Irradiated cross-linked collagen injectable filler and methods of making and using the same

Abstract

The present application provides a kind of irradiation crosslinking collagen injection filling agent and its preparation method and purposes.The collagen injection filling agent includes collagen gel microparticle, the collagen gel microparticle has porous structure;The collagen gel microparticle has connected fiber structure, the fiber structure is formed by collagen self-assembly, and the fiber structure includes fiber bundle and / or fiber mesh lamellar structure;The collagen gel microparticle in the collagen injection filling agent of the present application has the fiber structure formed after collagen self-assembly, i.e. collagen is spontaneously assembled from collagen molecule to fiber bundle and fiber mesh lamellar structure, and the degradation rate is delayed.The fiber network structure of the present application has good mechanical support and slow degradation rate, and simulates the three-dimensional microenvironment of natural extracellular matrix (ECM), promotes cell adhesion proliferation and tissue regeneration.The fiber structure of the present application is beneficial to the crawling proliferation of cells, and promotes the regeneration and repair of skin cells.

Description

Technical Field

[0001] This invention relates to an irradiated crosslinked collagen injection filler, its preparation method and uses, belonging to the field of medical materials. Background Technology

[0002] Injectable fillers used for soft tissue filling have been approved for marketing as medical devices. Currently, the four types of absorbable injectable materials used in plastic surgery both domestically and internationally include: (1) collagen; (2) sodium hyaluronate; (3) hydroxyapatite; and (4) polylactic acid. These four types of materials can also be combined with non-absorbable polymers (such as polymethyl methacrylate, abbreviated as "PMMA") as fillers. Among them, collagen is a type of natural protein and, as the main component of skin tissue, is a relatively ideal material for correcting facial soft tissue defects.

[0003] Collagen has the following advantages as a facial soft tissue filler: (1) good biocompatibility; (2) ability to interact with cells; (3) good mechanical properties; (4) moisturizing properties; (5) low immunogenicity; and (6) biodegradability. Due to its good biocompatibility, biodegradability, low immunogenicity, and feasibility for large-scale production, collagen occupies an important position in plastic surgery.

[0004] Currently, collagen is mainly derived from cowhide, pigskin, or human tissue. High-purity collagen is obtained through processing. Under sterile conditions, the purified collagen is uniformly dispersed in phosphate buffer to prepare collagen suspensions of different concentrations, which are then filled into sterile syringes.

[0005] However, existing collagen injection fillers require the use of chemical cross-linking agents such as glutaraldehyde and EDC for preparation, and the residue of these chemical cross-linking agents can cause cytotoxicity and immune risks. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] In view of the technical problems existing in the prior art, the present invention first provides a collagen injection filler. The gel microparticles have a fibrous structure formed by collagen self-assembly, and collagen spontaneously assembles from collagen molecules into fiber bundles and fibrous network lamellar structures under neutral conditions. The fibrous structure of the present invention has good mechanical support and simulates the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting cell adhesion, proliferation and tissue regeneration.

[0008] The present invention also provides a method for preparing a collagen injection filler, which is simple and easy to implement, uses readily available raw materials, and is suitable for mass production.

[0009] Solution for solving the problem

[0010] [1] A collagen injection filler, wherein the collagen injection filler includes collagen gel microparticles, the collagen gel microparticles having a porous structure;

[0011] The collagen gel microparticles have interconnected fibrous structures formed by collagen self-assembly, and...

[0012] The fiber structure includes fiber bundles and / or fiber mesh lamellar structures;

[0013] More than 90% of the collagen gel microparticles have a particle size of 50-300 μm;

[0014] The average pushing force of the collagen facial filler is no more than 15N, and the maximum pushing force is no more than 15N.

[0015] [2] The collagen injection filler according to [1] above, wherein the collagen gel microparticles are derived from collagen.

[0016] [3] The collagen injection filler according to [1] or [2] above, wherein the collagen injection filler has at least one of the following characteristics:

[0017] The collagen facial filler has a pH value of 6.0-8.0;

[0018] The collagen facial filler has an osmotic pressure of 200-400 mOsm / L;

[0019] The collagen facial filler has a melting point of 45-55℃;

[0020] The collagen facial filler is subjected to a shear rate of 2s. -1 The dynamic viscosity at a temperature of 25±0.1℃ is 50000-100000 mPa·s.

[0021] [4] A method for preparing a collagen injection filler according to any one of [1]-[3] above, comprising the following steps:

[0022] Collagen was dissolved in a solvent and then incubated once to obtain collagen gel.

[0023] After coarse homogenization, the collagen gel is incubated a second time to obtain a suspension;

[0024] The suspension is subjected to a single irradiation treatment to obtain a single irradiation product;

[0025] The product from the first irradiation was subjected to high-pressure homogenization to obtain a high-pressure homogenized product.

[0026] The high-pressure homogenized product was subjected to a second irradiation treatment to obtain collagen gel microparticles.

[0027] [5] According to the preparation method described in [4] above, the solvent includes an acidic solution, preferably, the concentration of the acidic solution is 0.05-0.5 mol / L;

[0028] The incubation process involves adjusting the pH to 6-9 using an alkaline reagent and incubating at 35-40°C for 4-24 hours.

[0029] [6] According to the preparation method described in [4] or [5] above, the time for coarse homogenization is 1-10 min, and the rotation speed of coarse homogenization is 3000-10000 rpm; preferably, the time for coarse homogenization is 1-5 min, and the rotation speed of coarse homogenization is 5000-9000 rpm.

[0030] The secondary incubation includes a stirring step using physiological saline and / or phosphate buffer at a temperature of 20-40°C; preferably, the stirring rate is 100-1000 rpm and the stirring time is 20-200 min.

[0031] [7] According to the preparation method described in [4] or [5] above, the first irradiation treatment is carried out in a low temperature environment by irradiation with a 5-45kGy electron beam or γ-ray, preferably, the low temperature environment is 1-15℃.

[0032] [8] According to the preparation method described in [4] or [5] above, the pressure of the high-pressure homogenization is 100-1000 bar, and the number of high-pressure homogenizations is 1-6 times.

[0033] [9] According to the preparation method described in [4] or [5] above, the secondary irradiation treatment is carried out in a low temperature environment by irradiation with a 5-45kGy electron beam or γ-ray; more preferably, the low temperature environment is 1-15℃.

