Topical product for intact or damaged skin, for cosmetic use and / or as medical device

Abstract

The invention relates to the field of beauty, in particular to a beauty product and application thereof. In particular, the present invention relates to a cosmetic product comprising a concentrate and ultrasonically treated hyaluronic acid, the hyaluronic acid having an average molecular weight in the range of from 100 kD to 500 kDa; wherein the concentrate comprises at least one growth factor and at least one cytokine. The invention also relates to the use of said cosmetic product for promoting tissue regeneration, improving skin quality, reducing skin wrinkles and scars, and reducing skin irritation and flaws.

Description

Technical Field

[0001] This invention relates to the field of beauty, and more particularly to a beauty product and its uses. Specifically, the present invention relates to a cosmetic product comprising a concentrate and ultrasonically treated hyaluronic acid, wherein the hyaluronic acid has an average molecular weight of 80kD-1200kDa; wherein the concentrate comprises at least one growth factor and at least one cytokine.

[0002] The present invention also relates to the use of the aforementioned beauty products for promoting tissue regeneration, improving skin texture, reducing skin wrinkles and scars, and reducing skin irritation and blemishes. Background Technology

[0003] Currently, there are no treatments specifically for mature skin. Treatments for skin showing signs of aging and / or wrinkles are temporary and have limitations and side effects. The decrease in skin strength and thickness is caused by the loss of collagen and elastin in the dermis, which may lead to the formation of fine lines and wrinkles. Several surgical methods are available for treating facial wrinkles, such as facelifts, laser surgery, skinpeels, and injectable treatments. However, surgical methods require repeated treatments over time and can lead to complications. Non-invasive treatments include topical preparations; however, these methods cannot completely eliminate wrinkles and require multiple, often expensive, treatments. Some topical preparations can act as skin irritants, triggering a wound-healing response, but they are not effective at replenishing thinned skin with sufficient protein to treat and / or prevent age-related defects.

[0004] Cell culture media are essential for cell growth and function in vitro, providing nutrients and growth factors such as essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids, and nucleosides. Currently, various cell culture media have been formulated to support the growth of various cell types and a wide range of experimental applications. Once cells come into contact with cell culture media, it is called "culturing" cells in cell culture media. The culture medium in which cells have been cultured is called a "conditioned medium." Conditioned media differ from the original culture medium in that it contains a large number of cellular metabolites, secreted proteins, and various original components of the culture medium. Examples of metabolites and proteins secreted into the culture medium during cell growth include growth factors, inflammatory mediators, and other extracellular proteins. These substances have beneficial biological effects on epidermal remodeling and are therefore receiving increasing attention in the field of cosmetic medicine.

[0005] However, there is a growing demand and importance for more effective cosmetic products for treating and / or preventing skin defects caused by aging and environmental factors.

[0006] Therefore, the purpose of this invention is to develop a beneficial skin care product that is effective not only on the skin surface but also on the dermis. Summary of the Invention

[0007] In fact, without being limited to any particular theory, the inventors unexpectedly discovered that the above problems could be solved by a beauty product containing: - A concentrate containing at least one growth factor and at least one cytokine, secreted from fibroblast cultures; and - Hyaluronic acid treated with ultrasound has an average molecular weight of 80kDa-1200kDa; The concentrate can be prepared by a method including the following steps: a. Provide fibroblast cultures in cell culture media; b. Stimulate 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. Separate the conditioned cell culture medium from step b from the cell culture to obtain the separated conditioned cell culture medium; d. Concentrate the isolated cell culture medium from step c to obtain a concentrate.

[0008] For example, the cosmetic product may be a topical preparation used to treat and / or prevent skin damage, wrinkles and / or other defects caused by aging and environmental factors.

[0009] This beauty product can be formulated to prevent, reduce, and / or eliminate wrinkles, frown lines, scars, and other age-related skin problems, as a complement or alternative to surgery, injections, silicone, or other products.

[0010] In a second aspect, the invention provides the use of the cosmetic product for promoting tissue regeneration. In a third aspect, the cosmetic products according to the present invention can be used to improve skin texture.

[0011] In a fourth aspect, the present invention relates to the use of the cosmetic product for reducing skin wrinkles.

[0012] In a fifth aspect, the present invention relates to the use of the cosmetic product for reducing scars. In the sixth aspect, this article describes the use of this beauty product to reduce skin irritation and blemishes. Attached Figure Description

[0013] Through the following detailed description, the embodiments provided for illustrative and non-limiting purposes, and the appended...Figures 1-8 The features and advantages of the present invention will become apparent.

[0014] Figure 1 Protein content was determined by electrophoresis gel electrophoresis. CCM samples were loaded into an acrylamide gel (4%-12% gradient): Lot 1 was observed at position 4, and Lot 2 at position 7. The gel was then stained with silver to highlight the proteins in brown.

[0015] Figure 2 The diagram shows a co-culture experiment in the Transwell system described in Example 6.

