Use of collagen particles in hair follicle neogenesis or angiogenesis

Abstract

Disclosed herein is the use of collagen particles for the preparation of a medicament for inducing hair follicle neogenesis or angiogenesis in a subject. The collagen particles of the present application have a diameter of about 10-200 μm. According to an embodiment of the present disclosure, the collagen particles have a concentration of about 0.1 mg / cm. 2 ~About 1000mg / cm 2 is administered to the subject in an amount of

Description

[Technical Field]

[0001] The present disclosure relates to the field of medicine. More specifically, the present disclosure relates to a method of inducing hair follicle neogenesis or angiogenesis by using collagen particles. [Background technology]

[0002] The skin is the largest organ in the human body, regulating body temperature and moisture, and forming the first line of defense against pathogens. Several skin appendages (e.g., hair follicles, sweat glands, etc.) reside in the dermis, and damage to the dermis often has serious consequences.

[0003] Specifically, hair grows from hair follicles in the dermis, which are composed of various types of cells, including hair follicle stem cells. The life cycle of a hair follicle is divided into three periods: anagen, catagen, and telogen. Hair (i.e., the hair shaft with a cuticle structure) is generated in the anagen phase, when hair follicle stem cells proliferate and differentiate into keratinocyte stem cells, which further differentiate into matrix keratinocytes. Derivatives of matrix keratinocytes form the outer and inner root sheaths of the hair follicle. Activated hair follicle stem cells can form epidermal stem cells that differentiate into epidermal cells. Furthermore, activated hair follicle stem cells can differentiate into sebaceous glands. During catagen and telogen, hair ceases to grow from the follicle, and the follicle begins to shrink. Therefore, severe dermal damage caused by abnormal hair follicle cycles or hair follicle damage can lead to irreversible hair loss or baldness.

[0004] Furthermore, skin damage caused by burns, ulcers, inflammation, radiation therapy, surgery, and diabetes leads to vascular atrophy and necrosis, resulting in chronic wounds in subjects. In clinical practice, chronic wounds will exhibit persistent inflammation and impaired angiogenesis. Therefore, skin damage and abnormalities not only cause infections, but also affect the normal function of skin appendages (e.g., hair loss or insufficient blood supply in local tissues), which has a significant impact on physical and mental health. However, none of the drugs currently used in clinics can solve the problem.

[0005] In view of the above, there is a need in the related art for pharmaceutical agents or methods that can effectively induce hair follicle neogenesis or angiogenesis. Summary of the Invention

[0006] The following presents a simplified summary of the disclosure in order to provide the reader with a basic understanding. This summary is not an exhaustive overview of the disclosure, and it does not identify key / critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

[0007] The present disclosure aims to provide a method for inducing hair follicle neogenesis and / or angiogenesis. As realized and broadly described in this disclosure, a first aspect of the present disclosure is directed to the use of collagen particles for the preparation of a medicament useful for inducing hair follicle neogenesis and / or angiogenesis in a subject, the medicament comprising a plurality of collagen particles.

[0008] According to some embodiments of the present disclosure, the collagen particles have a diameter of about 10-200 μm. Preferably, the collagen particles have a diameter of about 100-150 μm.

[0009] According to certain embodiments of the present disclosure, a pharmaceutical comprising a plurality of collagen particles is parenterally administered to a desired target site. Examples of parenteral routes include, but are not limited to, percutaneous administration, intradermal administration, and subcutaneous administration. The collagen particles are administered in a concentration of 0.1 to 1000 mg / cm. 2 Preferably, the collagen particles are present in the pharmaceutical preparation in an amount of 5 mg / cm 2 present in pharmaceuticals in amounts of

[0010] According to certain embodiments of the present disclosure, a pharmaceutical agent comprising a plurality of collagen particles is administered subcutaneously to a subject.