[0034]

[10] Use of the collagen injection filler according to any one of [1]-[3] above in the preparation of facial injection filler materials.

[0035] The effects of the invention

[0036] The collagen gel microparticles in the collagen injection filler of this invention possess a fibrous structure formed after collagen self-assembly. Specifically, collagen spontaneously assembles from collagen molecules into fiber bundles and fibrous network lamellar structures under neutral conditions, improving the mechanical properties of the fiber network and slowing its degradation rate. The fibrous network structure of this invention exhibits good mechanical support and a slow degradation rate, and mimics the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting cell adhesion, proliferation, and tissue regeneration. The fibrous structure of this invention facilitates cell proliferation and regeneration, promoting skin cell repair.

[0037] The collagen injection filler of this invention avoids the use of chemical cross-linking agents such as glutaraldehyde and EDC during preparation, fundamentally eliminating the cytotoxicity and immune risks caused by residual cross-linking agents. Irradiation cross-linking not only stabilizes the material structure but also provides terminal sterilization, integrating cross-linking and sterilization into a single process. This reduces complex cleaning and residue removal steps, significantly improving the simplicity and safety of the process. Furthermore, the method of this invention promotes cross-linking while avoiding excessive degradation, maximizing the preservation of collagen's structural characteristics and bioactivity, thus ensuring both stability and biocompatibility during use.

[0038] Furthermore, by utilizing the controllability of the irradiation dose, this invention allows for the flexible design of product models based on different clinical needs. Low cross-linking degree is suitable for superficial fine line repair, medium cross-linking degree is suitable for facial sculpting, and high cross-linking degree is suitable for deep contour filling. Attached Figure Description

[0039] Figure 1 Microscopic image (left) and scanning electron microscope image (right) of the collagen injection filler of Example 1 are shown.

[0040] Figure 2 The diagram shows a comparison of the compressive modulus and compressive strength of the collagen injection fillers of Comparative Examples 1 and 3-5 of the present invention.

[0041] Figure 3 The stress-strain curves of the collagen injection fillers of Comparative Examples 1, 6, and 4 of the present invention are shown, as well as a comparison graph of elastic modulus and compressive strength.

[0042] Figure 4 These images show comparative photographs of the degradation of collagen injection fillers in proteinase K solution in Comparative Examples 1, 6, and 4 of the present invention.

[0043] Figure 5 This diagram shows a comparison of the degradation rates of collagen injection fillers in proteinase K solution for Comparative Examples 1, 6, and 4 of the present invention.

[0044] Figure 6The diagram shows a comparison of the compressive modulus and compressive strength of the collagen injection fillers of Comparative Example 2, Comparative Example 7, and Example 5 of the present invention.

[0045] Figure 7 The following are comparative photographs and degradation rate comparison graphs of the collagen injection fillers of Comparative Examples 2, 7, and 5 of the present invention in proteinase K solution.

[0046] Figure 8 The cytotoxicity results of the collagen injection fillers of Comparative Examples 2, 7, and 5 of the present invention are shown.

[0047] Figure 9 The diagram shows a comparison of the compressive modulus and compressive strength of the collagen injection fillers of Comparative Example 2, Comparative Example 8, and Example 6 of the present invention.

[0048] Figure 10 The following are comparative photographs and degradation rate comparison graphs of the collagen injection fillers of Comparative Example 2, Comparative Example 8, and Example 6 of the present invention in proteinase K solution.

[0049] Figure 11 The cytotoxicity results of the collagen injection fillers of Comparative Examples 2, 8, and 6 of the present invention are shown.

[0050] Figure 12 This invention, according to Example 5, presents a gross observation and ultrasound photograph 30 days after the collagen injection filler was implanted subcutaneously in the back of a rabbit.

[0051] Figure 13 The images show H&E and Masson stained sections of the collagen injection filler from Example 5 of the present invention, 30 days after subcutaneous implantation in the back of a rabbit. Detailed Implementation

[0052] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0053] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0054] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0055] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0056] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0057] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0058] <First Aspect>

[0059] The present invention first provides a collagen injection filler, the collagen injection filler comprising collagen gel microparticles having a porous structure;

[0060] The collagen gel microparticles have interconnected fibrous structures formed by collagen self-assembly, and...

[0061] The fiber structure includes fiber bundles and / or fiber mesh lamellar structures;

[0062] More than 90% of the collagen gel microparticles have a particle size of 50-300 μm;

[0063] The average pushing force of the collagen facial filler is no more than 15N, and the maximum pushing force is no more than 15N.

[0064] In this invention, the fibrous structure formed by the self-assembly of collagen in the gel microparticles refers to the spontaneous assembly of collagen molecules into fiber bundles and fibrous network lamellar structures under neutral conditions. The fibrous structure of this invention possesses excellent mechanical support and mimics the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting cell adhesion, proliferation, and tissue regeneration.

[0065] This invention does not damage the essence of collagen fibers, but only shears them into small particles (e.g., 50-300 μm). Each particle retains the internal collagen fiber structure, providing better volume support and resistance to deformation. By controlling the particle size of the collagen gel microparticles in the collagen injectable filler of this invention within the range of 50-300 μm, it ensures both good injectability and a smooth injection feel, while also providing a uniform filling effect and ideal tissue support.

[0066] Furthermore, since the fiber structure includes fiber bundles and / or fiber mesh sheet structures, and they are interconnected, the collagen gel microparticles of the present invention have a porous structure, which is beneficial to cell adhesion and proliferation.

[0067] In some specific embodiments, the collagen injection filler includes the steps of dissolving collagen, incubating, homogenizing, and irradiating; the homogenization includes coarse homogenization and high-pressure homogenization; and irradiation is performed after each homogenization. The collagen injection filler prepared by the method of the present invention retains its fibrous structure, good mechanical support, and simulates the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting cell adhesion, proliferation, and tissue regeneration. In the present invention, the collagen gel microparticles are derived from collagen. The collagen can be derived from animal connective tissue. In the present invention, the collagen can be of natural origin, modified, or cross-linked, wherein the natural origin includes, but is not limited to, extracellular matrix biomaterials such as the submucosa, dermis, pericardium, and Achilles tendon. Specifically, the naturally sourced collagen may include one or more combinations of small intestinal submucosa collagen, bladder submucosa collagen, Achilles tendon collagen, pericardium collagen, and dermis collagen. In some embodiments of the present invention, the collagen is derived from the submucosa of the small intestine, preferably from the decellularized submucosa of the small intestine, that is, the collagen of the present invention can be prepared using the submucosa of the small intestine.

