Topical products for use on intact or damaged skin, for cosmetic purposes and / or as medical devices.

Abstract

This invention relates to the field of cosmetics, and more particularly to cosmetics and their use. Specifically, the present invention relates to a cosmetic product comprising a concentrate and sonicated hyaluronic acid having an average molecular weight in the range of 100 kDa to 500 kDa, wherein the concentrate comprises at least one growth factor and at least one cytokine. The present invention further relates to the use of cosmetics for promoting tissue regeneration, improving skin texture, reducing skin wrinkles and scars, and reducing skin irritation and blemishes.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and particularly to cosmetics and their use. Specifically, the present invention relates to a cosmetic comprising a concentrate and ultrasonicated hyaluronic acid having an average molecular weight in the range of 80 kDa to 1200 kDa, wherein the concentrate comprises at least one growth factor and at least one cytokine. The present invention further relates to the use of cosmetics for promoting tissue regeneration, improving the texture of the skin, reducing skin wrinkles and scars, and reducing skin irritation and blemishes.

Background Art

[0002] Currently, there is no fundamental treatment method for aging skin, and the treatment for skin showing signs of aging and / or wrinkles is temporary and has drawbacks and side effects. The decrease in the strength and thickness of the skin is due to the decrease in collagen and elastic protein present in the dermis layer, which may cause fine wrinkles and wrinkles. For the treatment of facial wrinkles, many surgical means such as facelift, laser surgery, skin peeling, injection therapy, etc. are available. However, surgical procedures need to be repeated over time and may cause complications. As non-invasive treatments, topical preparations are used, but none of the methods can completely remove wrinkles, and multiple and often expensive treatments are required. Some topical preparations cause skin irritation and induce a wound healing response, but do not sufficiently replenish the proteins necessary for the treatment and / or prevention of age-related defects into the thinning skin.

[0003] Cell culture media are essential for cell growth and function maintenance in vitro, and supply nutrients and growth factors such as essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, nucleosides, etc. Many cell culture media are designed to allow for the growth and experimental application of a wide range of cell types. When cells are brought into contact with a cell culture medium, the cells are said to be "incubated" in the medium.

[0004] The cell culture medium used to incubate cells is known as the "conditioning medium." Conditioning medium differs from the original medium because it contains numerous cell-derived metabolites and secreted proteins, as well as various components of the original medium. Examples of metabolites and proteins secreted into the medium by cells during growth include growth factors, inflammatory mediators, and other extracellular proteins. These may have beneficial biological effects on epidermal remodeling, and are therefore attracting increasing attention in the cosmetics field. However, the need for and importance of more effective cosmetics for treating and / or preventing skin defects caused by aging and environmental factors is increasing. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the objective of the present invention is to develop a useful skincare product that exerts effects not only on the skin surface but also on the dermis layer. [Means for solving the problem]

[0006] In fact, although not bound by any theory, the inventors have surprisingly found that the problem can be solved with the following cosmetics: - At least one growth factor and at least one cytokine secreted from fibroblast cultures, - Ultrasonically treated hyaluronic acid having an average molecular weight in the range of 80kDa to 1200kDa Cosmetics containing, The aforementioned concentrate is prepared by the following process: a. A step of providing fibroblast cultures in a cell culture medium, b. A step of stimulating the release of at least one growth factor and at least one cytokine into the cell culture medium of step a to obtain a conditioned cell culture medium, c. A step of isolating the conditioned cell culture medium from the cell culture to obtain the isolated conditioned cell culture medium, d. A step of concentrating the isolated conditioned cell culture medium from step c. Cosmetics that can be obtained by a method including the following.

[0007] Cosmetics may be topical formulations that are effective in treating and / or preventing skin damage, wrinkles, and / or other defects caused by aging or environmental factors. Cosmetics may be formulated to prevent, reduce and / or eliminate wrinkles, frown lines, scars and other skin conditions associated with aging, in addition to or as a substitute for surgery, injectable preparations, silicones or other products.

[0008] In a second embodiment, the present invention provides the use of cosmetics to promote tissue regeneration. In a third embodiment, the cosmetic according to the present invention can be used to improve the texture of the skin. In a fourth embodiment, the present invention relates to the use of cosmetics for reducing wrinkles on the skin. In a fifth aspect, the present invention relates to the use of cosmetics for reducing scarring. In a sixth aspect, the use of cosmetics to reduce skin irritation and discoloration is described herein.

