TWI791290B - Use of collagen particles in hair follicles formation or angiogenesis - Google Patents

Abstract

Disclosed herein is use of collagen particles for the manufacture of a medicament for use in stimulating hair follicles formation or angiogenesis in a subject. The collagen particles for use in the present application have a diameter of about 10–200 μm. According to some embodiments of the present disclosure, the collagen particles are administered to the subject in need in an effective amount of about 0.1 mg/cm ²to about 1000 mg/cm ².

Description

Application of Collagen Particles in Promoting Hair Follicle Generation or Angiogenesis

This disclosure is in the medical field, specifically methods of promoting folliculogenesis or angiogenesis by using collagen particles.

As the largest organ of the human body, the skin not only has the functions of regulating body temperature and moisture, but also the first line of defense of the human immune system. And because the dermis contains many appendages (such as hair follicles, sweat glands, etc.), once the dermis is damaged, it will often cause more serious results.

Specifically, the hair follicles in the dermis are used to grow hair and are composed of various types of cells including hair follicle stem cells. The life cycle of hair follicles consists of three stages: anagen stage, catagen stage and telogen stage. Generally recognized hair (that is, the structure of hair scales to form the visible hair shaft) will only proliferate and differentiate into keratinocyte stem cells (keratinocyte stem cells) through the activation of hair follicle stem cells during the activation period. Stem cells differentiate into matrix keratinocytes, and matrix keratinocyte derivatives form the outer root sheath and inner root sheath in the hair follicle structure; activated hair follicle stem cells can be derived into epidermal stem cells, and further differentiate into epidermal cells. In addition, the activated hair follicle stem cells can also rapidly differentiate into sebaceous glands, while in the catagen and telogen stages, the hair follicles stop growing and gradually shrink. Thus, abnormal hair follicle cycle or damaged hair follicles in severe dermis trauma often lead to irreversible severe hair loss or hairlessness.

In addition, skin trauma of burns, ulcers, inflammation, radiation therapy, surgery, and diabetes patients will cause blood vessels to shrink or die, making it difficult to heal the wound. Clinically, it is often found that this type of wound is not only accompanied by a continuous inflammatory reaction, but also has defects that are not easy to angiogenesis. In this way, long-term exposure to skin defects and abnormalities will not only easily cause infection, but also affect the normal function of skin accessory tissues (such as lack of hair covering or insufficient blood supply to local tissues), which will greatly affect physical and mental health. However, none of the current clinical drugs can effectively solve the above two defects.

Therefore, there is an urgent need in the art for a medicine or method that can effectively promote hair follicle formation and angiogenesis.

In order to provide the reader with a basic understanding, a brief summary of the disclosure is provided below. This summary is not an extensive overview of the disclosure and it is not intended to identify key/essential elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concepts of the disclosure in a simplified conceptual form as a prelude to the more detailed description that is presented later.

The present invention aims to provide a method for promoting hair follicle formation and/or angiogenesis. As embodied and broadly described herein, one aspect of the disclosure pertains to the use of a collagen particle for the manufacture of a medicament, wherein the medicament promotes folliculogenesis and/or angiogenesis in a subject, and the medicament The medicine contains an effective amount of collagen particles.

According to an embodiment of the present disclosure, the collagen particles have a particle size of 10-200 microns (μm); preferably a particle size of 100-150 microns.

According to an embodiment of the present disclosure, the collagen particles are administered to a suitable or desired location through parenteral (non-gastrointestinal) administration, and the administration methods include but are not limited to: transdermal, intradermal and subcutaneous administration . The aforementioned effective amount of collagen is 0.1 mg per square centimeter to 1000 mg per square centimeter. Preferably, the effective amount of the collagen particles is 5 mg per square centimeter.

According to an embodiment of the present disclosure, the collagen particles may be administered to the individual via subcutaneous injection.

According to the embodiment of the present disclosure, the collagen particles of the present invention are produced through a preparation method, and the preparation method includes the following steps: (1) using a first supercritical carbon dioxide (supercritical carbon dioxide, scCO ₂ ) at a pressure of 100- Treat an animal skin with a thickness of 0.1-2 mm for about 20 minutes to 10 hours at 500 bar and a temperature of 30-50° C. to decellularize the animal skin; (2) Treat step (1) with an alkaline solution The decellularized animal skin; (3) treating the animal skin treated with an alkaline solution in step (2) with a hydrogen peroxide solution; (4) using a second supercritical carbon dioxide at a pressure of 100-500 bar and at a temperature of 30-50°C, together with a co-solvent (or co-solvent), treat the animal skin treated with the hydrogen peroxide solution in step (3) for about 20 minutes to 10 hours to remove the residues on the animal skin and (5) drying and granulating the collagen matrix in step (4) to produce the collagen particles.

