Preparation method of compound collagen filler for injection

Abstract

The present invention relates to a preparation method of a collagen filler for injection. Non-crosslinked collagen, crosslinked collagen, and biodegradable polymer microspheres are compounded and mixed in a certain proportion to achieve an immediate filling effect. The biodegradable polymer microspheres play a long-term role, and the amino acid components degraded from collagen in the early and middle stages can become nutrients that stimulate the production of collagen regeneration by polyester microspheres. In the present invention, the crosslinked collagen has a high yield and less water loss through a gradient irradiation crosslinking technique. The non-crosslinked collagen gel can provide a microenvironment for nutritional repair and a filling effect in the early stage, the crosslinked extracellular matrix can provide a repair microenvironment in the middle stage of filling, and the degradation of polyester microspheres in the later stage promotes collagen regeneration, enabling the skin to have a good microenvironment throughout the repair cycle and increasing skin elasticity.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical aesthetic injectables, and particularly relates to a preparation method of a compound collagen filler for injection. Background Art

[0002] In terms of the selection of raw materials for injectable fillers, currently, sodium hyaluronate and collagen are mainly used. However, such materials are easily absorbed by the body tissues, have a short degradation period, and the filling effect is to temporarily occupy the skin volume and cause a limited local host tissue reaction (limited regenerative fibrosis), making it difficult to maintain the long-term filling and collagen regeneration effects. The toxic cross-linking agent components introduced for providing a long degradation time also limit their applications.

[0003] Due to these problems, several products for tissue repair treatment using biodegradable polymers have been developed recently, and these products have been developed and used as filler preparations of existing biocompatible polymers, or preparations in which insoluble polymers are dispersed into fine particles through a viscous medium (such as CMC, etc.) after treatment, such as polycaprolactone fillers. This type of material promotes collagen regeneration through the stimulating effect of degradation products to play a repair role. Since degradation takes a certain amount of time, the immediate effect is not obvious, and the nutritional effect of CMC is not strong. Collagen used alone has poor physical and mechanical properties, a single performance, and inevitable weaknesses such as strong hydrophilicity and being easily degraded by collagenase in the body, which limit its application. Although chemically cross-linked collagen can increase the degradation time of the gel, there is certain toxicity; polyester fillers cannot achieve an immediate collagen-stimulating effect. The adhesive CMC gel has a filling effect, but it is not a human component and does not have a nutritional and regenerative effect; there will also be residual toxic effects after hyaluronic acid gel is cross-linked.

[0004] CN118649289A discloses a collagen implant. Collagen is physically cross-linked, chemically cross-linked, and irradiated cross-linked in sequence to obtain collagen fibers, which are then dispersed and emulsified by mixing with a solution to obtain the collagen implant. In order to increase the cross-linking degree, this patent uses three cross-linking methods. The triple cross-linking makes the product have a high cross-linking degree and a low enzymatic hydrolysis rate, giving a more lasting filling effect. However, chemical cross-linking inevitably introduces chemical cross-linking agents, such as dialdehyde, carbodiimide, etc. The residue of the cross-linking agent brings more sensitization risks and even teratogenic risks to clinical use.

[0005] CN117618671A discloses a collagen filler, which has an average pore diameter D50 of 50 μm to 500 μm, a porosity of 80%-99.5%, a crosslinking degree of 10% to 70%, and an in vitro enzymatic hydrolysis loss rate of less than 40%. This patent controls the relative spacing of collagen fiber bundles during the crosslinking process by controlling the pore size with microspheres as a template, and further controls the pore size after the collagen fiber bundles form a crosslinked framework; on the other hand, by controlling the mechanical stirring and the microsphere addition method, a crosslinking process of a dynamic single template (or multi-template) is realized, so that each collagen fiber bundle can obtain an equal crosslinking opportunity, thereby achieving a more thorough and uniform crosslinking effect of the collagen implant.

[0006] CN117582549A discloses an injectable recombinant collagen soft tissue filling gel. By weight percentage, the injectable recombinant collagen soft tissue filling gel comprises the following raw materials in weight percentages: 5%-10% of type III recombinant collagen solution, 2%-3% of sodium hyaluronate solution for injection, 1%-3% of medicinal glycerol, 1%-5% of medical hydroxyapatite, and the balance is water. The injectable recombinant collagen soft tissue gel prepared by mixing the type III recombinant collagen solution, the sodium hyaluronate solution for injection, the medicinal glycerol and the medical hydroxyapatite uses the screened core functional region fragments with high activity and high water solubility, which are 100% identical to the corresponding fragments of human collagen, eliminating the immunogenicity, and at the same time having the characteristics of high biocompatibility, high biological activity, high water solubility and no virus reaction.

