HK1246358B - Preparation of high purity collagen particles and used thereof - Google Patents

Description

High-purity collagen particles and their preparation method and use

This application claims all rights of U.S. Provisional Application No. 62/203,904, filed August 11, 2015, the contents of which are incorporated herein in their entirety.

Technical Field

The present invention relates to a method for preparing collagen, and more particularly, an improved method for preparing collagen particles, wherein the collagen is suitable for use as a biological scaffold for cell growth, for example, as an injectable dermal filler for plastic surgery procedures (i.e., cosmetic surgery) or soft tissue augmentation (e.g., smoothing facial lines and wrinkles); or as a wound dressing to promote wound healing.

Background Art

Collagen is an insoluble fibrin that exists in vertebrates and is the main component of the fibrils of connective tissue (such as skin). Traditional methods for preparing collagen are not only time-consuming and inefficient, but also destroy the integrity of the collagen structure, making it unsuitable as a biological scaffold and thus unsuitable for use in cosmetic surgery or as a wound healing graft. In addition, the collagen available on the market (such as Zyderm and Zyplast) is taken from the skin of dairy cows, which cannot be used for all patients because bovine collagen is known to cause severe allergic reactions in many hosts, especially in individuals with a history of autoimmune diseases. In addition, bovine collagen does not exist at the injection site for a long time, and therefore, regular supplemental injections are required. The reason why collagen only exists briefly at the injection site is that during the process of extracting collagen from cowhide, the collagen structure is destroyed, causing the collagen to be easily absorbed by the host.

The field is currently focused on developing human-derived collagen (e.g., Cosmoderm and Cosmoplast), but sources are limited (e.g., from circumcised skin), resulting in high prices for human collagen. Alternatively, patients can obtain adipose tissue from their bodies through liposuction, which is then processed into injectable collagen for immediate reinjection or stored for future use. However, collagen obtained in this way suffers from the same drawbacks as bovine collagen: its integrity is destroyed during the isolation process.

Therefore, there is a need in the art for an improved method for preparing collagen, which can retain the natural structure and configuration of collagen during the process of separating collagen, so that the collagen prepared in this way can serve as a three-dimensional scaffold for host cell growth without inducing severe immune response in host cells.

Summary of the Invention

To solve the above-mentioned problem of preparing collagen particles, the inventors of this case disclosed a method for preparing collagen particles from animal skin (eg, cow or pig skin), and the collagen particles can retain the configuration of natural collagen.

Furthermore, the first aspect of the present invention is to provide a method for preparing collagen particles. The method comprises the following steps:

(1) applying a cell-removing treatment to an animal skin having a thickness of 0.1-1 mm;

(2) treating the decellularized animal skin in step (1) with an aqueous solution, wherein the aqueous solution comprises a nonionic surfactant;

(3) treating the animal skin treated with the aqueous solution in step (2) with a protease;

(4) treating the animal skin treated with the protease in step (3) with a nuclease;

(5) subjecting the animal skin treated with nuclease in step (4) to a deionization treatment;

(6) subjecting the animal skin subjected to the deionization treatment in step (5) to a de-chemical treatment to produce a collagen matrix; and

(7) Granulating the collagen matrix in step (6) to produce collagen particles with a particle size of 10-250 μm.

According to certain embodiments, in step (1), animal skin having a thickness of about 0.1-1 mm is treated with supercritical fluid (SCF) at a pressure of 100-500 bar (par) and a temperature of 30-50° C. for about 20 minutes to 5 days.

The SCF is supercritical carbon dioxide (scCO ₂ ), supercritical nitrous oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkanes, supercritical alkenes, supercritical alcohols, or supercritical acetone. In one embodiment, the SCF is scCO ₂ . In other embodiments, the SCF is scN ₂ O.

According to a preferred embodiment, the decellularization step is performed under the following conditions: a temperature of about 37° C., a pressure of about 350 bar, and a treatment time of 20 minutes.

According to certain embodiments, in step (2), the animal skin decellularized in step (1) is treated with an aqueous solution containing a surfactant, wherein the surfactant can be selected from the group consisting of: octylphenol ethoxylates (e.g., Triton X series), sorbitan monostearate, polysorbate, poloxomer, nonoxynols, cetyl alcohol, and alkyl polyglucoside. In an optional embodiment, the aqueous solution further comprises an anionic surfactant, such as laurylsulfonic acid, dodecylsulfonic acid, sodium dodecyl sulfate (SDS), dodecylbenzenesulfonic acid, tridecylbenzenesulfonic acid, alkyl-phenoxybenzenedisulfonic acid, naphthalenesulfonic acid, alkyl-naphthalenesulfonic acid, and alkenylnaphthalene. In another optional embodiment, the aqueous solution further comprises a salt, such as sodium chloride and potassium chloride. In a preferred embodiment, the anionic surfactant is sodium dodecylsulfonate (SDS).