As mentioned above, in one embodiment of the present disclosure, the preparation method does not use organic solvents and/or cross-linking agents.

According to some embodiments, in step (1), the first supercritical carbon dioxide is treated with the animal skin at a pressure of 350 bar and a temperature of 40° C. for 90 minutes.

According to a preferred embodiment, the alkaline solution in step (2) is sodium hydroxide (Sodium hydroxide, NaOH).

According to a preferred embodiment of the present disclosure, in step (4), the second supercritical carbon dioxide is treated with the animal skin for 90 minutes at a pressure of 350 bar and a temperature of 40°C.

In a preferred embodiment of the present disclosure, the co-solvent in step (4) is ethanol. In one embodiment, the volume ratio of co-solvent and supercritical carbon dioxide is 1:10.

According to yet another embodiment, the granulation step of step (5) is cutting or grinding the collagen matrix under liquid nitrogen to produce the collagen granules.

Another aspect of the disclosure relates to a method for treating hair follicle defect and/or vascular injury in an individual, comprising administering an effective amount of the collagen particles of the disclosure to the individual in need, wherein the collagen particles Has a particle size of 10-200 microns (μm).

According to an embodiment of the present disclosure, the collagen particles have a particle size of 100-150 microns.

According to some embodiments of the present disclosure, the effective amount of the collagen particles is 0.1 mg/cm2 to 1000 mg/cm2. Preferably, the effective amount of the collagen particles is 5 mg per square centimeter.

According to an embodiment of the present disclosure, the collagen particles may be administered to the individual via subcutaneous injection.

According to an embodiment of the present disclosure, the collagen particles of the present invention are produced through a preparation method, and the preparation method includes the following steps: (1) Treat an animal skin with a thickness of 0.1-2 mm with a first supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50°C for about 20 minutes to 10 hours to decellularize the animal skin; (2) Treating the decellularized animal skin in step (1) with an alkaline solution; (3) treating the animal skin treated with the alkaline solution in step (2) with a hydrogen peroxide solution; (4) Treat the animal skin treated with the hydrogen peroxide solution in step (3) with a second supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50° C. for about 20 minutes to 10 minutes. hours to remove chemicals remaining in the animal's skin and produce a collagen matrix; and (5) Drying the collagen matrix in step (4) and then granulating to produce the collagen particles.

As mentioned above, in one embodiment of the present disclosure, the preparation method does not use organic solvents and/or cross-linking agents.

According to some embodiments, in step (1), the first supercritical carbon dioxide is treated with the animal skin at a pressure of 350 bar and a temperature of 40° C. for 90 minutes.

According to a preferred embodiment, the alkaline solution in step (2) is sodium hydroxide.

According to a preferred embodiment of the present disclosure, in step (4), the second supercritical carbon dioxide is treated with the animal skin for 90 minutes at a pressure of 350 bar and a temperature of 40°C.

In a preferred embodiment of the present disclosure, the co-solvent in step (4) is ethanol. In one embodiment, the volume ratio of co-solvent and supercritical carbon dioxide is 1:10.

According to yet another embodiment, the granulation step of step (5) is cutting or grinding the collagen matrix under liquid nitrogen to produce the collagen granules.

Based on the above, the present disclosure can promote the generation of hair follicle cells and angiogenesis of the individual by administering an effective amount of a medicine containing collagen particles to an individual, thereby achieving the treatment of hair follicle defect and/or vascular injury.

After referring to the following embodiments, those with ordinary knowledge in the technical field of the present invention can easily understand the basic spirit and other invention objectives of the present invention, as well as the technical means and implementation modes adopted by the present invention.

In order to make the description of the present disclosure more detailed and complete, the following provides an illustrative description of the implementation aspects and specific embodiments of the present invention; but this is not the only form of implementing or using the specific embodiments of the present invention. The description covers features of various embodiments as well as method steps and their sequences for constructing and operating those embodiments. However, other embodiments can also be used to achieve the same or equivalent functions and step sequences.