[0007] However, among the above-mentioned existing technologies, there are products that combine collagen and microspheres for injection filling, but they still cannot achieve a long-term filling effect, and the risk of collagen allergy is relatively high. Summary of the Invention

[0008] In order to solve the defects of short action time, poor filling effect and short duration in the filler containing collagen, the present invention provides an injectable collagen filler, which increases the filling strength and duration by crosslinking collagen and adding microspheres for injection filling. Specifically, the present invention provides the following technical solutions to achieve the above object:

[0009] A preparation method of an injectable collagen filler, comprising the following steps:

[0010] (S1) The extracted collagen solution is subjected to salting-out precipitation, the precipitated matter is dialyzed and freeze-dried to obtain collagen;

[0011] (S2) After the collagen is dissolved with hydrochloric acid, it is homogenized with PBS buffer solution to prepare a non-crosslinked collagen gel;

[0012] (S3) Dissolve the collagen with hydrochloric acid, then homogenize it with PBS buffer solution. The obtained collagen gel is irradiated by a gradient irradiation cross-linking method. After irradiation, it is granulated through a sieve to obtain a cross-linked collagen gel; the gradient irradiation cross-linking is that the irradiation intensity gradually increases from 10 - 15 kGy to 25 - 30 kGy, and then the irradiation is maintained at this intensity;

[0013] (S4) Mix the non-cross-linked collagen gel obtained in step (S2), the cross-linked collagen gel obtained in step (S3), and the microspheres for injection filling, degas and fill it into containers to obtain a collagen filler for injection.

[0014] Further, in step (S1), the sources of collagen include but are not limited to animal tendons such as bovine tendon and porcine tendon; animal skins such as cowhide and pigskin; bovine pericardium and arachnoid membrane.

[0015] Further, when preparing the non-cross-linked collagen in (S2), the collagen is sourced from animal skins such as cowhide and pigskin; when preparing the cross-linked collagen in step (S3), the collagen is sourced from animal tendons such as bovine tendon and porcine tendon, preferably bovine tendon. Bovine tendon collagen is denser, the formed cross-linked collagen has higher strength, and the collagen content is also higher, making it more suitable as the raw material for cross-linked collagen. The inventors found that using collagen sourced from animal skins to prepare non-cross-linked collagen and collagen sourced from animal tendons to prepare cross-linked collagen results in the best filling effect of the obtained collagen filler product.

[0016] Further, the concentration of collagen in the non-cross-linked collagen gel is 20 - 35 mg / mL, and the concentration of collagen in the cross-linked collagen gel is 25 - 32 mg / mL. The limitation on the concentration of collagen in the cross-linked collagen gel is more stringent. A higher concentration can improve the yield after water loss of the product and also increase the productivity; however, the concentration cannot be too high, otherwise the strength will be too high, making granulation difficult and the pushing force of the formed filler relatively large, which is inconvenient to use.

[0017] Further, the extraction process of collagen is well-known in the art. For example, for collagen sourced from animal tendons, after removing the fascia from the animal tendons, washing, freezing, slicing, putting it into a pepsin solution for enzymatic hydrolysis, centrifuging the enzymatic hydrolysate, and salting out the supernatant, collagen sourced from animal tendons is obtained; for collagen sourced from animal skins, after removing the hair from the animal skin, removing the fat layer and the epidermis, leaving the dermis layer, washing with water, storing it frozen, soaking it in a Tris-NaCl buffer solution at 4 - 8 °C, washing with water, performing enzymatic hydrolysis in a pepsin solution, centrifuging to remove the residue, and salting out, collagen sourced from animal skins is obtained.