According to certain embodiments, the protease in step (3) can be selected from the group consisting of: pepsin, trypsin, chymotrypsin, papain, chymopapain, bromelain, actinidain, proteinase A, proteinase K, peptidase, ficin, calpain, caspase, and combinations thereof.

According to certain embodiments, in step (4), the animal skin treated with the protease in step (3) is treated with a nuclease, wherein the nuclease is a DNA nuclease or an RNA nuclease.

In an optional embodiment, the product of step (4) can be further treated with an aqueous solution from step (2), wherein the aqueous solution comprises a nonionic surfactant selected from the group consisting of octylphenol polyoxyethylene ether (e.g., Triton X series), sorbitan monostearate, polysorbate, poloxamer, nonoxynol, hexadecanol, and alkyl polyglucosides. In a preferred embodiment, the aqueous solution comprises 1% Triton X-100. In an optional embodiment, the aqueous solution further comprises an anionic surfactant, such as lauryl sulfonic acid, dodecyl sulfonic acid, dodecylbenzenesulfonic acid, tridecylbenzenesulfonic acid, alkylphenoxybenzenedisulfonic acid, naphthalenesulfonic acid, alkylnaphthalenesulfonic acid, and alkenylnaphthalene. In other optional embodiments, the aqueous solution further comprises a salt, such as sodium chloride and potassium chloride. In a preferred embodiment, the anionic surfactant is dodecyl sulfonic acid (SDS).

According to certain embodiments, in step (5), the animal skin treated with nuclease in step (4) is treated with hydrogen peroxide for 1 hour.

According to certain embodiments, in step (6), the chemical removal step comprises treating the deionized product in step (5) with a supercritical fluid (SCF) at a pressure of 100-500 bar and a temperature of 30-50° C. for 20 minutes to 5 days.

The supercritical fluid is supercritical carbon dioxide (scCO ₂ ), supercritical nitrous oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkane, supercritical alkene, supercritical alcohol, or supercritical acetone. In one embodiment, the SCF is scCO ₂ . In other embodiments, the SCF is scN ₂ O.

According to a preferred embodiment, the chemical removal step is performed in the presence of a co-solvent at a temperature of approximately 37°C and a pressure of approximately 350 bar for approximately 60 minutes. In one embodiment, the co-solvent is ethanol and is applied together with SCF in a volume ratio of 1:10.

According to another embodiment, in step (7), the granulation step is to cut or grind the collagen matrix in step (6) to form collagen particles with a particle size of 10-250 microns. The prepared collagen particles are composed of collagen and retain their natural structure and configuration. Therefore, such collagen particles can serve as a three-dimensional biological scaffold, and after administration to an individual, cells can grow on the collagen particles.

Therefore, a second aspect of the present invention provides a method for treating a skin condition in an individual, wherein the skin condition is a wound, lines, and/or depressions on the skin. The method further comprises administering a sufficient amount of collagen particles prepared by the method of the present invention to the subcutaneous tissue of the individual to alleviate or improve the skin condition. The skin condition is a wound, lines, and/or depressions on the skin. For example, wounds include, but are not limited to, surgical wounds (e.g., incisions), ulcers, and any other bodily injury that results in a break, cut, puncture, or tear in the skin or other tissue. Lines and/or depressions on the skin (e.g., on the face) include, but are not limited to, frown lines, lines around the mouth, worry lines, crow's feet, smile lines, and facial scars caused by acne or trauma. In a preferred embodiment, the collagen particles are used as a dermal filler to fill the soft tissue of the individual and are injected into the individual via a needle (e.g., a 32-gauge needle or smaller). In other embodiments, the collagen particles are used as a wound dressing, and an applicator is used to spread the collagen on a wound of an individual.

A third aspect of the present invention provides a kit for treating the aforementioned skin conditions during cosmetic surgery. The kit comprises the following components: a container; collagen particles produced by the present method, characterized by relatively intact collagen fibers and a diameter of approximately 10–250 microns; and instructions associated with the container to guide the user in using the collagen particles. The instructions may be in the form of a manual, cassette, CD, VCD, or DVD.

After referring to the following embodiments, those skilled in the art will be able to easily understand the basic spirit and other objectives of the present invention, as well as the technical means and embodiments adopted by the present invention.

It should be understood that the foregoing general description and the following detailed description are made with reference to the embodiments and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

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:

FIG1 is a photograph of the collagen particles of the present invention taken under an electron microscope (EM) at magnifications of (A) 50X and (B) 20,000X, respectively, according to one embodiment of the present invention; and

FIG2 shows electron microscope (EM) images of the collagen particles of the present invention taken at magnifications of (A) 2,000X and (B) 4,000X, respectively, after 3T3 cells were recellularized on the collagen particles of the present invention for (A) 12 hours and (B) 72 hours, according to one embodiment of the present invention.