[0011] In an embodiment of the present disclosure, the collagen particles comprise: (1) exposing animal skin having a thickness of about 0.1 to 2 mm to a first flow of supercritical carbon dioxide (scCO2) at a pressure of about 100 to 500 bar and a temperature of about 30 to 50°C for a period of about 20 minutes to 10 hours to decellularize the animal skin; (2) exposing the decellularized animal skin of step (1) to an alkaline solution; (3) exposing the alkaline solution-treated animal skin of step (2) to a hydrogen peroxide solution; (4) exposing the animal skin treated with the hydrogen peroxide solution of step (3) to a second flow of scCO2 in the presence of a co-solvent (or co-solvent) at a pressure of about 100-500 bar and a temperature of about 30-50°C for about 20 minutes to 10 hours to produce a collagen scaffold; (5) dehydrating and granulating the collagen scaffold of step (4) to produce collagen particles.

[0012] According to an embodiment of the present disclosure, the method is characterized by the absence of organic solvents and cross-linking agents.

[0013] According to one embodiment of the present disclosure, in step (1), the skin of the animal is exposed to a first flow of scCO2 at a pressure of 350 bar and a temperature of 40°C for 90 minutes.

[0014] According to one preferred embodiment, the alkaline solution in step (2) is a solution of sodium hydroxide (NaOH).

[0015] According to one preferred embodiment of the present disclosure, in step (4), the animal's skin is exposed to a second flow of scCO2 at a pressure of 350 bar and a temperature of 40°C for 90 minutes.

[0016] In a preferred embodiment of the present disclosure, the co-solvent in step (4) is ethanol. In one example, the co-solvent and the second flow of scCO2 are present in a volume ratio of 1:10.

[0017] In another embodiment of the present disclosure, in step (5), the collagen scaffold is cut or pulverized into a plurality of collagen particles in liquid nitrogen.

[0018] Another aspect of the present disclosure is directed to a method of inducing hair follicle neogenesis and angiogenesis in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical agent comprising a plurality of collagen particles of the present disclosure, the collagen particles having a diameter of 10 to 200 μm.

[0019] According to one embodiment of the present disclosure, each of the collagen particles is about 100-150 μm in diameter.

[0020] According to certain embodiments, the pharmaceutical product comprising a plurality of collagen particles has a concentration of about 0.1 to 1000 mg / cm. 2 Preferably, the pharmaceutical preparation comprising a plurality of collagen particles is administered in an amount of about 5 mg / cm. 2 is administered in an amount of

[0021] In certain embodiments of the present disclosure, a pharmaceutical agent comprising a plurality of collagen particles is administered subcutaneously to a subject.

[0022] According to certain embodiments of the present disclosure, the collagen particles comprise: (1) exposing animal skin having a thickness of 0.1 to 2 mm to a first flow of supercritical carbon dioxide at a pressure of about 100 to 500 bar and a temperature of about 30 to 50°C for about 20 minutes to 10 hours to decellularize the animal skin; (2) exposing the decellularized animal skin of step (1) to an alkaline solution; (3) exposing the alkaline solution-treated animal skin of step (2) to a hydrogen peroxide solution; (4) exposing the animal skin treated with the hydrogen peroxide solution of step (3) to a second flow of supercritical carbon dioxide in the presence of a co-solvent at a pressure of about 100-500 bar and a temperature of about 30-50°C for about 20 minutes to 10 hours to produce a collagen scaffold; (5) dehydrating and granulating the collagen scaffold of step (4) to produce collagen particles.

[0023] In one embodiment of the present disclosure, the method does not use organic solvents or cross-linking agents.

[0024] According to one embodiment, in step (1), the animal's skin is exposed to a first scCO2 under a pressure of 350 bar and a temperature of 40°C for 90 minutes.

[0025] In one preferred embodiment of the present disclosure, the alkaline solution is NaOH.

[0026] According to one preferred embodiment, in step (4), the animal's skin is exposed to a second scCO2 under a pressure of 350 bar and a temperature of 40°C for 90 minutes.