[0009] The features and advantages of the present invention will become apparent from the following detailed description, exemplary and non-limiting embodiments, and attached Figures 1-8. [Brief explanation of the drawing]

[0010] [Figure 1]Electrophoresis gel for determining protein content. CCM samples were loaded onto an acrylamide gel (4-12% gradient): Lot 1 was at position 4, and Lot 2 was at position 7. Subsequently, the gel was stained with silver to highlight proteins as brownish-red. [Figure 2] This is an explanatory diagram of the co-culture assay in the Transwell system described in Example 6. [Figure 3] RT-PCR results. The bar graphs show the gene expression levels of HMGB1 and type I collagen in fibroblasts: fibroblast population 1 (left), fibroblast population 2 (right), and the central bar graph corresponds to fibroblast population 1 cultured in a Transwell system in indirect contact with fibroblast population 2. The left and right bar graphs represent control cells, and the central bar graph represents treated cells. A: HMGB1 (48 hours), B: type I collagen (day 8). [Figure 4] Gel electrophoresis of HA samples after Stain-All staining. This dye causes hyaluronic acid to appear blue, making it easily identifiable. [Figure 5] Gaussian distribution of HA ultrasonically treated fragments described in Example 7. A: Cream, B: Serum [Figure 6] Stability testing of HA. Electrophoresis on agarose gel and staining with Stain All. Hyaluronic acid is identified by this dye, which causes it to turn blue. [Figure 7] Fibroblast scratch test. Images acquired at 100x magnification and graphs of the results obtained as follows: A.T0: Fibroblasts immediately after injury, B.T24: Spontaneous cell migration 24 hours after skin is untreated, C.T24: Cell migration 24 hours after skin is treated with the cosmetic of the present invention during 24-hour incubation, D: Graph of migration area % after 24 hours. [Figure 8] Keratinocyte scratch test. Images acquired at 100x magnification and graphs of results obtained over 24 hours of incubation. Results were obtained as follows: A. T0: Keratinocytes immediately after injury, B. T24: Natural cell migration after 24 hours in untreated skin, C. T24: Cell migration after 24 hours in treated skin with the cosmetic of the present invention, D. Graph of migration area % after 24 hours. Detailed Description of the Invention

[0011] The present invention relates to a cosmetic comprising: - at least one growth factor and at least one cytokine secreted from a fibroblast culture, and - sonicated hyaluronic acid having an average molecular weight in the range of 80 kDa to 1200 kDa wherein the concentrate is obtained by a method comprising the following steps: a. providing a fibroblast culture in a cell culture medium; b. stimulating the release of at least one growth factor and at least one cytokine into the cell culture medium of step a to obtain a conditioned cell culture medium; c. isolating the conditioned cell culture medium of step b from the cell culture to obtain an isolated conditioned cell culture medium; d. concentrating the isolated conditioned cell culture medium of step c. The obtained cosmetic contains a mixture of growth factors and cytokines secreted by fibroblasts together with matrix proteins. As used herein, the term "concentrate" or "concentrated conditioned medium" or "CCM" refers to the product obtained after step d. of the method of the present invention. Example 4 describes a possible embodiment for obtaining CCM by tangential flow filtration.

[0012]

[0013]

[0014] ​​In this specification, the terms “ultrasonically treated hyaluronic acid,” “HA,” or “ultrasonically treated sodium hyaluronate” refer to hyaluronic acid that has been ultrasonically treated and contains hyaluronic acid fragments of different sizes, as illustrated in Example 7. Depending on the formulation of the product, HA may exist as ultrasonically treated HA alone, or as a mixture of ultrasonically treated HA and unultrasonically treated low molecular weight HA. For example, the product may contain ultrasonically treated HA alone having a molecular weight in the range of 1200 kDa to 80 kDa, or a mixture of ultrasonically treated hyaluronic acid (in the range of 1200 kDa to 80 kDa) with the addition of unultrasonically treated low molecular weight hyaluronic acid (HA-LMW) having a molecular weight in the range of 1000 to 400 kDa. Typically, the HA in the products of the present invention has a size range of 1200 kDa to 80 kDa and is included in various amounts, for example, the following amounts: -5 to 20%, preferably about 9%, are fragments of 1200 to 800 kDa. -30-40%, preferably about 32-33%, are fragments of 800-400 kDa. -30-40%, preferably about 34-35%, are fragments of 400-200 kDa, and -5 to 20%, preferably about 15%, of fragments with a coherence of 200 to 80 kDa.

[0015] One of the most important properties of HA is represented by its molecular weight. Molecular weight defines the physical properties of HA and is essential in determining the molecular permeability to the skin. From a morphological standpoint, shorter polymer chain lengths offer advantages in terms of ease of unwinding and mobility (medium / low molecular weight HA). Longer polymer chain lengths result in bulkier and slower movement (high molecular weight HA).

[0016] High molecular weight HA (over 500 kDa) is not absorbed by the skin and exerts its effects at the epidermal level. In fact, when applied to the skin surface, high molecular weight HA acts by forming a hydrated viscoelastic film. Because this film is air permeable, skin respiration is not inhibited while the film "fixes" moisture on the skin surface. Indeed, the greatest advantage of high molecular weight hyaluronic acid is its excellent water-retention capacity, and therefore, when applied to the skin, HA primarily functions as a film-forming polymer, reducing water evaporation through an occlusive effect. Furthermore, this HA film-forming action reduces evaporation from the skin by mechanical means and limits interaction with environmental factors such as temperature, humidity, and UV irradiation. As a result, the protective barrier of HA improves the moisture content of the skin and promotes local healing of superficial wounds.