DETAILED DESCRIPTION

To provide a more detailed and complete description of the present invention, the following provides illustrative descriptions of the embodiments and examples of the present invention; however, these descriptions are not intended to be the only ways to implement or use the embodiments of the present invention. The embodiments cover features of various embodiments, as well as the method steps and sequences for constructing and operating these embodiments. However, other embodiments may also be used to achieve the same or equivalent functionality and step sequences.

The singular nouns "a" and "an" shown herein include the plural form unless otherwise defined.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values of the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. As used herein, "about" generally refers to the actual value being within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one skilled in the art. Except in the experimental examples, or unless expressly indicated otherwise, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the appended claims are approximate and may vary as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to apply normal rounding.

The present invention relates to a new method for preparing collagen particles, new collagen particles prepared by the method, and uses of the collagen particles.

The first aspect of the present invention is to provide a method for preparing collagen particles, which can retain the natural structure and configuration of collagen. Therefore, when the collagen particles of the present invention are injected into the host, a better microenvironment can be provided for the growth of host cell tissues thereon.

Accordingly, the method of the present invention comprises at least the following steps:

(1) applying a cell-removing treatment to an animal skin having a thickness of 0.1-1 mm;

(2) treating the decellularized animal skin in step (1) with an aqueous solution, wherein the aqueous solution comprises a surfactant;

(3) treating the animal skin treated with the aqueous solution in step (2) with a protease;

(4) treating the animal skin treated with the protease in step (3) with a nuclease;

(5) subjecting the animal skin treated with nuclease in step (4) to a deionization treatment;

(6) subjecting the animal skin subjected to the deionization treatment in step (5) to a de-chemical treatment to produce a collagen matrix; and

(7) Granulating the collagen matrix in step (6) to produce collagen particles with a particle size of 10-250 μm.

Prior to performing the present method, it is preferred to obtain the skin of an animal by skinning, and then clean, remove the hair, and defatted to obtain an animal skin suitable for use in the present method. In a preferred embodiment, the animals suitable for use in the present invention are commercial animals, including but not limited to pigs, cattle, dairy cows, bulls, sheep, goats, donkeys, rabbits, ducks, geese, and chickens. The dehairing and degreasing steps can be performed using any known physical or chemical dehairing or degreasing method. For example, the animal skin can be treated with acid to remove hair from the animal skin (e.g., pig skin), and the animal skin can be treated with an enzyme (e.g., lipase) or a chemical (e.g., detergent) to remove fat therefrom. Alternatively, the fat can be directly removed with a knife.

Next, the top layer of the hairless and defatted animal skin is removed with a dermaplaning knife, thereby producing an animal skin having a thickness of about 0.1 to 1 mm, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mm; preferably, about 0.2 to 0.6 mm, for example, 0.2, 0.3, 0.4, 0.5, and 0.6 mm; and most preferably, about 3 mm. The resulting animal skin can be used in the method described herein.

In an optional embodiment, the animal skin, approximately 0.1 to 1 mm thick, is treated with an alkaline agent at a temperature between 0°C and 55°C for approximately 0.1 to 24 hours to remove residual hair and/or fat. Examples of alkaline agents suitable for this method include, but are not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, urea, sodium sulfide, and calcium thioacetate. In a preferred embodiment, the animal skin is porcine skin, approximately 0.1 to 0.6 mm thick, and is treated with a 0.1-1N sodium hydroxide solution at 4°C for approximately 1 hour.

In step (1), animal skin with a thickness of 0.1-1 mm is subjected to a decellularization process. The purpose of the decellularization process is to remove cellular material from the animal skin while still retaining the physical and biochemical properties of collagen, so that it can be used as a tissue scaffold. Accordingly, in step (1), animal skin having a thickness of 0.1-1 mm is treated with a supercritical fluid (SCF) at a pressure of about 100-500 bar, for example, at a pressure of 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 and 500 bar; preferably about 150-450 bar, and 400 bar; and more preferably, at a pressure of about 200-400 bar, for example, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400 bar. In addition, step (1) is carried out at a temperature of 30-50°C, for example, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50°C; preferably between 35-45°C, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45°C; for about 20 minutes to 5 days, for example, 20, 30, 40, 50, and 60 minutes; 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 hours; 2, 3, 4 and 5 days. In certain embodiments, the animal skin having a thickness of 0.1-1 mm is treated with SCF for about 1 to 24 hours. In other embodiments, the animal skin having a thickness of 0.1-1 mm is treated with SCF for 2 to 5 days.