[0027] In a preferred embodiment of the present disclosure, in step (4), the co-solvent is ethanol. In one example, the co-solvent and scCO2 are present in a volume ratio of 1:10.

[0028] According to another embodiment, in step (5), the collagen scaffold is cut or crushed into collagen particles in liquid nitrogen.

[0029] Based on the above, the present method is useful for inducing hair follicle neogenesis and angiogenesis in a subject by administering to the subject an effective amount of a pharmaceutical agent comprising collagen particles to treat hair follicle loss and / or vascular damage in the subject.

[0030] Many of the attendant features and advantages of the present disclosure will become better understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0031] This description is better understood from the following detailed description read in light of the accompanying drawings. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows photographs of tissue staining according to one embodiment of the present disclosure. (A) Normal saline (Control 1), (B) Poly-L-lactic acid (Control 2), (C) Hyaluronic acid (Control 3), and (D) the present collagen particles were each subcutaneously injected into a rabbit's ear. After 30 days, the tissue was isolated from the ear and subsequently stained and detected under a microscope. Photographs were taken at a magnification of 40x. The black arrow indicates vascular tissue, and the red arrow indicates hair follicle cells. DETAILED DESCRIPTION OF THE INVENTION

[0033] The detailed description provided below in connection with the accompanying drawings is intended as a description of the present embodiment and is not intended to represent the only manner in which the present embodiment may be constructed or utilized. The description sets forth the functions of the present embodiment and the sequence of steps for constructing and operating the present embodiment, although the same or equivalent functions and sequences may be accomplished by different embodiments.

[0034] I. Definition For convenience of explanation, certain terms employed in the specification, examples, and appended claims are collected here. Unless otherwise defined, 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. The singular forms "a," "an," and "the" are used herein to include the plural forms unless the context clearly dictates otherwise.

[0035] The term "collagen particles" refers to granular collagen prepared by the methods disclosed herein, in which collagen particles are produced from an animal source by decellularization, sterilization, and milling to a specific size. Specifically, animal skin is decellularized with scCO2 to produce a collagen scaffold without the use of a crosslinking agent, and the collagen scaffold is then milled to produce collagen particles having a specific diameter while retaining the structure and organization of the native collagen.

[0036] The terms "administer," "administered," and "administration" refer to the mode of providing collagen particles to a subject to reduce and ameliorate symptoms associated with hair follicle loss and neovascularization.

[0037] The term "effective amount" refers to the amount of collagen particles or a pharmaceutical product containing collagen particles administered within a period of time that induces hair follicle neogenesis and angiogenesis, resulting in a desired effect on a disease, or delaying or minimizing symptoms associated with a disease. An effective amount of collagen particles or a pharmaceutical product of the present disclosure refers to an amount that can exhibit a beneficial effect on a disease when administered alone or in combination with other therapeutic agents. According to one example of the present disclosure, the amount of collagen particles or pharmaceutical product can induce hair follicle neogenesis and angiogenesis in the dermis of a subject.

[0038] The term "subject" refers to a mammal, including the human species, treatable by the methods of the present invention. Mammal refers to all members of the mammalian species, including humans, primates, domestic animals, livestock, captive animals, sport animals, pets, and rodents. Furthermore, the term "subject" or "patient" is intended to refer to both males and females, unless one gender is specifically specified.

[0039] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, as used herein, the term "about" generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the acceptable standard error of the mean as considered by one of ordinary skill in the art. Other than in the operating examples, or unless expressly stated otherwise, all numerical ranges, amounts, values, and ratios, such as those relating to amounts of materials thereof, durations, temperatures, operating conditions, ratios of amounts, and the like, disclosed herein, should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this disclosure and the appended claims are approximations that may be varied as desired. At the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0040] II. Detailed Description of the Invention The present disclosure is based, at least in part, on the discovery that collagen particles prepared by decellularization and pulverization without the use of crosslinking agents during the processing procedure can act as a three-dimensional biological scaffold in vivo on which cells can grow. Furthermore, the collagen particles can also induce hair follicle formation and angiogenesis. Accordingly, the present disclosure aims to provide a pharmaceutical product comprising the collagen particles, which is useful for repairing skin damage, such as by inducing hair follicle formation or angiogenesis in a subject.

