Application of acellular adipose tissue extract in promoting hair growth and hair solidification

Abstract

The present invention relates to the use of acellular adipose tissue extract to promote hair regeneration and hair fixation. The acellular adipose tissue extract of the present invention contains a variety of different natural factors, which have a significant synergistic effect in promoting hair growth, thereby safely, gently and efficiently promoting hair growth and / or hair fixation.

Description

Technical Field

[0001] The present invention relates to the biological field, and specifically to the use of acellular adipose tissue extracts in promoting hair growth and hair fixation. Background Art

[0002] Alopecia is a common and frequently occurring skin disease characterized by hair loss. It is roughly divided into two categories: non-scarring alopecia and scarring alopecia according to pathology. Non-scarring alopecia can be further divided into androgenic alopecia, alopecia areata, telogen effluvium, anagen effluvium, etc. Scarring alopecia can be further divided into chronic cutaneous lupus erythematosus, lichen planus pilaris, etc. The pathogenesis of alopecia is not fully understood, but is generally believed to be related to genetics, endocrine function, infection lesions, autoimmune function, mental factors, and nutritional status.

[0003] Currently, there are limited means of treating hair loss. Drug therapy and physical therapy are mainly aimed at promoting blood circulation, improving local blood supply to the hair loss area, promoting hair regeneration and prolonging the hair growth period. Since hair loss is more common in young and middle-aged people, it affects the appearance and usually brings mental distress and pain to patients.

[0004] Therefore, there is an urgent need to provide products that can effectively promote hair growth and / or hair solidification. Summary of the invention

[0005] The purpose of the present invention is to provide the use of acellular adipose tissue extract for preparing products for promoting hair growth and / or hair solidification.

[0006] In a first aspect, the present invention provides a use of a cell-free adipose tissue extract for preparing a composition or a product for promoting hair regeneration and / or hair fixation.

[0007] In another preferred embodiment, the cell-free tissue extract is extracted from adipose tissue, preferably, extracted from allogeneic adipose tissue, more preferably, human adipose tissue.

[0008] In another preferred embodiment, the cell-free adipose tissue extract contains one or more components selected from the following group: insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived factor (PDGF), epidermal growth factor (EGF), granulocyte colony-stimulating factor (G-CSF), or a combination thereof.

[0009] In another preferred embodiment, the acellular adipose tissue extract does not contain lipid droplets.

[0010] In another preferred embodiment, the lipid droplets are oil droplets released after the adipocytes are broken.

[0011] In another preferred embodiment, the “free of fat droplets” means that in the fat extract, the volume of oil droplets accounts for less than 1% of the total liquid, preferably less than 0.5%, and more preferably less than 0.1%.

[0012] In another preferred embodiment, the acellular adipose tissue extract does not contain cells.

[0013] In another preferred embodiment, the cells are selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

[0014] In another preferred embodiment, the “containing no cells” means that the average number of cells in 1 ml of fat extract is ≤1, preferably ≤0.5, more preferably ≤0.1, or 0.

[0015] In another preferred embodiment, the fat extract is not SVF.

[0016] In another preferred embodiment, the fat extract is a naturally obtained nano fat extract without any added ingredients.

[0017] In another preferred embodiment, the acellular adipose tissue extract has no added ingredients.

[0018] In another preferred embodiment, the “additive-free” means that except for the rinsing step, no solution, solvent, small molecule, chemical agent, and biological additive are added during the preparation of the fat extract.

[0019] In another preferred embodiment, the cell-free adipose tissue extract is prepared by mechanically cutting, emulsifying, and repeatedly freezing and thawing adipose tissue and then centrifuging it.

[0020] In another preferred embodiment, the acellular adipose tissue extract is prepared by the following method, comprising the following steps:

[0021] (1) providing a fat tissue material, chopping the fat tissue material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0022] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0023] (3) draining excess liquid from the bottom and the top layer of fat from the layered mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0024] (4) mechanically emulsifying the intermediate layer to obtain a mechanically emulsified fat mixture (also called nano fat);

[0025] (5) subjecting the mechanically emulsified fat mixture (which may or may not be combined) to centrifugation to obtain a transparent (or substantially transparent) intermediate liquid layer, which is the primary fat extract; and

[0026] (6) filtering and sterilizing the primary fat extract to obtain a fat extract without added ingredients.

[0027] In another preferred embodiment, before the centrifugation step in step (5), the method further comprises subjecting the mechanically emulsified fat mixture to freeze-thawing treatment.