I. Definition

For the convenience of description, specific terms described in this specification, examples, and appended claims are collectively described here. Unless otherwise defined herein, 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. Where there is no conflict with the context, the singular noun "a" used in this specification includes the plural form of the noun; and the plural noun used also includes the singular form of the noun.

The term "collagen particles" used in this disclosure refers to the granular collagen with a specific particle size that is decellularized, deactivated, sterilized, and ground from an animal source by known or disclosed preparation methods. For example, after decellularization of an animal's intact skin by supercritical carbon dioxide to produce a collagen matrix, the whole collagen matrix is ground without the application of any cross-linking agent, so that the resulting collagen particles can have a specific particle size, And it retains the natural structure and configuration of collagen on the microscopic scale.

The terms "administering", "administering", "administering", "administered" or "administration" are used interchangeably in this disclosure to refer to providing collagen particles to an individual for healing and improvement related to hair follicle damage or Symptoms associated with insufficient angiogenesis.

As used in this disclosure, the term "effective amount" refers to an amount of collagen particles in a dose and for the time necessary to provide a therapeutic effect or benefit in treating a condition, or to delay or minimize one or more Symptoms associated with the condition, whereby desired results associated with promoting follicular neogenesis or angiogenesis are achieved. The effective amount of collagen particles disclosed herein refers to the amount of collagen particles that can produce therapeutic benefits on pathological conditions, whether alone or in combination with other therapeutic agents. In certain embodiments of the present disclosure, the amount of collagen particles is sufficient to promote follicular neogenesis and angiogenesis in the dermis of an individual.

The term "subject" is used interchangeably herein and is meant to refer to mammals (including humans) capable of being treated by the methods of the present disclosure. The term "mammal" refers to all members of the class Mammalia, which includes humans, primates, domesticated and domesticated animals, as well as captive animals, animals used for sports or pets; and rodents. Further, the terms "subject" or "patient" include males (males) and females (females) unless gender is specifically indicated.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the relative numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently inherently contain standard deviations resulting from their individual testing methodology. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specified value or range. Alternatively, the term "about" means that the actual value falls within acceptable standard error of the mean, as considered by one of ordinary skill in the art to which this invention pertains. Except for experimental examples, or unless otherwise expressly stated, all ranges, quantities, values and percentages used herein should be understood to be Those) are modified by "covenant". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the appended patent claims are approximate values and may be changed as required. At least these numerical parameters should be understood as the value obtained by applying the normal rounding method to the indicated effective digits.

II. Specific implementation

This disclosure is based, at least in part, on the discovery that collagen particles prepared by decellularization and grinding without the use of any cross-linking agents can serve as three-dimensional bio-scaffolds that allow cells to be attached to collagen particles in vivo Growth, not only that, can also stimulate hair follicle cell proliferation and angiogenesis. Accordingly, the disclosure aims to provide a use of collagen particles to prepare a medicine, which can be used to promote hair follicle neogenesis or angiogenesis in an individual, so as to repair skin damage.

One aspect of the disclosure relates to a method for treating hair follicle defect or local blood vessel damage in an individual. The method includes administering an effective amount of collagen particles to the individual, and further includes administering the effective amount of collagen particles to the individual by subcutaneous injection, so as to promote local hair follicle neogenesis and angiogenesis.

Collagen particles can be prepared from animal tissue in a manner well known to those skilled in the art of the present invention, with or without the use of cross-linking agents. In the specific embodiment of this disclosure, an animal skin is decellularized by supercritical carbon dioxide, treated with several reagents (such as alkaline solution and hydrogen peroxide solution), and then ground into collagen with a specific particle size Protein particles, and this preparation method does not involve the use of any organic solvents (especially organic solvents known to be toxic to organisms) and cross-linking agents. Collagen particles may additionally be preserved in an in vitro environment, such as an aqueous solution environment, by means well known to those skilled in the art. The collagen particles prepared by the specific preparation method of the present disclosure still retain their original microscopic natural structural signal factors and configurations, which can provide a recipient or individual with a better microenvironment for the growth of the individual's cell tissue.

Collagen particles suitable for the therapeutic use of the present invention may be isolated from tissues of allogeneic and/or xenogeneic origin. Allogeneic collagen particles refer to collagen particles obtained from tissues or cells of the same species but different individuals; heterogeneous collagen particles refer to collagen particles obtained from individuals of different species. Specifically, collagen particles can be isolated from skin tissue, tendon tissue or any tissue rich in collagen tertiary structure among different individuals of the same species, or from different individuals of different species. Collagen sources suitable for the present disclosure include, but are not limited to, skin tissue, tendon tissue, and cartilage tissue. According to certain embodiments of the present disclosure, collagen particles are prepared from porcine skin tissue.