[0041] One aspect of the present disclosure is directed to a method of treating hair follicle loss or localized vascular damage in a subject, the method comprising administering to the subject an effective amount of collagen particles, preferably administered subcutaneously to the subject to induce localized hair follicle neogenesis and angiogenesis.

[0042] Collagen particles can be prepared from animal tissue by methods known to those skilled in the art or by the method disclosed herein, with or without the use of a crosslinking agent in the preparation process. In one embodiment of the present disclosure, animal skin is decellularized via scCO2, treated with an alkaline solution and a chemical agent containing hydrogen peroxide, and then milled to produce collagen particles of a specific size. Note that this process does not involve the use of organic solvents (especially those known to be toxic to living organisms) or crosslinking agents. The resulting collagen particles can be preserved in vitro by any method known in the art, for example, by storing them in solution. Because collagen particles produced by the method disclosed herein independently retain their native structure, signaling factors, and native collagen composition, they can act as a better microenvironment for cells and tissues to grow on.

[0043] Collagen particles suitable for use in the present disclosure can be isolated from allogeneic and / or xenogeneic tissues. Allogeneic collagen particles refer to those obtained from tissues or cells of the same species but different individuals, while xenogeneic collagen particles refer to those obtained from individuals of different species. Specifically, collagen particles may be isolated from collagen-rich skin tissue, tendon tissue, or any tissue, and these tissues may be from individuals of the same species or different species. Examples of tissue sources in the present disclosure include, but are not limited to, skin tissue, tendon tissue, and cartilage tissue. According to certain embodiments, the collagen particles are derived from pig skin tissue.

[0044] Specifically, the collagen particles of the present disclosure can be prepared by the following method without using an organic solvent or a crosslinking agent: (1) exposing animal skin having a thickness of about 0.1-2 mm to a first flow of scCO2 at a pressure of about 100-500 bar and a temperature of about 30-50°C for about 20 minutes to 10 hours to decellularize the animal skin; (2) exposing the decellularized animal skin of step (1) to an alkaline solution; (3) exposing the alkaline solution-treated animal skin of step (2) to a hydrogen peroxide solution; (4) exposing the animal skin treated with the hydrogen peroxide solution of step (3) to a second flow of scCO2 in the presence of a co-solvent at a pressure of about 100-500 bar and a temperature of about 30-50°C for about 20 minutes to 10 hours to produce a collagen scaffold; (5) dehydrating and granulating the collagen scaffold of step (4) to produce collagen particles.

[0045] Before starting the method, the animal's skin tissue is preferably subjected to washing, depilation, and lipid extraction processes. Animals that can serve as a source of skin tissue for use in the present invention are economic animals, including, but not limited to, pigs, cattle, cows, bulls, sheep, goats, donkeys, rabbits, ducks, geese, and chickens. Depilation and lipid extraction can be carried out by using any physical or chemical method known in the art. For example, the skin tissue can be subjected to acid treatment to remove hair thereon, or enzymatic (e.g., lipase) or chemical (e.g., detergent) treatment to remove lipids therefrom. Alternatively, lipids can be directly excised using a knife or cutting means.

[0046] The skin tissue is then preferably sliced ​​into skin having a thickness of about 0.1 to 2 mm, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm. Preferably, the animal skin is about 0.3 mm thick.

