Human Placenta-Derived Mesenchymal Stem Cells Combined With Artificial Dermal Scaffold Enhance Wound Healing in a Tendon-Exposed Wound of a Rabbit Model

Abstract

To overcome the difficulty of vascular regeneration in exposed tendon wounds, we combined human placenta-derived mesenchymal stem cells (hPMSCs) with an artificial dermal scaffold and assessed their role in promoting vascular regeneration and wound healing in vivo. hPMSCs were isolated from the human placenta and characterized based on their morphology, phenotypic profiles, and pluripotency. New Zealand rabbits were used to establish an exposed tendon wound model, and hPMSCs and artificial dermal scaffolds were transplanted into the wounds. The results of gross wound observations and pathological sections showed that hPMSCs combined with artificial dermal scaffold transplantation increased the vascularization area of the wound, promoted wound healing, and increased the survival rate of autologous skin transplantation. Following artificial dermal scaffold transplantation, hPMSCs accelerated the vascularization of the dermal scaffold, and the number of fibroblasts, collagen fibers, and neovascularization in the dermal scaffold after 1 week were much higher than those in the control group. Immunohistochemical staining further confirmed that the expression of the vascular endothelial cell marker, CD31, was significantly higher in the combined transplantation group than in the dermal scaffold transplantation group. Our findings demonstrated that hPMSCs seeded onto artificial dermal scaffold could facilitate vascularization of the dermal scaffold and improve tendon-exposed wound healing.

Keywords: angiogenesis; artificial dermal scaffold; placenta-derived mesenchymal stem cells; tendon exposure wound.

Figure 1.

Model preparation and treatment process. (A) Tendon-exposed wound preparation. (B) Artificial dermal scaffold or dermal scaffold with human placenta-derived mesenchymal stem cells placed to cover the wounds. (C) Surface of the dermal scaffolds covered with Vaseline gauze. (D) Gross examination. (E) Autograft harvested from the rabbits’ dorsum as split-thickness skin grafts with a roller knife. (F) Autologous skin transplantation.

Figure 2.

Characterization of hPMSCs. (A) Microscopic image showing spindle-shaped, third-passage hPMSCs. (B) Oil red O staining for adipocytes. (C) Alizarin red S staining for osteocytes. (D) Von Kossa staining for osteocytes. (E) hPMSCs characterization using flow cytometry showed high expression of CD13, CD44, CD73, CD90, and CD105 (positive) and very low expression of HLA-DR, CD34, and CD45 (negative) in the isolated hPMSCs. hPMSCs: human placenta-derived mesenchymal stem cells; PE-H: Phycoerythrin-Height.

Figure 3.

Characterization of hPMSCs on artificial dermal scaffold. (A) Autofluorescence of artificial dermal scaffold. (B) Light microscopy of artificial dermal scaffold. (C) Ultrastructures of artificial dermal scaffolds were examined under scanning electron microscopy. (D) Fluorescence microscopy showing large numbers of hPMSCs attached to the artificial dermal scaffold. (E) Light microscopy of hPMSCs on artificial dermal scaffold. (F) Scanning electron microscopy imaging showing hPMSCs consistently attached to the dermal scaffold. (G) CCK-8 assays were performed to evaluate the cellular growth curves. *P < 0.05, day 1 vs day 2; ^#P < 0.05, day 2 vs day 3; ^&P < 0.05, day 3 vs day 4. Scale bar: 200 μm (light microscopy and fluorescence microscopy). Scale bar: 20 μm (scanning electron microscopy). hPMSCs: human placenta-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8.

Figure 4.

Rabbits were observed grossly at days 7 and 14 after injury, and day 7 after autologous skin transplantation. (A) Representative images of the gross examination in control group. (B) Combined application of hPMSCs and artificial dermal scaffold group. hPMSCs: human placenta-derived mesenchymal stem cells.

Figure 5.

Combination of hPMSCs with artificial dermal scaffold facilitates vascularization degree of dermal scaffold and improves tendon-exposed wound healing. (A) The vascularization area of the wound was calculated 2 weeks after scaffold and cell transplantation. (B) hPMSCs improve tendon-exposed wound healing. Data are analyzed using independent-sample t test. hPMSCs: human placenta-derived mesenchymal stem cells.

Figure 6.

Histological analysis of wound tissues with H&E staining and Masson staining reveals the beneficial effect of hPMSCs on wound healing. (A) Representative images of wound sections with H&E staining on day 7 after dermal scaffold transplantation. (B) Representative images of wound sections with Masson staining (scale bar: 200 μm). H&E: hematoxylin and eosin; hPMSCs: human placenta-derived mesenchymal stem cells.

Figure 7.

Histological analysis of wound tissues showing the beneficial effect of hPMSCs on autologous skin survival. (A) Representative images of wound sections with H&E staining on day 7 after autologous skin transplantation. (B) Representative images of wound sections with Masson staining (scale bar: 200 μm). hPMSCs: human placenta-derived mesenchymal stem cells; H&E: hematoxylin and eosin.

Figure 8.

Expression of CD31 and human nuclei. (A) Paraffin sections of skin lesions from autologous skin transplantation for 1 week were obtained, and the endothelial cells CD31 and human nuclei were stained by immunohistochemistry. Scale bars: 100 μm (upper row), 20 μm (lower row). (B) Quantification of CD31-positive expression. hPMSCs: human placenta-derived mesenchymal stem cells.