Treatment of skin scars

Abstract

The present invention relates to a composition comprising a peripheral blood mononuclear cell (PBMC) cell culture supernatant for use in the treatment of cutaneous scarring.

Description

[Technical Field]

[0001] The present invention relates to the field of treating skin scars. [Background technology]

[0002] Administration of conditioned medium from mesenchymal stem cells (SCs) revealed that secreted factors, rather than the SCs themselves, exert beneficial paracrine effects, explaining most of the initial findings. Stressed peripheral blood mononuclear cells (PBMCs) have previously been shown to be an easily accessible and abundant source of APOSEC secretomes with robust regenerative activity. Several mechanisms of action have recently been elucidated, and various clinical indications for APOSEC have already been described, including wound healing, acute myocardial infarction, autoimmune myocarditis, cerebral ischemia, and spinal cord injury.

[0003] Pathological scar formation is a complex medical problem and represents a significant global disease burden. In Western countries, over 100 million people develop scars each year, and an estimated 11 million develop keloids. The risk of developing hypertrophic scars after surgery or burns exceeds 30%. In contrast to normal scars, hypertrophic scars are characterized by excessive fibrotic processes within the injured area. In contrast, keloids are progressive fibroproliferative malformations that form within scar tissue and overgrow the borders of the original injury. The molecular mechanisms leading to hypertrophic scar or keloid formation are not fully understood. In addition to imposing significant psychological challenges, hypertrophic scars and keloids are painful, pruritic, and cause movement limitations. Treatment options remain limited, and no gold standard exists for the treatment of pathological scars. Possible options include 1) administration of steroids to inhibit fibroblast proliferation, 2) radiation-induced cell cycle arrest and cell death of fibroblasts, 3) compression therapy to affect wound tension, and 4) surgical removal of scar tissue, which has an extremely high recurrence rate. Additionally, chemotherapeutic agents and other substances have been used without significant efficacy. The most promising treatment option to date is surgical removal combined with subsequent steroid therapy.

[0004] It is therefore an object of the present invention to provide a method for removing / treating skin scars that does not require surgery and / or drugs with undesirable side effects. Summary of the Invention

[0005] The present invention relates to a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment of skin scars.

[0006] Surprisingly, it has been found that the supernatant of PBMC cell cultures can be used to treat skin scars. It is already known that the supernatant of PBMC cell cultures can be used to treat damaged tissues such as skin wounds (see, for example, EP 2379085). However, completely different biological processes and cell types are involved in wound healing compared to mature skin scars.

[0007] Wound healing is a complex process consisting of four phases: hemostasis, inflammation, proliferation, and maturation. The primary function of wound healing is rapid closure of the injured area and establishment of a new protective barrier against the environment. Scars are fibrous tissues that remain after wound healing and replace normal skin. In contrast to normal skin, scar tissue is primarily characterized by the overproduction of extracellular matrix proteins, such as collagens COL1A1 and COL3A1, elastin (ELN), fibronectin 1 (FN1), and procollagen (PCOLCE) (Figure 2). Furthermore, several factors that modify the extracellular matrix, including cytokines (e.g., insulin-like growth factor binding protein 4 (IGFBP4)), proteases (e.g., matrix metalloproteinase-3 (MMP3)), and protease inhibitors (e.g., serpin family E member 1 (SERPINE1)), are significantly deregulated in scar tissue (Figure 2). Furthermore, myofibroblasts are highly active, leading to skin contraction. Therefore, the main goal of scar treatment is to reduce the overproduction of myofibroblasts and extracellular matrix components, and to normalize the deregulated production and expression of matrix-modifying factors.

[0008] Another aspect of the invention relates to a method for the cosmetic treatment of skin scars, comprising administering to the skin scar an effective amount of a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows that APOSEC antagonizes TGF-mediated myofibroblast differentiation. A) Western blot of primary human dermal fibroblasts stimulated with medium or APOSEC in the absence or presence of TGF-beta. APOSEC prevented TGF-beta-induced differentiation of fibroblasts into myofibroblasts, as indicated by the expression of alpha-SMA. ELISA of supernatants from TGF-beta-stimulated fibroblasts showed enhanced production of elastin. Addition of APOSEC reduced the enhanced secretion of elastin.