[0047] In step (1), hairless and fat-free animal skin of approximately 0.1 to 2 mm in thickness is decellularized via a first flow of scCO2. The purpose of step (1) is to remove cellular components from the animal skin while preserving the physical and biochemical properties of collagen so that it can be used as a tissue scaffold. Preferably, in step (1), the animal skin is decellularized at 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, The mixture is exposed to a first flow of scCO2 under a pressure of about 100 to 500 bar, such as 60, 470, 480, 490 or 500 bar, more preferably about 200 to 400 bar, such as 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 bar. Furthermore, step (1) is performed at a temperature of about 30-50°C, e.g., about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50°C, and preferably, step (1) is performed at a temperature of about 35-42°C. The treatment in step (1) lasts for about 20 minutes to 10 hours (e.g., 20, 30, 40, 50, or 60 minutes, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours). According to one embodiment of the present disclosure, animal skin having a thickness of about 0.1-2 mm is exposed to scCO2 at a temperature of 40°C and a pressure of 350 bar for 90 minutes. Essentially, decellularization is performed at a temperature close to body temperature (i.e., 37°C) to remove biologically active substances.

[0048] In step (2), the decellularized animal skin from step (1) is exposed to an alkaline solution to wash and remove any cellular debris. Specifically, the animal skin is immersed in the alkaline solution for 0.5 to 2 hours, for example, about 0.5, 1, 1.5, or 2 hours, preferably about 2 hours. Examples of alkaline solutions suitable for use in the present disclosure include, but are not limited to, sodium hydroxide solution, calcium hydroxide solution, potassium hydroxide solution, sodium carbonate solution, sodium bicarbonate solution, etc. According to a preferred embodiment of the present disclosure, the decellularized animal skin from step (1) is treated with a 1N sodium hydroxide solution.

[0049] In step (3), the animal skin treated with the alkaline solution in step (2) is further treated with a hydrogen peroxide solution for about 0.5 to 2 hours, for example, 0.5, 1, 1.5, or 2 hours. In a preferred embodiment, the hydrogen peroxide solution has a concentration of about 0.1 to 2% by weight, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, or 2% by weight. According to a preferred embodiment, the animal skin treated with the alkaline solution in step (2) is treated with a 1% hydrogen peroxide solution for about 1 hour.

[0050] Next, in step (4), the animal skin treated with the hydrogen peroxide solution produced in step (3) is exposed to a second flow of scCO in the presence of a co-solvent to produce a collagen scaffold essentially free of cells and bioactive components. Note that step (4) is performed under the same conditions as step (1), except that the animal skin is treated with scCO together with a co-solvent. That is, in step (3), the animal skin is treated with scCO at a pressure of 100-500 bar and a temperature of 30-50°C in the presence of a co-solvent for 20 minutes to 10 hours. According to a preferred embodiment of the present disclosure, in step (4), the animal skin treated with the hydrogen peroxide solution is exposed to scCO at a pressure of 350 bar in the presence of a co-solvent for 90 minutes.

[0051] The co-solvent is C 1-4The co-solvent may be an alcohol, examples of which include, but are not limited to, ethanol, propanol, isopropanol, butanol, isobutanol, 2-butanol, 2-methyl-2-propanol, and cyclobutanol. In certain preferred embodiments, the co-solvent is ethanol and is used with scCO2, and the co-solvent and scCO2 are present in a volume ratio of 1:20 to 1:4, e.g., 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, or 1:4. In one preferred embodiment, the ethanol and scCO2 are present in a volume ratio of 1:10.

[0052] Finally, in a granulation step (i.e., step (5)), the collagen scaffold from step (4) is cut or crushed in liquid nitrogen to produce collagen particles having a specific diameter. According to certain embodiments of the present disclosure, the collagen particles have a diameter of about 10-200 μm, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 μm. In a preferred embodiment, the collagen particles are about 100-150 μm in diameter, for example, about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 μm in diameter.

[0053] The above-mentioned method is characterized by using intact animal skin as the starting material, followed by decellularization to remove the cellular matrix and active ingredients, and then a pulverization treatment. The entire process does not use strong acids and / or strong bases, and therefore does not require the use of crosslinking agents to induce the polymerization of the main amino acids of collagen (i.e., glycine, proline, etc.) into secondary or tertiary structures. Therefore, the resulting collagen particles are characterized by collagen fibers that retain their native structure and conformation, allowing them to be used as biological scaffolds for cell growth.