The SCF can be supercritical carbon dioxide (scCO ₂ ), supercritical nitrous oxide (scN ₂ O), supercritical water (scH ₂ O), supercritical alkanes, supercritical alkenes, supercritical alcohols, or supercritical acetone. In one embodiment, the SCF is scCO ₂ . The mild supercritical conditions of scCO ₂ are 37°C and 350 bar. Therefore, decellularization can be performed at or near body temperature (i.e., 37°C) to remove biological material. In another preferred embodiment, the SCF is scN ₂ O.

Next, in step (2), the animal skin that has been decellularized in step (1) is cleaned with an aqueous solution containing a nonionic surfactant to remove any residual cellular material thereon. For example, nonionic surfactants suitable for use in the present invention include, but are not limited to, octylphenol polyoxyethylene ether (e.g., Triton X series), sorbitan monostearate, polysorbate, poloxamer, nonoxynol ether, hexadecanol, alkyl polyglucoside, and the like. In a preferred embodiment, the nonionic surfactant is octylphenol polyoxyethylene ether, i.e., Triton X series, which includes, but is not limited to, Triton X-15, Triton X-35, Triton X-45, Triton X-100, Triton X-102, Triton X-114, and the like. According to certain embodiments of the present invention, the nonionic surfactant is Triton X-100, and its concentration in the aqueous solution is 0.1-10% (weight %), 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, or 10% (weight %). In a preferred embodiment, the concentration of the ionic surfactant in the aqueous solution is 0.5-5% (weight %), for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, or 5% (weight %). According to a preferred embodiment, the aqueous solution contains about 1% (weight %) Triton X-100.

In an optional embodiment, the aqueous solution further comprises an anionic surfactant, such as lauryl sulfonic acid, dodecyl sulfonic acid, dodecylbenzenesulfonic acid, tridecylbenzenesulfonic acid, alkylphenoxybenzenedisulfonic acid, naphthalenesulfonic acid, alkylnaphthalenesulfonic acid, and alkenylnaphthalene. In another optional embodiment, the aqueous solution further comprises a salt, such as sodium chloride and potassium chloride. In a preferred embodiment, the anionic surfactant is dodecyl sulfonic acid (SDS).

Next, in step (3), the animal skin treated with the aqueous solution wash in step (2) is digested with a protease. This step uses a mild enzymatic digestion, so that the natural structure and configuration of the collagen fibers can be preserved. For example, the protease suitable for step (4) includes, but is not limited to, pepsin, trypsin, chymotrypsin, papain, chymopapain, bromelain, kiwifruit enzyme, proteinase A, proteinase K, peptidase, ficin, calpain, caspase or a combination thereof. In one embodiment, the protease is pepsin. In other embodiments, the protease is a mixture of trypsin and chymotrypsin. In a preferred embodiment, the concentration of the protease is about 0.001-0.1% (wt %), for example, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1% (wt %); in a preferred embodiment, about 0.002-0.05% (wt %), for example, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04 and 0.05% (wt %). According to a preferred embodiment, the animal skin washed with the aqueous solution in step (2) is treated with about 0.05% (weight %) pepsin for about 8-24 hours, for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 hours; preferably for about 12 to 20 hours, for example, 12, 13, 14, 15, 16, 17, 18, 19 and 20 hours.

In step (4), the product of step (3) is treated with a nuclease, for example, a DNA nuclease or an RNA nuclease, preferably a non-specific DNA/RNA nuclease. According to certain embodiments of the present invention, the product of step (3) is treated with the nuclease at 37° C. for 1 hour.

In an optional embodiment, the product of step (4) is further treated with a glycoside hydrolase (eg, α-galactosidase) to remove any remaining galactosyl moieties on the nuclease-treated product of step (3).

In another optional embodiment, the product of step (4) is further treated with the aqueous solution to remove any residual cellular material. In a preferred embodiment, the aqueous solution contains 1% Triton X-100 and also contains an anionic surfactant such as SDS.

In step (5), the animal skin treated with nuclease in step (4) is deionized, which comprises treating the animal skin treated with nuclease in step (4) with a hydrogen peroxide solution for about 0.5-5 hours, for example, about 0.5, 1, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5, and 5 hours. In a preferred embodiment, the concentration of hydrogen peroxide in the solution is 0.1-3% (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, 2, 2.5, or 3% (weight %). According to a preferred embodiment, the product of step (4) is treated with a 1% hydrogen peroxide solution for about 1 hour.