[0018] Furthermore, when extracting collagen from animal tendons, the freezing temperature is -15°C to 0°C, the slice thickness is 0.5 - 2 mm, the pH of the pepsin solution is 2 - 4, and the dosage ratio of the animal tendon slices to the pepsin solution is 1 kg: 100 - 200 L; the concentration of the pepsin solution is 1 g / 10 L to 1 g / 30 L, the enzymatic hydrolysis time is 3 - 7 days, saturated NaCl solution is used for salting out, and the precipitated precipitate is dialyzed with pH = 2 - 3, pH = 3 - 4 and purified water for 7 - 12 days, and then freeze-dried to obtain collagen from animal tendons; when extracting collagen from animal skins, the pH of the Tris-NaCl buffer solution is 7.5 - 8.5, the concentration of the animal skin in the pepsin solution is 1 - 5 g / L, the enzymatic hydrolysis time is 2 - 5 days, the flocculent precipitate obtained by salting out is redissolved in an acetic acid solution (acetic acid concentration 0.1 - 0.5 mol / L), the insoluble particles are removed by centrifugation, and the salting out and redissolution steps are repeated 2 - 5 times. The precipitate obtained by the last salting out is dialyzed in 0.1 - 0.2 M acetic acid solution for 2 - 4 days, and then dialyzed in purified water for 1 - 2 days, and freeze-dried to obtain collagen from animal skins.

[0019] Further, in step (S2) and step (S3), the pH of the hydrochloric acid is 3 - 4; the pH of the PBS buffer solution is 7.2 - 7.6, such as pH = 7.3, pH = 7.4, pH = 7.5.

[0020] Further, in step (S2), the ratio of collagen, hydrochloric acid, and PBS buffer is 1 g: 20 - 30 mL: 2 - 3 mL.

[0021] Further, in step (S3), the time of gradient irradiation is 15 - 30 min, and then the irradiation is maintained at the irradiation intensity for 3 - 5 min. The intensity of gradient irradiation gradually increases in an approximately linear manner. For example, the irradiation intensity gradually increases from 10 kgy to 30 ky in 20 min, and the rate of increase in irradiation intensity is about 1 kgy / min; another example is that the irradiation intensity gradually increases from 15 kgy to 25 ky in 20 min, and the rate of increase in irradiation intensity is about 0.5 kgy / min. After irradiation, granulation is carried out with a sieve, and the sieve aperture is 90 - 150 μm.

[0022] Further, in step (S4), the microspheres for injection filling are well-known in the art, including but not limited to PCL microspheres, PLLA microspheres, and PLGA microspheres. The particle size of the microspheres for injection filling is 30 - 50 μm. The microspheres for injection filling can be commercially purchased or self-made, and their preparation methods are well-known in the art. For example, for PCL microspheres, the PCL polymer is dissolved in dichloromethane and / or chloroform to prepare a 10 - 40 wt% polymer oil phase solution. After dissolving PVA at 70 - 90 °C, a 0.2 - 2 wt% PVA aqueous solution is made, and high-speed shearing is carried out. The volume ratio of the oil phase to the water phase is 1:2 - 10. The dichloromethane / chloroform solvent is stirred and volatilized, and sieved to obtain PCL microspheres with a particle size of 30 - 50 μm.

[0023] Further, in step (S4), the mass ratio of the non-crosslinked collagen gel, crosslinked collagen gel, and microspheres for injection filling is 2 - 4:3 - 5:1 - 2.

[0024] The present invention uses non-crosslinked collagen, crosslinked collagen, and biodegradable injectable polymer microspheres to be compounded and mixed in a certain proportion, so as to achieve an immediate filling effect. The polymer microspheres play a long-term role, and the amino acid components obtained by the degradation of collagen in the early and middle stages can become nutrients that stimulate the generation of collagen regeneration by polyester microspheres. However, the biggest problems of collagen for medical aesthetic injection filling at present are short duration and allergic reactions. The present invention has achieved the purpose of long filling duration and good filling effect by compounding crosslinked collagen and non-crosslinked collagen with different crosslinking degrees obtained by different irradiation intensities; the collagen allergen substances in the present invention are less than those extracted from other materials, reducing allergic reactions. The crosslinked collagen in the present invention uses the irradiation crosslinking technology without adding any chemical crosslinking agents and has no toxic effects of chemical agents. The gradient irradiation with gradually increasing irradiation intensity can have a higher yield and less water loss at the same concentration. The non-crosslinked collagen gel can provide a microenvironment for nutritional repair and filling effect in the early stage, the crosslinked extracellular matrix can provide a repair microenvironment in the middle stage of filling, and the degradation of polyester microspheres in the later stage promotes collagen regeneration, so that the skin has a good microenvironment throughout the repair cycle and increases skin elasticity. Description of the Drawings

[0025] Figure 1 It is a photograph of the crosslinked collagen gel obtained in step S3 of Example 1.