[0068] In some embodiments of the present invention, the collagen is derived from the submucosa of the small intestine, preferably from a decellularized submucosa of the small intestine, meaning the collagen of the present invention can be prepared using the submucosa of the small intestine. In some embodiments of the present invention, the collagen is derived from the dermis, preferably from a decellularized dermis, meaning the collagen of the present invention can be prepared using the dermis. In some embodiments of the present invention, the collagen is derived from the submucosa of the bladder, preferably from a decellularized submucosa of the bladder, meaning the collagen of the present invention can be prepared using the submucosa of the bladder. In some embodiments of the present invention, the collagen is derived from the pericardium, preferably from a decellularized pericardium, meaning the collagen of the present invention can be prepared using the pericardium. The submucosa (e.g., the submucosa of the small intestine), dermis, and pericardium are preferably derived from mammals, such as pigs, cattle, sheep, dogs, and cats.

[0069] In one embodiment of the present invention, the collagen is derived from decellularized porcine small intestinal submucosa. This porcine small intestinal submucosa is commercially available. It is widely available, economically accessible, and easy to process. Furthermore, the decellularized porcine small intestinal submucosa exhibits excellent biocompatibility, is rich in collagen and growth factors, and can induce cell diffusion, adhesion, growth, and proliferation, promoting the repair and regeneration of tissue at tissue defects. Therefore, it is suitable for use in preparing the collagen of the present invention. Thus, the present invention preferably uses porcine small intestinal submucosa as a raw material to prepare the porcine small intestinal submucosa material as the collagen.

[0070] The present invention does not impose any particular limitation on the preparation method of collagen, and it can be some preparation methods commonly used in the field. Of course, collagen can also be obtained by purchasing it commercially.

[0071] In some specific embodiments, over 90% of the collagen gel microparticles have a particle size of 50-300 μm, such as 50-200 μm or 50-150 μm. The suitable particle size of the collagen gel microparticles in this invention facilitates collagen injection, thereby maximizing the effectiveness of the collagen gel microparticles.

[0072] In some specific embodiments, the collagen injectable filler has at least one of the following characteristics: the pH value of the collagen facial filler is 6.0-8.0; the osmotic pressure of the collagen facial filler is 200-400 mOsm / L; the melting point of the collagen facial filler is 45-55℃; and the collagen facial filler has a shear rate of 2s. -1 The dynamic viscosity at a temperature of 25±0.1℃ is 50000-100000 mPa·s.

[0073] <Second aspect>

[0074] A second aspect of the present invention provides a method for preparing a collagen injection filler according to the first aspect of the present invention, comprising the steps of dissolving collagen and then incubating, homogenizing, and irradiating; wherein the homogenization includes coarse homogenization and high-pressure homogenization; and irradiation is performed after each homogenization.

[0075] Specifically, the preparation method includes the following steps:

[0076] Collagen was dissolved in a solvent and then incubated once to obtain collagen gel.

[0077] After coarse homogenization, the collagen gel is incubated a second time to obtain a suspension;

[0078] The suspension is subjected to a single irradiation treatment to obtain a single irradiation product;

[0079] The product from the first irradiation was subjected to high-pressure homogenization to obtain a high-pressure homogenized product.

[0080] The high-pressure homogenized product was subjected to a second irradiation treatment to obtain collagen gel microparticles.

[0081] In this invention, collagen is dissolved in a solvent and then incubated once to obtain collagen gel. By obtaining collagen gel, subsequent processing can be carried out without affecting the morphology of the collagen, which is beneficial for preserving the fibrous structure of collagen within the particles.

[0082] In this invention, the solvent is used solely to dissolve collagen, without denaturing it. To facilitate better dissolution of collagen, the solvent includes an acidic solution. Preferably, to prevent collagen denaturation, the concentration of the acidic solution is 0.05-0.5 mol / L, for example: 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, etc. Specifically, the acidic substance in the acidic solution can be one or a combination of two or more of hydrochloric acid, sulfuric acid, acetic acid, etc. To prevent collagen denaturation, acetic acid or hydrochloric acid is preferred.

[0083] The inventors of this invention have discovered that collagen self-assembly can be achieved through a single incubation, thereby giving the collagen gel an interconnected fibrous structure.

[0084] In some specific implementations, the incubation includes adjusting the pH to 6-9 using an alkaline reagent and incubating at 35-40°C for 4-24 hours. Specifically, the pH can be 6.5, 7, 7.5, 8, 8.5, etc.; the incubation temperature can be 35°C, 36°C, 37°C, 38°C, 39°C, etc.; and the incubation time can be 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, etc.

[0085] The present invention does not particularly limit the alkaline reagent, and it can be a commonly used alkaline reagent in the art, such as sodium hydroxide, potassium hydroxide, etc.

[0086] Furthermore, in this invention, the collagen gel is coarsely homogenized and then incubated a second time to obtain a suspension.

[0087] Coarse homogenization of the collagen gel yields collagen gel microparticles with larger particle sizes and wider particle size distribution, which is beneficial for subsequent high-pressure homogenization. Secondary incubation allows for the cleaning of the collagen gel without damaging its internal structure, facilitating subsequent steps.

[0088] In some specific implementations, the coarse homogenization time is 1-10 minutes, for example: 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, etc.; the coarse homogenization rotation speed is 3000-10000 rpm, for example: 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, etc. Preferably, the coarse homogenization time is 1-5 minutes, and the coarse homogenization rotation speed is 5000-9000 rpm. Specifically, the coarse homogenate can be prepared using a high-shear homogenizer.

[0089] In some specific embodiments, the secondary incubation includes a stirring step using physiological saline and / or phosphate buffer at a temperature of 20-40°C. The pH of the phosphate buffer can be 7-8, preferably 7.2-7.6. The stirring at 20-40°C is to prevent collagen denaturation.