Next, in step (6), the chemical substances of the product of step (5) are removed, which comprises treating the deionized product of step (5) with a supercritical fluid. The treatment is carried out under similar conditions as step (1). This step can use the same or different supercritical fluid, and the treatment is carried out at a pressure of about 100-500 bar and a temperature of 30-50°C for about 20 minutes to 5 days. In one embodiment, the SCF is _scCO2 . In other embodiments, the SCF is _scN2O . According to a preferred embodiment, the SCF is applied to the product of step (5) together with a co-solvent, and the treatment conditions of the chemical substance removal step are a temperature of about 37°C, a pressure of about 350 bar, and a treatment time of about 60 minutes. The co-solvent is a _C1-4 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, which can be administered together with SCF, wherein the volume ratio of the co-solvent to SCF is 1:20 to 1:4, for example, 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, and 1:4. In a preferred embodiment, the ethanol and SCF are administered at a volume ratio of about 1:19. In other embodiments, the ethanol and SCF are administered at a volume ratio of about 1:10. In yet another embodiment, the ethanol and SCF are administered at a volume ratio of about 1:4.

In the final step (7), the product of step (6) is granulated to produce collagen particles suitable for administration by injection. The granulation is performed by cutting, grinding or trimming the product of step (6) (i.e., the collagen matrix) in the presence of liquid nitrogen to produce collagen particles having a size of 10-250 microns, for example, about 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, 170, 175, 180, 185, 190, 195, 200, 205, 210, 220, 225, 230, 235, 240, 245 and 250 microns. In a preferred embodiment, the size of the collagen particles is about 50-100 microns, for example, about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 microns. In other embodiments, the size of the collagen particles is about 100-150 microns, for example, about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150 microns. In other embodiments, the size of the collagen particles is about 150-250 microns, for example, about 150, 155, 160, 170, 175, 180, 185, 190, 195, 200, 205, 210, 220, 225, 230, 235, 240, 245, and 250 microns. The collagen particles prepared according to the present method are characterized in that the collagen fibers in the collagen retain the natural collagen fiber structure and configuration, so that the collagen particles of the present invention can be used as a biological scaffold for cell growth.

Therefore, the collagen particles of the present invention can be used as dermal fillers and can be administered to a specific area (e.g., the face) of a subject using a 32-gauge needle or less, for example, a 32-gauge, 30-gauge, 29-gauge, 28-gauge, 27-gauge, 26-gauge, 26-gauge, 25-gauge, 24-gauge, 23-gauge, 22-gauge, 22-gauge, 21-gauge, 20-gauge, 19-gauge, 18-gauge, 17-gauge, 16-gauge, 15-gauge, 14-gauge, 13-gauge, 12-gauge, 11-gauge, 10-gauge, 9-gauge, 8-gauge, or 7-gauge needle. In one embodiment, a 30-gauge needle is used to inject the collagen particles of the present invention. In another embodiment, a 27-gauge needle is used to inject the collagen particles of the present invention.

Furthermore, the collagen particles are used as wound dressings, with an applicator evenly distributing the collagen onto individual wounds. The wounds include, but are not limited to, surgical wounds (e.g., incisions), ulcers, and any other injuries on the body that result in damage, cutting, puncture, or tearing of the skin or other tissues. Lines and/or depressions on the facial skin include, but are not limited to, frown lines, mouth lines, forehead wrinkles, crow's feet, nasolabial folds, and facial scars caused by acne or trauma.

The present method differs from existing techniques in that it does not require the use of crosslinking agents or the addition of salts to stabilize the collagen matrix, nor does it require extraction of collagen fibers from the collagen matrix. Instead, the present method pelletizes the intact collagen matrix to produce collagen particles, each with a diameter of approximately 10-250 microns. The collagen particles prepared using this method are composed of collagen fibers and retain the integrity of native collagen fibers. Therefore, the collagen particles of the present invention are suitable for use as biological scaffolds for the growth of host cells.

Therefore, another aspect of the present invention is to provide a method for treating an individual's skin condition; the skin condition refers to wounds, lines and/or depressions on the skin. The method comprises the following steps: applying a sufficient amount of collagen particles prepared by the present method to the skin of the individual to alleviate or improve the skin condition. The collagen particles of the present invention are suitable for promoting wound healing and smoothing lines and/or depressions on the face, in particular, the wounds include, but are not limited to, surgical wounds (such as incisions), ulcers, and any other injuries on the body, which cause damage, cutting, puncture or tearing of the skin or other tissues; and the lines and/or depressions on the facial skin include, but are not limited to, frown lines, mouth lines, forehead wrinkles, crow's feet, nasolabial folds, and facial scars caused by pimples or trauma.

To provide those skilled in the art with tools for applying the present invention, the collagen particles of the present invention can be packaged in a kit for treating the various skin conditions described above. In one embodiment, the present invention provides a kit for using the collagen particles of the present invention in facial cosmetic procedures. In other embodiments, the present invention provides a kit for using the collagen particles of the present invention in treating wounds in an individual.

