Patent Description:
Alopecia is a common and frequently-occurring skin disease characterized by hair loss. It can be roughly classified as two categories: non cicatricial alopecia and cicatricial alopecia according to the pathology. Among them, non cicatricial alopecia can be further classified as androgenic alopecia, alopecia areata, telogen alopecia, and anagen alopecia, etc.; cicatricial alopecia can be classified as chronic cutaneous lupus erythematosus, hair lichen planus, etc.. The pathogenesis of alopecia is not completely clear, but it is generally believed to be related to heredity, endocrine function, infection lesions, autoimmune function, mental factors, and nutritional status.

At present, there are limited means to treat alopecia. Drug therapy and physical therapy are mainly aimed at promoting blood circulation, improving local blood supply to the hair loss site, promoting hair regeneration and prolonging the growth period of hair. Because alopecia is more common in young adults, it affects the appearance, and usually brings mental distress and mental pain to patients.

Therefore, there is an urgent need to provide products that can effectively promote hair growth and/or retention.

<CIT> discloses a mesenchymal stem cell extract derived from an animal tissue comprising trophic factors, such as BMP-<NUM>, SDF-<NUM>, VEGF, CXCR4, BDNF and IL-<NUM>.

<CIT> relates to topical pharmaceutical compositions for promoting hair growth/reducing hair loss.

<CIT> discloses a method of preparing cell-free adipose tissue extract.

<NPL> discloses a cell-free therapeutic agent produced from human adipose tissue.

<CIT> discloses a concentrated nanofat, and a method of preparing the concentrated nanofat.

<NPL> discusses the relationship between adipose-derived stem cells and hair growth, and the interaction of this mechanism with hypoxia.

<NPL> reviews autologous adipose transplantation as a method of treating alopecia after trauma.

<CIT> discloses methods of promoting hair growth/preventing hair loss in a mammal by administering an agent containing an active ingredient comprising bFGF.

<CIT> relates to a formulation for promoting hair growth using a culture liquid of adipocyte stem cells.

<NPL> investigates the use of cell-free fat extract in treating chronic wound healing in mice with diabetes.

<NPL> evaluates the in vitro and in vivo effects of cell-free fat extract on the dermis, to assess the suitability of cell-free fat extract for use in skin rejuvenation.

The object of the present invention is to provide a use of an acellular adipose tissue extract for the preparation of a product for promoting hair growth and/or retention.

In the first aspect of the present invention, it provides an acellular adipose tissue extract or a composition comprising the acellular adipose tissue extract for use in the treatment or prevention of alopecia;
wherein the acellular adipose tissue extract is prepared by the following method, and the method comprises the following steps:.

In another preferred embodiment, the acellular adipose tissue extract or the composition comprising the acellular adipose tissue extract according to the first aspect of the invention contains one or more components selected from the group consisting of insulin-like growth factor (IGF-<NUM>), 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), and a combination thereof.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of IGF-<NUM> of <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of BDNF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract , the concentration of GDNF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of bFGF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of the VEGF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of TGF-β1 is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of the HGF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, in the acellular adipose tissue extract, the concentration of PDGF is <NUM>-<NUM> pg/ml.

In another preferred embodiment, the weight ratio of IGF-<NUM> to VEGF is <NUM>-<NUM>:<NUM>.

In another preferred embodiment, the weight ratio of BDNF to VEGF is <NUM>-<NUM>:<NUM>.

In another preferred embodiment, the weight ratio of GDNF to VEGF is <NUM>-<NUM>:<NUM>.

In another preferred embodiment, the weight ratio of bFGF to VEGF is <NUM>-<NUM>: <NUM>.

In another preferred embodiment, the weight ratio of TGF-β1 to VEGF is <NUM>-<NUM>:<NUM>.

In another preferred embodiment, the weight ratio of HGF to VEGF is <NUM>-<NUM>:<NUM>.

In another preferred embodiment, the weight ratio of PDGF to VEGF is <NUM>-<NUM>:<NUM>.

