Recombinant collagen gel material as well as preparation method and application thereof

Abstract

The invention provides a recombinant collagen gel material as well as a preparation method and application thereof. The preparation method of the material comprises the following steps: mixing recombinant collagen with a first water phase to obtain a recombinant collagen solution, and drying and granulating to obtain first recombinant collagen microspheres; performing physical crosslinking on the first recombinant collagen microspheres to obtain second recombinant collagen microspheres; mixing and stirring the second recombinant collagen microspheres and a second water phase, and swelling to obtain a white dilute solution with suspended particles; and carrying out low-temperature irradiation crosslinking on the white dilute solution with suspended particles to obtain the recombinant collagen gel material. According to the preparation method disclosed by the invention, the production period is shortened, the production cost is greatly reduced, and the gel material prepared by the method is high in biological safety and has good mechanical property and degradation property.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, relates to the field of injectables for medical aesthetic filling, and particularly relates to a recombinant collagen gel material, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogel is a three-dimensional network biomaterial composed of hydrophilic polymer chains, which can swell but not dissolve in water, and has advantages such as strong water absorption, water retention, and biocompatibility, and has been widely studied in the fields of biology and medical engineering. It includes applications in the field of biopharmaceuticals such as biomedical materials for implantable medical devices, medical wound dressings, drug release carriers, etc. Among them, hydrogel can be used as a soft tissue filling material to repair soft tissue injuries or cavities and defects caused by lesions, and to remove wrinkles or improve facial contours in the plastic and aesthetic industries.

[0003] Recombinant collagen is collagen produced by genetic engineering technology. Compared with collagen extracted from nature, it has higher purity, better biological safety, and a more controllable production process. The preparation methods of recombinant collagen hydrogel mainly include physical cross-linking, chemical cross-linking, enzymatic cross-linking methods, etc. The physical cross-linking method realizes physical cross-linking through repeated freeze-thaw cycles or by using non-covalent bonds (such as hydrogen bonds, hydrophobic interactions). The recombinant collagen hydrogel prepared in this way has good mechanical properties, but has disadvantages such as a cumbersome process and difficult cross-linking degree control. The hydrogel prepared by the chemical cross-linking method usually has residues of cross-linking agents and initiators, etc., and there is a risk of causing inflammation or cytotoxicity. The enzymatic cross-linking method is a method that uses specific enzyme-catalyzed reactions to achieve cross-linking between polymer or protein molecules. As a green and efficient cross-linking method, its production cost is relatively high, and there are certain difficulties in large-scale production.

[0004] In summary, the existing preparation methods of recombinant collagen hydrogel have problems such as a cumbersome process, difficult cross-linking degree control, residues of chemical cross-linking agents, and high costs. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a recombinant collagen gel material, a preparation method thereof, and an application thereof. The preparation method of the present invention can greatly reduce the production cost, shorten the production cycle, has high production efficiency, strong operability, and is easy to scale up production. The obtained material has good mechanical properties, is safe, non-toxic, biodegradable, has excellent biocompatibility, and has no virus hidden danger.

[0006] To achieve the above object, on the one hand, the present invention provides a preparation method of a recombinant collagen gel material, which includes:

[0007] Mix the recombinant collagen with the first aqueous phase to obtain a recombinant collagen solution, and obtain the first recombinant collagen microspheres after drying and granulation;

[0008] Perform physical cross-linking on the first recombinant collagen microspheres to obtain the second recombinant collagen microspheres;

[0009] Mix and stir the second recombinant collagen microspheres with the second aqueous phase and swell them to obtain a dilute white solution with suspended particles;

[0010] Perform low-temperature irradiation cross-linking on the dilute white solution with suspended particles to obtain a recombinant collagen gel material.

[0011] According to a specific embodiment of the present invention, the recombinant collagen gel material is a colorless and transparent jelly-like gel.

[0012] The recombinant collagen described in the present invention is the recombinant human-derived collagen disclosed in CN108070032B

A purification method of recombinant human-derived collagen

[0013] According to a specific embodiment of the present invention, the amino acid sequence of the recombinant collagen is as shown in SEQ ID No: 1.

[0014] In the specific examples of the present invention, the recombinant collagen raw materials are all proteins having the amino acid sequence shown in SEQ ID No: 1 in the sequence listing.