[0010] [Figure 2] Figure 2 shows scRNA seq of human hypertrophic scar and normal skin biopsies treated ex vivo with medium or APOSEC. Genes associated with dermal fibrosis are shown. Treatment with APOSEC normalized the expression of these genes to values observed in normal skin. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention relates to a composition comprising the supernatant of a PBMC cell culture for use in the treatment of skin scars.

[0012] Cells, particularly mammalian cells, are known to secrete numerous substances into cell culture media during culture. The conditioned culture media thus obtained can be used for the treatment and / or prevention of various diseases and disorders. For example, International Publication Nos. 2010 / 070105 and 2010 / 079086 disclose conditioned culture media ("supernatants") obtained by culturing PBMCs and used to treat various inflammatory conditions. Therefore, as used herein, "supernatants of peripheral blood mononuclear cells (PBMC) cell cultures" refers to any supernatant obtained by culturing PBMCs in vitro in a culture medium. After the culturing process, the cultured PBMCs are removed from the culture medium to obtain a substantially cell-free, preferably completely cell-free, supernatant. The supernatant of a PBMC culture contains substances produced and secreted by PBMCs and / or lysed PBMCs, apart from components of the culture medium. The term "supernatant" can be used interchangeably with conditioned culture medium obtained by culturing PBMCs.

[0013] The supernatant of the present invention can be obtained by culturing PBMCs, which are subjected to ionizing radiation before or during the culture, preferably gamma rays.

[0014] As used herein, "skin scar" refers to fibrous tissue that replaces normal skin tissue after skin injury. Skin scars are the result of the biological process of wound repair in the skin. Scar tissue is typically composed of the same protein (i.e., collagen) as the tissue it replaces. However, the fibrous composition of scar tissue is significantly different from that of unscarred tissue. In normal tissue, collagen forms a random basket weave, but in scar tissue, collagen is cross-linked and forms a unidirectional alignment. Due to this alignment of collagen scar tissue, scarred skin usually has inferior functional quality compared to the random alignment of normal collagen, so the skin is, for example, less resistant to ultraviolet light, and sweat glands and hair follicles do not regrow within the scar tissue. According to the present invention, "skin scar" refers to any type of skin scar, particularly hypertrophic scars, keloid scars, and stretch marks.

[0015] Hypertrophic scars are the result of an overproduction of collagen, which causes the scar to be elevated but remains within the boundaries of the original wound. They usually occur within 4 to 8 weeks after wound infection or wound closure.

[0016] Keloid scars are a more serious form of excessive scarring because they can grow uncontrollably into large tumor-like (albeit benign) neoplasms. Keloid scars usually extend outside the original wound area. They can be itchy or painful, and their surgical removal can exacerbate the condition and deterioration of the keloid.

[0017] Stretch marks, also known as striae, are a form of scarring. They are caused when the skin is stretched rapidly (such as during pregnancy, significant weight gain, or the height growth spurt during puberty) or when the skin is placed under tension during the healing process.

[0018] As used herein, the terms "treatment" and "treating" of cutaneous scars refer to a reduction in scar volume and / or scar size by at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and / or ideally reconstruction of the original tissue. Administration of the compositions of the invention should result in partial or complete conversion of scar tissue to normal tissue.

[0019] A further aspect of the invention relates to a method for the cosmetic treatment of skin scars, comprising administering to the skin scar an effective amount of a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture.

[0020] According to a preferred embodiment of the present invention, the composition of the present invention is applied to or injected into the skin scar.

[0021] The composition of the present invention can be applied topically to skin scars or injected.For smaller skin scars, it has been found that topical application of the composition of the present invention to the scar is sufficient.In particular, stretch marks can be treated topically.When scars include excessive accumulation of collagen, it is advantageous to inject the composition of the present invention into the scar and / or the tissue surrounding the scar.

[0022] When the compositions of the present invention are administered topically, they may be provided as a gel, preferably a hydrogel, an ointment, a paste, a cream, a powder, a liniment, or a lotion. These topical dosage forms may be formulated using an oily base, an absorbent base, a water-in-oil emulsion base, an oil-in-water emulsion base, a water-soluble or water-miscible base, and may also contain an antioxidant such as vitamin C.