The kit comprises: a container; collagen particles prepared according to any embodiment of the present invention, characterized by relatively intact collagen fibers and a diameter of approximately 10 to 250 microns per particle; and instructions accompanying the container describing how to use the collagen particles. The instructions may be in the form of a manual, cassette, CD, VCD, or DVD.

The following experimental examples illustrate certain embodiments of the present invention to facilitate practice by those skilled in the art. These experimental examples should not be construed as limiting the scope of the present invention. It is believed that after reading the description provided herein, a skilled artisan will be able to fully utilize and practice the present invention without undue interpretation. All publications cited herein are incorporated herein in their entirety as a part of this specification.

Experimental example

Materials and Methods

Cell culture

NIH-3T3 fibroblasts were cultured in high glucose DMEM (containing 10% fetal bovine serum (FBS), 100 units/ml of penicillin and 100 mg/ml of streptomycin) at 37°C and 5% CO2 in a humidified atmosphere.

Recellularization of collagen particles

When 3T3 cells reached approximately 80% confluent, attached cells were detached using an enzyme (0.25% trypsin in 1 mM EDTA) and collected by centrifugation at 500 x g for 5 minutes. The collected cells were resuspended in culture medium and plated onto SCF-treated collagen particles (concentration: 1 x ¹⁰ cells/ml). The particles were then plated in a 24-well culture plate and incubated in an incubator for 12, 24, 48, or 72 hours to ensure cell attachment to the collagen particle surface. The collagen particles with 3T3 cells were then fixed in 2.5% glutaraldehyde for 1.5 hours and 1% osmium tetraoxide for 1.5 hours, respectively, and then dehydrated in alcohol.

animal

New Zealand white rabbits (each weighing more than 0.5 or 2 kg) were used for pyrogen tests and intradermal irritation tests; ICR mice (BioLASCO Taiwan Co., Ltd) (each weighing about 17-23 grams) and guinea pigs (each weighing about 300-500 grams) were used for skin sensitivity tests. The experimental animals were raised in an animal facility, and the rabbits (one per cage), mice (5 per cage), and guinea pigs (5 per cage) were raised with free access to drinking water and food. The temperature and humidity in the animal facility were maintained at 18-26°C and 30-75%, respectively. The body temperature of the New Zealand white rabbits used in this pyrogen test did not exceed 39.8°C, and the difference between the highest and lowest body temperatures did not exceed 1°C. Animal quarantine was performed before each test, and the experimental animals were allowed to adapt to the experimental environment.

Example 1 Preparation and Characterization of Collagen Particles

1.1 Preparation of collagen matrix

Pig skin (0.2-0.4 mm) with hair and fat removed was dried at 4°C for 24 hours and then treated with supercritical carbon dioxide (scCO ₂ ) at 350 bar and 37°C for 40-180 minutes to remove any remaining cellular material.

The decellularized pig skin was subjected to the following treatments at room temperature (approximately 22-28°C), including sonication, washing, enzymatic digestion, and rinsing. Briefly, the decellularized pig skin was sonicated (0.1 M Tris) for 1 hour, shaken continuously for 22 hours (100 rpm), and then sonicated for 1 hour. The sonicated pig skin was then washed sequentially with water (10 minutes/wash, two washes), a 1% Triton X-100 solution (shaking at 100 rpm; treatment time: 24 hours), and water (10 minutes/wash, two washes) to remove any impurities and generate a collagen matrix. The collagen matrix was then treated with a pepsin solution (0.01% pepsin in 0.5 M acetic acid) for 1 hour and then washed with water (10 minutes/wash, two washes) while shaking at 100 rpm.

The collagen matrix was treated with a DNA nuclease solution (0.3 U/ ^cm² ) at 37°C for 1 hour, then washed twice with water (10 minutes/wash, 2 washes) at room temperature (approximately 22-28°C) using 1% Triton X-100 (shaking at 100 rpm for 24 hours). The collagen matrix was then treated with _a 1% _H₂O₂ solution at room temperature (approximately 22-28°C) for 1 hour (shaking at 65 rpm), then washed twice with water (10 minutes/wash, 2 washes), and then vacuum-dried at 37°C for approximately 8-30 minutes. The matrix was then treated with _scCO₂ at a pressure of 350 bar and a temperature of 37°C in the presence of 10% (vol%) ethanol for 60 minutes, then rehydrated with water at 25°C for 10 minutes. Finally, the matrix was vacuum-dried at 37°C for approximately 8-30 minutes.

1.2 Preparation of collagen particles

The rehydrated collagen matrix of Example 1.1 was cut and ground using a Freezer/Mill (6770/6870, 5-25 cycles) to produce collagen particles. The prepared collagen particles were irradiated with gamma rays (10-50 kGy) and stored in a sterile environment until use.