The acellular adipose tissue extract may contain no lipid droplet.

The lipid droplets may be oil droplets released after fat cells are disrupted.

The expression of "contain no lipid droplet" may mean that in the fat extract, the volume of oil droplets accounts for less than <NUM>% of the total liquid, preferably less than <NUM>%, more preferably less than <NUM>% of the total liquid.

The acellular adipose tissue extract may contain no cell.

The cells may be selected from the group consisting of endothelial cells, adipose stem cells, macrophages, and stromal cells.

The expression of "contain no cell" may mean that the average number of cells in <NUM> of the fat extract is ≤ <NUM>, preferably ≤ <NUM>, more preferably ≤ <NUM>, or <NUM>.

The fat extract may be a naturally-obtained nano-fat extract without added ingredients.

The expression of "without added ingredients" may mean that no solution, solvent, small molecule, chemical, and biological additive are added during the preparation of the adipose extract except rinsing step.

Before the centrifugation step of step (<NUM>), the method may further comprise subjecting the mechanically emulsified fat mixture to a freeze-thaw treatment.

In step (<NUM>), the mechanically emulsified fat mixture may be freeze-thawed one or more times (eg, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> times), and then the thawed mixture may be centrifuged, and the transparent liquid in the middle of the centrifuge tube may be collected, which is the adipose primary extract.

In step (<NUM>), the filtration and sterilization may be performed through a filter (eg, a <NUM> filter).

In step (<NUM>), the filtration and sterilization may be performed by first passing through a first filter that can filter out cells, and then passing through a second filter(such as a <NUM> filter) that can filter out pathogens (such as bacteria).

Step (<NUM>) may further comprise sub-packaging the adipose extract to form a sub-packed product. (The sub-packaged extract may be stored at -<NUM> for later use; may be thawed at room temperature then used directly, or thawed and stored at a low temperature (eg, <NUM>) for a period of time before use).

Step (a) of the method may further comprise providing an adipose tissue, and centrifuging the adipose tissue (preferably at <NUM>-<NUM> rpm, more preferably at <NUM>-<NUM> rpm, most preferably at <NUM> rpm) to obtain an intermediate fat layer located in the middle layer.

Step (b) of the method may further comprise freezing and thawing (preferably repeated freezing and thawing, such as repeated freezing and thawing <NUM>-<NUM> times) to obtain nano fat.

The emulsification may be mechanical emulsification by repeatedly blowing through a syringe for many times (eg, <NUM>-<NUM> times, preferably <NUM>-<NUM> times).

The emulsification may be a method of breaking up by a tissue homogenizer.

In another preferred embodiment, the alopecia includes cicatricial alopecia and/or non cicatricial alopecia.

In another preferred embodiment, the cicatricial alopecia is selected from the group consisting of hair lichen planus, postmenopausal alopecia, lupus erythematosus alopecia, Brocq pseudoalopecia areata, dissecting cellulitis of the scalp, or a combination thereof.

In another preferred embodiment, the non cicatricial alopecia is selected from the group consisting of androgenetic alopecia, alopecia areata, temporal triangle alopecia, trichotillomania, senile alopecia, telogen effluvium, anagen hair loss syndrome, infectious alopecia, lipoedema alopecia, salt alopecia, sugar alopecia, tumor-related alopecia, or a combination thereof, preferably androgenetic alopecia, temporal triangle alopecia, telogen effluvium, infectious alopecia, and/or psychogenic alopecia.

In another preferred embodiment, the composition is selected from the group consisting of ointment, compress, shampoo, conditioner, hair gel, mousse, lotion, hair cream, hair mask, hair dye, eyebrow rejuvenator, eyelash rejuvenator or eyelash nourisher, hair serum, scalp conditioner, or a combination thereof.

The composition or product may contain at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt %, at least <NUM> wt % %, at least <NUM> wt % or at least <NUM> wt % of the acellular adipose tissue extract, preferably <NUM> wt %, more preferably <NUM> wt %, most preferably <NUM> wt %, based on the total weight of the composition or product.