Specifically, the collagen particles of the present invention are prepared by a preparation method comprising the following steps without using organic solvents and cross-linking agents: (1) Treat an animal skin with a thickness of 0.1-2 mm with the first supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50°C for about 20 minutes to 10 hours to decellularize the animal skin; (2) Treating the decellularized animal skin in step (1) with an alkaline solution; (3) treating the animal skin treated with the alkaline solution in step (2) with a hydrogen peroxide solution; (4) Treat the animal skin treated with the hydrogen peroxide solution in step (3) for about 20 minutes to 10 hours with the second supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50°C together with a co-solvent , to remove chemicals remaining in the animal's skin and produce a collagen matrix; and (5) Drying the collagen matrix in step (4) and then granulating to produce the collagen particles.

Before carrying out the method, it is preferred to obtain the skin from an animal by skinning, and then wash, dehair and degrease to obtain animal skin suitable for the method of the present invention. In a preferred embodiment, animals suitable for use in the present disclosure are commercial animals including, but not limited to, pigs, cows, dairy cows, bulls, sheep, goats, donkeys, rabbits, ducks, geese, and chickens. The dehairing and degreasing steps may be performed using any known physical or chemical dehairing or degreasing method. For example, treatment of animal skin with acids to remove hair from animal skin, treatment of animal skin with enzymes (such as lipase) or chemicals (such as detergents) to remove fat from it, or through the use of a knife or instrument Fat is removed directly.

Next, the surface layer of the above-mentioned dehaired and degreased animal skin is removed with a dermatome, an instrument or a mechanical device, thereby producing a complete animal skin with 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 and 2.0 mm of animal skin; preferably about 0.3 mm. The animal skin thus obtained can be used in the methods described in this disclosure.

In step (1), decellularize the animal skin that has been dehaired and degreased and has a thickness of 0.1-2 mm with supercritical carbon dioxide. The purpose of the decellularization step is to remove cellular material from the animal's skin while still retaining the physical and biochemical properties of collagen that allow it to serve as a tissue scaffold. Accordingly, in step (1), at a pressure of about 100-500 bar (for example at 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 500 bar pressure), treat animal skin with a thickness of 0.1-2 mm with supercritical carbon dioxide; the aforementioned pressure is preferably about 200-400 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 pressure. In addition, step (1) is carried out at a temperature of 30 to 50° C., for example at 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , a temperature of 46, 47, 48, 49 or 50°C; preferably 35 to 42°C, for example at a temperature of 35, 36, 37, 38, 39, 40, 41 or 42°C; treatment for about 20 minutes to 10 hours , for example 20, 30, 40, 50 or 60 minutes, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. In some embodiments, animal skin with a thickness of 0.1-2 mm is treated with supercritical carbon dioxide for about 40 to 180 minutes. In one embodiment, the supercritical condition of supercritical carbon dioxide is a temperature of 40° C. and a pressure of 350 bar for 90 minutes. Accordingly, the decellularization process can be performed at a temperature close to the body temperature of the individual (ie, 37° C.) to remove the biologically active substances therein.

In step (2), the decellularized animal skin in step (1) is washed with an alkaline solution to remove any residual cellular material thereon. Specifically, soak the aforementioned animal skin in the alkaline solution for about 0.5-2 hours, such as about 0.5, 1, 1.5 and 2 hours; preferably 2 hours. For example, alkaline solutions suitable for the present invention include, but are not limited to: sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and the like. According to a specific embodiment of the present disclosure, the alkaline solution is sodium hydroxide at a concentration of 1N.

In step (3), the aforementioned animal skin is treated with hydrogen peroxide solution for about 0.5-2 hours, such as about 0.5, 1, 1.5 and 2 hours. In a preferred embodiment, the concentration of hydrogen peroxide in the solution is 0.1-2% (weight percent), such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5 or 2%. (percentage by weight). According to a preferred embodiment, the product of step (2) is treated with 1% hydrogen peroxide solution for about 1 hour.