[0054] According to certain embodiments of the present disclosure, administration of collagen particles prepared by the above-described method induces hair follicle neogenesis and angiogenesis in a subject. According to certain embodiments of the present disclosure, the collagen particles have a molecular weight of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg / cm of skin area 2 0.1 to 1000 mg / cm of skin area 2 Preferably, the collagen particles are administered to the subject in an amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / cm of skin area 2 0.1 to 500 mg / cm of skin area 2 More preferably, the collagen particles are administered to the subject in an amount of about 0.1 to 50 mg / cm of skin area. 2, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg / cm of skin area 2 In one particular embodiment of the present disclosure, the collagen particles are administered in an amount of about 5 mg / cm 2 The collagen particles are administered to a subject in need thereof in an effective amount. Furthermore, the collagen particles can be administered at a frequency ranging from once per day to once every three months. In certain embodiments, the collagen particles of the present disclosure are administered once per day, once every two days, once every three days, once per week, once every two weeks, once per month, once every two months, or once per three months. In certain embodiments, the collagen particles are administered at a frequency of once per week to once per month.

[0055] The collagen particles can be administered to the desired target site via an appropriate route. In some embodiments, the collagen particles are administered to the desired target site via non-oral administration or parenteral administration. Exemplary parenteral administration routes include, but are not limited to, transdermal, intradermal, and subcutaneous administration. Specifically, parenteral administration refers to subcutaneous administration (e.g., subcutaneous injection) and / or transdermal administration (i.e., topically applying the collagen particles to a wound in the subject's skin using an applicator) of the collagen particles to the target site. It should be understood that those skilled in the art will understand that the appropriate route will vary depending on the disease being treated, the severity of the disease, the subject's health status (including age, physiological state, body type, sex, and weight), the duration of treatment, coexisting medical conditions (if any), the dosage and nature of the active ingredient, genetic factors, and other similar factors commonly known and understood by medical professionals. These factors are known in the art and can be determined by those skilled in the art without undue experimentation. Generally, the best route of administration will depend on various factors, such as the stability of the pharmaceutical agent in the circulatory system and / or the physical condition of the subject (such as whether the subject can tolerate subcutaneous injection). According to certain embodiments of the present disclosure, collagen particles are administered to the subject via subcutaneous injection. In a preferred embodiment, the collagen particles are administered into the dermis of the subject.

[0056] The subject described in this disclosure refers to any animal that can be treated and benefit from the present methods. Exemplary animals include, but are not limited to, humans, rats, mice, guinea pigs, rabbits, monkeys, pigs, sheep, cows, horses, dogs, and cats. In an effective embodiment, the subject is a rabbit. In another embodiment, the subject is a human.

[0057] The present disclosure aims to repair skin damage by administering collagen particles composed of decellularized skin-derived collagen prepared by the disclosed method to a subject in need thereof. The collagen particles have a diameter of 10-200 μm and retain their native structure, signaling factors, and intact fibril conformation. Thus, after administration to the subcutaneous tissue of a subject, the collagen particles act as a 3D bioscaffold that provides an environment suitable for cell activation and proliferation, allowing cells (e.g., stem cells, fibroblasts, endothelial cells) to grow on. Thus, the collagen particles provide a means of inducing hair follicle formation and angiogenesis in the dermis of a subject to repair skin damage.

[0058] The present invention will now be more particularly described with reference to the following embodiments, which are provided for purposes of illustration and not limitation. [Example]

[0059] Materials and Methods animal New Zealand white rabbits (approximately 5 kg each) were used in the study. Each rabbit was housed individually in a cage in an animal facility with free access to food and water. The facility was maintained at a temperature of 19-25°C and humidity of approximately 50-60%. Body weight, body temperature, and other physiological values ​​were measured daily. Before each experiment, the animals were isolated and allowed to acclimate to the experimental environment.