The composition or product may further include a pharmaceutically or cosmetically acceptable carrier or excipient.

The cosmetically acceptable carrier or excipient may be selected from the group consisting of moisturizing agents, antioxidants, anti-ultraviolet agents, preservatives, film-forming agents, oil-soluble gelling agents, organic modified clay minerals, resins, antibacterial agents, fragrances, salts, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, ingredients for skin beautification, vitamins, amino acids, nucleic acids, hormones, inclusion compounds, and a combination thereof.

Also described herein but not explicitly claimed is a compress for promoting hair regeneration and/or retention, which comprises:
a substrate, and an array of microneedles immobilized on the substrate, and the compress contains acellular adipose tissue extract as active ingredient.

In another example, the acellular tissue extract is obtained by drying the acellular tissue extract.

In another example, the acellular adipose tissue extract is mixed with a carrier to form microneedles.

In another example, the carrier is selected from water-soluble macromolecules, liposomes, salts, or a combination thereof.

In another example, the acellular tissue extract is prepared into an elastic gel, and the elastic gel is coated on the surface of the substrate (and the microneedles) to form an elastic gel layer.

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

In another example, the elastic gel layer may further contain skin beautifying ingredients.

In another example, the distance between the top of the microneedle and the upper surface of the elastic hydrogel layer is <NUM>-<NUM>, preferably <NUM>-<NUM>.

In another example, the microneedles can reach the dermis, preferably the papillary layer of the dermis (also called the superficial dermis).

In another example, the length of the microneedles is <NUM>-<NUM>, preferably <NUM>-<NUM>, more preferably <NUM>-<NUM>.

In another example, the distance between adjacent microneedles is <NUM>-<NUM>, preferably <NUM>-<NUM>.

In another example, the area of the compress is <NUM>-<NUM><NUM>, preferably <NUM>-<NUM><NUM>, <NUM>-<NUM><NUM>.

In another example, the substrate is a flexible substrate.

In another example, the material of the substrate is a hyposensitive or desensitized material.

Also described herein but not explicitly claimed is a method for promoting hair regeneration and/or retention, the method comprising the step of: administering the acellular adipose tissue extract of the present invention or the compress in the second aspect of the present invention to a subject in need thereof.

In another example, the subject is a human or a non-human mammal.

In another example, the non-human mammal is rat, mouse, cat, dog, pig, cow, sheep or monkey.

It should be understood that, within the scope of the present invention, the above technical features of the present invention and the technical features specifically described in the following descriptions (such as the examples) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be repeated herein.

After extensive and in-depth research, the present inventors developed a use of acellular adipose tissue extract in promoting hair regeneration and/or retention through extensive screening and testing. In the present invention, after the adipose tissue is subjected to a series of treatments such as mechanical cutting, chylolysis, repeated freezing and thawing, and the like, and further by the method of centrifugation, the adipose extract liquid without oil droplets and living cell components is extracted. Unexpectedly, the inventors found that there is an obvious synergistic effect of multiple cytokines in the acellular adipose tissue extract in promoting hair growth/retention, which has excellent hair regeneration and/or hair retention promotion effects. The present invention has been completed on this basis.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value may vary by no more than <NUM>% from the recited value. For example, as used herein, the expression "about <NUM>" includes all values between <NUM> and <NUM> (eg, <NUM>, <NUM>, <NUM>, <NUM>, etc.).

As used herein, the terms "contain" or "comprise (include)" may be open form, semi-closed form, and closed form. In other words, the terms also include "substantially consisting of" or "consisting of".

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

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

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

The tests show that the acellular adipose tissue extract contains the following components (but not limited to): insulin-like growth factor (IGF-<NUM>), 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 cells Growth factor (EGF), granulocyte colony stimulating factor (G-CSF).

The acellular adipose tissue extract is obtained by subjecting the adipose tissue to mechanically cutting, chylolysis, repeating freezing and thawing, and then centrifuging.