[0016] Figure 3: RT-PCR results. The bars correspond to the gene expression levels of HMGB1 and type I collagen in fibroblasts: fibroblast population 1 (left), fibroblast population 2 (right), and the middle bar corresponds to fibroblast population 1 cultured in the Transwell system and indirectly contacted with fibroblast population 2. The bars on the left and right are control cells, and the middle bar is treated cells. A. HMGB1, 48 hours; B. Type I collagen, day 8. Figure 4 Gel electrophoresis image of HA samples after Stain-All staining. This dye makes hyaluronic acid appear blue.

[0017] Figure 5: Gaussian distribution of the HA fragments subjected to ultrasonic treatment as described in Example 7. A. Cream, B. Serum.

[0018] Figure 6 Stability testing of HA. Agarose gel electrophoresis was performed and stained with Stain-All. This dye gives hyaluronic acid a blue color.

[0019] Figure 7 Fibroblast scratch assay. Images were acquired at 100x, and the results were obtained at the following time points: A. T0: Immediate state of fibroblasts after injury; B. T24: Natural cell migration after 24 hours of untreated skin; C. T24: Cell migration after 24 hours of skin treatment with the cosmetic product of this invention during a 24-hour culture period; D: Graph of migration area % after 24 hours.

[0020] Figure 8Scratch assay of keratinocytes. Images were acquired at 100x and the results were obtained after 24 hours of incubation. Results were obtained at the following time points: A. T0: Immediate state of keratinocytes after damage; B. T24: Natural cell migration after 24 hours of untreated skin; C. T24: Cell migration after 24 hours of skin treatment with the cosmetic product of this invention; D: % of migration area after 24 hours. Detailed Implementation

[0021] This invention relates to a beauty product comprising: - A concentrate containing at least one growth factor and at least one cytokine, secreted from fibroblast cultures; and - Hyaluronic acid treated with ultrasound has an average molecular weight of 80kDa-1200kDa; The concentrate can be prepared by a method including the following steps: a. Provide fibroblast cultures in cell culture media; b. Stimulate 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. Separate the conditioned cell culture medium from step b from the cell culture to obtain the separated conditioned cell culture medium; d. Concentrate the isolated cell culture medium from step c to obtain a concentrate.

[0022] The resulting beauty products contain a mixture of growth factors and cytokines secreted by fibroblasts, as well as matrix proteins.

[0023] As used herein, the terms "concentrate," "concentrated conditioned medium," or "CCM" refer to the product obtained after step d of the method of the present invention. Example 4 describes a possible embodiment of obtaining CCM by tangential flow filtration.

[0024] As used herein, the terms "ultrasonicated hyaluronic acid" or "HA" or "ultrasonicated sodium hyaluronate" refer to hyaluronic acid that has undergone ultrasonic treatment (as shown in Example 7), which comprises hyaluronic acid fragments with different molecular weights. Depending on the product formulation, HA may be present solely as ultrasonicated HA or as a mixture of ultrasonicated HA and unultrasonicated low molecular weight HA. For example, the product may contain only ultrasonicated HA with a molecular weight of 1200 kDa-80 kDa; or a mixture of ultrasonicated hyaluronic acid (1200 kDa-80 kDa) and added low molecular weight hyaluronic acid (HA-LMW) with a molecular weight of 1000 kDa-400 kDa, which has not undergone ultrasonic treatment. Typically, the HA in the products of this invention has a molecular weight of 1200 kDa-80 kDa and varies in content, for example, the following contents: - 5%-20%, with approximately 9% of the fragments preferably having a value of 1200-800 kDa; - 30%-40%, preferably about 32-33% of the fragments are 800-400 kDa; - 30%-40%, preferably about 34-35% of the fragments are 400-200 kDa; and - 5%-20%, preferably about 15% of the fragments are 200-80 kDa.

[0025] One of the most important characteristics of HA is its molecular weight. Molecular weight determines the properties of hyaluronic acid and is key to its molecular skin permeability.

[0026] From a morphological perspective, shorter polymers are more prone to chain breaking and have an advantage in mobility (medium / low molecular weight HA). The longer the polymer, the larger its volume and the slower its mobility (high molecular weight HA).

[0027] High molecular weight hyaluronic acid (HA) (greater than 500 kDa) cannot be absorbed by the skin and exerts its effects only in the epidermis. In fact, when applied to the skin surface, high molecular weight HA works by forming a hydrated viscoelastic film. This film is breathable, thus not hindering skin respiration, while simultaneously "locking" moisture in the skin. Indeed, the most significant advantage of high molecular weight hyaluronic acid lies in its excellent water-retention capacity; therefore, once applied to the skin, HA primarily functions as a film-forming polymer: it reduces water evaporation, acting as a occlusive agent. Furthermore, HA's film-forming effect mechanically reduces skin moisture evaporation and limits interactions with environmental factors such as temperature, humidity, and UV radiation. In this way, the protective barrier of HA improves skin hydration and promotes local healing of superficial wounds.

[0028] Medium / low molecular weight HA can exert its effects in the deepest layers of the skin, reaching the dermis. Due to their smaller size, they encounter less resistance when passing through the epidermis, thus enabling them to function effectively in the dermis.