[0026] Figure 2 It is a photograph of the crosslinked collagen gel obtained in Comparative Example 1.

[0027] Figure 3 It is a photograph of the crosslinked collagen gel obtained in Comparative Example 2.

[0028] Figure 4It is a stained section photograph of the filler in Example 1 after 12 months of subcutaneous implantation in rabbits.

[0029] Figure 5 It is a stained section photograph of the filler in Comparative Example 1 after 12 months of subcutaneous implantation in rabbits.

[0030] Figure 6 It is a stained section photograph of the filler in Comparative Example 3 after 12 months of subcutaneous implantation in rabbits.

[0031] Figure 7 It is a stained section photograph of the filler in Comparative Example 3 after 12 months of subcutaneous implantation in rabbits. Detailed implementation manners

[0032] The technical solution of the present invention will be further explained and illustrated through specific embodiments below.

[0033] Example 1

[0034] (S1) Extraction of collagen:

[0035] (1.1) Cut the bovine Achilles tendon in a Y shape and keep the main trunk part while removing the fascia of the bovine Achilles tendon. Wash it with purified water no less than 3 times, drain the water, freeze it in the refrigerator at a temperature of (-15 to 0 °C). Take out the cross-section slices (thickness 0.5 - 2 mm) from the refrigerator and add the slices to a pepsin solution with pH = 2 - 4 (bovine Achilles tendon slices: enzyme digestion solution = 1 kg / 100 L - 1 kg / 200 kg), the concentration of the enzyme digestion solution is (1 g / 10 L - 1 g / 30 L), and the enzyme digestion time is 3 - 7 days. Then centrifuge to collect the supernatant, and perform saturated sodium chloride salting-out on the supernatant. The precipitated white floccules are dialyzed in pH = 3, pH = 4 and purified water for 7 days respectively, and then the dialysate is freeze-dried to obtain bovine Achilles tendon collagen.

[0036] (1.2) Select fresh pigskin, remove hair and dirt, use a knife to remove the subcutaneous fat layer and the epidermis, leaving the dermis layer close to the epidermis. Cut it into pieces of 2 cm × 2 cm size and rinse 3 times with deionized water. Store the treated pigskin at -20 °C. Soak the pigskin in a Tris-NaCl (Tris: 0.05 mol / L; NaCl: 1 mol / L; pH 7.5) buffer solution at 4 °C (material-liquid ratio 1:20) for 12 h, and then rinse 3 times with deionized water. Take the defatted pigskin and hydrolyze it in a pepsin solution with a concentration of one in a hundred thousand and a pH value of 2-3 at room temperature for 48 h. The concentration of pigskin is 1 mg / mL. Centrifuge to remove unreacted residues. Add saturated NaCl to the above reaction solution and salt out at 4 °C until there are flocculent precipitates in the slurry. Centrifuge (4 °C, 10000 rpm, 15 min), collect the lower precipitate, redissolve it in a solution with a pH value of 2.2 (0.5 mol / L acetic acid), and centrifuge to remove insoluble particles (4 °C, 10000 rpm, 15 min). Repeat this process 3 times. Put the last salted-out precipitate into a dialysis bag, dialyze it in a 0.1 M acetic acid solution for 48 h, and then dialyze it in deionized water for 24 h. Subsequently, perform vacuum freeze-drying to obtain pigskin collagen.

[0037] (S2) Preparation of non-crosslinked collagen gel: Dissolve 700 mg of freeze-dried pigskin collagen in 18 ml of hydrochloric acid with a pH of 3, and then homogenize and mix it with 2 ml of PBS to prepare a non-crosslinked collagen gel, where the collagen concentration is 35 mg / ml.

[0038] (S3) Preparation of crosslinked collagen: Dissolve 1400 mg of freeze-dried bovine Achilles tendon collagen in 45 ml of hydrochloric acid with a pH of 3, and then homogenize and mix it with 5 ml of PBS to prepare a gel with a collagen concentration of 28 mg / ml. Crosslink it by cobalt-60 irradiation. The irradiation intensity increases from 10 kGy to 30 kGy at a rate of 1 kGy / min within 20 min, and then continue to irradiate for 5 min. Granulate it using a 150-mesh sieve.