The preparation method of the acellular adipose tissue extract of the present invention comprises the following steps:.

The composition or product of the present disclosure includes the above-mentioned acellular adipose tissue extract.

The acellular adipose tissue extract may have the effect of promoting hair growth and/or retention. For example, the promotion of hair growth and/or retention may include promoting proliferation and/or activation of dermal papilla cells, delaying degeneration of hair follicle cells, promoting telogen follicles into the anagen phase, or a combination thereof.

The promoting hair growth may include promoting hair regeneration and/or hair growth and/or hair thickening.

The hair retention may include reducing hair loss and/or strengthening hair roots.

The compositions or products of the present disclosure can be used to treat and/or prevent alopecia.

Typically, the alopecia includes cicatricial and/or non cicatricial alopecia.

The cicatricial alopecia is selected from the group consisting of hair lichen planus, postmenopausal alopecia, alopecia lupus erythematosus, Brocq pseudoalopecia areata, dissecting celhlitis of the scalp, and a combination thereof.

The non cicatricial alopecia is selected from the group consisting of androgenetic alopecia, alopecia areata, temporal triangle alopecia, trichotillomania, senile alopecia, telogen effluvium, anagen hair loss syndrome, infectious alopecia, lipoedema alopecia, salt alopecia, sugar alopecia, tumor-related alopecia, or a combination thereof, preferably androgenetic alopecia, temporal triangle alopecia, telogen effluvium, infectious alopecia, and/or psychogenic alopecia.

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) during the preparation of the extract of the present invention, some or small amounts of safe substances that do not negatively or adversely affect the activity of the extract of the present invention (such as small amount of water) may also be added. For example, the extract obtained after preparation can be used directly without added ingredients, for example (but not limited to), smeared on the skin or injected into the dermis at multiple points through a syringe.

The acellular adipose tissue extract of the present invention can be formulated into a pharmaceutical composition or a cosmetic composition with pharmaceutically or cosmetically acceptable carriers or excipients.

To the extent that the effects of the present invention are not hindered, other ingredients commonly used in cosmetics may be added into the cosmetics of the present invention, such as film formers, oil-soluble gelling agents, organically modified clay minerals, resins, moisturizers, preservatives, antibacterial agents, fragrances, salts, antioxidants, pH adjusters, chelating agents, cooling agents, anti-inflammatory agents, ingredients for skin beautification (whitening agents, cytoactive agents, skin roughness improving agents, blood circulation promoters, skin firming agents, anti-lipid leakage agents, etc.), vitamins, amino acids, nucleic acids, hormones, inclusion compounds and the like.

The oil-soluble gelling agent is selected from metal soaps such as aluminum stearate, magnesium stearate, and zinc myristate; amino acid derivatives such as N-lauroyl-L-glutamic acid, α,γ-di-n-butylamine; cyclodextrin fatty acid esters such as cyclodextrin palmitate, cyclodextrin stearate, and cyclodextrin <NUM>-ethylhexanoic acid palmitate; sucrose fatty acid esters such as sucrose palmitate and sucrose stearate; benzylidene derivatives of sorbitol such as monobenzylidene sorbitol and dibenzylidene sorbitol; gelling agent of organically modified clay minerals such as dimethylbenzyldodecylammonium montmorillonite clay and dimethyldioctadecylammonium montmorillonite clay. One, two or more types of agents may be used as required.

Moisturizers include glycerin, sorbitol, propylene glycol, dipropylene glycol, <NUM>,<NUM>-butanediol, glucose, xylitol, maltitol, polyethylene glycol, hyaluronic acid, chondroitin sulfate, pyrrolidone carboxylate, polyoxyethylene methyl glucoside, polyoxypropylene methyl glucoside and the like.

Antibacterial preservatives include alkyl p-hydroxybenzoate, benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, phenoxyethanol, etc.. Antibacterial agents include benzoic acid, salicylic acid, carbolic acid, sorbic acid, alkyl p-hydroxybenzoate, p-chloro-m-cresol, hexachlorophenol, benzalkonium chloride, chlorhexidine chloride, trichloro-N-carbanilide, triclosan, photosensitizer, phenoxyethanol and the like.

