Evaluation of epithelial progenitor cells and growth factors in a preclinical model of wound healing induced by mesenchymal stromal cells

Abstract

Background: Skin wounds continue to be a global health problem. Several cellular therapy protocols have been used to improve and accelerate skin wound healing. Here, we evaluated the effect of transplantation of mesenchymal stromal cells (MSC) on the wound re-epithelialization process and its possible relationship with the presence of epithelial progenitor cells (EPC) and the expression of growth factors.

Methods: An experimental wound model was developed in C57BL/6 mice. Human MSCs seeded on collagen membranes (CM) were implanted on wounds. As controls, animals with wounds without treatment or treated with CM were established. Histological and immunohistochemical (IH) studies were performed at day 3 post-treatment to detect early skin wound changes associated with the presence of EPC expressing Lgr6 and CD34 markers and the expression of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF).

Results: MSC transplantation enhanced skin wound re-epithelialization, as compared with controls. It was associated with an increase in Lgr6+ and CD34+ cells and the expression of KGF and bFGF in the wound bed.

Conclusion: Our results show that cutaneous wound healing induced by MSC is associated with an increase in EPC and growth factors. These preclinical results support the possible clinical use of MSC to treat cutaneous wounds.

Keywords: EPC; FGF; KGF; MSC; re-epithelialization; wound healing.

Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

© 2020 The Author(s).

Figures

Figure 1. Schematic representation of the skin wound model

C57BL/6 mice were anesthetized and hair was removed from the dorsal surface area (A). A 4-mm full thickness excisional skin wound was created on the dorsal area (B). A sterile band-aid was placed so that the wound was centered within it, and a transparent film dressing (Tegaderm 3M) was placed over the wound using an immediate-bonding adhesive to better fix the dressing to the skin (C).

Figure 2. Phenotypical and functional characterization of MSC

Microscopical observation shows the fibroblast-like morphology of MSC in culture (A). Flow cytometry analysis of MSC marker expression shows the expression of CD73 and CD90 (arrows). Negative controls were stained with the respective isotype (arrows) (B). Multipotent differentiation assays show the osteogenic (C), adipogenic (D) and chondrogenic (E) potential of MSC.

Figure 3. Implant of MSC on cutaneous wounds

Culture medium containing MSC (head arrow, A) was added on CM transwell (arrow, A). After 72 h, MSC reached 100% confluence and exhibited fibroblast-like typical morphology on CM (B). MSC/CM were removed from the insert (arrow, C). CM (arrow) were cut and implanted on the bed of cutaneous wounds (D). The wound was covered with a band-aid and Tegaderm (E).

Figure 4. Evaluation of wound closure after MSC transplantation

Wounds were evaluated before (d0) and after (d3) MSC transplantation. Wound closure was compared between the same experimental group (circle of the same size) (A). Wound closure was determined by using the ImageJ program. It was expressed as the percentage (means ± SE) of wound closure at day 3 as compared with d0 in each group (% of wound closure = [wound area d0 − wound area d3/area d0] × 100). There were not a statistically significant difference in wound closure between day 3 and d0 post-wounding in all groups (control, n=5; CM, n=5 and MSC/CM, n=4) (B). Signs of early re-epithelialization (whitish areas covering the wound surface) were observed in wounds (higher magnification) in all groups (C, arrows).

Figure 5. MSC transplantation enhances wound re-epithelialization

Histological studies of wounds were performed in untreated wounds (control, A), CM-treated wounds (B) and MSC/CM-treated wounds (C), at day 3 post wounding. H&E-stained sections show NS and NE in the edges of wounds (W). Higher magnification of the newly formed epidermis is shown in each section (control, A1–A2; CM, B1–B2; and MSC/CM C1–C2). Histologic sections of wounds treated with MSC/CM show a larger area of re-epithelialization (C), as compared with those treated with CM alone (B) or control (A). Image analysis from histological sections show a significant increase in the percentage of re-epithelialization in wound treated with MSC/CM, as compared with control groups (D). Scale bar = 100 μm. Results are presented as means ± SE (Control, n=7; CM, n=7; MSC/CM, n=8). *P<0.05; ***P<0.001.

Figure 6. Evaluation of PMN in wounds

PMN were evaluated before (d0) and after (d3) MSC implantation in control, MC- and MSC/MC-treated wounds. Analysis of PMN infiltrating wounds was performed by two independent observers (low powered field microscopy) by using a semiquantitative cross-scoring system (low = +, moderate = ++, high = +++). Results are presented as means ± SE. There was not a statistically significant difference in PMN infiltration between all experimental groups (control, n=8; CM, n=6; MSC/CM, n=9).

Figure 7. Detection of Lgr6+ progenitor cells in cutaneous wounds after 3 days of MSC implantation

IH studies to detect Lgr6+ cells were performed in untreated wounds (control, A), CM-treated wounds (B) and MSC/CM-treated wounds (C). Tissue sections show NS and NE in the edges of wounds (W). Lgr6+ cells were present at the NS and NE of the control group and treated with CM (A,B, respectively). Most of the Lgr6+ cells were detected at the NE adjacent to the wound treated with MSC/CM (C). They were also detected in HFs close to the wound (head arrows). Each picture is representative of three different experiments, all with similar results. Scale bar = 50 μm.

Figure 8. Detection of CD34+ cells in cutaneous wounds after 7 days of MSC implantation

IH studies to detect CD34+ were performed in untreated wounds (control, A), CM-treated wounds (B) and MSC/CM-treated wounds (C). Tissue sections show NS and NE in the edges of wounds (W). A larger number of CD34+ cells are observed in epidermis adjacent to the wound, NE, HFs (head arrow) and SGs (asterisks) in the group treated MSC/CM (C), as compared with the control and the group treated with CM (A,B, respectively). Scale bar = 50 μm.

Figure 9. Increased expression of KGF and bFGF in cutaneous wounds after 3 days of MSC implantation

Wounds treated with MSC/CM show an increased expression of KGF and bFGF (C,F, respectively) in NS, HF and SGs (asterisks), as compared with untreated (A,D, respectively) and CM-treated groups (B,E, respectively). Arrows indicate the edge of wound. Each picture is representative of three different experiments, all with similar results. Scale bar = 50 μm.