[0090] Preferably, in order not to damage the fiber structure of the self-assembled collagen, the stirring rate is 100-1000 rpm, for example: 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, 900 rpm, etc.; the stirring time is 20-200 min, for example: 50 min, 80 min, 100 min, 120 min, 150 min, 180 min, etc.

[0091] Furthermore, the suspension is subjected to a single irradiation treatment to obtain a single irradiation product. This invention avoids the use of chemical crosslinking agents such as glutaraldehyde and EDC by using irradiation crosslinking, fundamentally eliminating the cytotoxicity and immune risks caused by residual crosslinking agents, and effectively preserving the fiber structure formed by the self-assembly of collagen molecules.

[0092] In some specific implementations, the irradiation treatment is performed under low-temperature conditions using a 5-45 kGy electron beam or gamma rays, preferably at 1-15°C. The inventors of this invention have discovered that irradiation under low-temperature conditions promotes cross-linking while avoiding excessive degradation, maximizing the preservation of collagen's structural characteristics and bioactivity, thus achieving both stability and biocompatibility during use. In this invention, the adjustable irradiation dose allows for flexible product design based on different clinical needs. Specifically, low cross-linking is suitable for superficial fine line repair, medium cross-linking is suitable for facial contouring, and high cross-linking is suitable for deep contour filling.

[0093] Furthermore, the irradiated product is subjected to high-pressure homogenization to obtain a high-pressure homogenized product. The inventors of this invention have discovered that high-pressure homogenization can reduce the particle size of over 90% of the collagen gel microparticles to 50-300 μm. This ensures good injectability and a smooth injection feel, while also providing uniform filling effect and ideal tissue support.

[0094] In some specific implementations, the pressure of the high-pressure homogenization is 100-1000 bar, for example: 200 bar, 300 bar, 400 bar, 500 bar, 600 bar, 700 bar, 800 bar, 900 bar, etc.; the number of high-pressure homogenizations is 1-6 times, for example: 2 times, 3 times, 4 times, 5 times, etc.

[0095] Furthermore, the high-pressure homogenized product is subjected to secondary irradiation to obtain collagen gel microparticles. This invention, through secondary irradiation, not only achieves further stabilization of the collagen injection filler structure but also provides terminal sterilization, integrating cross-linking and sterilization. This reduces complex cleaning and residue removal steps, significantly improving the simplicity and safety of the process.

[0096] In some specific implementations, the secondary irradiation treatment may be performed under the same or different conditions as the primary irradiation treatment. Specifically, the secondary irradiation treatment may also be performed in a low-temperature environment using an electron beam or gamma rays of 5-45 kGy, such as 10 kGy, 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy, 40 kGy, etc. More preferably, the low-temperature environment is 1-15℃, such as 3℃, 5℃, 7℃, 9℃, 11℃, 13℃, etc.

[0097] <Third aspect>

[0098] A third aspect of the present invention provides the use of the collagen injection filler according to the first aspect of the present invention in the preparation of facial injection filler materials.

[0099] Example

[0100] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0101] The collagen preparation method used in this example is as follows:

[0102] 1. Cleaning and pretreatment: At room temperature, scrape off the mucosa and fascia of the small intestine, leaving the submucosa of the middle small intestine. Wash the material with purified water for later use.

[0103] 2. Virus inactivation: Prepare a 1% peracetic acid virus inactivation solution, and soak the cleaned submucosal material of the small intestine in the virus inactivation solution at room temperature for 2 hours. After soaking, wash with purified water.

[0104] 3. Degreasing: Soak the virus-killed casings in isopropanol solution, then ultrasonically soak them for 30 minutes in an ultrasonic cleaner. Replace the degreasing solution and repeat the process 3 times. Rinse the casings with purified water.

[0105] 4. Decellularization: Soak the sausage casing in trypsin phosphate buffer, sonicate in an ultrasonic cleaner for 30 minutes, rinse with purified water after sonication, and dry the sausage casing in a 37°C oven for 24 hours.

[0106] 5. Acid soaking: Cut the dried sausage casings into pieces and soak them overnight in a 0.5 mol / L acetic acid solution.

[0107] 6. Enzymatic hydrolysis: After soaking, the sausage casings are pulverized and stirred at low temperature for 5 minutes using a pulverizer. They are then soaked in a pepsin solution and mechanically stirred at low temperature. After enzymatic hydrolysis, the mixture is centrifuged, and the supernatant is taken as the crude collagen extract solution.

[0108] 7. Purification: Salt out the crude collagen solution using neutral sodium chloride or ammonium sulfate. After salting out, centrifuge to collect the precipitate particles, wash with distilled water, dissolve in 0.5 mol / L acetic acid, and then concentrate by ultrafiltration using an ultrafiltration membrane with a pore size of 0.05-0.1 micrometers to remove excess small molecule impurities and obtain a high-purity collagen solution.

[0109] 8. Drying: Freeze-dry the high-purity collagen solution to obtain collagen.

[0110] Example 1

[0111] 1. Dissolve collagen in 0.05 mol / L acetic acid to achieve a collagen concentration of 10 mg / mL; after dissolution, adjust the pH to 7.0 with 1.0 mol / L sodium hydroxide solution, and incubate at 37℃ for 12 h to obtain collagen gel.

[0112] 2. The collagen gel was coarsely homogenized at 6000 rpm / min for 5 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 800 rpm and the stirring time was 20 min to obtain a suspension.

[0113] 3. The suspension was loaded into a radiation-resistant reagent bottle and irradiated once with an electron beam at a dose of 25 kGy at a low temperature of 10°C to obtain the irradiated product.

[0114] 4. The product from the first irradiation was homogenized using a high-pressure homogenizer. The homogenized product was subjected to 100 bar pressure four times to obtain a high-pressure homogenized product. A laser particle size analyzer was used for testing, and it was found that over 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0115] 5. Use a filling machine to fill the high-pressure homogenized product; use aseptic packaging after filling;

[0116] 6. The packaged product is subjected to secondary irradiation with an electron beam of 25 kGy to obtain collagen injection filler.

[0117] Example 2

[0118] 1. Dissolve collagen in 0.25 mol / L hydrochloric acid to achieve a collagen concentration of 20 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 8 hours to obtain collagen gel.