Antioxidants include tocopherol, butylhydroxyanisole, dibutylhydroxytoluene, phytic acid and the like. PH regulators include lactic acid, citric acid, glycolic acid, succinic acid, tartaric acid, dl-malic acid, potassium carbonate, sodium bicarbonate, ammonium bicarbonate and the like. Chelating agents include alanine, sodium ethylenediaminetetraacetate, sodium polyphosphate, sodium metaphosphate, phosphoric acid and the like. Cooling agents include L-menthol, camphor and the like. Anti-inflammatory agents include allantoin, glycyrrhetinic acid, glycyrrhizic acid, tranexamic acid, azulene and the like.

Ingredients for skin beautification include whitening agents such as placenta extract, arbutin, glutathione and saxifrage extract; cytoactive agents such as royal jelly, photoreceptor, cholesterol derivatives, calf blood extract; skin roughness improving agents; blood circulation promoters such as valeramide pelargonate, benzyl nicotinate, β-butoxyethyl nicotinate, capsaicin, gingerone, cantharidin tincture, ichthyol, caffeine, tannic acid, α-borneol, tocopherol nicotinate, inositol hexanicotinate, cyclomandelate, cinnarizine, tolazoline, acetylcholine, verapamil, stephane and γ-oryzanol; skin firming agents such as zinc oxide, tannic acid; anti-lipid leakage agents such as sulfur. Vitamins include vitamin A such as vitamin A oil, rosin oil, rosin acetate, rosin palmitate; vitamin B2 such as riboflavin, riboflavin butyrate and flavin adenine nucleotides; vitamin B6 such as pyridoxine hydrochloride, pyridoxine dicaprylate, pyridoxine tripalmitate; vitamin B such as vitamin B12 and its derivatives, vitamin B15 and its derivatives; vitamin C such as L-ascorbic acid, L-ascorbyl dipalmitate, sodium L-ascorbate-<NUM>-sulfate and dipotassium L-ascorbate phosphate diester; vitamin D such as ergocalciferol and cholecalciferol; vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, dl-α-tocopherol succinate; vitamin H; vitamin P; niacin such as nicotinic acid, benzyl nicotinate, niacinamide; pantothenic acid such as calcium pantothenate, D-panthenol, pantothen ethyl ether and acetyl pantothen ethyl ether; biotin and the like.

Amino acids include glycine, valine, leucine, isoleucine, serine, threonine, phenylalanine, arginine, lysine, aspartic acid, glutamic acid, cystine, cysteine, methionine and tryptophan. Nucleic acids include deoxyribonucleic acid and the like, and hormones include estradiol, vinyl estradiol and the like.

There is no particular limitation on the form of the product, and it may be liquid, emulsion, cream, solid, paste, gel, powder, multilayer, mousse, spray, and the like.

Preferred examples of the composition or product of the present disclosure include (but not limited to) hair growth and/or retention lotion, hair growth and/or retention ointment, hair growth and/or retention compress, shampoo, conditioner, hair gel, mousse, lotion, hair cream, hair mask, hair dye, eyebrow rejuvenator, eyelash rejuvenator or eyelash nourisher, hair serum, scalp conditioner.

The acellular adipose tissue extract of the present invention directly reaches the dermis layer to maximize its effect. It is usually injected directly into the scalp through a syringe, but the method is cumbersome, time-consuming, and very dangerous, and need high requirement for operators.

Therefore, also described herein but not explicitly claimed is compress with arrays of microneedles. For example, the compress includes a substrate, and an array of microneedles immobilized on the substrate, and the compress contains acellular adipose tissue extract as an active ingredient.

The acellular adipose tissue extract can be prepared into microneedle shape together with carrier and the microneedles are fixed on a substrate. For example, it can be prepared by the method disclosed in <CIT>. The acellular tissue extract as drug was mixed with a carrier.

