Pre-Vascularized 3-Dimensional Skin Substitutes Promote Angiogenesis and Tissue Repair in a Murine Model of Refractory Skin Ulcers

Abstract

Restoring blood flow is crucial for treating refractory ulcers. Despite advancements in various biomaterials, none incorporating pre-formed blood vessels have been commercialized. To address this, we developed a pre-vascularized three-dimensional (3D) skin substitute (PV-3D skin) designed to enhance healing when treating refractory ulcers. This study aimed to evaluate the therapeutic role of PV-3D skin transplantation in refractory ulcer models, induced by applying mitomycin C to wounds in severe immunodeficient mice. The wounds were then treated with PV-3D skin, non-vascularized 3D skin, skin grafts, or wound dressings. The PV-3D skin group demonstrated healing dynamics comparable to those of the skin graft group, with similar tissue morphology and wound temperature changes. Furthermore, at day 7 post-transplantation, the PV-3D skin group demonstrated significantly higher hypoxia-inducible factor 1-alpha expression levels compared to the 3D skin group. By day 14, the PV-3D skin group exhibited a significantly larger vascular area compared to the 3D skin group. Notably, PV-3D skin treatment stimulated host-derived angiogenesis, thereby enhancing wound healing and reducing the recurrence of refractory ulcers. These results suggest that PV-3D skin transplantation offers a promising therapeutic approach for refractory ulcers, especially in terms of angiogenesis.

Keywords: angiogenesis; regional blood flow; skin substitutes; skin ulcer; wound healing.

Figure 1

Schematic illustration of the treatment of refractory skin ulcers in mice with severe combined immunodeficiency. MMC: mitomycin C; PV-3D: pre-vascularized three-dimensional.

Figure 2

Post-transplantation macroscopic images of the wound area in the recipient mice.

Figure 3

Evaluation of the therapeutic response in experimental models using infrared thermographic imaging. (A) Representative observation of the wound temperature. (B) Comparative analysis of wound temperatures across groups at each time point (n = 4–8 animals/time point). Data are represented as mean ± standard error of the mean. * p < 0.05, *** p < 0.001.

Figure 4

Histological analysis of the skin. (A) Hematoxylin and eosin staining performed on days 7 and 14 post-transplantation. (B) Quantitative analysis of wound diameter and the thickness of the epidermis, dermis, and subdermal layer on day 14 (n = 6–12 animals). Data are represented as mean ± standard error of the mean. * p < 0.05, ** p < 0.01. (C) Masson’s Trichrome staining on day 14.

Figure 5

Immunohistochemical analysis of transplanted tissues on day 14. (A) Human leukocyte antigen and (B) Fibroblast activation protein staining. Dashed black lines indicate the interface between the transplanted tissue and the mouse wound bed.

Figure 6

Blood flow monitoring in the pre-vascularized three-dimensional (3D) skin substitute and 3D skin groups following transplantation. (A) Representative laser speckle contrast images. (B) Quantitative analysis of blood flow changes at each time point (n = 6 animals/time point). Data are represented as mean ± standard error of the mean. * p < 0.05, *** p < 0.001.

Figure 7

Immunohistochemical analysis at the wound site. (A) Human-specific CD31 and mouse-specific CD31 staining (n = 11–12 animals) on days 7 and 14 post-transplantation. (B) Quantification of the area occupied by mouse-derived blood vessels on day 14. (C) Hypoxia-inducible factor 1-alpha (HIF-1α) staining at the wound site on day 7. (D) Quantification of the HIF-1α stained cells (n = 5 animals). Data are represented as mean ± standard error of the mean. * p < 0.05, ** p < 0.01.

Figure 8

Wound site images on day 28. (A) Macroscopic images of the wound area and (B) Histological images at the wound site.