[0119] 2. The collagen gel was coarsely homogenized at 8000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring rate during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0120] 3. The suspension was loaded into a radiation-resistant reagent bottle and irradiated once with γ-rays at a dose of 25 kGy at a low temperature of 10°C to obtain the irradiated product.

[0121] 4. The product irradiated once was homogenized twice with a pressure of 200 bar to obtain a high-pressure homogenized product. The product was tested with a laser particle size analyzer and found that more than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0122] 5. Use a filling machine to fill the high-pressure homogenized product, and then use aseptic packaging after filling;

[0123] 6. The packaged product is subjected to secondary irradiation with 25kGy gamma rays to obtain collagen injection filler.

[0124] Example 3

[0125] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 30 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 8 hours to obtain collagen gel.

[0126] 2. The collagen gel was coarsely homogenized at 8000 rpm / min for 1 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring rate during the second incubation was 300 rpm and the stirring time was 60 min to obtain a suspension.

[0127] 3. The suspension was loaded into a radiation-resistant reagent bottle and irradiated once with a 25 kGy electron beam at a low temperature of 10°C to obtain the irradiated product.

[0128] 4. The product irradiated once was homogenized twice with a pressure of 300 bar to obtain a high-pressure homogenized product. The product was tested with a laser particle size analyzer and found that more than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0129] 5. Use a filling machine to fill the high-pressure homogenized product; use aseptic packaging after filling;

[0130] 6. The packaged product is subjected to secondary irradiation with a 25kGy electron beam to obtain collagen injection filler.

[0131] Example 4

[0132] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 20 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0133] 2. The collagen gel was coarsely homogenized at 7000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0134] 3. The suspension was loaded into a radiation-resistant reagent bottle and irradiated once with 25 kGy γ rays at a low temperature of 10°C to obtain the first irradiation product;

[0135] 4. The product irradiated once was homogenized using a high-pressure homogenizer. The product was homogenized four times at a pressure of 100 bar to obtain a high-pressure homogenized product. The product was tested using a laser particle size analyzer. More than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0136] 5. Use a filling machine to fill the high-pressure homogenized product, and then use aseptic packaging after filling;

[0137] 6. The packaged product is subjected to secondary irradiation with 25kGy gamma rays to obtain collagen injection filler.

[0138] Example 5

[0139] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 30 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0140] 2. The collagen gel was coarsely homogenized at 7000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0141] 3. The suspension was loaded into a radiation-resistant reagent bottle and irradiated once with a 10 kGy electron beam at a low temperature of 10°C to obtain the irradiated product.

[0142] 4. The product irradiated once was homogenized using a high-pressure homogenizer. The product was homogenized four times at a pressure of 100 bar to obtain a high-pressure homogenized product. The product was tested using a laser particle size analyzer. More than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0143] 5. Use a filling machine to fill the high-pressure homogenized product, and then use aseptic packaging after filling;

[0144] 6. The packaged product is subjected to secondary irradiation with a 25kGy electron beam to obtain collagen injection filler.

[0145] Example 6

[0146] The intensity of the first irradiation in Example 5 was adjusted to 30 kGy, and the remaining steps were exactly the same as in Example 5, to obtain the collagen injection filler.

[0147] Comparative Example 1

[0148] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 20 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0149] 2. The collagen gel was coarsely homogenized at 7000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0150] 3. The suspension was homogenized using a high-pressure homogenizer. The suspension was homogenized four times at a pressure of 100 bar to obtain the high-pressure homogenized product. The product was tested using a laser particle size analyzer. More than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0151] 4. The high-pressure homogenized product is filled using a filling machine; after filling, it is aseptically packaged to obtain collagen injection filler.

[0152] Comparative Example 2

[0153] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 30 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0154] 2. The collagen gel was coarsely homogenized at 7000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0155] 3. The suspension was homogenized twice with a high pressure homogenizer at 200 bar to obtain the high pressure homogenized product. The product was tested with a laser particle size analyzer. More than 90% of the collagen gel particles in the high pressure homogenized product had a diameter of 50-300 μm.

[0156] 4. The high-pressure homogenized product is filled using a filling machine, and then aseptically packaged to obtain collagen injection filler.

[0157] Comparative Example 3

[0158] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 20 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0159] 2. The collagen gel was coarsely homogenized at 7000 rpm / min for 2 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring speed during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0160] 3. The suspension was homogenized using a high-pressure homogenizer. The suspension was homogenized 4 times at a pressure of 100 bar to obtain the high-pressure homogenized product. The product was tested using a laser particle size analyzer. More than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0161] 4. Use a filling machine to fill the high-pressure homogenized product; then use aseptic packaging after filling;

[0162] 5. The packaged product is irradiated with 5kGy gamma rays to obtain collagen injection filler.

[0163] Comparative Example 4

[0164] The irradiation intensity in Comparative Example 3 was adjusted to 10 kGy, and the remaining steps were exactly the same as in Comparative Example 3, to obtain the collagen injection filler.

[0165] Comparative Example 5

[0166] The irradiation intensity in Comparative Example 3 was adjusted to 15 kGy, and the remaining steps were exactly the same as in Comparative Example 3, to obtain the collagen injection filler.

[0167] Comparative Example 6

[0168] The irradiation intensity in Comparative Example 3 was adjusted to 25 kGy, and the remaining steps were exactly the same as in Comparative Example 3, to obtain the collagen injection filler.

[0169] Comparative Example 7

[0170] 1. Dissolve collagen in 0.5 mol / L acetic acid to achieve a collagen concentration of 30 mg / mL; after dissolution, adjust the pH to approximately 7 using 1.0 mol / L sodium hydroxide solution, and incubate at 37°C for 12 hours to obtain collagen gel.

[0171] 2. The collagen gel was coarsely homogenized at 8000 rpm / min for 1 min using a homogenizer, and then incubated a second time with phosphate buffer solution at pH 7.4. The stirring rate during the second incubation was 500 rpm and the stirring time was 30 min to obtain a suspension.

[0172] 3. The suspension was homogenized under high pressure using a high-pressure homogenizer. The suspension was homogenized twice at a pressure of 200 bar to obtain the high-pressure homogenized product. The product was tested using a laser particle size analyzer. More than 90% of the collagen gel particles in the high-pressure homogenized product had a diameter of 50-300 μm.