[0028] In another preferred embodiment, in step (5), the mechanically emulsified fat mixture is freeze-thawed once or multiple times (e.g., 1, 2, 3, 4, 5 times), and the thawed mixture is centrifuged to collect the transparent liquid in the middle of the centrifuge tube, which is the primary fat extract.

[0029] In another preferred embodiment, in step (6), the filtration and sterilization are performed through a filter (such as a 0.22 μm filter).

[0030] In another preferred embodiment, in step (6), the filtration and sterilization are first performed through a first filter that can filter out cells, and then through a second filter (such as a 0.22 μm filter) that can filter out pathogens (such as bacteria).

[0031] In another preferred embodiment, step (6) further comprises packaging the fat extract to form packaged products. (The packaged extract can be stored at -20°C for later use; can be used directly after thawing at room temperature, or can be stored at a low temperature (such as 4°C) for a period of time after thawing before use).

[0032] In another preferred embodiment, step (a) of the method further includes: providing adipose tissue, and centrifuging the adipose tissue (preferably at a rotation speed of 800-2500 rpm, more preferably 1000-1500 rpm, and most preferably 1200 rpm) to obtain an intermediate fat layer located in the middle layer.

[0033] In another preferred embodiment, step (b) of the method further comprises: freezing and thawing (preferably repeated freezing and thawing, such as repeated freezing and thawing 1-3 times) to obtain nano fat.

[0034] In another preferred embodiment, the emulsification is mechanical emulsification.

[0035] In another preferred embodiment, the emulsification is performed by repeatedly blowing through a syringe for multiple times (such as 30-200 times, preferably 50-150 times) for mechanical emulsification.

[0036] In another preferred embodiment, the emulsification is a method of breaking up with a tissue homogenizer.

[0037] In another preferred embodiment, the promotion of hair growth and / or hair fixation includes: promoting the proliferation and / or activation of hair papilla cells, delaying the regression of hair follicle cells, promoting hair follicles in the resting phase to enter the growth phase, or a combination thereof.

[0038] In another preferred embodiment, the promoting hair growth includes promoting hair regeneration and / or hair growth.

[0039] In another preferred embodiment, the hair strengthening includes reducing hair loss and / or strengthening hair roots.

[0040] In another preferred embodiment, the promotion of hair growth and / or hair fixation includes treating and / or preventing hair loss. Preferably, the hair loss includes scarring alopecia and / or non-scarring alopecia.

[0041] In another preferred embodiment, the scarring alopecia is selected from the group consisting of lichen planus pilaris, postmenopausal alopecia, lupus erythematosus alopecia, Brocq pseudoalopecia, scalp cellulitis dissecans, or a combination thereof.

[0042] In another preferred embodiment, the non-scarring alopecia is selected from the group consisting of androgenic alopecia, alopecia areata, temporal triangular baldness, trichotillomania, senile alopecia, telogen effluvium, loose anagen hair syndrome, infectious alopecia, lipedema alopecia, salt alopecia, sugar alopecia, tumor-related alopecia, or a combination thereof, preferably androgenic alopecia, temporal triangular baldness, telogen effluvium, infectious alopecia and / or psychogenic alopecia.

[0043] In another preferred embodiment, the composition or product is selected from the following group: hair growth and / or hair strengthening lotion, hair growth and / or hair strengthening ointment, hair growth and / or hair strengthening patch, shampoo, conditioner, hair gel, mousse, lotion, hair cream, hair mask, hair dye, eyebrow regeneration agent, eyelash regeneration agent or eyelash nutrition agent, hair care essence, scalp care agent, or a combination thereof.

[0044] In another preferred embodiment, the composition or product contains at least 1wt%, at least 5wt%, at least 20wt%, at least 30wt%, at least 40wt%, at least 50wt%, at least 60wt%, at least 70wt%, at least 80wt% or at least 90wt% of the acellular adipose tissue extract, preferably 95wt%, more preferably 98wt%, and most preferably 99wt%, based on the total weight of the composition or product.

[0045] In another preferred embodiment, the composition or product further comprises a pharmaceutically or cosmetically acceptable carrier or excipient.

[0046] In another preferred embodiment, the cosmetically acceptable carrier or excipient is selected from the following group: moisturizers, antioxidants, UV inhibitors, preservatives, film formers, oil-soluble gelling agents, organic modified clay minerals, resins, antibacterial agents, flavors, salts, pH regulators, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients, vitamins, amino acids, nucleic acids, hormones, inclusion compounds or combinations thereof.