Then, in step (4), the animal skin produced in step (3) is treated with supercritical carbon dioxide for the second time to remove the chemical substances remaining in the animal skin and produce a cell and bioactive component collagen matrix. This step utilizes similar treatment conditions to step (1), the difference is that together with a co-solvent, the animal skin treated with hydrogen peroxide in step (3) is treated for about 20 minutes at a pressure of 100-500 bar and a temperature of 30-50°C to 10 hours. According to a preferred embodiment, the treatment conditions of the second supercritical carbon dioxide removal step are a temperature of about 40° C., a pressure of about 350 bar, and a treatment time of about 90 minutes.

Following the foregoing, the co-solvent is a C _1-4 alcohol, which includes, but is not limited to: ethanol, propanol, isopropanol, butanol, isobutanol, sec-butanol, tert-butanol, and cyclobutanol. In certain preferred embodiments, the co-solvent is ethanol and is administered together with supercritical carbon dioxide, wherein the volume ratio of co-solvent and supercritical carbon dioxide is from 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 and 1:4. In a preferred embodiment, the volume ratio of ethanol and supercritical carbon dioxide is about 1:10.

In the final granulation step (i.e. step (5)), the product (i.e., collagen matrix) in step (4) is processed under liquid nitrogen in a manner of cutting, grinding or trimming, so as to produce a Collagen particles with specific particle size. According to an embodiment of the present disclosure, the particle size of the collagen particles is 10-200 microns, including 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 and 200 micron particle size . In preferred embodiments, the collagen particles have a size of about 100-150 microns, such as about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 microns.

The feature of the aforementioned preparation method is that relatively intact animal skin is used as a source, and the intercellular substance and active ingredients are completely removed by decellularization treatment before grinding. Since the process does not involve strong acid and/or strong alkali treatment, there is no need to apply additional cross-linking agents to promote the repolymerization of the main amino acids of collagen (ie: glycine, proline, etc.) level structure. Therefore, the characteristic of the collagen particles prepared by the aforementioned method in this case is that the collagen fibers retain the structure and configuration of the natural collagen fibers, so that the collagen particles of the present invention can be used as biological scaffolds for cell growth.

According to an embodiment of the present disclosure, the collagen particles prepared according to the aforementioned method can be administered to an individual, which is sufficient to promote hair follicle neogenesis and angiogenesis in the individual. According to certain embodiments of the present disclosure, the collagen particles are administered to the individual in an amount of 0.1 mg/cm2 of skin area to 1000 mg/cm2 of skin area. For example, the aforementioned amount can be about 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, 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. The preferred amount of collagen particles administered to the individual is about 0.1 mg to 500 mg per square centimeter of skin area, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 per square centimeter of skin area , 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, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg. A more preferred amount of collagen particles is about 0.1 to 50 milligrams per square centimeter of skin area, such as about 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, or 50 mg. In certain embodiments of the present disclosure, an effective amount of about 5 mg/ ^cm2 of collagen particles can be administered to a subject in need thereof. In addition, the collagen particles can also be administered at a frequency of once a day to once every three months. In some embodiments, the collagen particles of the present disclosure are administered in the form of once daily, once every two days, once every three days, once a week, once every other week, once a month, once every other month, or every three months One frequency dosing. In some embodiments, the collagen particles may be administered to the individual on a weekly to monthly basis.

The collagen particles of the present disclosure can be delivered to the desired site of action through a suitable or desired mode of administration. In some embodiments, the collagen particles are administered parenterally (parenterally) to a suitable or desired location. Suitable exemplary parenteral routes of administration include, but are not limited to, transdermal, intradermal, and subcutaneous administration. Specifically, the contemplated routes are via subcutaneous administration (eg, subcutaneous injection) and/or transdermal topical administration to the affected site (ie, using an applicator to spread collagen particles over a skin wound in an individual). The appropriate route, as understood by those of ordinary skill in the art, will depend on the particular condition being treated, the severity of the condition, the physical condition of the individual patient (including age, physiological condition, body size, sex and weight, duration of treatment, concomitant therapy The type of disease (if any), the dose and nature of the active ingredient, genetic factors and other similar factors in the general knowledge and professional knowledge of health practitioners. These factors have ordinary knowledge in the technical field are known techniques and can be implemented without undue experimentation. Often the most appropriate route of administration will vary with factors such as the stability of the agent in the circulatory environment, and/or The physiological condition of the individual (e.g., whether the individual can tolerate subcutaneous injections). According to some embodiments of the present disclosure, collagen particles may be administered to an individual via subcutaneous injection. In some preferred embodiments, it is via subcutaneous injection Collagen particles are administered to the dermis of an individual.