[0173] 4. Filling is carried out using a filling machine, and aseptic packaging is used after filling;

[0174] 5. The packaged product is irradiated with a 10kGy electron beam to obtain collagen injection filler.

[0175] Comparative Example 8

[0176] The irradiation intensity in Comparative Example 7 was adjusted to 30 kGy, and the remaining steps were exactly the same as in Comparative Example 7, to obtain the collagen injection filler.

[0177] Performance testing

[0178] 1. Microscopic observation and scanning electron microscopy observation

[0179] The collagen injection filler obtained in Example 1 of this invention was dispersed at a low concentration in deionized water and observed using an electron serial upright microscope (Ci-s, Nikon, Japan). The results are as follows: Figure 1 As shown.

[0180] Depend on Figure 1 (Left image) As can be seen, collagen gel microparticles are uniformly dispersed in water, with a particle size of 50-300μm.

[0181] The collagen injection filler obtained in Example 1 of this invention was frozen at -80°C. It was then freeze-dried in a vacuum dryer. The sample was adhered to a sample stage with conductive adhesive, sputtered with gold, and its morphology was observed using a scanning electron microscope (S-4800, HITACHI, Japan).

[0182] Depend on Figure 1 (Right figure) As can be seen, the collagen self-assembles into fibrous structures within the collagen gel microparticles, i.e., collagen spontaneously self-assembles from collagen molecules into fiber bundles and fibrous network lamellar structures under neutral conditions. These fibrous structures provide excellent mechanical support and mimic the three-dimensional microenvironment of the natural extracellular matrix (ECM), promoting cell adhesion and proliferation. This invention does not destroy the essence of collagen fibers; it merely shears them to form small-sized particles (e.g., 50-300 μm). Each particle retains the internal collagen fibrous structure, providing better volume support and resistance to deformation.

[0183] 2. Mechanical property testing

[0184] The compression properties of collagen injection fillers (made into cylindrical shapes with a diameter of 1.5 cm and a height of 1 cm using a mold) from Examples 4-6 and Comparative Examples 1-8 were tested using an electronic universal testing machine (EUT6502, Sansi, China). The compression speed was set to 5 mm / min, and the measurements were taken at room temperature. The results are as follows: Figure 2 , 3 As shown in 6 and 9.

[0185] First, the collagen injection fillers of Comparative Examples 1 and 3-5 were subjected to the above-mentioned compression performance test, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the compressive modulus and strength of collagen injection fillers gradually increase with the increase of gamma-ray irradiation intensity, and basically reach their maximum at 15 kGy. Figure 2 The results showed that gamma-ray irradiation can effectively improve the strength of collagen injection fillers, but the overall mechanical strength of Comparative Examples 1 and 3-5 was relatively low.

[0186] To further verify the change in crosslinking degree of collagen injection fillers under gamma-ray irradiation, compressive performance tests were also conducted on the collagen injection fillers of Comparative Example 1, Comparative Example 6, and Example 4, and stress-strain curves were provided; changes in the strength of the collagen injection fillers were also detected. Experimental results showed that the compressive strength and modulus of the collagen injection filler in Example 4 were significantly improved, and the strength of the collagen injection filler after two irradiations was significantly higher than that after one irradiation and without irradiation. Figure 3 ).

[0187] To verify the change in crosslinking degree of collagen injection fillers under electron beam irradiation, mechanical property tests were performed on Comparative Examples 2, 7, and 5; changes in the strength of the collagen injection fillers after irradiation were also detected. Experimental results showed that the compressive modulus and strength of the collagen injection filler in Comparative Example 7 after 10 kGy electron beam irradiation were improved to some extent compared to Comparative Example 2. However, the compressive modulus and strength of the collagen injection filler in Example 5 after an initial irradiation of 10 kGy and a secondary irradiation of 25 kGy were significantly higher than those of the collagen injection fillers in Comparative Examples 2 and 7. Figure 6 ).

[0188] In Comparative Example 8, the collagen injection filler irradiated with 30 kGy electron beam showed significantly improved compressive modulus and strength compared to Comparative Example 2. However, in Example 6, after the collagen injection filler underwent a first irradiation of 30 kGy and a second irradiation of 25 kGy, its compressive modulus and strength decreased compared to Comparative Example 8. Figure 9 It can be demonstrated that appropriate doses of electron beam irradiation can effectively improve the strength of collagen injection fillers, but when the dose is too high, it will lead to a certain degree of decrease in the mechanical properties of the material.

[0189] 3. Degradation performance test

[0190] A 0.004 mg / mL proteinase K solution was prepared using phosphate buffer (pH 7.4). 0.5 g of each of the collagen injection fillers from Comparative Examples 1-2, 6-8, and 4-6 were placed in centrifuge tubes and immersed in 5 mL of proteinase K solution (n=3). The tubes were then placed in a 56°C water bath for enzymatic hydrolysis. Samples were centrifuged, weighed, and photographed every 10 minutes until complete degradation. The degradation rate was calculated based on the mass loss. The results are as follows: Figure 4 , 5 As shown in 7 and 10.

[0191] The degradation rate of collagen injection fillers after gamma ray irradiation was verified. The gel degradation rate decreased significantly after irradiation. As can be seen from Comparative Examples 1, 6, and 4, the gel that underwent two irradiations had a lower degradation rate than the gel that was sterilized by a single irradiation. Figures 4-5 This indicates that gamma-ray irradiation can effectively improve the cross-linking degree of collagen injection fillers, and the cross-linking strength is even higher after two irradiations.

[0192] The degradation rate of collagen injection fillers after electron beam irradiation was verified. In Example 5, the collagen injection fillers subjected to an initial irradiation of 10 kGy and a secondary irradiation of 25 kGy showed a significantly lower degradation rate compared to the collagen injection fillers in Comparative Example 7 subjected to 10 kGy electron beam irradiation. Figure 7 However, after the collagen injection filler in Example 6 underwent an initial irradiation of 30 kGy and a secondary irradiation of 25 kGy, its degradation rate was higher than that of the collagen injection filler in Comparative Example 8 after irradiation with a 30 kGy electron beam. Figure 10 Similarly, it has been demonstrated that appropriate doses of electron beam irradiation can effectively reduce the degradation rate of collagen injection fillers, but excessive doses can lead to an accelerated degradation rate.