[0029] HA is a fundamental component of the dermal extracellular matrix because it stimulates the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes, and promotes angiogenesis. Thanks to its deep-seated effects on the skin, HA helps maintain a high level of hydration in the deep epidermis, thereby improving skin tone and elasticity.

[0030] The contribution of medium / low molecular weight HA is essential for the physiological maintenance of the extracellular matrix in the dermis.

[0031] To enable HA to function in both layers of the skin, a portion of the HA in the product of this invention is fragmented by ultrasonic treatment.

[0032] As used in this article, “cosmetic product” refers to a product containing two components: hyaluronic acid (HA may be in the form of HA-LMW, or a mixture of HA-LMW and ultrasonically treated HA, depending on the product formulation) and CCM. Preferably, the beauty product of the present invention may further include excipients suitable for the final formulation.

[0033] The two main components, CCM and HA, enhance each other's efficacy through synergistic effects. Without being limited to any particular theory:

[0034] The synergistic effect of CCM and HA in beauty products promotes the natural regeneration of the epidermis and dermis.

[0035] In the method of the present invention, the concentrate does not contain animal protein and is obtained from a culture of expanded fibroblasts.

[0036] In an embodiment, the cosmetic product of the present invention is prepared by a method wherein, in step b, the stimulation of at least one growth factor and at least one cytokine to release into the culture medium of the method is carried out by subjecting the fibroblast culture to a nutrient deprivation step.

[0037] In a preferred aspect, at the end of the method, in step d, the concentrated conditioned medium may be sterile filtered.

[0038] In a further embodiment, the cosmetic product of the present invention is prepared by a method wherein the fibroblasts are isolated from a tissue sample, preferably selected from the group consisting of free skin and umbilical cord.

[0039] To isolate cells from the selected tissue, the skin biopsy sample was digested. As described in Example 1, the tissue was first treated with a neutral protease, washed, and then digested with collagenase. Subsequently, the resulting cell suspension was seeded onto a complete culture medium and cultured. In a further embodiment, the cosmetic product of the present invention is thus prepared by a method wherein the fibroblasts are isolated from the tissue sample by digestion with a protease followed by digestion with a collagenase.

[0040] In a further embodiment, the beauty product of the present invention is prepared by a method wherein the concentrate comprises a mixture of growth factors, cytokines and matrix proteins.

[0041] 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.

[0042] In a further embodiment, the beauty product of the present invention can be prepared by a method wherein the concentrate contains one or more growth factors, such as those listed in Table 1, which lists the main components of the fibroblast secretome. The cosmetic product of the present invention is prepared by a method wherein the concentrate contains one or more growth factors selected from the group consisting of: vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), fibroblast growth factor (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), longevity protein 1 (SIRT1), longevity protein 2 (SIRT2), tissue inhibitor of metalloproteinases 1 (TIMP1), macrophage colony-stimulating factor 1 (M-CSF or CSF1), stem cell factor (SCF), insulin-like growth factor (IGF-1), insulin-like growth factor 2 (IGF2), interleukin-10 (IL-10), and hepatocyte growth factor (HGF).

[0043] Table 1

[0044]

[0045]

[0046]

[0047]

[0048] Based on current research, we can confidently say that FGF2 and type I collagen are present in the CCM, as shown in Tables 2A and 2B: Table 2A:

[0049] Table 2B:

[0050] Concentrated conditioned medium (CCM) is a conditioned medium obtained from fibroblast cultures and contains no animal-derived components. At the end of the method of this invention, steps c and d may optionally involve aseptic filtration of the collected concentrated conditioned medium. The final product contains a mixture of growth factors and cytokines secreted by fibroblasts, as well as matrix proteins that contribute to tissue regeneration.

[0051] CCM contains approximately 10-50 ug / mL of protein, which have different molecular weights.

[0052] Table 3 shows the quantitative analysis of protein content in CCM using the micro-BCA method.

[0053] Table 3

[0054] This heterogeneous group of proteins is referred to in the literature as the fibroblast secretome. It contains numerous proteins, some of which have not yet been identified.

[0055] Figure 1 The electrophoresis results of fibroblast protein content are shown.

[0056] The main molecules present in the fibroblast secretory group and subsequently in the extracellular matrix (CCM) are vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), and fibroblast growth factor (FGF). These factors play a crucial role in collagen deposition in the extracellular matrix and in anti-aging treatments, as the synthesis of collagen and elastin determines the skin's elasticity and resilience, properties that diminish with skin aging.

[0057] Other growth factors, such as platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (GCSF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF), participate in dermal remodeling by stimulating the synthesis of new collagen, new elastin, and new glycosaminoglycans, as well as by mediating angiogenesis.

[0058] To elucidate the mechanism of action of CCM without being limited to any particular theory, co-culture experiments were conducted to verify whether components of the secretomic group are released from fibroblasts into the culture medium, and whether these components affect a second group of fibroblasts. This study aimed to simulate a physiological state in which different fibroblast populations coexist in the same tissue (skin).

