*3.3. Histological Examination*

In all groups, an open wound defect covered with a purulent-necrotic crust was formed on the 28th day after irradiation. Weak, mainly perivascular lymphocytic-plasmocytic infiltration with an admixture of single neutrophilic granulocytes and moderate vascular proliferation of the microcirculatory bed was detected in the underlying dermis in the area of the defect bottom. Moderate thickening of the adjacent epidermis was noted along the edges of the wound defect, and few intraepidermal lymphocytes were recorded. At the same time, only in the CMPL group was the "creeping" of regenerating epithelium from one of the wound edges in the form of a strip 3–4 epithelial cells thick noted (Figure 3).

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**Figure 1.** MSCs derived from the placenta immunophenotype: CD90+/CD105+73+/CD45−/CD34−/HLA-DR−, 7−ADD (99.5%). **Figure 1.** MSCs derived from the placenta immunophenotype: CD90+/CD105+73+/CD45−/CD34−/ HLA-DR *Cells* −**<sup>20</sup>** , 7 **<sup>20</sup>**−, *<sup>9</sup>* ADD (99.5%). , x FOR PEER REVIEW 6 of 12

**Figure 2.** Dynamics of the LRI of animals: (**a**) dynamics of development of visible region of rat skin changes that occurred under X-ray radiation; (**b**) dynamics of development of an open wound surface of rat skin after exposure to X-ray radiation; (**c**) dynamics of healing of an open wound surface of the skin in animals (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from a culture of the MSCs). **Table 1.** The area of the total changed surface of animal skin in LRI (cm<sup>2</sup>). **Figure 2.** Dynamics of the LRI of animals: (**a**) dynamics of development of visible region of rat skin changes that occurred under X-ray radiation; (**b**) dynamics of development of an open wound surface of rat skin after exposure to X-ray radiation; (**c**) dynamics of healing of an open wound surface of the skin in animals (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from a culture of the MSCs).

 6.76 ± 0.34 4.59 ± 0.21 5.92 ± 0.19 6.04 ± 0.21 6.54 ± 0.35 4.62 ± 0.24 5.80 ± 0.21 6.06 ± 0.39 6.50 ± 0.41 4.18 ± 0.26 <sup>1</sup> 5.71 ± 0.24 <sup>2</sup> 6.05 ± 0.41 <sup>2</sup> 6.56 ± 0.60 3.88 ± 0.23 5.64 ± 0.37 <sup>2</sup> 6.43 ± 0.54 <sup>2</sup> 6.33 ± 0.72 3.81 ± 0.26 <sup>1</sup> 5.33 ± 0.29 6.26 ± 0.58 6.49 ± 1.04 3.59 ± 0.43 5.44 ± 0.53 6.06 ± 0.41 <sup>2</sup> 6.41 ± 1.21 3.50 ± 0.54 5.45 ± 0.58 5.36 ± 0.47 <sup>2</sup> 5.50 ± 0.88 3.38 ± 0.47 5.60 ± 0.52 <sup>2</sup> 5.07 ± 0.58 <sup>2</sup> 5.19 ± 0.79 3.05 ± 0.71 6.13 ± 0.95 4.43 ± 0.31 5.47 ± 0.69 3.08 ± 0.67 6.06 ± 0.81 4.04 ± 0.23 4.35 ± 0.42 3.07 ± 0.60 5.75 ± 0.61 3.57 ± 0.30 Notes: <sup>1</sup> Significant differences in all groups compared to the control (C) (*p* ≤ 0.05). <sup>2</sup> Significant

differences between the PL and CMPL groups compared to the CM group (*p* ≤ 0.05).

From the 14th day of the experiment, the open wound surface of the skin was recorded in all groups of animals. The dynamics of reducing the area of the open wound surface was the same for

**Day C CM Pl CMPL** 7 7.76 ± 0.47 6.60 ± 0.42 10.5 ± 0.31 1,2 10.27 ± 0.19 1,2


**Table 1.** The area of the total changed surface of animal skin in LRI (cm<sup>2</sup> ).

Notes: <sup>1</sup> Significant differences in all groups compared to the control (C) (*<sup>p</sup>* <sup>≤</sup> 0.05). <sup>2</sup> Significant differences between the PL and CMPL groups compared to the CM group (*p* ≤ 0.05).