[0017] Medium / low molecular weight HA can reach the dermis and exert its function in the deepest layers of the skin. This is because smaller molecules experience less obstruction during epidermal passage, allowing them to function effectively in the dermis. HA is an essential component of the extracellular matrix of the dermis, stimulating the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes, and promoting neovascularization. Through its action in the deep layers of the skin, HA can maintain high moisture retention levels in the deep epidermis, improving skin tone and elasticity. The contribution of medium- and low-molecular-weight HA is essential for assisting in the physiological maintenance of the extracellular matrix in the dermis. In order for HA to exert its effects at both skin levels, a portion of the HA in the product of this invention is fragmented by ultrasonic treatment.

[0018] In this specification, "cosmetic" means a product containing two components: hyaluronic acid (HA in the form of HA-LMW alone, or a mixture of HA-LMW and ultrasonically treated HA, depending on the product formulation) and CCM. Preferably, the cosmetic of the present invention may further contain an excipient suitable for the final formulation. These two main components, CCM and HA, work synergistically to enhance each other's potential.

[0019] Not bound by any theory, CCM acts as a stabilizer for HA, • HA interacts with CCM-derived proteins, allowing these proteins to pass through the epidermal barrier and exert their effects more easily in the dermis.

[0020] The synergistic action of CCM and HA in cosmetics promotes the natural regeneration of the epidermis and dermis. The concentrate does not contain animal-derived proteins and is obtained from a culture of fibroblasts cultured in the method of the present invention.

[0021] In one embodiment, the cosmetic of the present invention can be obtained by a method in which step b., which stimulates the release of at least one growth factor and at least one cytokine into the cell culture medium, is carried out by subjecting the fibroblast culture to a nutrient depletion step.

[0022] In a preferred embodiment, the concentrated conditioned product may be sterile filtered in the final step d of the method. In a further embodiment, the cosmetic of the present invention can be obtained by isolating the fibroblasts from a tissue sample, wherein the tissue is preferably selected from the group consisting of skin and umbilical cord.

[0023] To isolate cells from the selected tissue, the skin biopsy sample is subjected to digestion. As described in Example 1, the tissue can be treated with a neutral protease, washed, and then digested with collagenase. The resulting cell suspension is seeded in complete culture medium and incubated. Therefore, in further embodiments, the cosmetic of the present invention can be obtained by isolating the fibroblasts from the tissue sample by digestion with a protease, followed by digestion with collagenase.

[0024] In a further embodiment, the cosmetic of the present invention can be obtained by a method in which the concentrate comprises a mixture of growth factors, cytokines, and matrix proteins.

[0025] The protein content of CCM can be quantified using the Bicinchoninic Acid (BCA) protein assay, also known as the Smith assay, as shown in Example 5.

[0026] In a further embodiment, the cosmetic of the present invention may be obtained by a method in which the concentrate contains one or more growth factors (for example, those listed in Table 1 which enumerate the major components of the fibroblast secretome). The cosmetic of the present invention can be obtained by a method wherein the concentrate contains one or more growth factors selected from the group including: vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β) and fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (GCSF), keratinocyte growth factor (KGF), transforming growth factor β3 (TGF-β3), nerve growth factor β (NGF-β), sirtuin 1 (SIRT1), sirtuin 2 (SIRT2), metalloproteinase 1 tissue inhibitor (TIMP1), macrophage colony-stimulating factor 1 (M-CSF or CSF1), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF2), interleukin 10 (IL-10), and hepatocyte growth factor (HGF).

[0027] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0028] Based on the studies conducted to date, we have definitively confirmed the presence of FGF2 and type I collagen in CCM, as shown in Tables 2A and 2B. [Table 2-1] [Table 2-2]

[0029] The concentrated conditioned medium (CCM) is a conditioned medium obtained from fibroblast culture that does not contain animal-derived components. In the final steps c. and d. of the method of the present invention, the collected concentrated conditioned medium was optionally subjected to sterile filtration. The final product contained a mixture of growth factors and cytokines secreted by fibroblasts, and matrix proteins useful for tissue regeneration. CCM contains approximately 10-50 μg / mL of proteins with different molecular weights. Table 3 shows the quantification of CCM protein content using microBCA.

[0030] [Table 3]

[0031] This heterogeneous group of proteins is referred to in the literature as the fibroblast secretome. It contains countless proteins, some of which have yet to be identified. Figure 1 shows the electrophoresis results of fibroblast-derived proteins.

[0032] The main molecules present in the fibroblast secretome and consequently in CCM are vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), and fibroblast growth factors (FGFs). These last factors play a role in collagen deposition in the extracellular matrix, and furthermore, collagen and elastin synthesis determine skin elasticity and toughness, which are characteristics of skin that decrease with age, making them useful in anti-aging therapies.

[0033] Other growth factors, such as platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (GCSF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), are involved in dermal remodeling by stimulating the synthesis of new collagen, elastin, and glycosaminoglycans and mediating angiogenesis.

[0034] While not bound by any particular theory, a co-culture test was conducted to clarify the mechanism of action of CCM, verifying whether components of the secretome are released from fibroblasts into the culture medium and whether they affect a second population of fibroblasts. This assay was intended to mimic the physiological conditions under which different populations of fibroblasts coexist within the same tissue as skin.

[0035] We investigated the behavior of fibroblasts using the Transwell system. We were able to examine the indirect interactions between two different cell populations that were incubated together in the same culture medium (Example 6).