[0015] According to a specific embodiment of the present invention, in the recombinant collagen solution, the mass fraction of the recombinant collagen is 10%-20%.

[0016] According to a specific embodiment of the present invention, the pH of the recombinant collagen solution is <7, preferably, the pH is 4-6. According to a specific embodiment of the present invention, in the dilute white solution with suspended particles, the mass fraction of the second recombinant collagen microspheres is 2%-4%.

[0017] In the preparation method of the present invention, the content of the second recombinant collagen microspheres in the dilute white solution with suspended particles is the content of the recombinant collagen in the recombinant collagen gel material of the present invention.

[0018] According to a specific embodiment of the present invention, the drying and granulation is carried out using a spray dryer, the spray temperature is 140-260°C, the feeding speed is 200-500 ml / h, and the nozzle diameter is 0.75-1.5 mm.

[0019] According to a specific embodiment of the present invention, the temperature of the physical cross-linking is 120-160°C, and the time is 2-5 h; preferably, the temperature of the physical cross-linking is 120-140°C or 140-160°C, and the time is 2-3 or 3-5 h.

[0020] According to a specific embodiment of the present invention, the vacuum degree of the physical crosslinking is 0.08 MPa - (-0.1 MPa).

[0021] According to a specific embodiment of the present invention, the rotation speed of the mixing and stirring is 80 - 120 r / min, and the time is 30 - 60 min.

[0022] According to a specific embodiment of the present invention, the temperature of the swelling is 2 - 8 °C, and the time is 6 - 10 h.

[0023] According to a specific embodiment of the present invention, the radiation crosslinking uses electron beam or 60 Co-γ ray, and the radiation dose is 10 - 25 kGy.

[0024] According to a specific embodiment of the present invention, when the dilute white solution with suspended particles is subjected to radiation crosslinking, the temperature of the dilute white solution with suspended particles is 2 - 8 °C, preferably 2 - 4 °C.

[0025] According to a specific embodiment of the present invention, the first aqueous phase or the second aqueous phase is selected from one or a combination of two or more of ultrapure water, water for injection, and phosphate buffer solution.

[0026] On the other hand, the present invention also provides a recombinant collagen gel material prepared by the above preparation method.

[0027] The present invention provides a recombinant collagen gel material, wherein, based on the total mass of the recombinant collagen gel material being 100%, the content of the recombinant collagen is 2% - 4%, and the content of the aqueous phase is 96% - 98%;

[0028] The amino acid sequence of the recombinant collagen is as shown in SEQ ID No: 1;

[0029] The recombinant collagen gel material is a colorless and transparent jelly-like gel with a porous network structure inside.

[0030] After the recombinant collagen gel material of the present invention is freeze-dried, the cross-section of the gel material is a honeycomb-like and dense porous network structure, which is beneficial to the diffusion of nutrients and promotes cell proliferation.

[0031] According to a specific embodiment of the present invention, the aqueous phase is selected from one or a combination of two or more of ultrapure water, water for injection, and phosphate buffer solution;

[0032] According to a specific embodiment of the present invention, the pushing force of the recombinant collagen gel material through a 27G needle is 15 - 20 N, more preferably 16 - 18 N.

[0033] In the present invention, the conditions for measuring the injection force are as follows: filling a 2 mL sample of the recombinant collagen gel material, attaching a 27G needle, and injecting at a constant speed of 10 mm / min until the sample is completely ejected with a constant force value, at which point the experiment ends. During the experiment, the maximum force value measured is the injection force.

[0034] According to a specific embodiment of the present invention, the osmotic pressure of the recombinant collagen gel material is 270 - 310 mOsm / L, which is close to the ideal state of human physiology and activity.

[0035] According to a specific embodiment of the present invention, the rotational viscosity of the recombinant collagen gel material is 6000 - 9000 cp.

[0036] In the present invention, the conditions for measuring the rotational viscosity are as follows: taking 200 ml of the sample to be measured, using a No. 5 rotor, rotating at 25 rpm, and detecting the viscosity at 25°C using a rotational viscometer.

[0037] On the other hand, the present invention also provides the application of the above preparation method, or the recombinant collagen gel material prepared by the above preparation method, or the above recombinant collagen gel material in the preparation of medical materials.