**Table 2.** Area of open wound surface of animal skin in LRI (cm<sup>2</sup> ).

Notes: <sup>1</sup> Significant differences in all groups compared to the control (C) (*<sup>p</sup>* <sup>≤</sup> 0.05). <sup>2</sup> Significant differences between the PL and CMPL groups compared to the CM group (*<sup>p</sup>* <sup>≤</sup> 0.05). <sup>3</sup> Significant differences between the PL and CMPL groups (*p* ≤ 0.05).

In group C, on day 56, the bottom of the skin defect reached the large subcutaneous muscle, and, in some cases, the subcutaneous fat. Pronounced edema and lymphocytic-plasmocytic infiltration of muscle tissue were determined. In the connective tissue of the dermis in the area of the edges of the wound defect, lymph-histiocytic infiltration with an admixture of neutrophils, granulation, and proliferation of microvessels were detected. The epidermis adjacent to the wound defect was thickened to 10–12 layers of cells, and focal hyperkeratosis, acanthosis, and degenerative changes in keratinocytes were noted. Areas of epithelial regeneration with a thickness of 4–8 epithelial cells were determined in the area of the defect edges (Figure 3(1b)); by the 112th day, pronounced purulent-necrotic changes in soft tissues appeared in the area of the defect bottom. Underlying connective tissue and moderate lymphocytic-plasmocytic infiltration were observed, with an admixture of neutrophilic granulocytes, moderate vascular proliferation of the microcirculatory bed, focal edema, and pronounced fibrotic changes. In most cases, areas of fibrosis and weakly expressed lymphoplasmocytic infiltration

in the area of the large subcutaneous muscle and Hypoderma were detected. At the edges of the wound defect, there were large areas of regeneration of the integumentary epithelium in the form of a layer of cells 1–2 epithelial cells thick. The adjacent epidermis was thickened (up to 6–11 layers of cells), with signs of vacuole dystrophy and acanthosis (Figure 3(1c)). *Cells* **2020**, *9*, x FOR PEER REVIEW 8 of 12

**Figure 3.** Histological examination of animal skin defects. Hemotoxylin‒eosin staining, X40 magnification for C and CM groups, X20 for PL and CMPl groups (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSCs derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from the culture of the MSCs). **Figure 3.** Histological examination of animal skin defects. Hemotoxylin-eosin staining, ×40 magnification for C and CM groups, ×20 for PL and CMPl groups (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSCs derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from the culture of the MSCs).

In group C, on day 56, the bottom of the skin defect reached the large subcutaneous muscle, and, in some cases, the subcutaneous fat. Pronounced edema and lymphocytic‒plasmocytic infiltration of muscle tissue were determined. In the connective tissue of the dermis in the area of the edges of the wound defect, lymph‒histiocytic infiltration with an admixture of neutrophils, granulation, and proliferation of microvessels were detected. The epidermis adjacent to the wound defect was thickened to 10‒12 layers of cells, and focal hyperkeratosis, acanthosis, and degenerative changes in keratinocytes were noted. Areas of epithelial regeneration with a thickness of 4‒8 epithelial cells were determined in the area of the defect edges (Figure 3, 1b); by the 112th day, pronounced purulent‒necrotic changes in soft tissues appeared in the area of the defect bottom. Underlying connective tissue and moderate lymphocytic‒plasmocytic infiltration were observed, In the CM group, signs of epithelization along the edges of the defect were noted on day 56 in most samples; in some cases a deep skin defect remained, which reached the subcutaneous fat and was covered with a purulent-necrotic crust. In all preparations, the large subcutaneous muscle was of the usual histological structure with moderate edema, subcutaneous fat with moderate edema, or moderate lymph with plasmocytic infiltration. In the underlying dermis, there were areas of fibrosis and weak perivascular lymphocytic infiltration (Figure 3(2b)). By the 112th day, only one observation revealed a large skin defect covered with a purulent-necrotic crust, which reached the subcutaneous fat with necrosis in the superficial parts and clusters of hemosiderophages in the deeper parts. In other preparations, the skin defect was partially or completely epithelized. In the dermis, there were areas of fibrosis and mild perivascular lymphocytic infiltration. In all cases, the adjacent epidermis was thickened (up to 10–11 layers of cells), with signs of severe dystrophy (Figure 3(2c)).