[0059] The Transwell system was used to study fibroblast behavior. Indirect interactions between two different cell populations cultured together in the same culture medium can be investigated (Example 6).

[0060] In a further embodiment, the cosmetic product of the present invention is prepared by a method wherein the ultrasonically treated hyaluronic acid comprises ultrasonically treated hyaluronic acid fragments with different contents and molecular weights of 1200 kDa-80 kDa, for example, approximately 9% of 1200 kDa-800 kDa fragments, approximately 32%-33% of 800 kDa-400 kDa fragments, approximately 34%-35% of 400 kDa-200 kDa fragments, and approximately 15% of 200 kDa-80 kDa fragments.

[0061] One of the most important characteristics of HA is its molecular weight. Molecular weight determines the properties of HA and is key to determining its molecular skin permeability.

[0062] From a morphological perspective, shorter polymers are more prone to chain breaking and have an advantage in mobility (medium / low molecular weight HA). The longer the polymer, the larger its volume and the slower its mobility (high molecular weight HA).

[0063] High molecular weight hyaluronic acid (HA) (greater than 500 kDa) cannot be absorbed by the skin and exerts its effects only in the epidermis. In fact, when applied to the skin surface, high molecular weight HA works by forming a hydrated viscoelastic film. This film is breathable, thus not hindering skin respiration, while simultaneously "locking" moisture in the skin. Indeed, the most significant advantage of high molecular weight hyaluronic acid lies in its excellent water-retention capacity; therefore, once applied to the skin, HA primarily functions as a film-forming polymer: it reduces water evaporation, acting as a occlusive agent. Furthermore, the film-forming effect of HA mechanically reduces skin moisture evaporation and limits interactions with environmental factors such as temperature, humidity, and UV radiation. In this way, the protective barrier of HA improves skin hydration and promotes local healing of superficial wounds.

[0064] Medium / low molecular weight HA can exert its effects in the deepest layers of the skin, reaching the dermis. Due to their smaller size, they encounter less resistance when passing through the epidermis, thus enabling them to function effectively in the dermis.

[0065] HA is a fundamental component of the dermal extracellular matrix because it stimulates the proliferation and migration of fibroblasts, endothelial cells, and keratinocytes, and promotes angiogenesis. Thanks to its deep-seated effects on the skin, HA helps maintain a high level of hydration in the deep epidermis, thereby improving skin tone and elasticity.

[0066] The contribution of medium / low molecular weight HA is essential for the physiological maintenance of the extracellular matrix in the dermis.

[0067] To ensure that HA (hyaluronic acid) functions effectively in both layers of the skin (outer and inner layers), a portion of the HA in the cosmetic product of this invention is fragmented through ultrasonic treatment. The device used for HA ultrasonic treatment is a Sonicated Sonics Vibracell, model VCX 130 SN 40225L, operating at 100% frequency for 10 minutes using an intermittent mode of 10 seconds on and 10 seconds off. Other devices can be used to obtain HA fragments of different sizes; however, it is important to note that the device parameter settings need to be adjusted according to the applicable scope.

[0068] The fragmentation process is carried out in a physical, chemical-free manner to avoid the generation of harmful reaction intermediates or processing residues (Example 7).

[0069] In a further embodiment, the beauty product of the present invention can be prepared by a method, wherein the beauty product is in the form of a cream or serum and further comprises cosmetically acceptable excipients.

[0070] The cosmetic products can be formulated as topical cosmeceutical creams, lotions, and / or serums, with or without growth factors, peptides, and / or other proteins and bioactive substances. In addition to active ingredients, the products may contain cosmetically acceptable excipients such as emollients, water-binding agents, moisturizers, vitamins, antioxidants, anti-irritants, and soothing agents.

[0071] The beauty product of this invention unexpectedly exhibits a positive effect in promoting the natural regeneration of the dermis under various conditions, such as after laser treatment and radiation therapy, scars, wrinkles, and various types of irritation (redness and sun exposure).

[0072] Therefore, in a second aspect, the present invention provides the use of the cosmetic product for promoting tissue regeneration.

[0073] In a third aspect, the cosmetic products according to the present invention can be used to improve skin texture.

[0074] In a fourth aspect, the present invention relates to the use of the cosmetic product for reducing skin wrinkles.

[0075] In a fifth aspect, the present invention relates to the use of the cosmetic product for reducing scars.

[0076] In the sixth aspect, this article describes the use of this beauty product to reduce skin irritation and blemishes.

[0077] The various embodiments and aspects of the invention described above, as well as the claims made in the claims section below, are all experimentally supported in the following examples.

[0078] Example The following embodiments, together with the above description, illustrate some implementation methods of the present invention.

[0079] The donor's informed consent was obtained before the tissue samples were acquired.

[0080] The preparation of concentrated conditioned medium (CCM) involves screening suitable skin or umbilical cord donors (discarded tissues) in accordance with European guidelines on the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells (2004 / 23 / EC, 2006 / 17 / EC and subsequent updates and national conversions).

[0081] All human tissue samples were preserved in specific transport media and delivered to authorized cell factories by qualified courier companies under controlled transport conditions of 2-8°C.