In accordance with the present disclosure, an individual refers to an animal that can be treated and benefits from the methods of the present disclosure. Examples of animals include, but are not limited to: humans, rats, mice, guinea pigs, rabbits, monkeys, pigs, sheep, cows, horses, dogs and cats. In an exemplary embodiment, the individual is a rabbit. In another exemplary embodiment, the individual is a human.

The present disclosure is prepared by administering collagen particles to an individual through the method described in the present disclosure, mainly composed of decellularized skin collagen, which has a particle size of 10-200 microns, and retains natural structural signaling factors and complete fibril configuration. Therefore, such collagen particles can be used as a three-dimensional biological scaffold. After being administered to an individual, the subcutaneous tissue provides an environment conducive to cell activation and proliferation, allowing stem cells, fibroblasts, vascular endothelial cells and other cell groups to grow on the collagen particles. It can effectively promote hair follicle cell regeneration and angiogenesis in the individual dermis, thereby repairing skin damage.

A number of experimental examples are provided below to illustrate certain aspects of the present invention, so as to facilitate those skilled in the art to implement the present invention, and these experimental examples should not be considered as limiting the scope of the present invention. It is believed that one skilled in the art can, after reading the description presented herein, fully utilize and practice the present invention without undue interpretation. All publications cited here are considered as a part of this specification in their entirety.

Experimental example

Materials and Methods

animal

New Zealand white rabbits (about 5 kg each) were used for the subcutaneous injection test. Rabbits were housed one per cage in an animal facility with free access to drinking water and food. Temperature and humidity in the animal facility were maintained at 19-25 °C and 50-60%, respectively. The basic physiological values such as body weight and body temperature of New Zealand white rabbits were recorded daily. Before each experiment, animal quarantine will be carried out and the experimental animals will be allowed to adapt to the experimental environment.

Embodiment 1: the preparation of collagen

Pigskin (0.3mm) with hair and fat removed was dried at low temperature (4°C) for 24 hours, and then treated with carbon dioxide supercritical fluid (hereinafter referred to as scCO ₂ ) at a pressure of 350 bar and a temperature of 40°C 90 minutes, all cellular material is removed through this step.

Then, at room temperature (about 19-25°C), the decellularized pigskin is sequentially soaked in sodium hydroxide (NaOH) aqueous solution, hydrogen peroxide aqueous solution, low-temperature drying and a second carbon dioxide supercritical fluid cleaning . Specifically, the decellularized pigskin was first soaked in 1N sodium hydroxide for 2 hours, and then soaked in 1% hydrogen peroxide solution for 1 hour. The second carbon dioxide supercritical fluid (scCO ₂ ) is treated with 10% (volume concentration percentage) ethanol (that is, the volume ratio of ethanol to supercritical carbon dioxide is 1:10) at a pressure of 350 bar and a temperature of 40°C. The aforementioned collagen matrix was 90 minutes to remove any impurities. At this time, the pigskin after the decellularization treatment and the aforementioned cleaning treatment has formed a collagen matrix.

After the aforementioned collagen matrix was vacuum-dried at 4°C for about 8-30 hours, it was ground with a grinder (model: Retsch, ZM200) to produce collagen particles of different particle sizes, about 100-150 μm in size. The prepared collagen particles are irradiated with gamma rays (10-50 kGy) and stored in a sterile environment for future use. Observation under an electron microscope shows that the collagen particles prepared by the aforementioned method all have a complete fibril structure.

Embodiment 2: the collagen particle of embodiment 1 promotes hair follicle hyperplasia and angiogenesis

In order to evaluate whether the collagen particles of the present invention have good bioscaffold properties in the skin tissue, in this experiment, the aqueous solution of collagen particles (particle size 100-150 μm) in Example 1 was used at a concentration of 35 mg/mL, and 100 μL was added to the Inject subcutaneously in a specific area of the rabbit ear (a skin area with an area of about 1 cm x 1 cm). In addition, 100 μL of polylactic acid (concentration 30 mg/mL), 100 μL of hyaluronic acid (concentration 20 mg/mL) and 100 μL of normal saline were injected into the same ear but in different areas as a control group. After 30 days of injection, ear skin tissue sections were removed and stained to observe whether there were any changes in these injection areas and evaluate the degree of change. The results are presented in Table 1 below and Figure 1 .

