Assessment of the quality of the healing process in experimentally induced skin lesions treated with autologous platelet concentrate associated or unassociated with allogeneic mesenchymal stem cells: preliminary results in a large animal model

Abstract

Regenerative medicine for the treatment of skin lesions is an innovative and rapidly developing field that aims to promote wound healing and restore the skin to its original condition before injury. Over the years, different topical treatments have been evaluated to improve skin wound healing and, among them, mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) have shown promising results for this purpose. This study sought to evaluate the quality of the healing process in experimentally induced full-thickness skin lesions treated with PRP associated or unassociated with MSCs in a sheep second intention wound healing model. After having surgically created full-thickness wounds on the back of three sheep, the wound healing process was assessed by performing clinical evaluations, histopathological examinations, and molecular analysis. Treated wounds showed a reduction of inflammation and contraction along with an increased re-epithelialization rate and better maturation of the granulation tissue compared to untreated lesions. In particular, the combined treatment regulated the expression of collagen types I and III resulting in a proper resolution of the granulation tissue contrary to what was observed in untreated wounds; moreover, it led to a better maturation and organization of skin adnexa and collagen fibers in the repaired skin compared to untreated and PRP-treated wounds. Overall, both treatments improved the wound healing process compared to untreated wounds. Wounds treated with PRP and MSCs showed a healing progression that qualitatively resembles a restitutio ad integrum of the repaired skin, showing features typical of a mature healthy dermis.

Keywords: healing process; mesenchymal stem cells; platelet rich plasma; sheep; skin lesion.

Figure 1

Representative macroscopic images of the skin wounds during the wound healing process.

Figure 2

Clinical score for the presence of granulation tissue. Data are shown as mean ± SD. Different letters within time points means statistically significant different values for p < 0.05.

Figure 3

(A) Percentage of contraction and (B) re-epithelialization of skin wounds during the experimentation. Data are shown as mean ± SEM.

Figure 4

Histopathological microphotographs of the skin biopsies obtained at different time points during the wound healing process. (A–C) Skin wounds at 7 days; (D–F) wounds at 14 days; (G–I) wounds at 21 days; (J–L) wounds at 42 days after wounding. GT, granulation tissue; NE, neoepidermis; NS, neoskin; F, fibrosis. Scalebar, 200 μm.

Figure 5

Epidermal thickness index (ETI) at 42 days of all wounds respect to unwounded skin. Data are shown as mean ± SEM; **** p < 0.0001.

Figure 6

(A) Quantitative analysis of the percentage of positive area of each wound for the proliferation marker Ki67 and (B) histological score for the alpha smooth muscle actin (α-SMA). Data are shown as mean ± SEM; * p < 0.05; ** p < 0.01; *** p < 0,001; **** p < 0.0001.

Figure 7

Real Time PCR for collagen type I and III. (A) Relative expression of collagen type I (Col1α1) gene and (B) collagen type III (Col3α1) gene at 7, 14, 21, and 42 days post-wounding. Data are shown as mean ± SEM. Unwounded skin was used as the calibrator sample. Statistical differences were measured between the three experimental groups at the same time point. * p < 0.05; *** p < 0.001; **** p < 0.0001.

Figure 8

Real Time PCR for vascular endothelial growth factor A (VEGF) and hair-Keratin (hKER) genes. (A) Relative gene expression of the VEGF-A and (B) hKER at 7, 14, 21, and 42 days after wounding. Data are shown as mean ± SEM. Unwounded skin was used as the calibrator sample. Statistical differences were measured between the two experimental groups at the same time point; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.