[0082] Upon arrival, after verifying the integrity of the packaging, serological reports, and temperature records, the tissue was digested using specific GMP-grade enzymes (neutral protease, collagenase, and hyaluronidase).

[0083] Enzymes used for digesting tissues are typically lyophilized and then reconstituted according to the manufacturer's instructions.

[0084] Example 1: Isolation of cells from skin neutral protease digestion Digestion size 1 cm 2 Organizational blocks: A 7 mL digest was prepared by adding a volume equivalent to 6 DMC (1.2 mL) of neutral protease and bringing the volume to a final volume with Hanks' balanced salt solution (HBSS). A 2 mM solution was prepared by adding 14 µL of 1 M CaCl2 solution. One DMC unit catalyzes the cleavage of 1 µmol of peptide bonds in dimethylcasein per minute at 25°C and pH 7.0, expressed as the newly generated terminal amino groups, and determined using TNBS1.

[0085] Before performing neutral protease digestion, wash twice with DPBS solution containing antibiotics (penicillin-streptomycin), and then wash twice with Dulbecco's phosphate buffered saline (DPBS).

[0086] After washing, the skin sample was placed in a tube containing digestive fluid and incubated overnight at 2°C -8°C.

[0087] For larger tissue samples, use a scalpel to cut them into smaller segments, and take a proportional volume for the digestion method described above.

[0088] Dermal / epidermal separation, collagenase digestion, and inoculation into culture flasks. After removing neutral proteases, the samples were washed with DPBS and then transferred to culture dishes for dermal / epidermal separation. After removing the epidermis, the dermis was cut into small fragments. The fragments were placed in tubes containing 10 mL of collagenase solution (0.3 PZU / mL, dissolved in HBSS) and 20 µL of 1M CaCl2 solution (final concentration 2 mM). The samples were incubated at 37°C for 2 h. PZU: According to Wünsch's definition, at 25°C and pH 7.1, 1 U catalyzes the hydrolysis of 1 µmol of 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucylglycyl-L-prolyl-D-arginine per minute.

[0089] After 2 hours of digestion, the digest was filtered through a 100 µm membrane. The membrane was washed with 10 mL of HBSS and then centrifuged at 1500 rpm for 10 min.

[0090] Discard the supernatant, resuspend the precipitate in 10 mL of complete medium (CM), seed all the cell suspension into T25 culture flasks, and incubate at 37°C and 5% CO2 for 2-4 days.

[0091] Material: a. Preparation of neutral protease: First, resuspend the lyophilized enzyme to obtain a solution with a concentration of 100 DMCU / mL, then further dilute to 5 DMCU / mL. The solution is aseptically filtered and then aliquoted into 1.5 mL portions. Label each bottle with the product name, batch number, concentration, and expiration date (1 year after reconstitution).

[0092] b. Preparation of collagenase: First, resuspend the lyophilized enzyme by 1 mL to obtain a stock solution (PZU / mL, according to the concentration shown in the analytical certificate).

[0093] To prepare the working solution, dilute the stock solution and aseptically filter to obtain aliquots of 3 PZU. The solution must always be stored on ice until use. Label the bottle with the product name, batch number, concentration, and expiration date (1 year after reconstitution).

[0094] c. Preparation of complete culture medium (CM): A culture medium consisting of DMEM, glutamine, sodium pyruvate, and fetal bovine serum (irradiated and EDQM certified) is called complete culture medium (CM), and its formula is as follows: - 500 mL DMEM (high sugar) - 50 mL FBS - 10 mL 200 mM glutamine - 5 mL 1M sodium pyruvate.

[0095] All culture medium components were sterile, and the preparation process was carried out in a laminar flow hood (Grade A) surrounded by Grade B laminar flow hoods; each component was aseptically added to a culture flask containing DMEM.

[0096] Example 2: Cell isolation from umbilical cord Hyaluronidase and collagenase were used to digest and inoculate the culture flasks. Umbilical cord samples were washed with DPBS, transferred to culture dishes, and divided into small pieces, which were then crushed into fine fragments. The tissue fragments were placed in 50 mL tubes, and an equal volume of DPBS was added. Then, 1% hyaluronidase and collagenase (equivalent to the final volume) and 0.04% calcium gluconate (equivalent to a 10% calcium gluconate solution) were added. The samples were incubated at 37°C for 2 h.

[0097] After digestion, the sample was diluted with DPBS (1:1), centrifuged at 300 g for 10 seconds to precipitate all undigested fragments, and then the digest was filtered through a 1 mm filter and subsequently through a 100 µm filter membrane. The solution was centrifuged at 450 g rpm for 7 min.

[0098] Discard the supernatant, resuspend the precipitate in 25 mL of complete culture medium (CM), inoculate all the cell suspension into T150 culture flasks, and incubate at 37°C and 5% CO2 for 2-4 days.