[0036] In a further embodiment, the cosmetic of the present invention may be obtained by comprising ultrasonically treated hyaluronic acid fragments having a size of 1200 kDa to 80 kDa in various amounts, for example, about 9% of 1200 to 800 kDa fragments, about 32 to 33% of 800 to 400 kDa fragments, about 34 to 35% of 400 to 200 kDa fragments, and about 15% of 200 to 80 kDa fragments.

[0037] One of the most important properties of HA is represented by its molecular weight. Molecular weight defines the physical properties of HA and is essential for determining the molecular permeability to the skin. From a morphological standpoint, shorter polymer chain lengths result in more easily unraveled chains and improved mobility (medium / low molecular weight HA). Longer polymer chain lengths result in bulkier polymers and slower diffusion (high molecular weight HA).

[0038] High molecular weight hyaluronic acid (over 500 kDa) is not absorbed by the skin and acts at the epidermal level. In fact, when applied to the skin surface, high molecular weight hyaluronic acid acts by forming a hydrated viscoelastic film. Because this film is air permeable, skin respiration is not inhibited while the film "fixes" moisture to the skin surface. In fact, the greatest advantage of high molecular weight hyaluronic acid is its excellent water-retention capacity, and therefore, when applied to the skin, HA primarily functions as a film-forming polymer, reducing water evaporation through an occlusive effect. Furthermore, this HA film-forming action reduces evaporation from the skin by mechanical means and limits interaction with environmental factors such as temperature, humidity, and UV irradiation. As a result, the protective barrier of HA improves the moisture content of the skin and promotes local healing of superficial wounds.

[0039] Medium / low molecular weight HA can reach the dermis, the deepest layer of the skin, and exert its function there. This is because smaller molecules experience less obstruction during passage through the epidermis, allowing them to function effectively in the dermis.

[0040] HA is an essential component of the extracellular matrix of the dermis because it stimulates the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes, and promotes neovascularization. Through its action in the deeper layers of the skin, HA can maintain high levels of moisture retention in the deep epidermis, improving skin tone and elasticity.

[0041] The contribution of medium- and low-molecular-weight HA is essential for assisting in the physiological maintenance of the extracellular matrix in the dermis.

[0042] To ensure that HA acts on both layers of the skin (outer and inner layers), a portion of the HA in the cosmetic product of this invention is fragmented by sonication. The device used for sonication of the HA was a Sonicated Sonics Vibracell model VCX 130 SN40225L, and the process was performed for 10 minutes at 100% output with intermittent conditions of 10 seconds ON / 10 seconds OFF. Note that other devices can be used to obtain fragments of different size ranges, in which case the device settings must be adjusted to suit the purpose.

[0043] The fragmentation process is carried out using physical and non-chemical agents to avoid generating potentially harmful reaction intermediates or processing residues (Example 7).

[0044] In a further embodiment, the cosmetic of the present invention may be obtained by a method in which the cosmetic is in the form of a cream or serum and further comprises an excipient that is acceptable as a cosmetic. Cosmetics can be formulated as functional cosmetic facial creams, lotions, and / or serums for topical application, and may or may not contain additional growth factors, peptides, and / or other proteins and bioactive substances. In addition to active ingredients, products may contain cosmetic-acceptable excipients such as emollients, water binders, moisturizers, vitamins, antioxidants, anti-irritants, and sedatives.

[0045] The cosmetics of the present invention have shown remarkable effectiveness in promoting the natural regeneration of the dermis in various situations, including post-laser and post-radiation therapy treatments, scarring, wrinkles, and various types of irritation (redness and sun irritation). Therefore, in a second embodiment, the present invention provides the use of cosmetics to promote tissue regeneration. In a third embodiment, the cosmetic according to the present invention can be used to improve the texture of the skin. In a fourth embodiment, the present invention relates to the use of cosmetics for reducing wrinkles on the skin. In a fifth aspect, the present invention relates to the use of cosmetics for reducing scarring. In a sixth aspect, the use of cosmetics to reduce skin irritation and discoloration is described herein.

[0046] Various embodiments and aspects of the present invention, as described above in this specification and in the claims below, are experimentally supported in the following examples. [Examples]

[0047] Herein, we refer to the following examples illustrating some embodiments of the present invention, along with the above description. Tissue samples were obtained after obtaining informed consent from the donor. In the preparation of concentrated culture medium (CCM), appropriate skin or umbilical cord donor (discarded tissue) was selected in accordance with the European Directives on the Provision, Procurement, Testing, Processing, Preservation, Storage and Distribution of Human Tissues and Cells (2004 / 23 / EC, 2006 / 17 / EC, and subsequent amendments and national transitions).

[0048] All human tissue samples were stored in dedicated transport culture media and transported to certified cell processing facilities by authorized transporters under temperature control of 2-8°C. Upon arrival, the integrity of the packaging, the serological test report, and the temperature history were checked, and then the tissue was digested using GMP-grade enzymes (neutral protease, collagenase, and hyaluronidase). The enzymes used for tissue digestion were typically freeze-dried and then reconstituted according to the manufacturer's instructions.