In addition, the acellular adipose tissue extract can be prepared into an elastic gel, which is then coated on the substrate with an array of microneedles. For example, it can be prepared by the methods disclosed in <CIT>.

The length of the microneedles preferably makes it reach the dermis, especially the papillary dermis. Preferably, the length of the microneedles is <NUM>-<NUM>, preferably <NUM>-<NUM>, more preferably <NUM>-<NUM>.

The area of the compress can be adjusted according to the site of use, preferably, the area of the compress is <NUM>-<NUM><NUM>, preferably <NUM>-<NUM><NUM>, <NUM>-<NUM><NUM>.

The substrate is preferably a flexible substrate.

The material of the substrate may be a hyposensitive or desensitized material.

The present invention will be further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are generally performed under the conventional conditions or according to the manufacturer's instructions. Unless indicated otherwise, percentage and parts are calculated by weight.

Adipose was obtained from volunteers with informed consent. The extraction method was as follows:.

The obtained adipose extract was tested for growth factor content using ELISA immunosorbent assay kit, including cytokines such as IGF-<NUM>, BDNF, GDNF, bFGF, VEGF, TGF-β, HGF and PDGF.

ELISA analysis was performed on biological materials prepared from six donor-derived fats, and the results are shown in Table <NUM>: (unit: pg/mL).

The average concentrations of <NUM> samples detected are as follows: IGF-<NUM> (<NUM>±<NUM> pg/ml), BDNF (<NUM>± <NUM> pg/ml), GDNF (<NUM>± <NUM> pg/ml), bFGF (<NUM>± <NUM> pg/ml), VEGF (<NUM>±<NUM> pg/ml), TGF-β1 (<NUM>±<NUM> pg/ml), HGF (<NUM>±<NUM> pg/ml), PDGF (<NUM>±<NUM> pg/ml).

Growth factors IGF-<NUM>, VEGF, and bFGF contained in acellular adipose tissue extract (FE) have been reported to be related to hair growth. To compare the efficacy of the combination administration of these known ingredients with that of acellular adipose tissue extract, a hair growth-related growth factor solution (containing IGF-<NUM> (12000pg/mL), VEGF (200pg/mL) and bFGF (250pg/mL)) with a concentration close to acellular adipose tissue extract was used as the growth factor group; acellular adipose tissue extract (sample <NUM>) was used as the experimental group; normal saline was used as the control group.

There were <NUM> C57 mice, <NUM> week-old, wherein <NUM> mice were in the experimental group and <NUM> mice were in the growth factor group. The back was shaved using a razor a day in advance, and the hair was removed completely by using a depilatory cream. From the first day, the mice in the experimental group were injected subcutaneously with acellular adipose tissue extract on the left side of the back and normal saline was injected on the right side; the mice in the growth factor group were injected subcutaneously with growth factor solution on the left side of the back and normal saline was injected on the right side. The injection site is about <NUM> away from the midline, and multiple injections were performed parallel to the midline. The injection dose was <NUM> microliters per point. The injection frequency was once a day for <NUM> consecutive days. Photographs were taken at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days to observe hair growth.

The experimental results are shown in <FIG>. After the back of the C57 mice in the experimental group was shaved with a razor, the left side was subcutaneously injected with acellular adipose tissue extract, and the right side was injected with normal saline. After the back of the C57 mice in the growth factor group was shaved with a razor, growth factors were injected subcutaneously on the left side and normal saline was injected on the right side.

Photographs were taken on days <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, respectively. The experimental results are shown in <FIG>. It can be seen that the skin on the side injected with acellular adipose tissue extract (left side) of the experimental group became darker on the 9th day, and the hair erupted on the 11th day. The skin on the side injected with normal saline (right side) became darker on the 11th day, and the hair erupted on the 13th day. The new hair area of the acellular adipose tissue extract side was significantly larger than that of the saline side, suggesting the acellular adipose tissue extract accelerated hair regeneration; however, there was no significant difference in hair growth area and speed between the growth factor injection side (left side) and the normal saline injection side (right side) in the growth factor group.