[0193] 4. Cytotoxicity test

[0194] The cytotoxicity of the collagen injection fillers used in Comparative Examples 2, 7-8, and 5-6 was evaluated using mouse fibroblasts (L929). 1 × 10⁶ cells were seeded per well in 96-well plates. 4L929 cells were cultured in serum-free recombinant cell cryopreservation (RPMI) medium at 37°C for 24 hours. Collagen injection filler was added to RPMI medium at a concentration of 0.5 g / mL and incubated at 37°C for 24 hours. The culture medium in the 96-well plates was replaced with the extract. The plates were then cultured at 37°C for another 24 hours. After 24 hours, the supernatant was aspirated from the plates, and 100 μL of culture medium containing thiazolyl blue (MTT, 0.5 mg / mL) was added to each well. The plates were incubated in the dark for 4 hours, and then the supernatant was replaced with an equal volume of dimethyl sulfoxide (DMSO) to dissolve the formazan. OD was read at 490 nm using a microplate reader. Cell viability was calculated using the formula: (A...) 材料 -A0)×100% / (A 对照 -A0), where A 材料 A0 is the absorbance of the cell culture wells containing the extract, and A0 is the absorbance of the culture wells containing sterile water. 对照 The absorbance of the culture wells containing the culture medium is shown. The results are as follows: Figure 8 , Figure 11 As shown.

[0195] Depend on Figure 8 and Figure 11 It can be seen that the survival rate of L929 cells co-cultured with the collagen injection filler of Comparative Examples 2, 7-8, and 5-6 remained above 95%, and no obvious cytotoxicity was observed, indicating that it has good biocompatibility.

[0196] 5. Application Test: Subcutaneous Implantation Experiment in Rabbits

[0197] All animal experiments complied with the Tianjin Municipal Medical Laboratory Animal Management Regulations, and the animal experiment protocols were approved by the animal management agency and committee of Yishengyuan Gene Technology (Tianjin) Co., Ltd. A New Zealand rabbit subcutaneous implantation model was established to evaluate the volume retention and tissue regeneration capacity of the material. Anesthesia was administered via intravenous injection of 3% sodium pentobarbital (30 mg / kg). The rabbits were then fixed in a prone position on the operating table. The back surgical area was prepared, and aseptic techniques were followed during the surgery. The skin of the surgical area was disinfected with povidone-iodine. Markings were made symmetrically on both sides of the back, using the spine as the midline. The injection needle was inserted at the marked injection sites, and 500 μL of the collagen injection filler from Example 5 was injected subcutaneously. After each injection, a small clump could be felt with gentle finger pressure; excessive pressure was avoided, and the injection site was kept relatively still to prevent frequent animal movement. Gross and ultrasound observations of the skin tissue at the injection sites were performed 30 days post-surgery. Results are as follows: Figure 12 As shown, samples were subsequently taken for histological examination (H&E, Masson), and the results are as follows. Figure 13 As shown.

[0198] Depend on Figure 12-13It can be seen that 30 days after the collagen injection filler was implanted into the skin, the raised structure still remained, the overall shape was stable, and there was no obvious collapse, spread, or inflammatory reaction. Figure 12 Left). Ultrasound examination ( Figure 12 (Right) This further validates the conclusions of the gross observations. The implant in the image appears as a well-defined hypoechoic dark area, suggesting that its internal structure is homogeneous and clearly demarcated from the surrounding tissue. This indicates that the collagen filler has good volume retention in vivo and has not experienced significant volume loss due to rapid degradation or tissue fluid infiltration.

[0199] Hematoxylin and eosin (H&E) staining showed ( Figure 13 (Left) The implant is a well-defined clump of tissue. Magnified images show a large number of cell nuclei inside the implant, evenly distributed in the matrix, indicating that host cells have infiltrated the collagen injection filler to participate in tissue regeneration. Vascular structures are visible inside the implant matrix, suggesting that the collagen injection filler can induce vascularization and promote the integration of new tissue.

[0200] Masson staining shows ( Figure 13 (Right) The collagen fibers inside the implant are densely distributed and extend into the surrounding host tissue, indicating that the collagen filler not only has a stable structure but also maintains its volume through interaction with the host collagen. The collagen fibers are distributed in a network within the implant and gradually merge with the collagen fibers of the surrounding host tissue, indicating that the collagen filler, acting as a scaffold, effectively guides the regeneration and deposition of host collagen and promotes the integration of new tissue.

[0201] 6. Physical property testing

[0202] 6.1 Average pushing force, maximum pushing force

[0203] After equilibrating the samples from Examples 1-6 and Comparative Examples 1-8 at room temperature for 1 hour, they were placed on a syringe mold and the pushing force was tested using a universal testing machine at a pushing speed of 30 mm / min. The average pushing force and the maximum pushing force in the plateau region were recorded and calculated. The results are shown in Table 1-2 below.

[0204] As can be seen from Table 1, the collagen injection filler of the present invention can effectively prevent the aggregation between gel particles and maintain the injectability of the gel.

[0205] Table 2 shows that Comparative Examples 1 and 2, without irradiation crosslinking, have low crosslinking degrees and therefore exhibit good pushing force. Comparing the average and maximum pushing forces of Comparative Examples 3, 4, and 5-6, when no irradiation was performed before high-pressure homogenization and only a single irradiation crosslinking was used after high-pressure homogenization, the pushing force gradually increased with increasing gamma-ray irradiation intensity. When the irradiation intensity exceeded 10 kGy, further crosslinking occurred between collagen gel particles, leading to particle aggregation and clogging of the needle, preventing injection. This was particularly true for Comparative Examples 5 and 6, which lacked injectable properties. Comparing Comparative Examples 7-8 shows that when the collagen concentration is higher, similarly, when the irradiation intensity exceeds 10 kGy, further crosslinking occurs between collagen gel particles, causing particle aggregation and clogging of the needle, preventing injection.

[0206] 6.2 pH value

[0207] Samples from Examples 1-6 and Comparative Examples 1-8 were diluted with purified water in equal mass ratios, and the pH value of the collagen facial filler was measured using a pH meter. The results are shown in Table 1-2 below.