[0099] Material: a. Preparation of hyaluronidase: Resuspend the lyophilized enzyme to obtain a 10 KU / mL solution. Filter the solution aseptically and then aliquot it into 1 mL portions. Label each bottle with the product name, batch number, concentration, and expiration date (1 year after reconstitution).

[0100] b. Preparation of collagenase: The lyophilized enzyme was resuspended to obtain a 50 mg / mL solution. The solution was aseptically filtered and then aliquoted into 1 mL portions.

[0101] The bottle should be labeled with the product name, batch number, concentration, and expiration date (1 year after reconstitution).

[0102] c. Preparation of complete culture medium (CM): This culture medium consists of low-glucose DMEM, irradiated and EDQM certified fetal bovine serum, human platelet lysate (HPL), and heparin. The complete culture medium (CM) formulation is as follows: - 500 mL DMEM (low sugar) - 15 mL FBS, - 15 mL HPL, - 2.5 mL heparin (1000 U / mL).

[0103] All culture medium components were sterile, and the preparation process was carried out in a laminar flow hood (Grade A) surrounded by Grade B laminar flow hoods; each component was aseptically added to a culture flask containing DMEM.

[0104] Example 3: Cell Expansion and Inoculation in a Bioreactor Amplification: After isolating fibroblasts, the amplification steps for the two different tissues in Examples 1 and 2 were the same, only the culture media used for cell amplification differed. The culture medium for dermal fibroblasts is the same as in Example 1, and the culture medium for umbilical cord fibroblasts is the same as in Example 2.

[0105] The starvation culture medium was the same for both tissue samples.

[0106] When cell confluence was observed to be approximately 80% under a microscope, fibroblasts were isolated from the culture flasks using trypsin, counted, and seeded into new T150 culture flasks. Through a series of expansion steps, from 1 T150 to 3 T150, and then from 3 T150 to 12 T150, the cell volume required for seeding 2-3 bioreactors was obtained, with 1 L of complete culture medium used in each reactor. All steps were performed under identical culture conditions (37°C and 5% CO2), with the culture medium changed every 2-4 days until cell confluence was achieved.

[0107] hunger: Once cell confluence in the bioreactors is achieved, remove the complete culture medium (DMEM, glutamine, sodium pyruvate, FBS), wash twice with DPBS, and add 1 L of incomplete culture medium (DMEM, glutamine, and sodium pyruvate without phenol red) to each bioreactor. Incubate for 48 h. This 48 h nutrient deprivation stimulates the release of regeneration factors from the culture medium. After incubation, collect the culture medium in sterile bottles and centrifuge to remove cell debris. Filter the supernatant using a 0.22 μm filtration system (500 mL), label the bottles with the code and batch number, and store frozen at -20°C.

[0108] Example 4: Concentrating conditioned medium using tangential flow filtration (TFF) This concentration method can accelerate the processing speed and can be used to process large volumes of supernatant in quantities reaching liter levels.

[0109] The TFF concentration process is performed by setting the following operating parameters: Phase I: 10X Concentration Phase II: Percolation with 10 times the volume of buffer solution (0.9% NaCl). Phase III: 20X Concentration (Final) Transmembrane pressure (TMP): 0.3 bar Flow rate: 500 mL / min Molecular weight cutoff: 10 kDa.

[0110] The parameters were set to achieve an average permeation rate of approximately 54 mL / min or approximately 20 L / m. 2 / h, shear stress is approximately 2300 / 2400 s -1 The concentration in stage I, the percolation in stage II, and the concentration in stage III lasted for 32', 30', and 2', respectively.

[0111] The system was then emptied, all product from the selected bag / container was recovered and filtered through a 0.22 mm syringe filter, and then aliquoted. All aliquots were stored frozen at -20°C.

[0112] Example 5: Release test of concentrated conditioned medium: protein determination The protein content in CCM was quantitatively analyzed using the dioctanine acid (BCA) protein assay. The dioctanine acid (BCA) protein assay, also known as the Smith assay, is a highly sensitive colorimetric method suitable for dissolved protein solutions.

[0113] The protein content in a solution can be quantified by measuring its absorption spectrum and comparing it with a protein solution of known concentration.

[0114] A standard albumin curve was used in the test. After 2 hours of incubation, the absorbance of the samples was read at 562 nm. The average absorbance measurement of each standard and each sample at 562 nm was subtracted from the average absorbance measurement of the blank standard at 562 nm.

[0115] Plot a standard curve with the concentration of the standard as the X-axis and the absorbance as the Y-axis.

[0116] The resulting standard curve was used to determine the protein concentration for each unknown sample.

[0117] Example 6: Characterization of conditioned medium To investigate the indirect interaction between two different cell populations, the behavior of fibroblasts was studied using the Transwell system (i.e., the co-culture method). In this system, cells are cultured together with only a common culture medium.

[0118] The Transwell system provides: an outer chamber at the bottom for culturing a population of fibroblasts; and an inner chamber with a porous membrane for placing a second population of fibroblasts within the inner chamber.

[0119] The two cell populations do not come into direct contact; the Transwell system allows molecules released by cells in the upper compartment to be transferred to cells in the lower compartment.