[0047] In a second aspect, the present invention provides a patch for promoting hair regeneration and / or hair fixation, comprising:

[0048] A substrate and a microneedle array fixed on the substrate, wherein the patch contains acellular adipose tissue extract as an active ingredient.

[0049] In another preferred embodiment, the cell-free tissue extract is obtained by drying the cell-free tissue extract solution.

[0050] In another preferred embodiment, the acellular adipose tissue extract is mixed with a carrier to prepare microneedles.

[0051] In another preferred embodiment, the carrier is selected from: water-soluble polymers, liposomes, salts, or combinations thereof.

[0052] In another preferred embodiment, the cell-free tissue extract is prepared into an elastic gel, and the elastic gel covers the surface of the substrate (and the microneedles) to form an elastic gel layer.

[0053] In another preferred embodiment, the elastic gel layer includes a coagulant, which is a natural, semi-synthetic or synthetic polymer material, such as polysaccharides (starch, cellulose, alginate, hyaluronic acid or chitosan) and polypeptides (collagen, poly-L-lysine or poly-L-glutamic acid), synthetic polymers (povidone, polyethylene, polyacrylic acids (such as carbomer, polyacrylic acid)), or a combination thereof.

[0054] In another preferred embodiment, the elastic gel layer may further contain skin beautifying ingredients.

[0055] In another preferred embodiment, the distance between the tip of the microneedle and the upper surface of the elastic hydrogel layer is 200-500 um, preferably 200-300 um.

[0056] In another preferred embodiment, the microneedle can reach the dermis, preferably the papillary layer of the dermis (also called the superficial dermis).

[0057] In another preferred embodiment, the length of the microneedle is 0.05 mm-1.5 mm, preferably 0.1 mm-1.0 mm, and more preferably 0.2 mm-0.5 mm.

[0058] In another preferred embodiment, the spacing between adjacent microneedles is 0.8-1.5 mm, preferably, 1-1.2 mm.

[0059] In another preferred embodiment, the area of ​​the patch is 0.0001-0.1 m 2 , preferably, 0.005-0.05m 2 , 0.001-0.02m 2 .

[0060] In another preferred embodiment, the substrate is a flexible substrate.

[0061] In another preferred embodiment, the substrate is made of a hypoallergenic or desensitizing material.

[0062] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a diagram showing the effect of mouse hair growth in Example 2.

[0064] Figure 2 This is a diagram showing the effect of mouse hair growth in Example 3. DETAILED DESCRIPTION

[0065] After extensive and in-depth research, the inventors have developed the use of acellular adipose tissue extract in promoting hair regeneration and / or hair fixation through a large number of screenings and tests. The present invention further extracts a fat extract that does not contain oil droplets and living cell components by centrifugation after a series of treatments such as mechanical cutting, emulsification, and repeated freezing and thawing of adipose tissue. Unexpectedly, the inventors found that the various cytokines in the acellular adipose tissue extract have obvious synergistic effects in promoting hair growth / hair fixation, and have excellent effects in promoting hair regeneration and / or hair fixation. The present invention was completed on this basis.

[0066] the term

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0068] As used herein, when used in reference to a specific recited value, the term "about" means that the value may be

[0069] The values ​​recited do not vary by more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (eg, 99.1, 99.2, 99.3, 99.4, etc.).

[0070] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0071] Cell-free adipose tissue extract

[0072] The acellular adipose tissue extract of the present invention is an extract obtained by removing lipid droplets and cells from adipose tissue.

[0073] Preferably, the acellular adipose tissue extract is extracted from allogeneic adipose tissue, such as human.

[0074] In another preferred embodiment, the fat extract is not SVF.

[0075] After testing, the cell-free adipose tissue extract contains the following components (but not limited to): insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived factor (PDGF), epidermal growth factor (EGF), and granulocyte colony-stimulating factor (G-CSF).

[0076] Preparation method

[0077] The cell-free adipose tissue extract can be prepared by mechanically cutting, emulsifying, and repeatedly freezing and thawing adipose tissue followed by centrifugation.

[0078] Typically, the preparation of the acellular adipose tissue extract of the present invention comprises the following steps:

[0079] (1) providing a fat tissue material, chopping the fat tissue material, and rinsing it (e.g., using physiological saline) to obtain rinsed fat tissue;

[0080] (2) centrifuging the rinsed adipose tissue to obtain a layered mixture;

[0081] (3) draining excess liquid from the bottom and the top layer of fat from the layered mixture, and collecting the middle layer (i.e., the fat layer containing fat cells);

[0082] (4) mechanically emulsifying the intermediate layer to obtain a mechanically emulsified fat mixture (also called nano fat);

[0083] (5) subjecting the mechanically emulsified fat mixture (which may or may not be combined) to centrifugation to obtain a transparent (or substantially transparent) intermediate liquid layer, which is the primary fat extract; and

[0084] (6) filtering and sterilizing the primary fat extract to obtain a fat extract without added ingredients.