[0023] According to another preferred embodiment of the present invention, the composition is injected into the skin scar by means of a needle or a microneedle assembly comprising a plurality of microneedles.

[0024] The composition of the present invention can be injected into a skin scar using a needle. Depending on the size of the scar, the composition is injected into the scar at one or more injection sites. The use of a microneedle assembly containing multiple microneedles is particularly advantageous. The use of such a microneedle assembly allows the composition of the present invention to be injected at two or more injection sites simultaneously. Furthermore, the microneedle assembly is particularly suitable for introducing the composition of the present invention into a skin scar by penetrating a scar that has previously been covered with the composition of the present invention. In such cases, the microneedle assembly is moved in and out of the skin several times.

[0025] The microneedle assembly may also be part of a transdermal patch containing the composition of the present invention that is applied to a skin scar. Such transdermal patches are particularly suitable as they allow for continuous administration of the composition of the present invention over an extended period of time.

[0026] According to another preferred embodiment of the invention, the composition is at least partially injected into the dermal scar during removal of the needle or microneedle.

[0027] In order to spread the compositions of the present invention more evenly within the skin scar, it is preferred to inject the composition while removing the needle or needles from the skin scar.

[0028] The skin scar is preferably a hypertrophic scar, a keloid scar or a stretch mark.

[0029] According to another preferred embodiment of the invention, the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells.

[0030] According to a further preferred embodiment of the present invention, the PBMC cells are cultured in a cell growth medium, preferably a CellGro medium, more preferably a cell culture medium selected from the group consisting of Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

[0031] The PBMCs of the PBMC cell culture are subjected to ionizing radiation before or during culture. In addition to these stress-inducing conditions, the PBMCs may be subjected to additional stresses. Thus, according to a preferred embodiment of the present invention, the PBMCs are subjected to one or more additional stress-inducing conditions before or during culture.

[0032] As used herein, the term "under stress-inducing conditions" refers to culture conditions that result in stressed cells. Conditions that cause stress to cells include, among others, heat, chemicals, radiation, hypoxia, osmotic pressure, etc.

[0033] Further stress on the cells of the present invention results in further increased expression and secretion of substances beneficial for treating inflammatory skin conditions, particularly skin conditions associated with ischemia.

[0034] According to a preferred embodiment of the present invention, the stress-inducing conditions comprise hypoxia, ozone, heat (e.g., more than 2°C, preferably more than 5°C, more preferably more than 10°C above the optimal culture temperature for PBMCs, i.e., 37°C), radiation (e.g., ultraviolet light, gamma rays), chemicals, osmotic pressure (i.e., osmotic pressure conditions increased by at least 10% compared to those normally occurring in body fluids, in particular blood), or a combination thereof.

[0035] Therefore, according to a further preferred embodiment of the present invention, the stress-inducing conditions are selected from the group consisting of radiation, in particular ionizing radiation or ultraviolet light, hypoxia, ozone, heat, osmotic pressure and pH shift.

[0036] According to another preferred embodiment of the present invention, the PMC is subjected to a dose of ionizing radiation, preferably gamma radiation, of at least 10 Gy, preferably at least 20 Gy, more preferably at least 40 Gy, more preferably at least 50 Gy.

[0037] According to a preferred embodiment of the present invention, the PBMCs are cultured for at least 4 hours, preferably at least 6 hours, more preferably at least 12 hours before isolating the supernatant thereof.

[0038] According to a preferred embodiment of the present invention, PCBMC cell cultures are 1x10 5 ~1x10 8 PBMCs / ml, preferably 1x10 6 ~1x10 7 PBMCs / ml, more preferably 2x10 6 ~5x10 6 Contains PBMCs / ml.

[0039] The compositions of the present invention may contain pharmaceutically acceptable excipients such as diluents (e.g., buffers such as phosphate buffered saline (PBS), NaCl solution, or Hank's balanced salt solution (HBSS)), stabilizers (e.g., butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), vitamin C, vitamin E), carriers, etc. Methods for preparing these are well known to those skilled in the art.

[0040] To extend the shelf life of the compositions according to the invention, the supernatant may be lyophilized. Methods for lyophilizing such preparations are well known to those skilled in the art.

[0041] The lyophilized preparation can be contacted with water or an aqueous solution containing buffers, stabilizers, salts, etc. prior to its use.