[0039] (S4) Preparation of the filler: Weigh 100 g of polycaprolactone with a molecular weight of 25,000 and a polydispersity index (PDI) = 2.3, and dissolve it in 250 ml of dichloromethane to prepare a polymer oil-phase solution; dissolve 3.2 g of PVA raw material in 70 - 90 °C of water for injection, and let it cool to prepare a 0.4% (W / V) PVA aqueous solution; the oil-phase solution and the PVA aqueous solution enter an in-line shear mixer simultaneously through a pipeline equipped with a flow pump for mixing, shear emulsification is carried out at a rotation speed of 3200 rpm, then stir to volatilize dichloromethane, use 30 μm and 50 μm sieves, and finally perform freeze-drying to prepare PCL microspheres with a size of 30 - 50 μm. Mix non-crosslinked collagen gel, crosslinked collagen gel, and PCL microspheres in a mass ratio of 3:5:2, degas and fill them into a prefilled syringe for use.

[0040] Example 2

[0041] Other conditions are the same as those in Example 1, the difference is that in step (S2) and step (S3), the collagen used is porcine skin collagen.

[0042] Example 3

[0043] Other conditions are the same as those in Example 1, the difference is that in step (S2) and step (S3), the collagen used is bovine tendon collagen.

[0044] Example 4

[0045] Other conditions are the same as those in Example 1, the difference is that in step (S2), the collagen concentration of the non-crosslinked collagen gel is 20 mg / mL; in step (S2), the collagen concentration of the crosslinked collagen gel is 32 mg / mL.

[0046] Example 5

[0047] Other conditions are the same as those in Example 1, the difference is that in step (S4), the mass ratio of non-crosslinked collagen gel, crosslinked collagen gel, and PCL microspheres is 4:4:2.

[0048] Example 6

[0049] Other conditions are the same as those in Example 1, the difference is that in step (S4), the mass ratio of non-crosslinked collagen gel, crosslinked collagen gel, and PCL microspheres is 2:3:1.

[0050] Example 7

[0051] Other conditions are the same as those in Example 1, the difference is that in step (S3), the collagen concentration of the crosslinked collagen gel is 25 mg / mL.

[0052] Example 8

[0053] Other conditions are the same as those in Example 1, except that in step (S3), for the gradient irradiation, the irradiation intensity is increased from 15 kGy to 25 kGy at a rate of 0.5 kGy / min within 20 min, and then irradiation continues for 5 min.

[0054] Comparative Example 1

[0055] Other conditions are the same as those in Example 1, except that in step (S3), the collagen concentration of the crosslinked collagen gel is 20 mg / mL.

[0056] Comparative Example 2

[0057] Other conditions are the same as those in Example 1, except that in step (S3), the irradiation is changed to a fixed irradiation intensity of 20 kGy for 20 min.

[0058] Comparative Example 3

[0059] Other conditions are the same as those in Example 1, except that in step (S4), the filler is a mixture of crosslinked collagen gel and PCL microspheres with a mass ratio of 8:2, that is, non-crosslinked collagen gel is not added.

[0060] Comparative Example 4

[0061] Other conditions are the same as those in Example 1, except that in step (S4), the filler is a mixture of non-crosslinked collagen gel and PCL microspheres with a mass ratio of 8:2, that is, crosslinked collagen gel is not added.

[0062] Application Example 1

[0063] The telopeptide detection (ELISA), DNA residue (fluorescence method), and α-antigen detection were performed on the bovine Achilles tendon collagen and porcine skin collagen prepared in step S1 of Example 1, and the results are shown in Table 1 below.

[0064] Table 1 Residual Detection

[0065]

[0066] Analysis: For the severity of allergens, DNA residue > α-antigen > telopeptide, and the animal-derived collagen obtained in the present invention has a lower immunological reaction.

[0067] Application Example 2

[0068] Test the mass of the collagen gel before and after crosslinking in step S3 in the above examples and comparative examples, calculate the yield after water loss, and the yield after water loss = mass of the gel after crosslinking / mass of the gel before crosslinking. The results are shown in Table 2 below. Figure 1 It is a photo of the crosslinked collagen gel obtained in step S3 of Example 1; Figure 2It is a photograph of the crosslinked collagen gel obtained in Comparative Example 1; Figure 3 It is a photograph of the crosslinked collagen gel obtained in Comparative Example 2.