[0085] Composition or product, use

[0086] The composition or product of the present invention comprises: the above-mentioned acellular adipose tissue extract.

[0087] The acellular adipose tissue extract of the present invention has the effect of promoting hair growth and / or hair fixation. For example, the hair growth and / or hair fixation promotion includes (but is not limited to): promoting the proliferation and / or activation of hair papilla cells, delaying the regression of hair follicle cells, promoting the hair follicles in the resting phase to enter the growth phase, or a combination thereof.

[0088] In another preferred embodiment, the promoting hair growth includes promoting hair regeneration and / or hair growth.

[0089] In another preferred embodiment, the hair strengthening includes reducing hair loss and / or strengthening hair roots.

[0090] The composition or product of the present invention may be used to treat and / or prevent hair loss.

[0091] Typically, the hair loss comprises scarring alopecia and / or non-scarring alopecia.

[0092] The scarring alopecia is selected from the group consisting of lichen planus pilaris, postmenopausal alopecia, lupus erythematosus alopecia, Brocq pseudoalopecia, cellulitis dissecans, or a combination thereof.

[0093] The non-scarring alopecia is selected from the group consisting of androgenic alopecia, alopecia areata, temporal triangular baldness, trichotillomania, senile alopecia, telogen effluvium, loose anagen hair syndrome, infectious alopecia, lipedema alopecia, salt alopecia, sugar alopecia, tumor-related alopecia, or a combination thereof, preferably androgenic alopecia, temporal triangular baldness, telogen effluvium, infectious alopecia and / or psychogenic alopecia.

[0094] The acellular adipose tissue extract of the present invention can be used directly. In addition, it should be understood that although it is not necessary to add any additives (or added ingredients) to the extract of the present invention during the preparation process, some or a small amount of safe substances (such as a small amount of water) that have no negative or adverse effects on the activity of the extract of the present invention can also be added. For example, the extract obtained after preparation is used directly without adding ingredients, for example (but not limited to), applied to the skin or injected into the dermis at multiple points through a syringe.

[0095] The acellular adipose tissue extract of the present invention can be prepared into a pharmaceutical composition or a cosmetic composition with a pharmaceutically or cosmetically acceptable carrier or excipient.

[0096] To the extent that the effects of the present invention are not hindered, other ingredients commonly used in cosmetics may be added to the cosmetics of the present invention, such as film formers, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, flavors, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, skin beautifying ingredients (whitening agents, cell activators, rough skin improving agents, blood circulation promoters, skin astringents, anti-seborrheic agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds, etc.

[0097] The oil-soluble gelling agent is a gelling agent selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid and α, γ-di-n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin 2-ethylhexanoate palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzylidene sorbitol derivatives such as monobenzylidene sorbitol and dibenzylidene sorbitol; organic modified clay minerals such as dimethylbenzyldodecyl ammonium montmorillonite clay and dimethyldioctadecyl ammonium montmorillonite clay, and the like. One or two or more kinds of gelling agents can be used as needed.

[0098] Moisturizers include: glycerin, sorbitol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside, etc.

[0099] Antibacterial preservatives include: alkyl p-hydroxybenzoates, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc. Antibacterial agents include: benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl p-hydroxybenzoates, para-chlorometacresol, hexachlorophene, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbanilide, triclosan, photosensitizer, phenoxyethanol, etc.

[0100] Antioxidants include: tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, phytic acid, etc., pH adjusters include: lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate, etc., chelating agents include alanine, sodium salt of ethylenediaminetetraacetic acid, sodium polyphosphate, sodium metaphosphate, phosphoric acid, etc., coolants include: L-menthol, camphor, etc., anti-inflammatory agents include: allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene, etc.