with an admixture of neutrophilic granulocytes, moderate vascular proliferation of the microcirculatory bed, focal edema, and pronounced fibrotic changes. In most cases, areas of fibrosis and weakly expressed lymphoplasmocytic infiltration in the area of the large subcutaneous muscle and Hypoderma were detected. At the edges of the wound defect, there were large areas of regeneration of the integumentary epithelium in the form of a layer of cells 1‒2 epithelial cells thick. The adjacent epidermis was thickened (up to 6‒11 layers of cells), with signs of vacuole dystrophy and acanthosis (Figure 3, 1c). In the CM group, signs of epithelization along the edges of the defect were noted on day 56 in most samples; in some cases a deep skin defect remained, which reached the subcutaneous fat and was covered with a purulent‒necrotic crust. In all preparations, the large subcutaneous muscle was In the PL group on day 56, the bottom of the skin defect showed necrotic dermis, striated muscle, and underlying adipose tissue with pronounced neutrophilic infiltration. At the edges of the defect, there was moderate lymph-plasmocytic infiltration with an admixture of neutrophilic granulocytes, moderate proliferation of microcirculatory vessels, granulation, and fibrosis of striated muscle tissue (Figure 3(3b)). By the 112th day, the extensive skin defect was covered with a purulent-necrotic crust in all cases. Its bottom is represented by fibrotic connective tissue with angiomatosis, granulations, moderate lymph-plasmocytic infiltration with an admixture of neutrophilic granulocytes, and microvessel proliferation. The striated tissue at the bottom of the defect was not detected. There were extensive areas of fibrosis of the underlying adipose tissue. The epidermis at the edge of the wound defect was

other preparations, the skin defect was partially or completely epithelized. In the dermis, there were

of the usual histological structure with moderate edema, subcutaneous fat with moderate edema, or moderate lymph with plasmocytic infiltration. In the underlying dermis, there were areas of fibrosis and weak perivascular lymphocytic infiltration (Figure 3, 2b). By the 112th day, only one observation thickened (up to 8–10 layers of cells), with signs of vacuole dystrophy and proliferation of hair follicles (Figure 3(3c)). defect, there was moderate lymph‒plasmocytic infiltration with an admixture of neutrophilic granulocytes, moderate proliferation of microcirculatory vessels, granulation, and fibrosis of striated

muscle, and underlying adipose tissue with pronounced neutrophilic infiltration. At the edges of the

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areas of fibrosis and mild perivascular lymphocytic infiltration. In all cases, the adjacent epidermis

In the CMPL group, on day 56, the open wound skin defect was covered with a purulent-necrotic crust, marginal epithelization was recorded over a longer length in most samples, and the thickness of the epithelial layer was 5–8 cells. The underlying dermis was moderately fibrotic with focal subepithelial edema and the presence of hair follicle rudiments in the amount of 1–3 in the field of vision. In the area of the defect bottom, muscle and adipose tissue were completely replaced by fibrous tissue with granulations with moderate lymph-plasmocytic infiltration with an admixture of neutrophilic granulocytes and pronounced microvessel proliferation (Figure 3(4b)). By the 112th day in all samples, the skin defect was completely epithelized and the thickness of the epithelial layer was 5–7 cells. The underlying dermis was focally fibrotic. There were rudiments of hair follicles (1–3) in the field of vision with focal proliferation of microcirculatory vessels. Large subcutaneous muscle was not detected in the central parts; it was replaced by connective tissue. There were no inflammatory changes (Figure 3(4b)). muscle tissue (Figure 3, 3b). By the 112th day, the extensive skin defect was covered with a purulent‒necrotic crust in all cases. Its bottom is represented by fibrotic connective tissue with angiomatosis, granulations, moderate lymph‒plasmocytic infiltration with an admixture of neutrophilic granulocytes, and microvessel proliferation. The striated tissue at the bottom of the defect was not detected. There were extensive areas of fibrosis of the underlying adipose tissue. The epidermis at the edge of the wound defect was thickened (up to 8‒10 layers of cells), with signs of vacuole dystrophy and proliferation of hair follicles (Figure 3, 3c). In the CMPL group, on day 56, the open wound skin defect was covered with a purulent‒necrotic crust, marginal epithelization was recorded over a longer length in most samples, and the thickness of the epithelial layer was 5‒8 cells. The underlying dermis was moderately fibrotic with focal subepithelial edema and the presence of hair follicle rudiments in the amount of 1‒3 in the field of vision. In the area of the defect bottom, muscle and adipose tissue were completely replaced by fibrous tissue with granulations with moderate lymph‒plasmocytic infiltration with an

