, and C Young’s module of pre- and post-expanded skin were evaluated using a universal testing system 5965 (Instron). Maximum load and tensile strength were increased but Young’s module was decreased in the expanded skin (n = 3). *p < 0.05

Fig. 4

Blood flow assessment during wound healing. Bright-field images showed skin wound healing after autografting. Blood plasma distribution was visualized with indocyanine green (ICG) fluorescence by Spy system. Red intensity indicates concentration of blood albumin tagged with ICG. At 3 weeks after grafting, blood flow was detected in the grafted site and then remained during wound healing. (Color figure online)

Fig. 5

RT-PCR. mRNA levels of the skin were analyzed by RT-PCR. A COL1A2 expression was elevated by expansion and transplantation (at 5 and 11 weeks), and COL3A1 was upregulated at 5 weeks after transplantation. B The ratio of expression levels to GAPDH was quantitated using ImageJ. The measurements were replicated four times. Statistical significance: ***p < 0.001 vs. Normal inguinal skins. Abbreviations: Col. I; COL1A2, Col. III; COL3A1

Fig. 6

Hematoxylin and eosin (H&E), Masson’s trichrome, and elastin staining. A–E H&E, F–J Mason’s trichrome, and K–O elastin staining were performed with pre- (A, F, and K) and postexpanded inguinal (B, G, and L), dorsal (C, H, and M), transplanted skin at 5 (D, I, and N) and 11 weeks (E, J, and O) after surgery. Scale bar is 100 µm

Fig. 7

TUNEL assay. TUNEL assay was performed with A pre- and B post-expanded inguinal, C dorsal, transplanted skin at D 5 and E 11 weeks after surgery. F Sections digested using DNase I were used as a positive control. Scale bar is 100 μm

Fig. 8

Immunohistochemistry (PCNA). Immunohistochemistry of PCNA was performed with A pre- and B post-expanded inguinal, C dorsal, transplanted skin at D 5 and E 11 weeks after surgery. F A section labeled with IgG only was used as a negative control. Scale bar is 50 μm

Fig. 9

Immunohistochemistry (E-cadherin). Immunohistochemistry of E-cadherin was performed with A pre- and B post-expanded inguinal, C dorsal, transplanted skin at D 5 and E 11 weeks after surgery. F A section labeled with IgG only was used as a negative control. Scale bar is 50 μm

Fig. 10

May-Grünwald stain. To visualize immune cells, May-Grünwald staining was performed with A pre- and B post-expanded inguinal, C dorsal, transplanted skin at D 5 weeks and E 11 weeks after surgery. Inlets show magnified image of each *. D, E The cells stained in dark blue were presented in the dermal matrix at 5 and 11 weeks after transplantation. E Stained cells decreased at 11 weeks after grafting. Scale bar is 200 μm