[0208] As can be seen from Tables 1 and 2, the pH values ​​of the samples in Examples 1-6 and Comparative Examples 1-8 are all within the range of 7.00-7.50, indicating that the acid-base neutralization process and buffer system can effectively regulate and stabilize the pH of the collagen solution. After the samples are implanted in the body, they can effectively avoid inflammatory reactions or tissue damage caused by pH imbalance.

[0209] 6.3 Osmotic pressure

[0210] Samples from Examples 1-6 and Comparative Examples 1-8 were used as extraction media with phosphate buffer solution and soaked in a sealed container at 37℃±1℃ for 24 hours. The absorption capacity of the extraction medium (the amount absorbed per 0.1g of material) was measured. During extraction, an extraction mixture was prepared at 0.1g / mL. The extraction medium absorption capacity was added to the extraction mixture for every 0.1g of material. The mixture was soaked in a sealed container at 37℃±1℃ for 24 hours. The supernatant was taken and its osmotic pressure was measured using an ice-point osmometer. The results are shown in Tables 1-2 below.

[0211] As can be seen from Tables 1 and 2, the osmotic pressure of the samples in Examples 1-6 and Comparative Examples 1-8 is in the range of 260-290 mOsm / L, which is close to the normal osmotic pressure range of human blood plasma and interstitial fluid, indicating that they can provide a stable physicochemical environment for cells and tissues.

[0212] 6.4 Melting point

[0213] Differential scanning calorimetry (DSC) was used for testing. Samples from Examples 1-6 and Comparative Examples 1-8 were placed in a test aluminum dish and tested in a nitrogen atmosphere at a heating rate of 2℃ / min within a temperature range of 40-80℃. The temperature values ​​corresponding to the peak absorption values ​​of the samples were recorded. The results are shown in Table 1-2 below.

[0214] As can be seen from Table 1, the melting point of the collagen injection filler of the present invention is between 45-55℃, which indicates that its cross-linking degree is appropriate and the degradation rate of the collagen injection filler is slow.

[0215] As shown in Table 2, in Comparative Examples 1-2, due to the lack of irradiation crosslinking, the degree of crosslinking was low, resulting in a low melting point of the collagen injection filler and a rapid degradation rate, which is consistent with the degradation performance. Although Comparative Examples 3-8 had suitable melting points and also possessed a high degree of crosslinking, there were many crosslinks between collagen gel particles in Comparative Examples 3-8, making them difficult to inject.

[0216] 6.5 Dynamic viscosity

[0217] Using a rotational viscometer at a shear rate of 2 s -1 The dynamic viscosity of collagen facial filler was determined at a temperature of 25±0.1℃, and the results are shown in Table 1-2 below.

[0218] As can be seen from Table 1, the collagen facial filler of the present invention exhibits performance at a shear rate of 2s. -1 The dynamic viscosity at a temperature of 25±0.1℃ is 50000-100000 mPa·s, which is suitable and has excellent injectability.

[0219] As shown in Table 2, in Comparative Examples 1-2, due to the lack of irradiation crosslinking, the degree of crosslinking was low, resulting in low viscosity and rapid degradation of the collagen injection filler, consistent with melting point testing and degradation performance. In Comparative Example 3, the dynamic viscosity was too high, making injection difficult. In Comparative Examples 4-8, because only a single irradiation was performed after high-pressure homogenization, crosslinking between collagen gel particles caused them to aggregate into a single gel mass, rendering the gel injectable and making dynamic viscosity undetectable.

[0220] Table 1

[0221]

[0222] Table 2

[0223]

[0224] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0225] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a collagen injection filler, characterized in that, Includes the following steps: Collagen was dissolved in a solvent and then incubated once to obtain collagen gel. After coarse homogenization, the collagen gel is incubated a second time to obtain a suspension; The suspension is subjected to a single irradiation treatment to obtain a single irradiation product; The product from the first irradiation was subjected to high-pressure homogenization to obtain a high-pressure homogenized product. The high-pressure homogenized product was subjected to a second irradiation treatment to obtain collagen gel microparticles; The secondary incubation includes a stirring step using physiological saline and / or phosphate buffer at a temperature of 20-40°C; The collagen injection filler includes collagen gel microparticles, which have a porous structure. The collagen gel microparticles have interconnected fibrous structures formed by collagen self-assembly, and... The fiber structure includes fiber bundles and / or fiber mesh lamellar structures; More than 90% of the collagen gel microparticles have a particle size of 50-300 μm; The average pushing force of the collagen injection filler is no more than 15N, and the maximum pushing force is no more than 15N.

2. The preparation method according to claim 1, characterized in that, The collagen gel microparticles are derived from collagen.

3. The preparation method according to claim 1 or 2, characterized in that, The collagen injection filler also has at least one of the following characteristics: The pH value of the collagen injection filler is 6.0-8.0; The osmotic pressure of the collagen injection filler is 200-400 mOsm / L; The melting point of the collagen injection filler is 45-55℃; The collagen injection filler is subjected to a shear rate of 2s. -1 The dynamic viscosity at a temperature of 25±0.1℃ is 50000-100000 mPa·s.

4. The preparation method according to claim 1 or 2, characterized in that, The solvent includes an acidic solution; the concentration of the acidic solution is 0.05-0.5 mol / L; The incubation process involves adjusting the pH to 6-9 using an alkaline reagent and incubating at 35-40°C for 4-24 hours.

5. The preparation method according to claim 1 or 2, characterized in that, The coarse homogenization time is 1-10 min, and the coarse homogenization speed is 3000-10000 rpm; The stirring rate is 100-1000 rpm, and the stirring time is 20-200 min.

6. The preparation method according to claim 1 or 2, characterized in that, The irradiation treatment is carried out in a low-temperature environment using a 5-45 kGy electron beam or gamma rays; the low-temperature environment is 1-15℃.

7. The preparation method according to claim 1 or 2, characterized in that, The pressure of the high-pressure homogenization is 100-1000 bar, and the number of high-pressure homogenization cycles is 1-6.

8. The preparation method according to claim 1 or 2, characterized in that, The secondary irradiation treatment is carried out in a low-temperature environment using a 5-45 kGy electron beam or gamma rays; the low-temperature environment is 1-15℃.

9. The use of a collagen injection filler prepared by the preparation method according to any one of claims 1-8 for the preparation of facial injection filler materials.

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