[0069] Table 2 Crosslinked Collagen Gel Yield Test

[0070]

[0071]

[0072] It can be seen that in Comparative Example 1, after crosslinking with a concentration of 20 mg / ml, the gel strength is weak, it is easy to break in the middle, the yield after crosslinking is also low (more water loss), and after being formulated into an injection, the elastic modulus (filling performance) is weak. In Comparative Example 1, with a fixed irradiation intensity, the yield and elastic modulus also decreased to a certain extent. While the crosslinked collagen in Example 1 is not easily broken when pressed and has a high elasticity.

[0073] Application Example 3

[0074] The fillers obtained in the examples and comparative examples were measured for viscosity and viscoelastic modulus using a rheometer, and a subcutaneous implantation experiment was conducted on rabbits to observe the degradation and filling effects. The results are shown in Table 3 below. The degradation performance is that there are microsphere residues and sufficient filling performance is maintained. The viscosity of the injectable filler is preferably in the range of 200 - 350 Mpa·s at 0.25 Hz. With a suitable viscosity, good injectability and filling effect can be achieved. If the viscosity is too low, the filling effect is average; if the viscosity is too high, it is not easy to inject and the needle is easily blocked.

[0075] Table 3 Filler Effect Test

[0076]

[0077]

[0078] Figure 4 It is a photograph of the section staining of the filler in Example 1 after 12 months of subcutaneous implantation experiment on rabbits; Figure 5 It is a photograph of the section staining of the filler in Comparative Example 1 after 12 months of subcutaneous implantation experiment on rabbits; Figure 6 It is a photograph of the section staining of the filler in Comparative Example 3 after 12 months of subcutaneous implantation experiment on rabbits; Figure 7 It is a photograph of the section staining of the filler in Comparative Example 3 after 12 months of subcutaneous implantation experiment on rabbits.

Claims

1. A preparation method of a collagen filler for injection, characterized in that, It includes the following steps: (S1) The extracted collagen solution is subjected to salting-out precipitation, and the precipitated matter is dialyzed and freeze-dried to obtain collagen; (S2) The collagen is dissolved with hydrochloric acid and then homogenized with PBS buffer solution to prepare a non-crosslinked collagen gel; the concentration of collagen in the non-crosslinked collagen gel is 20 - 35 mg / mL; when preparing the non-crosslinked collagen, the collagen is sourced from animal skin; (S3) The collagen is dissolved with hydrochloric acid and then homogenized with PBS buffer solution, and the obtained collagen gel is irradiated by a gradient irradiation crosslinking method. After irradiation, it is granulated through a sieve to obtain a crosslinked collagen gel; the gradient irradiation crosslinking is that the irradiation intensity gradually increases from 10 - 15 kGy to 25 - 30 kGy, and then the irradiation intensity is maintained for irradiation; when preparing the crosslinked collagen, the collagen is sourced from animal tendon; the concentration of collagen in the crosslinked collagen gel is 25 - 32 mg / mL; the time of gradient irradiation is 15 - 30 min, and then the irradiation intensity is maintained for irradiation for 3 - 5 min; the intensity of gradient irradiation increases gradually in a linear manner; (S4) The non-crosslinked collagen gel obtained in step (S2), the crosslinked collagen gel obtained in step (S3), and microspheres for injection filling are mixed, degassed and filled to obtain a collagen filler for injection; the mass ratio of the non-crosslinked collagen gel, the crosslinked collagen gel, and the microspheres for injection filling is 2 - 4:3 - 5:1 - 2; the microspheres for injection filling include PCL microspheres, PLLA microspheres or PLGA microspheres, and the particle size of the microspheres for injection filling is 30 - 50 μm.

2. The preparation method according to claim 1, wherein In step (S2) and step (S3), the pH of the hydrochloric acid is 3 - 4; the pH of the PBS buffer solution is 7.2 - 7.

6.

3. The preparation method according to claim 1, characterized in that, In step (S2), the ratio of collagen, hydrochloric acid, and PBS buffer solution is 1 g:20 - 30 mL:2 - 3 mL.

4. The preparation method according to claim 1, wherein In step (S3), after irradiation, it is granulated through a sieve, and the sieve aperture is 90 - 150 μm.

5. The preparation method according to claim 1, characterized in that In step (S2), the animal skin is selected from cowhide and pigskin.

6. The preparation method according to claim 1, characterized in that, In step (S3), the animal tendon is selected from cow tendon and pig tendon.

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