[0049] Example 1: Cell isolation from skin Neutral protease digestion 1cm 2 To digest tissue fragments of this size: A 7 mL digestion solution was prepared by adding a volume of neutral protease equal to 6 DMCU (1.2 mL), and the volume was adjusted with Hanks equilibrium salt solution (HBSS). A 2 mM solution was prepared by adding 14 μL of 1 M CaCl2 solution. 1 DMC unit is represented by the catalyzed cleavage of peptide bonds from dimethylcasein at 25°C and pH 7.0, and quantified by TNBS1 as nascent terminal amino groups.

[0050] Before proceeding with neutral protease digestion, the samples were washed twice with a DPBS solution containing antibiotics (pen-strep), and then twice with Dulbecco's phosphate-buffered saline (DPBS) alone. After washing, the skin samples were placed in tubes containing digestive solution and incubated overnight at 2-8°C. For larger tissue samples, they were cut into smaller pieces with a scalpel, and a proportional volume was used for the digestion method described above.

[0051] Dermis / epidermis separation, collagenase digestion, and seeding into flasks After removing the neutral protease, the sample was washed with DPBS and transferred to a Petri dish for dermal / epidermal separation. After removing the epidermis, the dermis was fragmented into small pieces. The obtained fragments were placed in tubes prepared with 10 mL of collagenase solution (0.3 PZU / mL in HBSS) and 20 μL of 1 M CaCl2 solution (final 2 mM solution). The sample was incubated in a 37°C incubator for 2 hours. According to PZU:Wunsch, 1 U catalyzes the hydrolysis of 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine at 1 μmol per minute at 25°C and pH 7.1.

[0052] At the end of the 2-hour digestion period, the digested solution was filtered through a 100 μm filter. The filter was washed with 10 mL of HBSS and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, the pellet was resuspended in 10 mL of complete medium (CM), and the resulting whole cell suspension was seeded into a T25 flask and incubated at 37°C in a 5% CO2 incubator for 2–4 days.

[0053] material: a. Preparation of neutral proteases: The lyophilized enzyme was first resuspended to obtain a concentration of 100 DMCU / mL, and then further diluted to 5 DMCU / mL. The solution was filtered sterile and aliquoted into 1.5 mL portions. Each vial was labeled with the product name, lot number, concentration, and expiration date (1 year after reconstitution).

[0054] b. Preparation of collagenase: The freeze-dried enzyme was first resuspended in 1 mL to obtain a stock solution (PZU / mL according to the concentration indicated on the certificate of analysis). For the preparation of the working solution, the stock solution was diluted and sterile filtered to obtain aliquots with a concentration of 3 PZU. This solution was always stored on ice until use. Each vial was labeled with the product name, lot number, concentration, and expiration date (1 year after reconstitution).

[0055] c. Preparation of complete medium (CM): The complete culture medium (CM) consisted of DMEM, glutamine, sodium pyruvate, and fetal bovine serum (irradiated, EDQM certified), and its composition was as follows: - DMEM (high glucose) 500mL -FBS 50mL - Glutamine 200 mM 10 mL - Sodium pyruvate 1M 5mL

[0056] All culture medium components were sterile and prepared under laminar flow hood (grade A) and ambient environment grade B conditions. Each component was aseptically added to a bottle containing DMEM.

[0057] Example 2: Cell isolation from umbilical cord Digestion of hyaluronidase and collagenase and seeding into flasks The umbilical cord sample was washed with DPBS, transferred to a Petri dish, separated into small pieces, and then crushed into fine tissue clumps. These tissue clumps were placed in a 50 mL tube, an equal volume of DPBS was added, and then hyaluronidase and collagenase were added in amounts totaling 1% of the final volume, along with calcium gluconate in an amount equivalent to 0.04% of a 10% calcium gluconate solution. The sample was incubated at 37°C for 2 hours.

[0058] After digestion was complete, the sample was diluted with DPBS (1:1), centrifuged at 300g for 10 seconds to settle all undigested debris, and the digested solution was passed through a 1mm strainer and then through a 100μm filter. The solution was centrifuged at 450g rpm for 7 minutes. The supernatant was discarded, the pellet was resuspended in 25 mL of complete medium (CM), and the resulting whole cell suspension was seeded into a T150 flask and incubated at 37°C in a 5% CO2 incubator for 2–4 days.

[0059] material: a. Preparation of hyaluronidase: The lyophilized enzyme was resuspended to obtain a 10 KU / mL solution. The solution was filtered sterile and aliquoted into 1 ml portions. Each vial was labeled with the product name, lot number, concentration, and expiration date (1 year after reconstitution).

[0060] b. Preparation of collagenase: The lyophilized enzyme was resuspended to obtain a 50 mg / ml solution. The solution was filtered sterile and dispensed into 1 ml aliquots. Each vial was labeled with the product name, lot number, concentration, and expiration date (1 year after reconstitution).

[0061] c. Preparation of complete medium (CM): The culture medium consisted of DMEM low glucose, fetal bovine serum (irradiated, EDQM certified), human platelet lysate (HPL), and heparin. The complete medium (CM) formulation was as follows: - DMEM (low glucose) 500mL -FBS 15mL -HPL 15mL - Heparin (1000 U / mL) 2.5 mL

[0062] All culture medium components were sterile and prepared under laminar flow hood (grade A) and ambient environment grade B conditions. Each component was aseptically added to a bottle containing DMEM.