[0101] Skin beautification ingredients include: whitening agents such as placenta extract, arbutin, glutathione, and saxifrage extract; cell activators such as royal jelly, photosensitizer, cholesterol derivatives, and calf blood extract; skin roughness improvers; blood circulation promoters such as valeric acid amide, benzyl nicotinate, nicotinate β-butoxyethyl ester, capsaicin, ginger ketone, blister beetle tincture, ichthyol, caffeine, tannic acid, α-borneol, nicotinic acid tocopherol, hexanicotinate inositol ester, cycloamycin, cinnarizine, tolazoline, acetylcholine, verapamil, cepharanthin, and γ-oryzanol; skin astringents such as zinc oxide and tannic acid; anti-seborrheic acid agents such as sulfur, etc. Vitamins include: vitamin A oil, rosin oil, rosin oil acetate, rosin oil palmitate, etc.; riboflavin, riboflavin butyrate, flavin adenine nucleotide, etc. Vitamin B2; Vitamin B6 such as pyridoxine hydrochloride, pyridoxine dioctanoate, and pyridoxine tripalmitate, vitamin B12 and its derivatives, vitamin B15 and its derivatives, vitamin C such as L-ascorbic acid, L-ascorbyl dipalmitate, sodium L-ascorbic acid-2-sulfate, and dipotassium L-ascorbyl diester phosphate; Vitamin D such as ergocalciferol and cholecalciferol; Vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinic acid, and dl-α-tocopherol succinate; Vitamin H; Vitamin P; Nicotinic acid such as niacin, benzyl nicotinate, and niacinamide; Pantothenic acid such as calcium pantothenate, D-panthenol, pantothenic ethyl ether, and acetyl pantothenic ethyl ether; Biotin, etc.

[0102] Amino acids include: glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine, tryptophan, etc.; nucleic acids include deoxyribonucleic acid, etc.; hormones include estradiol, vinyl estradiol, etc.

[0103] There is no particular limitation on the form of the product, and it may be in the form of liquid, emulsion, cream, solid, paste, gel, powder, multi-layered, mousse, spray, or the like.

[0104] Preferred examples of the compositions or products of the present invention include (but are not limited to): hair growth and / or hair strengthening lotions, hair growth and / or hair strengthening ointments, hair growth and / or hair strengthening patches, shampoos, conditioners, hair gels, mousses, lotions, hair creams, hair masks, hair dyes, eyebrow regeneration agents, eyelash regeneration agents or eyelash nutrients, hair care essences, and scalp care agents.

[0105] The acellular adipose tissue extract of the present invention can reach the dermis directly to exert its effect to the maximum extent. It is usually injected directly into the scalp through a syringe, but this method is cumbersome, time-consuming, and very dangerous, and has high requirements on the operator.

[0106] Therefore, a more preferred product of the present invention is a patch with a microneedle array. For example, the patch includes: a substrate, and a microneedle array fixed on the substrate, and the patch contains acellular adipose tissue extract as an active ingredient.

[0107] The acellular adipose tissue extract can be prepared into a microneedle shape together with a carrier and fixed on a substrate. For example, it can be prepared by the method disclosed in publication number CN 109069817 A, all of which are incorporated herein by reference, and the acellular tissue extract is mixed with a carrier as a drug.

[0108] In addition, the acellular adipose tissue extract can be prepared into an elastic gel, and then the elastic gel can be covered on a substrate with a microneedle array. For example, it can be prepared by the method disclosed in CN 109077938 A, all of which are incorporated herein by reference.

[0109] The length of the microneedle is preferably enough to reach the dermis, especially the papillary dermis. Preferably, the length of the microneedle is 0.05 mm to 1.5 mm, more preferably 0.1 mm to 1.0 mm, and more preferably 0.2 mm to 0.5 mm.

[0110] The area of ​​the patch can be adjusted according to the application site. Preferably, the area of ​​the patch is 0.0001-0.1 m 2 , preferably, 0.005-0.05m 2 , 0.001-0.02m 2 .

[0111] The substrate is preferably a flexible substrate.

[0112] In another preferred embodiment, the substrate is made of a hypoallergenic or desensitizing material.

[0113] The main advantages of the present invention include:

[0114] 1. The present invention is the first to discover that multiple cytokines in acellular adipose tissue extract have obvious synergistic effects in promoting hair growth / hair fixation, and have excellent effects in promoting hair regeneration and / or hair fixation.

[0115] 2. Acellular adipose tissue can be prepared directly from adipose tissue, contains a variety of growth factors, is of natural origin, and is simple and convenient to prepare and use.

[0116] 3. The cell-free adipose tissue extract removes the fat droplet components, reducing possible side effects; it removes the cell components, thus removing the immunogenicity; it is mild and highly safe;

[0117] 4. Cell-free adipose tissue extract can realize the application of allogeneic fat extract, and achieve batch production and quality control;

[0118] 5. The cell-free adipose tissue extract is easy to store by freezing and maintains biological activity. No protective agent needs to be added during cryopreservation, thus avoiding contamination by other chemical components. It is simple to operate and highly practical.