#### *3.4. Immunohistochemical Study* admixture of neutrophilic granulocytes and pronounced microvessel proliferation (Figure 3, 4b). By

As a result of an immunohistochemical study, it was found that the number of newly formed vessels in whose endothelial cells the expression of CD31 was determined increased from day 28 to day 112 in the PL and CMPL groups (from 2.6 ± 1.0 to 10.97 ± 1.6 and from 4.1 ± 0.6 to 8.2 ± 1.8, respectively, *p* ≤ 0.05), which indicated an increase in neoangiogenesis by the end of the experiment. Such changes were not detected in groups C and CM (Figure 4), nor for the vascular endothelial growth factor (VEGF) in endothelial cells and stroma cells in all the studied groups, except for CMPL (Figures 4d and 5b). In groups C, CM, and PL, an increase in FVIII expression in vascular endothelial cells was observed by day 112 of the experiment (*p* ≤ 0.05) (Figures 4b and 5a). the 112th day in all samples, the skin defect was completely epithelized and the thickness of the epithelial layer was 5‒7 cells. The underlying dermis was focally fibrotic. There were rudiments of hair follicles (1‒3) in the field of vision with focal proliferation of microcirculatory vessels. Large subcutaneous muscle was not detected in the central parts; it was replaced by connective tissue. There were no inflammatory changes (Figure 3, 4b). *3.4. Immunohistochemical Study* As a result of an immunohistochemical study, it was found that the number of newly formed

In the course of the experiment, we noted an increase in the number of CD68-positive macrophages in the tissues surrounding the wound defect in groups C and PL (from 11.7 ± 1.4 and 12.9 ± 3.6 at 28 days to 24.73 ± 2.4 and 29.3 ± 3.5 at 112 days, respectively, *p* ≤ 0.05), while in the CM group it was determined by the decrease in the number of these cells (22.1 ± 1.6 and 13.07 ± 1.8, *p* ≤ 0.05), and in the CMPL group their number did not change (Figures 4a and 5a). vessels in whose endothelial cells the expression of CD31 was determined increased from day 28 to day 112 in the PL and CMPL groups (from 2.6 ± 1.0 to 10.97 ± 1.6 and from 4.1 ± 0.6 to 8.2 ± 1.8, respectively, *p* ≤ 0.05), which indicated an increase in neoangiogenesis by the end of the experiment. Such changes were not detected in groups C and CM (Figure 4), nor for the vascular endothelial growth factor (VEGF) in endothelial cells and stroma cells in all the studied groups, except for CMPL

The number of regenerating nerve fibers expressing PGP9.5 increased by the end of the experiment in the C, CM, and PL groups (*p* ≤ 0.05), and remained unchanged in the CMPL group (Figures 4a and 5a). (Figures 4d and 5b). In groups C, CM, and PL, an increase in FVIII expression in vascular endothelial cells was observed by day 112 of the experiment (*p* ≤ 0.05) (Figures 4b and 5a).

(**a**) (**b**)

**Figure 4.** *Cont*.

full expression.).

(Figures 4a and 5a).

(Figures 4a and 5a).

**Figure 4.** Immunohistochemical study of animal skin defects: (**a**) CD31, CD68, PGP9.5; (**b**) Ki67 and FVIII; (**c**) TIMP2, Collagen I, Collagen III, and FVIII; (**d**) VEGF, MMP2, and MMP9. (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from the culture of the MSCs). **Figure 4.** Immunohistochemical study of animal skin defects: (**a**) CD31, CD68, PGP9.5; (**b**) Ki67 and FVIII; (**c**) TIMP2, Collagen I, Collagen III, and FVIII; (**d**) VEGF, MMP2, and MMP9. (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from the culture of the MSCs). **Figure 4.** Immunohistochemical study of animal skin defects: (**a**) CD31, CD68, PGP9.5; (**b**) Ki67 and FVIII; (**c**) TIMP2, Collagen I, Collagen III, and FVIII; (**d**) VEGF, MMP2, and MMP9. (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from the culture of the MSCs).