[0063] Example 3: Cell proliferation and inoculation in a biofactory Proliferation: After isolating fibroblasts, the procedure for scaling up proliferation was the same for both the two different tissues in Example 1 and Example 2, with only the cell proliferation medium differing. The medium for skin-derived fibroblasts was as described in Example 1, and the medium for umbilical cord-derived fibroblasts was as described in Example 2.

[0064] The starvation medium is the same for both tissue samples. When the cells reached approximately 80% confluence under microscopic observation, the fibroblasts were detached from the flask using trypsin, the number of cells was counted, and they were seeded into new T150 flasks. The number of cells was gradually increased from one T150 to three T150s, and from three T150s to twelve T150s, to obtain the number of cells necessary for inoculation into two to three biofactories, each using 1 liter of complete culture medium. All steps were carried out while maintaining the same culture conditions (37°C, 5% CO2), with the culture medium being changed every 2 to 4 days until confluence was reached.

[0065] hunger: After reaching confluence in the biofactory, the complete medium (DMEM, glutamine, sodium pyruvate, FBS) was removed, and after two washes with DPBS, 1 L of incomplete medium (DMEM, glutamine, sodium pyruvate without phenol red) was added to each biofactory over 48 hours. The 48-hour nutrient depletion showed an effect of stimulating the release of regeneration-related factors into the culture medium. After this incubation, the medium was collected in sterile bottles and centrifuged to remove cell debris. The supernatant was filtered through a 0.22 μm filter system (500 mL), the bottles were coded for identification, and then frozen and stored at -20°C.

[0066] Example 4: Concentration of acclimatized medium by tangential flow filtration (TFF) This type of concentration allows for faster processing and enables the use of larger quantities of supernatant, on the order of liters.

[0067] The TFF enrichment process was carried out with the following operating parameters: Phase I: 10x concentrated Phase II: Diafiltration with 10 volumes of buffer (NaCl 0.9%) Phase III: 20x concentrated (final) Transmembrane pressure difference (TMP): 0.3 bar Flow rate: 500ml / min Molecular weight cutoff: 10 kDa

[0068] With the above settings, the average permeation flow rate is approximately 54 ml / min or approximately 20 L / m³. 2 A shear stress of approximately 2300-2400 s⁻¹ was obtained at a rate of / h. The required times for Phase I of enrichment, Phase II of diafiltration, and Phase III of enrichment were 32 minutes, 30 minutes, and 2 minutes, respectively. The system was then emptied to recover the entire product into a selected bag / container, filtered through a 0.22 mm syringe filter, and dispensed into aliquots. All aliquots were frozen at -20°C.

[0069] Example 5: Release test of concentrated conditioned medium: Protein quantification The protein content of CCM is quantified using the bicinchoninic acid (BCA) protein assay. The bicinchoninic acid (BCA) protein assay, also known as the Smith assay, is a highly sensitive colorimetric method suitable for solubilized protein solutions. The amount of protein in the solution could be quantified by measuring the absorbance spectrum and comparing it with that of a protein solution of known concentration. A standard albumin curve was used for the experiment. After a 2-hour incubation, samples were measured at 562 nm. The average 562 absorbance of the blank standard was subtracted from the average 562 absorbance of each standard and each sample. A standard curve was plotted with the concentration of the standard substance on the X-axis and absorbance on the Y-axis. The protein concentration of each unknown sample was determined using the obtained standard curve.

[0070] Example 6: Characterization of conditioned culture medium To investigate the indirect interactions between two different cell populations, we analyzed the behavior of fibroblasts using the Transwell system, a co-culture method. In this system, cells are incubated together, but the only thing they have in common is the culture medium.

[0071] The Transwell system provides a configuration in which one population of fibroblasts is cultured on the bottom surface of an outer chamber, and a second population of fibroblasts is placed on an inner chamber having a porous membrane. The two groups do not come into direct contact, and the Transwell system allows molecules released from cells in the upper compartment to pass through to cells in the lower compartment.

[0072] The tests were conducted under two conditions: one using two populations of unfrozen "normal" fibroblasts (referred to as population 1 and population 2), and another using one population of "normal" fibroblasts and one population of "frozen BLAST fibroblasts" (frozen in modified Tyrode solution). At 48 hours and 8 days, the expression levels of two markers related to tissue regeneration, namely HMGB1 and type I collagen, were analyzed.

[0073] This book only includes the results for populations 1 and 2 of normal fibroblasts. When population 1 was co-cultured with population 2, increased production of the signaling molecule HMGB1 was observed in fibroblasts from population 1 after 48 hours, and increased production of type I collagen was observed after 8 days. This indicates that fibroblasts can release factors into the culture medium, which can stimulate the surrounding environment through paracrine effects (Figure 3). Similar results were obtained in the co-culture assay of "normal" fibroblasts and "frozen BLAST fibroblasts."

[0074] Example 7: Method for ultrasonic treatment of hyaluronic acid The fragmentation process is carried out using physical and non-chemical agents to avoid generating potentially harmful reaction intermediates or processing residues.