[0060] Example 1: Production of collagen particles Hairless and fat-free porcine skin (0.3 mm thick) was dehydrated for 24 hours at 4°C and decellularized by exposure to scCO2 at 350 bar pressure and 40°C temperature for 90 minutes to remove cellular components.

[0061] The decellularized porcine skin was then sequentially treated with NaOH, hydrogen peroxide solution, low-temperature dehydration, and a second scCO2 treatment. Specifically, the decellularized porcine skin was immersed in a 1N NaOH solution for 2 hours and then in a 1% hydrogen peroxide solution for 1 hour. The porcine skin was then subjected to a second scCO2 treatment at 350 bar and 40°C for 90 minutes in the presence of ethanol as a cosolvent, with the ethanol and scCO2 present in a volume ratio of 1:10, to produce a pure collagen scaffold.

[0062] The collagen scaffolds were dehydrated at 4°C for 8–30 h and then independently ground into collagen particles approximately 100–150 μm in diameter using a grinder (Retsch, ZX200). The resulting collagen particles were irradiated with gamma rays (10–50 kGy) and stored under sterile conditions until further use. The collagen particles produced by the above method were observed under an electron microscope, and EM images revealed that the fibril structure of each collagen particle remained intact after grinding.

[0063] Example 2: Induction of hair follicle formation or angiogenesis by the collagen particles of Example 1 To investigate whether the collagen particles of Example 1 could act as a biological scaffold in skin tissue, a specific area (approximately 1 cm x 1 cm) of a rabbit's ear was injected with 100 μL of a collagen solution (35 mg / mL) containing the collagen particles of Example 1 (100-150 μm diameter). The other area of ​​the same ear was injected with the same volume of polylactic acid (30 mg / mL), hyaluronic acid (20 mg / mL), or normal saline as controls. Thirty days after injection, skin tissue was harvested from the ear and examined by immunohistochemical staining. The results are shown in Table 1 and Figure 1. Table 1. Hair follicle neogenesis and angiogenesis in rabbit subcutaneous tissue after various treatments [Table 1]

[0064] As shown in Figure 1, compared to treatments with normal saline (control group, Panel (A) of Figure 1), polylactic acid (Panel (B) of Figure 1), and hyaluronic acid (Panel (C) of Figure 1), hair follicle formation (Panel (D) of Figure 1, red arrow) and angiogenesis (Panel (D) of Figure 1, black arrow) were detected in the ears of rabbits injected with this collagen particle solution. On the other hand, no hair follicle formation or angiogenesis was detected in the areas injected with polylactic acid or hyaluronic acid.

[0065] As a result, the collagen particles with intact fibrillar structure can be used in vivo as a biological scaffold to maintain subcutaneous tissue that supports stem cell and fibroblast growth, allowing stem cells to differentiate into specific cells, inducing fibroblasts to secrete more growth factors and extracellular matrix, including de novo collagen, stimulating hair follicle growth via paracrine signaling, and inducing fibroblast angiogenesis.

[0066] It should be understood that the above description of the embodiments is given by way of example only, and that various modifications may be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the invention. While various embodiments of the invention have been described above with a certain degree of particularity or with reference to one or more specific embodiments, those skilled in the art could make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. A pharmaceutical composition for inducing hair follicle neogenesis or angiogenesis in a subject, said pharmaceutical composition comprising collagen particles, said collagen particles having a diameter of about 10-200 μm.

2. 2. The pharmaceutical composition of claim 1, wherein the collagen particles are about 100-150 μm in diameter.

3. The pharmaceutical composition is administered in an amount of 0.1 to 1000 mg / cm ２ 10. The pharmaceutical composition of claim 1, wherein the composition is administered to the subject in an amount of

4. The pharmaceutical composition is administered at a concentration of 5 mg / cm ２ 4. The pharmaceutical composition of claim 3, wherein the composition is administered to the subject in an amount of

5. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition is administered subcutaneously to the subject.

Images