[0119] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0120] Example 1

[0121] 1. Preparation and component analysis of acellular adipose tissue extract

[0122] Preparation method

[0123] The fat was obtained with the informed consent of the volunteers. The extraction method was as follows

[0124] (1) Take the fat obtained by suction or surgical resection, cut it into small pieces, and rinse it with saline three times.

[0125] (2) The washed adipose tissue was placed in a 50 ml centrifuge tube (about 30-50 ml per tube), and centrifuged at 1200 rpm for 3 minutes to obtain a layered mixture.

[0126] (3) For the layered mixture, the excess liquid at the bottom and the uppermost layer of fat are drained, and the middle layer (i.e., the fat layer containing fat cells) is collected.

[0127] (4) The intermediate layer is mechanically emulsified by connecting two 10 ml syringes to a three-way tube for about 60-120 times to obtain a mechanically emulsified fat mixture (also called nano fat).

[0128] (5) The mechanically emulsified fat mixture (which may or may not be combined) is placed in a 50 ml test tube, centrifuged at 1500 rpm for 5 minutes, and the transparent liquid in the middle of the test tube is collected, which is the initial fat extract. Alternatively, the mechanically emulsified fat mixture is placed in a -80°C refrigerator (or liquid nitrogen) for freezing, and then thawed in a water bath (such as a 20-37°C water bath), and repeated freezing and thawing 1-2 times. The thawed mixture is centrifuged at 1500 rpm for 5 minutes, and the transparent liquid in the middle of the centrifuge tube is collected, which is the initial fat extract.

[0129] (6) The primary fat extract is filtered through a 0.22 μm filter to sterilize and remove any living cells that may be present, thereby obtaining a fat extract without any additives. The fat extract is packaged and stored at -20°C for later use. It can be used directly after thawing at room temperature, or after thawing, it can be stored at a low temperature (e.g., 4°C) for a period of time before use.

[0130] Content analysis

[0131] The obtained fat extract was used to detect the content of growth factors, including cytokines such as IGF-1, BDNF, GDNF, bFGF, VEGF, TGF-β, HGF and PDGF, using an ELISA immunosorbent assay kit.

[0132] ELISA analysis was performed on the biomaterials prepared from fat from six donor sources. The results are shown in Table 1: (Unit: pg / mL)

[0133] Table 1

[0134] sample 1 2 3 4 5 6 IGF-1 14834.37 12394.01 9916.26 12454.56 11666.78 7343.9 BDNF 1942.6 2329.63 1192.41 1620.71 1438.56 2642.01 GDNF 1487.5 2024.76 1250.52 1934.82 1643.64 2598.16 TGF-β 650.26 861.91 388.47 585.66 1175.98 2456.05 HGF 388.65 1108.36 1287.66 673.82 324.5 329.84 FGF 192.18 245.21 333.68 284.71 127.35 192.46 VEGF 192.38 208.49 193.09 152.84 206.69 230.16 PDGF 150.3 172.29 142.66 191.15 163.1 232.06 EGF 63.11 83.27 47.98 173.23 125.2 339.14 NT-3 59.5 71.77 45.12 40.24 67.17 71.14 G-CSF 19.8 25.47 20.46 27.85 22.96 27.32

[0135] The average concentrations of the 6 samples tested are as follows: IGF-1 (9840.6±2960.9pg / ml), BDNF (1764.5±583.5pg / ml), GDNF (1831.9±467.7pg / ml), bFGF (242.3±73.0pg / ml), VEGF (202.9±28.4pg / ml), TGF-β1 (954.5±806.5pg / ml), HGF (898.4±545.5pg / ml), and PDGF (179.9±30.3pg / ml).

[0136] Example 2

[0137] FE promotes hair growth in normal C57 mice

[0138] The growth factors IGF-1, VEGF, and bFGF contained in acellular adipose tissue extract (FE) have been reported to be associated with hair growth. In order to compare the therapeutic effects of the combined use of these known components with acellular adipose tissue extract, a hair growth-related growth factor solution with a growth factor concentration close to that of acellular adipose tissue extract (containing IGF-1 (12000pg / mL), VEGF (200pg / mL), and bFGF (250pg / mL)) was used as the growth factor group; acellular adipose tissue extract (sample 4) was used as the experimental group; and normal saline was used as the control group.