[0075] High molecular weight hyaluronic acid (HMW-HA, approximately 2200-1800 kDa) in physiological saline solution was sonicated for 10 minutes at 100% power setting under intermittent conditions of 10 seconds ON / 10 seconds OFF (Sonicated Sonics Vibracell model VCX 130 SN 40225L). The time, setting, and frequency selected for the sonication method of HA are also important in avoiding the formation of pro-inflammatory small fragments that may occur due to enzymatic fragmentation.

[0076] The product of the present invention may contain a mixture of HA with different molecular weights (either ultrasonically treated or untreated). The cream contains 0.25% ultrasonically treated HMW, while the serum contains 0.15% ultrasonically treated high molecular weight (HMW) HA and 0.1% LMW-HA. To confirm HA fragmentation, the sample was loaded onto a 1% agarose gel along with a reference sample and electrophoresis was performed (Figure 4).

[0077] Each sample is distributed and forms a smear. The smear is composed of HA fragments of different lengths; the upper part of the smear corresponds to larger fragments (which have a larger volume and do not easily propagate within the gel), while the lower part contains smaller fragments. For example, the smear of 1000 kDa HA (sample 6) remains in the upper part of the gel. HA in Blast cream (Sample 1) exhibits a smear similar to that of the HA250kDa standard marker (Sample 5).

[0078] To obtain more precise information on the distribution of HA fragments, smears of HA in Blast cream (Sample 1) and HA in Blast serum (Sample 4) were divided into square sections of the same size, and the color intensity of each section was quantified relative to a standard sample (as pixel values ​​using the ImageJ program). The darker the color, the greater the amount of fragments. The obtained values ​​were graphed, and the percentages were calculated (Figure 5). The molecular weight of HA in the cream and serum follows a Gaussian curve characterized by both medium-to-high molecular weight HA and medium-to-low molecular weight HA. The composition of HA in the cream and HA in the serum is shown in Table 4.

[0079] [Table 4]

[0080] This distribution ensures the advantages of both high-to-medium molecular weight and low-to-medium molecular weight polysaccharides. Depending on the size of the polysaccharides, at the epidermal level (fragments greater than 500 kDa), they impart skin properties such as elasticity, moisture content, and flexibility, while at the dermal level (fragments less than 500 kDa), they contribute to the physiological maintenance of the extracellular matrix and its anti-inflammatory effects.

[0081] Example 8: HA+ conditioned medium: Stability and protection The cosmetic product of this invention is derived from a combination of hyaluronic acid (HA) and concentrated conditioned medium (CCM).

[0082] Stability tests were conducted to show how the quality and stability of the components change over time under the influence of environmental factors (temperature, humidity, light) and other components / raw materials. In this experiment, the stability of HA in the presence of CCM, a concentrated conditioned medium obtained from in vitro cultured human fibroblasts, was evaluated. The integrity of HA was assessed by gel electrophoresis after 14 days.

[0083] The results clearly show no difference in size between HA alone and HA mixed with CCM. That is, the polysaccharides were the same size, no small fragments were formed, and no visible aggregates were observed. It can be concluded that CCM does not affect the integrity of HA. The stability of sonicated HA fragments is crucial for their effectiveness at both the epidermal and dermal levels. During their migration to the epithelial layer, HA undergoes a series of interactions with the surrounding environment. Hyaluronidase, a potent inhibitor of HA activity that catalyzes polypeptide degradation, is present in the skin. While this may be advantageous in some respects, as it breaks down high molecular weight HA (over 1000 kDa) into smaller fragments more easily absorbed by the skin, it can also lead to the formation of small HA fragments where random degradation can be pro-inflammatory.

[0084] To understand how CCM functions in the presence of this physiologically present enzyme in the skin, we prepared an enzyme assay. This test aims to mimic the physiological conditions of the epidermis. The objective of this study was to obtain not only qualitative but also quantitative data to demonstrate that the presence of CCM has the ability to stabilize HA from hyaluronidase attack. This experiment utilizes the ability of long-chain hyaluronic acid to cause turbidity in albumin-containing solutions. Turbidity is a function of HA concentration. Higher turbidity indicates a greater abundance of HA. If HA is cleaved by hyaluronidase, it can no longer form a network with albumin, and the solution becomes clear. The decrease in turbidity is linked to enzymatic fragmentation of HA.

[0085] [Table 5]

[0086] The data shown in Table 5 indicates that HA alone was almost completely degraded in the presence of the enzyme (97.5%), while HA combined with CCM was only minimally degraded (3.17%). Competitive assays demonstrated the protective ability of CCM against HA under enzymatic action.

[0087] The protective effect of CCC on HA stability may be due to the sum of several factors. In fact, hyaluronidase inhibitors and / or antioxidants appear to be present in CCM, resulting in less fragmentation of HA. Furthermore, it is thought that small enzyme proteins exist in CCM that can chemically modify the structure of polysaccharides to make them more stable. From the two tests conducted, it can be concluded that sonicated HA has a size suitable for action in both the epidermis and dermis, and can interact with CCM proteins (via hydrophobic interactions (charge-charge) and / or hydrogen bonding), forming a novel complex exhibiting new properties that are more stable and less sensitive to hyaluronidase activity. The interaction between HA and proteins was also utilized for protein transport at the subepithelial level. As a result, intradermal action of the mixture occurs. These data support the robustness and effectiveness of the cosmetic product of the present invention.