[0120] The tests were conducted using two unfrozen “normal” fibroblast populations (referred to as cell population 1 and cell population 2), as well as one “normal” cell population and one “frozen BLAST fibroblast” population (frozen in a modified Tyrode solution).

[0121] The expression levels of two biomarkers associated with tissue regeneration were analyzed at 48 hours and 8 days: HMGB1 and type I collagen.

[0122] The results for normal fibroblast population 1 and cell population 2 are only listed in the article.

[0123] When cell population 1 was co-cultured with cell population 2, the production of the signaling molecule HMGB1 increased after 48 hours and the production of type I collagen increased after 8 days. This indicates that fibroblasts release factors into the culture medium, which can stimulate the surrounding environment through paracrine effects (Figure 3).

[0124] Similar results were found in co-culture experiments of "normal" fibroblasts and "frozen BLAST fibroblasts".

[0125] Example 7: Hyaluronic Acid Ultrasound Method The fragmentation process is carried out in a physical, chemical-free manner to avoid the generation of harmful reaction intermediates or processing residues.

[0126] 0.25% high molecular weight hyaluronic acid (HMW-HA, approximately 2.2000-1.800 kDa) was dissolved in physiological solution and sonicated at 100% frequency for 10 minutes using an intermittent mode of 10-second on / 10-second off (Sonicated Sonics Vibracell model VCX 130 SN 40225L). The time, settings, and frequency of the sonication method for HA are also important to avoid the formation of small fragments with pro-inflammatory properties that can be produced by enzymatic cleavage. 0.25% of the product of this invention may contain a mixture of HAs of different molecular weights and may be sonicated or unsonicated.

[0127] The cream contains 0.25% ultrasonically treated HMW, while the serum contains 0.15% high molecular weight (HMW) ultrasonically treated HA and 0.1% LMW-HA.

[0128] To verify the HA fragment, it was loaded onto a 1% agarose gel along with a reference sample, and then electrophoresis was performed. Figure 4 ).

[0129] Each sample distribution forms a smear. This smear consists of HA fragments of varying lengths: the upper part corresponds to larger fragments, which have a larger volume and poorer flowability in the gel; while the lower part of the smear contains smaller fragments. For example, the 1000 kDa HA (sample 6) smear is located at the top of the gel.

[0130] The HA in Blast cream (sample 1) had a similar tailing to the 250 kDa HA standard (sample 5).

[0131] To obtain more precise information on the distribution of HA fragments, the HA tails in Blast cream (sample 1) and Blast serum (sample 4) were divided into squares of the same size, and the color intensity of each segment was quantified (in pixels using ImageJ software) with reference to a standard. The darker the color, the higher the fragment content. The values ​​were plotted on a graph to obtain the percentage (Figure 5).

[0132] The molecular weight distribution of HA in creams and serums conforms to a Gaussian curve, characterized by the presence of both medium-to-high molecular weight and medium-to-low molecular weight HA.

[0133] The composition of HA in creams and serums is detailed in Table 4.

[0134] Table 4

[0135] This distribution combines the advantages of both medium-high to medium molecular weight and low to medium molecular weight. The specific size of the polysaccharide allows it to function both in the epidermis (fractions larger than 500 kDa), imparting elasticity, hydration, and softness to the skin, and in the dermis (fractions smaller than 500 kDa), helping to maintain the physiological state of the extracellular matrix and exerting its anti-inflammatory effects.

[0136] Example 8: HA+ conditioned medium: stability and protection The beauty product of the present invention is composed of hyaluronic acid (HA) and concentrated conditioned medium (CCM).

[0137] To demonstrate how the quality and stability of the components change over time under the influence of environmental factors (temperature, humidity, light) and other components / ingredients, stability tests were conducted.

[0138] In this experiment, the stability of HA in the presence of CCM (concentrated conditioned medium obtained from cultured human fibroblasts) was evaluated. The integrity status of HA was assessed by gel electrophoresis after 14 days.

[0139] The results clearly show that there is no difference in size between HA alone and the HA-CCM mixture: the polysaccharides are the same size, with no small fragments formed and no visible aggregates. Therefore, it can be concluded that CCM does not affect the integrity of HA.

[0140] The stability of HA fragments after ultrasound treatment is crucial for their function in the epidermis and dermis. For HA to penetrate the epidermis, it needs to interact with its surrounding environment. Hyaluronidase, a potent inhibitor of HA activity, catalyzes the degradation of peptides and is present in the skin. On the one hand, the action of hyaluronidase may be beneficial for high molecular weight HA (greater than 1000 kDa) because it can break down large HA molecules into smaller fragments that are more easily absorbed by the skin; on the other hand, random degradation can lead to the formation of small HA fragments, which may trigger pro-inflammatory responses.

[0141] To understand how the CCM functions in the presence of this enzyme (which is physiologically present in the skin), an enzymatic assay was prepared. This test was designed to simulate the physiological state of the epidermis.

[0142] This study aims to obtain quantitative, not just qualitative, data to demonstrate that the presence of CCM stabilizes HA, protecting it from hyaluronidase attack.