It shows that in addition to the factors related to hair growth, the acellular adipose tissue extract also exerts the effect of promoting hair growth through other ways, and its components have a significant synergistic effect in promoting hair growth.

There were <NUM> C57 mice, <NUM> week-old. The back was shaved using a razor a day in advance, and the hair was removed completely by using a depilatory cream. <NUM> of dihydrotestosterone (DHT) was injected subcutaneously in the neck to simulate androgenetic alopecia. After modeling, the mice were divided into the FE group and the control group. The mice in the FE group were injected with acellular adipose tissue extract FE (sample <NUM>) at multiple points on the back, and the control group was injected with normal saline. The injection dose was <NUM> microliters per point. The frequency of injection was once a day for <NUM> consecutive days. Photographs were taken every week to observe hair growth, the observation period was <NUM> days.

In the androgenetic alopecia model simulated by dihydrotestosterone, the mice were divided into acellular adipose tissue extract group (FE group) and normal saline group (control group). As shown in <FIG>, the skin color of the mice became darker and a small amount of hair sprouted locally on the 14th day in control group (A). On the 14th day, hair sprouting in most areas of the mice could be observed in FE group (B), and the area and hair length were significantly larger than those of the control group, indicating that the acellular adipose tissue extract can promote hair regeneration in mice with androgenetic alopecia.

In conclusion, the acellular adipose tissue extract has an excellent effect of promoting hair growth. Surprisingly, compared with the case where only known hair growth-related factors are contained, the acellular adipose tissue extract contains many different natural factors, which has a significant synergistic effect in promoting hair growth, so as to promote hair growth safely, mildly and efficiently, and experiments have proved that acellular adipose tissue extract can be used for alopecia caused by a variety of reasons.

Tonnard first proposed the concept of nano-fat. Nano-fat containing lipid droplets, vascular matrix components (SVF) and growth factors can be obtained after mechanical emulsification of the fat obtained by liposuction. Nano-fat mainly works 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.

The difference from the previous study is that the living cells and lipid droplets in the nano-fat are removed by centrifugation and filtration in the present invention, and the growth factor components are retained. The growth factor components therein are rich in type, including factors related to angiogenesis, factors related to nerve regeneration, factors related to inflammation, and factors related to hair regeneration, etc. Animal experiments have shown that local injection of acellular adipose tissue extract can promote hair regeneration and accelerate hair growth in both normally shaved mice and in mouse model of androgen-induced depilation. Because acellular adipose tissue extract contains a variety of growth factors, its mechanism of action can play a role in many aspects: <NUM>. promoting new blood vessel formation and improving local blood supply; <NUM>. regulating local inflammatory response; <NUM>. promoting nerve regeneration and improving nerve nourishes; <NUM>. stimulating the proliferation and differentiation of dermal papilla cells. Therefore, the acellular adipose tissue extract can improve the condition of hair follicle cells and dermal papilla while promoting hair growth, thus playing a role in hair retention.

Claim 1:
An acellular adipose tissue extract or a composition comprising the acellular adipose tissue extract for use in the treatment or prevention of alopecia;
wherein the acellular adipose tissue extract is prepared by the following method, and the method comprises the following steps:
(<NUM>) providing a human adipose tissue raw material, shredding the adipose tissue raw material, and rinsing, thereby obtaining a rinsed adipose tissue;
(<NUM>) centrifuging the rinsed adipose tissue to obtain a layered mixture;
(<NUM>) discharging the excess liquid at the bottom and the grease on top from the layered mixture and collecting the intermediate layer;
(<NUM>) subjecting the intermediate layer to mechanical emulsification to obtain a mechanically emulsified fat mixture;
(<NUM>) centrifuging the mechanically emulsified fat mixture to obtain a transparent or substantially transparent intermediate liquid layer, which is an adipose primary extract; and
(<NUM>) subjecting the adipose primary extract to filtration and sterilization to obtain the adipose extract without added ingredients.