Electron microscopy (EM) analysis results show that each collagen particle prepared by the present invention has a relatively complete fibril structure (see FIG1 ).

1.3 Example 1.2 Collagen particles can support the growth of 3T3 cells

Following the procedures described in the "Materials and Methods" section above, the collagen particles from Example 1.2 were used as growth scaffolds to test their ability to support cell regrowth. The porous nature of the collagen particles from Example 1.2 made them a suitable biological scaffold for supporting the growth of newly seeded 3T3 cells on these collagen particles (see Figure 2, (A)). After 72 hours of culture, the entire collagen particle was covered with 3T3 cells (see Figure 2, (B)).

Example 2 Allergy test of collagen particles in Example 1

To assess the potential risk of the collagen particles of the present invention causing other host immune responses, this experiment utilized collagen extracts prepared from the collagen matrix of Example 1.1. Various allergy tests were conducted in accordance with relevant approved procedures (particularly ISO 0993-10 and 11), including pyrogen testing, skin sensitization testing, acute systemic injection testing, and subcutaneous irritation testing.

2.1 Preparation of collagen extract

The collagen matrix (3 x 4 cm) from Example 1.1 was immersed in 0.9% saline or cottonseed oil at 50°C for 72 hours with continuous stirring (150 rpm) to dissolve the collagen in the saline or cottonseed oil, thereby producing a collagen extract. The surface area ratio of the collagen matrix to the 0.9% saline or cottonseed oil was approximately 1 cm²/1 ml.

2.2 Pyrogen test

Here, a pyrogen test was performed according to the procedures specified in the United States Pharmacopoeia (Pharmacopoeia National Formulary USP36/NF31 (151)). Briefly, this test used 6 male New Zealand white rabbits (>1.5 kg, control group: 3 rabbits, and experimental group: 3 rabbits). The experimental process was to inject 10 ml/kg of the collagen extract of Example 2.1 (in normal saline) into the ear vein of the rabbits. The rabbits in the control group were injected with only 0.9% normal saline. The injection was completed within 10 minutes. Then, the body temperature of the experimental animals was measured at 1, 1.5, 2, 2.5 and 3 hours after the administration of the collagen extract.

The body temperatures of the rabbits in the control group were 39.1, 38.8, and 38.8°C, respectively (Table 1). In the animals in the experimental group, after administration of the collagen extract (in saline), the body temperatures of the experimental animals fluctuated slightly, but such fluctuations were within an acceptable range (Table 2). These results indicate that the collagen extract prepared from the collagen matrix of Example 1 is essentially non-pyrogenic.

Table 1 Body temperature of experimental animals before administration of collagen extract

Table 2 Body temperature of experimental animals after administration of collagen extract

HBT: Maximum body temperature

CT: Control temperature of experimental animals in Table 1

2.3 Skin sensitization test

A skin sensitization test was conducted according to the procedures outlined in ISO 10993-10. Thirty male guinea pigs were randomly assigned to four groups: control group 1 (N=5), control group 2 (N=5), treatment group 1 (N=10), and treatment group 2 (N=10). Prior to the test, the animals' hair was removed from their backs, necks, and shoulder blades using an electric scalpel. Three hair-removed areas, A, B, and C, were created on the left side of each animal. The same procedure was followed on the right side of the animals, creating three hair-removed areas, A, B, and C. Each area was approximately 2 x 2 cm square.

On the first day of treatment, according to the experimental conditions of "Induction (I)" shown in Table 3 or Table 4, 0.1 ml of the corresponding solution was injected intradermally into the hair-removed area of each group of experimental animals. One week later, if the experimental animals did not produce an irritation reaction, 10% dodecylsulfonic acid (SDS) was injected into the injected area. Then, according to the conditions of "Induction (II)" shown in Table 3 or Table 4, patches pre-soaked in 0.2 ml of the corresponding solution were respectively covered on the same injected areas. Two weeks after the "Induction (II)" treatment, the hair on the lower back of the experimental animals from the scapula to the buttocks was removed, and appropriate locations were selected and patches pre-soaked in 0.1 ml of the corresponding treatment solution were respectively covered on the appropriate locations according to the conditions of "Excitation Period" shown in Table 3 or Table 4.

Table 3

FCA: Freund’s complete adjuvant Table 4

The hair-removed skin areas were observed 24 and 48 hours after challenge for irritation and/or allergic reactions. The results showed that neither the control nor the treated animals showed visible irritation in the test areas. Therefore, the collagen extract of the present invention did not cause delayed-type hypersensitivity reactions in the guinea pigs tested.