**Figure 5.** Immunohistochemical study of animal skin defects: (**a**) Absolute number of IHC markers in excised animal skin defect tissue samples per 10 visual fields; (**b**) semiquantitative assessment of the expression of IHC markers in excised animal skin defect samples (with a score from 0 to 3, where 0 is the absence of expression and 3 is full expression). (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from culture of the MSCs. Blue: 28 days; orange: 112 days from the start of therapy. The score is from 0 to 3, where 0 is the absence of expression and 3 is **Figure 5.** Immunohistochemical study of animal skin defects: (**a**) Absolute number of IHC markers in excised animal skin defect tissue samples per 10 visual fields; (**b**) semiquantitative assessment of the expression of IHC markers in excised animal skin defect samples (with a score from 0 to 3, where 0 is the absence of expression and 3 is full expression). (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from culture of the MSCs. Blue: 28 days; orange: 112 days from the start of therapy. The score is from 0 to 3, where 0 is the absence of expression and 3 is full expression.). **Figure 5.** Immunohistochemical study of animal skin defects: (**a**) Absolute number of IHC markers in excised animal skin defect tissue samples per 10 visual fields; (**b**) semiquantitative assessment of the expression of IHC markers in excised animal skin defect samples (with a score from 0 to 3, where 0 is the absence of expression and 3 is full expression). (C: irradiated rats without subsequent therapy, CM: irradiated rats that received intradermal administration of culture medium (MesenCult) concentrate, PL: therapy of LRI using MSC derived from the placenta, CMPL: therapy of LRI using a concentrate of conditioned medium collected from culture of the MSCs. Blue: 28 days; orange: 112 daysfrom the start of therapy. The score is from 0 to 3, where 0 is the absence of expression and 3 is full expression).

(**a**) (**b**)

In the course of the experiment, we noted an increase in the number of CD68-positive macrophages in the tissues surrounding the wound defect in groups C and PL (from 11.7 ± 1.4 and 12.9 ± 3.6 at 28 days to 24.73 ± 2.4 and 29.3 ± 3.5 at 112 days, respectively, *p* ≤ 0.05), while in the CM group it was determined by the decrease in the number of these cells (22.1 ± 1.6 and 13.07 ± 1.8, *p* ≤ 0.05), and in the CMPL group their number did not change (Figures 4a and 5a). In the course of the experiment, we noted an increase in the number of CD68-positive macrophages in the tissues surrounding the wound defect in groups C and PL (from 11.7 ± 1.4 and 12.9 ± 3.6 at 28 days to 24.73 ± 2.4 and 29.3 ± 3.5 at 112 days, respectively, *p* ≤ 0.05), while in the CM group it was determined by the decrease in the number of these cells (22.1 ± 1.6 and 13.07 ± 1.8, *p* ≤ 0.05), and in the CMPL group their number did not change (Figures 4a and 5a). The number of regenerating nerve fibers expressing PGP9.5 increased by the end of the Expression of matrix metalloproteinases (MMP) 2 and 9, which led to the destruction of extracellular matrix proteins and stimulated cell migration and reproduction, decreased in all groups at the end of the experiment, with the exception of the CMPL group (Figure 4b,d), while expression of TIMP2, which is a tissue inhibitor of MMP, increased by day 112 in the C and CM groups, decreased in the PL group, and remained unchanged in the CMPL group (Figure 4b,c). The expression of "mature" type I collagen in the stroma increased in all groups from day 28 to day 112, with the exception of the PL

experiment in the C, CM, and PL groups (*p* ≤ 0.05), and remained unchanged in the CMPL group

The number of regenerating nerve fibers expressing PGP9.5 increased by the end of the experiment in the C, CM, and PL groups (*p* ≤ 0.05), and remained unchanged in the CMPL group group, while the expression of "immature" type III collagen in the stroma decreased by day 112 in the C, CM, and PL groups, but did not change in the CMPL group (Figure 4b,c).