[0042] According to another preferred embodiment of the present invention, the composition of the present invention can be used to treat skin scars in all kinds of mammals, including humans and horses, dogs, cats and camels.

[0043] The present invention is further illustrated by, but not limited to, the following examples. [Example]

[0044] example Materials and Methods

[0045] patient material

[0046] Excised scar tissue was obtained from three patients undergoing elective scar excision. The scars had previously been classified by plastic surgeons as hypertrophic pathological scars according to the POSAS standard (Fearmonti RM et al. Plastic and Reconstructive Surgery 127, 242 (2011)). All scars were mature, i.e., at least 2 years old, had not undergone surgery, and had not previously been treated with corticosteroids, 5-FU, irradiation, or similar. All scar samples were obtained from male and female patients under 45 years of age who had no chronic diseases or chronic medications. Healthy skin was obtained from excess abdominal skin excised during elective abdominoplasty from three healthy female donors aged 25–45 years.

[0047] animal

[0048] All animal experiments used 8- to 12-week-old female Balb / c mice (Medical University of Vienna Animal Breeding Facility, Austria). Mice were housed in a selected pathogen-free environment under a 12 / 12-h light / dark cycle according to enriched standard housing protocols and provided with food and water ad libitum.

[0049] A full-thickness wound and scarring model in mice

[0050] For the full-thickness skin wound and scarring model, mice were deeply anesthetized with intraperitoneal ketamine 80–100 mg / kg and xylazine 10–12.5 mg / kg, and postoperative analgesia was administered with a subcutaneous injection of 0.1 ml / 10 mg of buprenorphine and 7.5 mg / ml of piritramide in drinking water. A 9 × 9 mm square area was marked on the dorsum and excised with sharp scissors. The wound was left uncovered without further intervention for 4 weeks to heal, and the resulting scar tissue was observed and recorded.

[0051] Generation of APOSEC

[0052] PBMC secretomes were generated according to Good Manufacturing Practice (GMP) by the Austrian Red Cross, Blood Transfusion Service for Upper Austria (Austria) as described (Wagner T et al. Sci Rep 8, 18016 (2018); Laggner M et al. Stem cell research & therapy 11, 9 (2020)). PBMCs were obtained by Ficoll-Paque PLUS (GE Healthcare, USA)-assisted density gradient centrifugation and 25 × 10 6The secretomes were adjusted to a concentration of 1000 cells / mL (25 U / mL, 1 unit = 1 million secretome cells) and exposed to 60 Gy of cesium-137 gamma irradiation (IBL 437C, Isotopen Diagnostik CIS GmbH, Germany). Cells were cultured in phenol red-free CellGenix GMP DC medium (CellGenix GmbH, Germany) for 24 ± 2 hours. Cells and cell debris were removed by centrifugation, and the supernatant was passed through a 0.2 μm filter. Viral clearance was performed using methylene blue treatment as described (Gugurell A et al., Blood Transfus 18(2020):30-39). Secretomes were lyophilized, terminally sterilized by high-dose gamma irradiation, and stored at -80°C. All experiments were performed using the following batches of secretome produced under GMP: A000918399086, A000918399095, and A000918399098, A000918399101, A000918399102, and A000918399105. Immediately before the experiments, the lyophilisates were resuspended in 0.9% NaCl to an original concentration of 25 U / ml.

[0053] APOSEC stimulation of mouse scars

[0054] Starting 29 days after skin wounding, mice were injected every 2 days for 2 weeks with 100 yl of 0.9% NaCl, medium (phenol red-free CellGenix GMP DC medium), or APOSEC prepared as described above, after which half of the mice in each group were sacrificed and subjected to analysis, while the other half were left without further intervention for an additional 2 weeks and then sacrificed.

[0055] Ex vivo skin and scar stimulation

[0056] Six-mm punch biopsies were taken from human skin and scar tissue, and subcutaneous adipose tissue was removed. The biopsies were placed in a 12-well plate containing 400 yl DMEM (Gibco, Thermo Fisher, USA, containing 10% fetal bovine serum and 1% penicillin / streptomycin) and 100 μl of CellGenix medium or 100 yl of APOSEC. An additional 100 μl of medium or APOSEC was injected into the upper dermis at the center of the biopsy. The biopsies were incubated for 24 hours and then harvested for scRNAseq analysis.