[0088] Example 9: HA+ conditioned medium: Stability and protection: In vitro efficacy Wound healing assays are in vitro techniques for studying collective cell migration. They are also called scratch assays because they involve scratching a cell monolayer, imaging it at the start and at regular intervals to track the wound closure process, and comparing the resulting images to quantify the cell migration rate.

[0089] Because this mechanism involves many different physiological aspects, scratch assays are an excellent tool for studying cell migration. Cell migration plays a major role in skin re-epithelialization, and therefore, studying cell migration can provide advances in understanding non-healing wounds. This assay was used to verify the ability of a system in which component CCM was added to fragmented HA to promote the natural cell migration (mimicking wound healing) of fibroblasts or keratinocytes.

[0090] On day 1, a scratch is introduced into a cell monolayer. The cells are incubated with essential media that mimic physiological conditions or a combination of HA and CCM. The scratch appears as a cell-free area, and the same location is imaged at time 0 and 24 hours. During this time, cells migrate into the void and attempt to close the gap. Image analysis is performed using the TScratch program to calculate the percentage of cell migration area (Figures 6 and 7).

[0091] Fibroblasts (Figures 6A-D) and keratinocytes (Figures 7A-D) incubated with the cosmetic of the present invention showed more rapid closure of the injury space compared to cells treated with essential media. The cosmetic promoted natural cell migration after injury. Scratch tests suggested that using the two components together enhances and synergistically improves the individual effects of each component. Specifically, the mechanical viscoelasticity of HA supports physiological dermal regeneration, and combining this with CCM demonstrates a stabilizing effect on HA.

[0092] Example 10: Product INCI The following table shows the components of some of the cosmetics of the present invention. Cream Rich (Table 6), Cream Light (Table 7), Serum (Face and / or Body) (Table 8), and Hair Serum (Table 9).

[0093] [Table 6] [Table 7] [Table 8] [Table 9]

[0094] From the above description and examples, the advantages achieved by the cosmetic described and obtained in accordance with the present invention are clear.

Claims

1. - At least one growth factor and at least one cytokine secreted from a fibroblast culture, - Ultrasonically treated hyaluronic acid having an average molecular weight in the range of 80 kDa to 1200 kDa Cosmetics containing, The aforementioned concentrate is produced by the following process: a. A step of providing fibroblast cultures in a cell culture medium, b. A step of stimulating the release of at least one growth factor and at least one cytokine into the cell culture medium of step a to obtain a conditioned cell culture medium, c. A step of isolating the conditioned cell culture medium from the cell culture to obtain the isolated conditioned cell culture medium, d. A step of concentrating the isolated conditioned cell culture medium from step c. It can be obtained by a method including, The cosmetic product wherein step b., which stimulates the release of at least one growth factor and at least one cytokine into the cell culture medium, is carried out by subjecting the fibroblast culture to a nutrient depletion step.

2. The cosmetic according to claim 1, wherein the fibroblasts are isolated from a tissue sample selected from the group consisting of skin and umbilical cord.

3. The cosmetic according to claim 2, wherein the fibroblasts are isolated from the tissue sample by digestion with protease, followed by digestion with collagenase.

4. The cosmetic according to any one of claims 1 to 3, wherein the concentrate comprises a mixture of growth factors, cytokines, and matrix proteins.

5. The cosmetic according to any one of claims 1 to 4, wherein the concentrate contains growth factors selected from the group including vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β) and fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (GCSF), keratinocyte growth factor (KGF), transforming growth factor β3 (TGF-β3), nerve growth factor β (NGF-β), sirtuin 1 (SIRT1), sirtuin 2 (SIRT2), metalloproteinase 1 tissue inhibitor (TIMP1), macrophage colony-stimulating factor 1 (M-CSF or CSF1), stem cell factor (SCF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF2), interleukin 10 (IL-10), and hepatocyte growth factor (HGF).

6. The cosmetic according to any one of claims 1 to 5, wherein the ultrasonically treated hyaluronic acid comprises ultrasonically treated hyaluronic acid fragments having sizes of 1200 to 800 kDa (5 to 20%), 800 to 400 kDa (30 to 40%), 400 to 200 kDa (30 to 40%), and 200 to 80 kDa (5 to 20%).

7. The cosmetic according to any one of claims 1 to 6, wherein the cosmetic is in the form of a cream or serum and further comprises an excipient that is acceptable as a cosmetic.

8. Use of the cosmetic product according to any one of claims 1 to 7 for promoting tissue regeneration.

9. Use of the cosmetic product according to any one of claims 1 to 7 for improving the texture of the skin.

10. Use of the cosmetic product according to any one of claims 1 to 7 for reducing wrinkles on the skin.

11. Use of the cosmetic product according to any one of claims 1 to 7 for reducing scarring.

12. Use of the cosmetic product according to any one of claims 1 to 7 for reducing skin irritation and blemishes.

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