[0139] Twelve 7-week-old C57 mice, 6 in each of the experimental group and growth factor group. The back was shaved with a razor one day in advance, and the hair was completely removed with a depilatory cream. Starting from the first day, the experimental group mice were subcutaneously injected with acellular adipose tissue extract on the left side of the back, and saline on the right side; the growth factor group mice were subcutaneously injected with growth factor solution on the left side of the back, and saline on the right side. The injection site was about 1 cm away from the midline, and multiple injections were parallel to the midline. The injection dose was 25 microliters per point. The injection frequency was once a day for 7 consecutive days. Photos were taken on days 1, 3, 5, 7, 9, 11, 13, and 15 to observe hair growth.

[0140] The experimental results are as follows Figure 1 As shown, after the back of C57 mice in the experimental group was shaved with a razor, acellular adipose tissue extract was injected subcutaneously on the left side, and saline was injected on the right side. After the back of C57 mice in the growth factor group was shaved with a razor, a growth factor was injected subcutaneously on the left side, and saline was injected on the right side.

[0141] Take photos on days 1, 3, 5, 7, 9, 11, 13, and 15. The experimental results are as follows: Figure 1 As shown, it can be seen that the local skin of the experimental group injected with acellular adipose tissue extract (left side) became darker on the 9th day, and hair sprouted on the 11th day. The skin of the side injected with normal saline (right side) became darker on the 11th day, and hair sprouted on the 13th day. The new hair area on the acellular adipose tissue extract side was significantly larger than that on the normal saline side, indicating that the acellular adipose tissue extract accelerated the regeneration of hair. However, there was no significant difference in the hair growth area and speed between the growth factor injected side (left side) and the normal saline injected side (right side) in the growth factor group.

[0142] This indicates that in addition to the factors related to hair growth, the cell-free adipose tissue extract also promotes hair growth through other pathways, and its components have a significant synergistic effect in promoting hair growth.

[0143] Example 3

[0144] FE promotes hair growth in androgenic alopecia mouse model

[0145] Six 7-week-old C57 mice were used. The backs were shaved with a razor one day in advance, and the hair was completely removed with a depilatory cream. 3 mg of dihydrotestosterone (DHT) was injected subcutaneously in the neck to simulate androgenic alopecia. After modeling, the mice were divided into FE group and control group. The FE group mice were injected with acellular adipose tissue extract FE (sample 4) at multiple points on the back, and the control group was injected with normal saline. The injection dose was 25 microliters per point. The injection frequency was once a day. The injection was continued for 7 consecutive days, and photos were taken every week to observe the hair growth. The observation period was 14 days.

[0146] In the dihydrotestosterone-simulated androgenic alopecia model, mice were divided into a cell-free adipose tissue extract group (FE group) and a normal saline group (control group). Figure 2 In the control group (A), on the 14th day, it was observed that the skin color of the mice darkened and a small amount of hair sprouted locally. In the FE group (B), on the 14th day, hair sprouted in most areas of the mice, and the area and length of hair were significantly greater than those in the control group, indicating that the cell-free adipose tissue extract can promote hair regeneration in mice with androgenic alopecia.

[0147] In summary, acellular adipose tissue extract has an excellent effect in promoting hair growth. Surprisingly, compared with the case where it contains only known hair growth-related factors, the acellular adipose tissue extract contains multiple different natural factors, which have a significant synergistic effect in promoting hair growth, thereby promoting hair growth safely, gently and efficiently. Experiments have shown that acellular adipose tissue extract can be used for hair loss caused by various reasons.

[0148] Discussion

[0149] Tonnard first proposed the concept of nano fat. After mechanical emulsification of fat obtained by liposuction, nano fat containing lipid droplets, vascular stromal components (SVF) and growth factors can be obtained. Nano fat mainly produces different effects through SVF. SVF contains endothelial cells, adipose stem cells, macrophages, etc. On the one hand, cells can directly participate in tissue formation, and on the other hand, cells can promote tissue regeneration by secreting cytokines.

[0150] The difference from previous studies is that the present invention removes the living cells and lipid droplet components in nano fat by centrifugal filtration, retains the growth factor components, and the growth factors are rich in variety, including factors related to angiogenesis, factors related to nerve regeneration, factors related to inflammation, and factors related to hair regeneration. Animal experiments show that local injection of acellular adipose tissue extract can promote hair regeneration and accelerate hair growth, whether in normal shaved mice or in androgen-induced hair loss mouse models. Because the acellular adipose tissue extract contains a variety of growth factors, its mechanism of action can play a role in many aspects: 1. Promote angiogenesis and improve local blood supply; 2. Regulate local inflammatory response; 3. Promote nerve regeneration and improve nerve nourishment; 4. Stimulate the proliferation and differentiation of hair papilla cells. Therefore, the acellular adipose tissue extract can improve the condition of hair follicle cells and hair papilla while promoting hair growth, thereby playing a role in hair fixation.