[0057] Skin and scar APOSEC stimulation, cell isolation and droplet-based scRNAseq

[0058] Mouse scar and human irritated skin and scar samples were digested for 2.5 hours using a Miltenyi Whole Skin Dissociation Kit (Miltenyi Biotec, Germany) according to the manufacturer's protocol and processed using a GentleMACS OctoDissociator (Miltenyi). The cell suspension was passed through 100 μm and 40 μm filters, centrifuged at 1500 rpm for 10 minutes, washed twice, and resuspended in 0.04% FBS in phosphate-buffered saline (PBS). 1 μl of DAPI per million cells was added for 30 seconds, and the cells were washed twice again and sorted for viability using a MoFlo Astrios high-speed cell sorter (Beckman-Coulter, USA). Only clearly DAPI-negative cells were used for further processing. Immediately after sorting, live cells were loaded onto a 10X Chromium instrument (Single cell gene expression 3'v 2 / 3, 10X Genomics, USA) to generate gel beads in emulsion (GEMs). GEM generation, library preparation, RNA sequencing, demultiplexing, and counting were performed by the Biomedical Sequencing Core Facility at the Center for Molecular Medicine (CeMM, Austria). Sequencing was performed on an Illumina HiSeq 3000 / 4000 (Illumina, USA) with 2 × 75 bp paired-end sequences.

[0059] result

[0060] APOSEC prevents TGF-beta-mediated destruction of dermal elastic fibers

[0061] Ex vivo biopsies were injected with TGF-beta 1, TGF-beta 1 and medium, and TGF-beta 1 and APOSEC. Normal human skin biopsies were injected with TGF-beta 1 to induce scar-like tissue remodeling. Two days after TGF-beta 1 injection, destruction of the dense extracellular matrix (ECM) and elastic fibers was observed. Similar morphological changes were observed when TGF-beta 1 and medium were injected into the skin. The combination of TGF-beta 1 and APOSEC inhibited ECM deposition and elastic fiber degradation.

[0062] APOSEC prevents TGF-beta-induced ECM accumulation in mouse skin

[0063] Next, we investigated whether similar effects would be observed when APOSEC was injected into mouse skin in vivo. Analysis showed that APOSEC significantly reduced collagen production and deposition after TGF-beta 1 injection. Furthermore, we observed a decrease in the expression of alpha-smooth muscle actin, a protein that represents myofibroblasts.

[0064] APOSEC prevents TGF-beta-induced differentiation of fibroblasts into myofibroblasts.

[0065] To further investigate the effect of APOSEC on the differentiation of fibroblasts into contractile myofibroblasts, human primary fibroblasts were stimulated in vitro with TGF-beta. Stimulation with TGF-beta strongly induced the expression of alpha-smooth muscle actin (alpha-SMA), a protein marker of myofibroblasts. While the addition of medium alone had no effect on alpha-SMA expression, the addition of APOSEC completely abolished TGF-beta-induced differentiation of fibroblasts into myofibroblasts (Figure 1A). Furthermore, elastin protein expression was significantly induced by TGF-beta and inhibited by APOSEC (Figure 1B).

[0066] Intradermal injection of APOSEC into ex vivo human scars modulates RNA expression of scar-associated genes

[0067] Punch biopsies of human skin and scar tissue were injected intradermally with APOSEC or control medium. After 24 hours, the biopsies were enzymatically dissociated and single cells were analyzed by single-cell RNA sequencing (Figure 2). Several mRNAs associated with scar formation showed significant modulation after injection of APOSEC. Administration of APOSEC to scar tissue clearly shifted the mRNA profile to levels occurring in healthy skin (i.e., non-scarred skin).