Table 1. Comparison of hyperplasia of subcutaneous hair follicles and blood vessels in rabbit ears under four different treatment conditions control group polylactic acid hyaluronic acid Collagen in this case hair follicle hyperplasia -- + + +++ degree of angiogenesis -- + + +++

As shown in the experimental results in Figure 1, compared with the control group injected with saline (Figure 1 (A) panel) and the area where only polylactic acid or hyaluronic acid was applied (see Figure 1 (B) or ( C) small picture), through the rabbit ear area injected with collagen in Example 1 of the present invention, new hair follicle cells can be observed in the subcutaneous (dermis) area (the small picture of Figure 1 (D), indicated by the red arrow) and New vascular proliferation (panel 1 (D) panel, indicated by black arrows). In addition, no obvious new hair follicle cells or vascular tissue were observed in the area where only polylactic acid or hyaluronic acid was applied.

According to the above, the collagen particles of the present invention have a complete fibril structure, and can be used as a biological scaffold in vivo, which can maintain the structure of subcutaneous tissue to provide an excellent growth environment for stem cells and fibroblasts, so that stem cells can be differentiated into specific cells, making fibroblasts The cells secrete more growth factors and extracellular matrix components (including new collagen) to stimulate hair follicle cells and stimulate fibroblasts to promote the growth of vascular tissue by paracrine.

Although the specific embodiments of the present invention have been disclosed in the above embodiments, they are not intended to limit the present invention. Those who have ordinary knowledge in the technical field of the present invention, without departing from the principle and spirit of the present invention, when Various alterations and modifications can be made to it, so the protection scope of the present invention should be defined by the appended patent scope.

none

In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

Figure 1 is a subcutaneous injection of (A) normal saline for injection (control group 1) and (B) polylactic acid (Poly-L-lactic acid, control group 1) in rabbit ears according to an embodiment of the present disclosure. 2), (C) hyaluronic acid (hyaluronic acid, control group 3) and (D) collagen particles of the present invention 30 days later, tissue staining photographs taken at a magnification of 40× (Fig. 1 (D), black arrows: blood vessels tissue; red arrows: hair follicle cells).

Claims (10)

A kind of collagen particle is used for the purpose of preparing the medicine that promotes the generation of a human hair follicle, wherein the medicine comprises an effective amount of collagen particle, and the collagen particle has a particle size of 10-200 micrometers (μm), wherein The collagen particles are prepared by a method comprising the following steps: (1) treating an animal with a thickness of 0.1-2 mm with a first supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50°C Skin for about 20 minutes to 10 hours to decellularize the animal skin; (2) treat the decellularized animal skin in step (1) with an alkaline solution; (3) treat the animal skin with a hydrogen peroxide solution (2) the animal skin treated with alkaline solution; (4) treat the animal skin in step (3) with a second supercritical carbon dioxide at a pressure of 100-500 bar and a temperature of 30-50°C together with a co-solvent Animal skin treated with hydrogen peroxide solution for about 20 minutes to 10 hours, to remove the chemical substances remaining in the animal skin and produce a collagen matrix; and (5) dry the collagen matrix in step (4) and proceed Granulating to produce the collagen particles; wherein the method does not use organic solvents and cross-linking agents. The use as claimed in claim 1, wherein the collagen particles have a particle size of 100-150 microns. The use according to claim 1, wherein the collagen particles have the effective amount of 0.1 mg/cm2 to 1000 mg/cm2. The use as described in claim 3, wherein the collagen particles have the effective amount of 5 mg per square centimeter. The use as described in claim 1, wherein the collagen particles are administered to the individual through subcutaneous injection. The use as claimed in claim 5, wherein in step (1) of the method, the first supercritical carbon dioxide is treated with the animal skin for 90 minutes at a pressure of 350 bar and a temperature of 40°C. The use as described in claim 5, wherein in the step (2) of the method, the alkaline solution is sodium hydroxide. The use as claimed in item 5, wherein in step (4) of the method, the second supercritical carbon dioxide is treated with the animal skin for 90 minutes at a pressure of 350 bar and a temperature of 40°C. The use as described in claim 5, wherein in step (4) of the method, the co-solvent is ethanol. The use as described in claim 5, wherein the step (5) of the method is to cut or grind the collagen matrix under liquid nitrogen to produce the collagen particles.

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