2.4 Intradermal irritation test

The irritation test was performed according to the steps shown in ISO 10993-10. Briefly, this test used 6 male New Zealand white rabbits (>2 kg, control group: 3 rabbits, and experimental group: 3 rabbits). Before the test, the hair on the back of the experimental animals was removed using an electric knife. On the day of treatment, 0.2 ml of the collagen extract of Example 2.1 (in normal saline) was injected into 5 areas on the left side of each experimental rabbit; and 0.2 ml of the collagen extract of Example 2.1 (in cottonseed oil) was injected into 5 areas on the right side of each experimental rabbit. The experimental rabbits in the control group were injected with 0.2 ml of 0.9% normal saline or cottonseed oil to the same positions. 24, 48 and 72 hours after application, the treated areas of the experimental animals were observed for skin reactions.

The experimental results showed that no animals in the control or treatment groups showed obvious clinical signs of intradermal irritation, and no animals died. Therefore, a single topical application of collagen extract does not cause intradermal irritation in New Zealand white rabbits.

2.5 Acute systemic injection study

Here, an acute systemic injection test was conducted according to the procedure of ISO 10993-11. Twenty male guinea pigs were randomly divided into four groups: control group 1 (N=5), control group 2 (N=5), treatment group 1 (N=5), and treatment group 2 (N=5).

On the first day of treatment, animals in treatment group 1 received a single intravenous dose of collagen extract (50 ml/kg) dissolved in saline. Animals in treatment group 2 received a single intraperitoneal dose of collagen extract (50 ml/kg) dissolved in cottonseed oil. Control group 1 and control group 2 animals were administered 0.9% saline and cottonseed oil, respectively. Mice were observed for toxic reactions 4, 24, 48, and 72 hours after administration.

The experimental results showed that there were no obvious clinical signs of toxicity in either the control or treatment groups, and no animals died. Therefore, a single topical application of collagen extract did not cause toxic reactions in the experimental animals.

Although the above embodiments disclose specific embodiments of the present invention, they are not intended to limit the present invention. Those skilled in the art may make various changes and modifications thereto without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on that defined by the appended claims.

Claims (5)

1. A method for preparing collagen particles, characterized in that it comprises: Step (1) Decellularize a pig skin with hair and fat removed and a thickness of 0.1-1 mm using supercritical carbon dioxide (scCO2) at a pressure of 350 bar and a temperature of 37°C for 40 to 180 minutes. Step (2) treats the pig skin decellularized by supercritical carbon dioxide (scCO2) in step (1) with an aqueous solution, wherein the aqueous solution contains a nonionic surfactant selected from the group consisting of: octylphenol polyoxyethylene ether, sorbitan monostearate, polysorbate, poloxomer, nonyl alcohol ether, cetyl alcohol and alkyl polyglucoside; Step (3) treat the pig skin treated with the aqueous solution in step (2) with a protease, wherein the protease is selected from the group consisting of: pepsin, trypsin, chymotrypsin, papain, papain, bromelain, kiwi, protease A, protease K, peptidase, figase, calpain, caspase or a combination thereof. Step (4) involves treating the protease-treated pig skin in step (3) with a nuclease, wherein the nuclease is a DNA nuclease or an RNA nuclease; Step (5) The pig skin treated with nuclease in step (4) is treated with a hydrogen peroxide solution for one hour; Step (6) involves applying the hydrogen peroxide-treated pig skin from step (5) to supercritical carbon dioxide (scCO2) along with a co-solvent at a pressure of 350 bar and a temperature of 37°C for 60 minutes to remove residual chemicals and produce a collagen matrix; and Step (7) involves granulating the collagen matrix from step (6) by cutting or grinding it under liquid nitrogen to produce collagen particles with a particle size of 10-250 micrometers. The method is characterized by not using crosslinking agents or salts. 2. The method of claim 1, wherein the aqueous solution in step (2) further comprises an anionic surfactant selected from the group consisting of: lauryl sulfonic acid, dodecyl sulfonic acid, sodium dodecyl sulfate (SDS), dodecylbenzene sulfonic acid, tridecylbenzene sulfonic acid, alkylphenoxybenzene disulfonic acid, naphthalene sulfonic acid, alkylnaphthalene sulfonic acid and alkenylnaphthalene. 3. The method of claim 1, further comprising treating the product of step (4) with the aqueous solution of step (2). 4. The method of claim 3, wherein the aqueous solution further comprises an anionic surfactant selected from the group consisting of: lauryl sulfonic acid, dodecyl sulfonic acid, sodium dodecyl sulfate (SDS), dodecylbenzene sulfonic acid, tridecylbenzene sulfonic acid, alkylphenoxybenzene disulfonic acid, naphthalene sulfonic acid, alkylnaphthalene sulfonic acid, and alkenylnaphthalene. 5. The method according to claim 1, wherein the co-solvent is ethanol.