[0068] conclusion

[0069] These analyses demonstrate that administration of APOSEC, particularly intradermal injection, can improve scar quality by inhibiting myofibroblast differentiation and the production and deposition of extracellular matrix components. APOSEC clearly improves existing mature scars (as opposed to the development of new scars). The present disclosure includes the following preferred aspects. (1) A composition for use in the treatment of skin scars, comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture. (2) A method for the cosmetic treatment of skin scars, comprising administering to the skin scar an effective amount of a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture. (3) The composition for use according to (1) or the method according to (2), wherein the composition is applied to or injected into a skin scar. (4) The composition for use according to (3) or the method according to (3), wherein the composition is injected into the skin scar by means of a needle or a microneedle assembly comprising a plurality of microneedles. (5) The composition for use according to (4) or the method according to (4), wherein the composition is injected at least partially into the skin scar during removal of the needle or microneedle. (6) The composition for use according to any one of (1) or (3) to (5) or the method according to any one of (2) to (5), wherein the skin scar is a hypertrophic scar, a keloid scar, or a stretch mark. (7) The composition for use according to any one of (1) or (3) to (6), or the method according to any one of (2) to (6), wherein the PBMC cell culture comprises monocytes, T cells, B cells, and / or NK cells. (8) The composition for use according to any one of (1) or (3) to (7), or the method according to any one of (2) to (7), wherein the PBMCs are cultured in a cell culture medium selected from the group consisting of a cell growth medium, preferably CellGro medium, more preferably Cellgro GMP DC medium, RPMI, DMEM, X-vivo, and Ultraculture. (9) The composition for use according to any one of (1) or (3) to (8) or the method according to any one of (2) to (8), wherein the PBMCs are subjected to one or more stress-inducing conditions before or during culture. (10) The composition for use according to (9) or the method according to (9), wherein the stress-inducing conditions are selected from the group consisting of radiation, particularly ionizing radiation or ultraviolet light, hypoxia, ozone, heat, osmotic pressure, and pH shift. (11) The composition for use according to (10) or the method according to (10), wherein the PMCs are subjected to a dose of ionizing radiation of at least 10 Gy, preferably at least 20 Gy, more preferably at least 40 Gy, more preferably at least 50 Gy. (12) The composition for use according to any one of (1) or (3) to (11) or the method according to any one of (2) to (11), wherein the PBMCs are cultured for at least 4 hours, preferably at least 6 hours, more preferably at least 12 hours before isolating the supernatant thereof. (13) PCBMC cell cultures were 1 x 10 5 ~1×10 8 PBMCs / ml, preferably 1 x 10 6 ~1×10 7 PBMCs / ml, more preferably 2 x 10 6 ~5×10 6 The composition for use according to any one of (1) or (3) to (12) or the method according to any one of (2) to (12), comprising PBMCs / ml.

Claims

1. 1. A composition for use in the treatment of skin scars comprising a supernatant of a peripheral blood mononuclear cell (PBMC) cell culture, the skin scar is a hypertrophic scar or a keloid scar, and the PBMC cell culture comprises monocytes, T cells, B cells and NK cells; the PBMC cell culture is subjected to a dose of at least 10 Gy of ionizing radiation prior to or during culture; The above composition.

2. 1. A composition for use in a method for the cosmetic treatment of skin scars comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture, the method comprising the step of administering to the skin scar an effective amount of the composition, the PBMC cell culture comprises monocytes, T cells, B cells and NK cells; the PBMC cell culture is subjected to a dose of at least 10 Gy of ionizing radiation prior to or during culture; The above composition.

3. 3. A composition for use according to claim 1 or 2, wherein the composition is applied to or injected into a skin scar.

4. 4. The composition for use according to claim 3, wherein the composition is injected into the skin scar by means of a needle or by means of a microneedle assembly comprising a plurality of microneedles.

5. 5. The composition for use according to claim 4, wherein the composition is injected at least in part into the skin scar during removal of the needle or microneedle.

6. 3. The composition for use according to claim 2, wherein the skin scar is a stretch mark.

7. 3. The composition for use according to claim 1 or 2, wherein the PBMCs are subjected to a dose of ionizing radiation of at least 20 Gy, at least 40 Gy, or at least 50 Gy.

8. 3. The composition for use according to claim 1 or 2, wherein the PBMCs are cultured for at least 4 hours, at least 6 hours, or at least 12 hours before isolating the supernatant thereof.

9. PBMC cell cultures were 1 × 10 ５ ~1 x 10 ８ PBMCs / ml, 1 x 10 ６ ~1 x 10 ７ PBMCs / ml, or 2 x 10 ６ ~5 x 10 ６ 3. The composition for use according to claim 1 or 2, comprising PBMCs / ml.

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