Prevascularized mesenchymal stem cell-sheets increase survival of random skin flaps in a nude mouse model

Abstract

A random skin flap is commonly applied in plastic and reconstructive surgery. The distal part of the random skin flap often risks necrosis because the blood flow may be compromised. Prevascularization is a widely used technology to intensify the vascularization function of biomaterials. In fact, human mesenchymal stem cell (hMSC) sheets promote neoangiogenesis. We speculated that prevascularized hMSC cell sheets (PHCS) would further improve neovascularization by producing more angiogenic growth factors in a random skin flap animal model. In this study, PHCS were set up by co-culturing human umbilical vein endothelial cells (HUVECs) with hMSC cell sheets (HCS). In vitro, we observed microvessel formation and significantly increased production of angiogenesis-related factors. Thus, we analyzed the microvessel networks, vascular maturation, and angiogenic growth factors of the cell sheet. In vivo, PHCS and HCS were implanted in a murine ischemic random skin flap model. Implanted PHCS significantly increased blood perfusion and improved skin flap survival when compared to untreated control skin flaps. The survival rate of the prevascularized and control skin flaps was assessed after 3, 5, and 7 days via analysis of macroscopic images and Laser Doppler Perfusion Imaging (LDPI). Additionally, the numbers of skin appendages, collagen fibers deposition, and epidermal thickness were evaluated. Moreover, the PHCS group also induced the most intense neovascularization, the upshot of which was a robust blood microcirculation supporting skin flap survival. Therefore, PHCS implantation can effectively enhance local neoangiogenesis and hence increase the survival of otherwise ischemic skin flaps. Hence, local administration of PHCS may serve as an alternative approach in improving random skin flap survival.

Keywords: Skin flap; angiogenic growth factors; human mesenchymal stem cell; prevascularized cell sheet.

Figure 1

Complex microvessel network formation of PHCS at day 7 and 14. A. Mosaic image of CD31 (green) Immunofluorescence staining and analysis result with Angiotool (yellow: vessel outline, red: skeleton, blue: branch points). B. Quantitative analysis of microvessel networks of prevascularized cell sheets. *p < 0.05. Bar: 1 mm.

Figure 2

Microvessel maturation of PHC. A. CD31 (green) and collagen IV (red) staining of PHC at day 14, and the latter showing the basement membrane (yellow box) formed around the HUVECs. B. Dextran permeability assay of PHCS at day 14. Some low dextran (red) signal regions (yellow arrow and dashed line) formed in the microvessel (green) regions. Bar: 10 µm.

Figure 3

Human proteome profiler antibody array of conditioned medium. A. Representative images of proteome profiler arrays probe. (1: PIGF; 2: Leptin; 3: Ang2). B. The quantitative data of the three growth factors with the greatest difference were obtained from three biological replicates (*P < 0.05). The PHCS secreted more proangiogenic growth factors than HCS.

Figure 4

Morphology of different skin flaps: control, HCS, and PHCS. A. Morphology of skin flaps over 7 days post surgery. B. Quantification of percent survival skin flap. *P < 0.05. The PHCS treated group had a better survival rate of skin flap than the other groups.

Figure 5

Laser doppler perfusion image analysis. A. Representative LDPI images obtained on post-operation day 1, 3, 5, and 7. In multiple color-coded images, high blood flow is depicted in red, while low perfusion is displayed as blue to dark blue. In blue-white images, blood flow lower than 100RFU is depicted in blue. Bar: 5 mm. B. Mean blood flow perfusion of flaps of three groups. *P < 0.05. C. Ischemic area of skin flaps of three groups. *P < 0.05. PHCS promoted the recovery of blood perfusion, and decreased ischemic areas of the skin flaps.

Figure 7

Morphological changes of epidermis and dermis in the normal skin and skin flaps. A. Masson’s trichrome staining on post operative day 7. Bar: 50 μm. B. Quantification of collagen intensity in the image. *P < 0.05. C. HE staining of epidermis. Bar: 50 μm. D. Thickness of epidermis on day 14 and 28. *P < 0.05. In the PHCS group, the morphological features of epidermis and dermis are very close to those proper of normal skin.

Figure 8

Vascular density analysis. A. Representative results of anti-CD31 staining. Blue: DAPI, Red: CD31. B. The extent of CD31-positive areas per HPF was determined and analyzed. The PHCS group showed the highest vascular density. *P < 0.05. Scale bars: 100 μm.

Figure 6

The changes of skin appendages in different groups. A. HE staining of skin flaps showing hair follicles (indicated by yellow arrows). B. The PHCS group had the best recovery of hair follicles. *P < 0.05. Bar: 200 μm.