[0143] This test utilizes the property of long-chain hyaluronic acid (HA) to produce turbidity in albumin-containing solutions. Turbidity is a function of HA concentration. Higher turbidity indicates a higher concentration 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 related to the enzyme's breakdown of HA.

[0144] Table 5 Enzyme competition test for hyaluronidase

[0145] The data shown in Table 5 indicate that, in the presence of the enzyme, HA alone was almost completely degraded (97.5%), while the degradation of HA in combination with CCM was minimal (3.17%).

[0146] Competitive assays showed that CCM protects against HA when subjected to enzyme attack.

[0147] The protective effect of CCC on HA stability is likely the result of several factors working together. Indeed, CCM appears to contain hyaluronidase inhibitors and / or antioxidants, thereby reducing the breakdown of hyaluronic acid. Furthermore, CCM may contain small-molecule enzymes capable of chemically modifying the polysaccharide structure, thus enhancing its stability.

[0148] Two tests conducted concluded that ultrasound-treated HA has a suitable size for functioning in both the epidermis and dermis; and that it can interact with proteins in the CCM (through hydrophobic interactions (charge-charge) and / or hydrogen bonds) to form new compounds with novel properties (i.e., greater stability and lower sensitivity to hyaluronidase activity). The interaction between HA and proteins was also used for protein transport in the subendothelial layer. The result is that this mixture functions effectively within the dermis.

[0149] Furthermore, these data highlight the robustness and effectiveness of the beauty products of this invention. Example 9: HA+ conditioned medium: stability and protective effect: in vitro efficacy The wound healing assay is an in vitro technique used to study collective cell migration. It is also known as a scratch assay because it involves creating scratches on a monolayer of cells and taking images periodically at the start of the scratch and during the process of cell migration to heal the wound. The images are then compared to quantify the rate of cell migration.

[0150] Scratch assays are an effective tool for studying cell migration because the mechanism involves many different physiological processes. Cell migration plays a significant role in skin re-epithelialization, therefore studying cell migration can help provide insights into non-healing wounds.

[0151] This experiment was designed to verify the ability of fragmented HA and the additional component CCM to promote the natural cell migration of fibroblasts or keratinocytes, thereby mimicking the wound healing process.

[0152] On day one, scratches were created on a monolayer of cells. Cells were cultured in basal medium simulating physiological conditions or in a medium containing a combination of HA and CCM. The scratched areas appeared as cell-free regions, and images were taken at the same location at 0 and 24 hours. During this period, cells migrated to the blank areas in an attempt to close the scratches. Image analysis was performed using the TScratch program, and the percentage of cell migration area was calculated (…). Figure 6 and Figure 7 ).

[0153] Compared to cells treated with basal culture medium, fibroblasts cultured using the cosmetic products of this invention ( Figure 6 AD) and keratinocytes ( Figure 7 AD) can close damaged areas more quickly. The cosmetic product promotes natural cell migration after injury.

[0154] The scratch test suggests that the two components have an enhancing and synergistic effect when used together, relative to their individual effects: the mechanoviscoelasticity of HA supports physiological dermal regeneration, while the combination with CCM shows a stabilizing effect on HA.

[0155] Example 10: Product INCI The following shows some of the components of the beauty products of the present invention.

[0156] Moisturizing creams (Cream Rich, Table 6), lightweight creams (Cream Light, Table 7), serums (face and / or body) (Table 8), and hair serums (Table 9).

[0157] Table 6

[0158] Table 7

[0159] Table 8

[0160] Table 9

[0161] The advantages of the beauty products described and prepared according to the present invention are obvious from the above description and the examples listed.

Claims

1. A beauty product comprising: - A concentrate containing at least one growth factor and at least one cytokine, secreted from fibroblast cultures; and - Hyaluronic acid treated with ultrasound has an average molecular weight of 80kDa-1200kDa; in, The concentrate can be prepared by a method comprising the following steps: a. Provide fibroblast cultures in cell culture media; b. Stimulate 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. Separate the conditioned cell culture medium from step b from the cell culture to obtain the separated conditioned cell culture medium; d. Concentrate the cell culture medium used for separation in step c; In step b, the stimulation of at least one growth factor and at least one cytokine to release into the cell culture medium is carried out by subjecting the fibroblast culture to a nutrient deprivation step.

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

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

5. The cosmetic product according to any one of claims 1-4, wherein the concentrate comprises a growth factor selected from the group consisting of: vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), fibroblast growth factor (FGF), 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), longevity protein 1 (SIRT1), longevity protein 2 (SIRT2), tissue inhibitor of metalloproteinases 1 (TIMP1), macrophage colony-stimulating factor 1 (M-CSF or CSF1), stem cell factor (SCF), insulin-like growth factor (IGF-1), insulin-like growth factor 2 (IGF2), interleukin-10 (IL-10), and hepatocyte growth factor (HGF).

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

7. The beauty product according to any one of claims 1-6, wherein the beauty product is in the form of a cream or serum, and further comprises beauty product-acceptable excipients.

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 skin texture.

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

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

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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