[0151] Compared with nanofat, acellular adipose tissue extract has a wider range of application prospects. First, the lipid droplet components are removed, reducing possible side effects; second, the cell components are removed, thus removing immunogenicity, and in the future, the application of allogeneic fat extracts can be realized, achieving mass production and quality control; third, it is easy to freeze and maintain biological activity, and no protective agent needs to be added during freezing, avoiding contamination by other chemical components.

[0152] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of acellular adipose tissue extract in the preparation of a composition or product for promoting hair growth and / or hair fixation, wherein the acellular adipose tissue extract is extracted from human adipose tissue; The acellular adipose tissue extract is prepared by the following method, comprising the following steps: (1) providing a fat tissue material, cutting the fat tissue material into pieces, and rinsing the pieces with physiological saline to obtain rinsed fat tissue; (2) centrifuging the rinsed adipose tissue to obtain a layered mixture; (3) draining excess liquid from the bottom and the top layer of fat from the layered mixture, and collecting the middle layer, i.e., the fat layer containing fat cells; (4) mechanically emulsifying the intermediate layer to obtain a mechanically emulsified fat mixture; (5) subjecting the mechanically emulsified fat mixture to centrifugation to obtain a transparent or substantially transparent intermediate liquid layer, which is the primary fat extract; and (6) filtering and sterilizing the primary fat extract to obtain a fat extract without additives; The cell-free adipose tissue extract includes 9840.6±2960.9 pg / ml of insulin-like growth factor (IGF-1), 1764.5±583.5 pg / ml of brain-derived neurotrophic factor (BDNF) and 1831.9±467.7 pg / ml of glial-derived neurotrophic factor (GDNF).

2. The use according to claim 1, characterized in that The acellular adipose tissue extract is free of added ingredients.

3. The use according to claim 1, characterized in that The acellular adipose tissue extract further contains a component selected from the group consisting of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), platelet-derived factor (PDGF), epidermal growth factor (EGF), granulocyte colony stimulating factor (G-CSF), or a combination thereof.

4. The use according to claim 1, characterized in that Prior to the centrifugation in step (5), the mechanically emulsified fat mixture is subjected to freeze-thaw treatment.

5. The use according to claim 1, characterized in that The method of promoting hair growth and / or hair fixation includes: promoting the proliferation and / or activation of hair papilla cells, delaying the regression of hair follicle cells, promoting hair follicles in the resting phase to enter the growth phase, or a combination thereof.

6. The use according to claim 1, characterized in that The promoting hair growth includes promoting hair regeneration and / or hair growth.

7. The use according to claim 1, characterized in that The hair strengthening includes reducing hair loss and / or strengthening hair roots.

8. The use according to claim 7, characterized in that The hair loss is non-scarring hair loss, and the non-scarring hair loss is selected from the group consisting of androgenic alopecia, alopecia areata, temporal triangular hair loss, trichotillomania, telogen effluvium, or a combination thereof.

9. The use according to claim 1, characterized in that The composition or product is selected from the following group: hair growth and / or hair strengthening lotion, hair growth and / or hair strengthening ointment, hair growth and / or hair strengthening patch, shampoo, conditioner, hair gel, mousse, lotion, hair cream, hair mask, hair dye, eyebrow regeneration agent, eyelash regeneration agent or eyelash nutrition agent, hair care essence, scalp care agent, or a combination thereof.

10. The use according to claim 1, characterized in that The composition or product may further include a pharmaceutically or cosmetically acceptable excipient.

11. The use according to claim 1, characterized in that The acellular adipose tissue extract does not contain lipid droplets and cells; The lipid droplets are oil droplets released after the fat cells are broken; the cells are selected from the following group: endothelial cells, adipose stem cells, macrophages, and stromal cells.

12. The use according to claim 1, characterized in that The composition or product is a patch for promoting hair growth and / or hair solidification, comprising: A substrate and a microneedle array fixed on the substrate, wherein the patch contains acellular adipose tissue extract as an active ingredient.

13. The use according to claim 1, characterized in that The acellular adipose tissue extract also includes 242.3±73.0 pg / ml basic fibroblast growth factor (bFGF), 202.9±28.4 pg / ml vascular endothelial growth factor (VEGF), 954.5±806.5 pg / ml transforming growth factor β (TGF-β), 898.4±545.5 pg / ml hepatocyte growth factor (HGF) and 179.9±30.3 pg / ml platelet-derived factor (PDGF).

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