[0038] According to a specific embodiment of the present invention, preferably, the medical material is used for aesthetic medical skin hydration, aesthetic medical filling, soft tissue repair, or anti-adhesion.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] First, the present invention uses two cross-linking steps. Among them, the first physical cross-linking is semi-cross-linking. Through semi-cross-linking, the denaturation temperature, that is, the temperature at which part of the collagen unwinds its helix structure, can be increased. After collagen dehydration, only some of the collagen molecular chains form cross-linking points. The second cross-linking is carried out by low-temperature irradiation cross-linking, which causes free radicals to be generated from amino acid residues in the recombinant collagen and recombine within an extremely short time. Low temperature can slow down the diffusion rate of free radicals and promote the formation of a cross-linking network to achieve secondary cross-linking, thereby preparing the recombinant collagen gel material. No additional chemical cross-linking agent needs to be introduced during the production process, ensuring high biological safety, being safe, non-toxic, and having excellent biocompatibility.

[0041] Second, it is possible to achieve the effect of obtaining a material prepared from a high-concentration raw material with low-concentration recombinant collagen microspheres, greatly reducing the production cost, having strong operability, and being easy to scale up production.

[0042] Third, spray drying shortens the production cycle and improves production efficiency compared to freeze drying.

[0043] Fourth, using low-temperature irradiation cross-linking can have good effects of preventing bacteria, extending the storage time, and ensuring the quality of the hydrogel.

[0044] Fifthly, the use of secondary cross-linking can greatly enhance the mechanical properties and biodegradability of the material.

[0045] Sixthly, the composition is single, containing only one recombinant collagen component, and has high biological safety. Description of the Drawings

[0046] Figure 1 It is the electron micrograph of the material prepared in Example 1. Among them, (A) is the scanning electron micrograph (5000 times) of the first recombinant collagen microsphere, and (B) is the scanning electron micrograph (100,000 times) of the internal porous structure of the first recombinant collagen microsphere; the scanning electron micrograph (200 times) of the cross-section after freeze-drying of the recombinant collagen gel material prepared in Example 1 after the first cross-linking (C) and the second cross-linking (D);

[0047] Figure 2 It is the comparison diagram of the appearance of the samples prepared from the recombinant collagen gel material prepared in Example 1 after the first cross-linking and the second cross-linking;

[0048] Figure 3 It is the recombinant collagen gel material sample prepared and filled into a syringe and then injected into a glass dish to observe the morphology of the sample after injection. Among them, A is the morphology of the sample in Example 1 after injection, B is the morphology of the sample in Comparative Example 4 after injection, and C is the morphology of the sample in Comparative Example 7 after injection;

[0049] Figure 4 It is the test of the recombinant collagen gel material prepared in Example 1;

[0050] Figure 5 It is the effect diagram of the treatment of the photoaging model with the recombinant collagen gel material prepared in Example 1;

[0051] Figure 6 It is the effect diagram of the treatment of the photoaging model with the recombinant collagen gel material prepared in Comparative Example 1;

[0052] Figure 7 It is the statistical results of skin elasticity, collagen content, and moisture of the recombinant collagen gel material prepared in Example 1 for the photoaging model mice at 0w, 1w, and 2w;

[0053] Figure 8 It is the statistical results of skin elasticity, collagen content, and moisture of the recombinant collagen gel material prepared in Comparative Example 1 for the photoaging model mice at 0w, 1w, and 2w;

[0054] Figure 9 It is the hematoxylin-eosin staining picture of the recombinant collagen gel material prepared in Example 1 for the aging model mice at 2w. Detailed Implementation Modes

[0055] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the following detailed description of the technical solution of the present invention is provided, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0056] In actual production, those skilled in the art can understand that, on the premise of obtaining qualified products, some relevant process steps can be adjusted or increased or decreased.

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0058] The amino acid sequence of the recombinant collagen used in the following embodiments is:

[0059] GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP(SEQ ID No:1).

[0060] Example 1

[0061] This example provides a method for preparing a recombinant collagen gel material:

[0062] (1) Add 100 g of recombinant human collagen (a protein with the amino acid sequence shown in SEQ ID No: 1) to 900 g of water for injection, and stir with a magnetic stirrer for 2 h to obtain a 10% recombinant collagen solution, where the pH of the recombinant collagen solution is 5.15.

[0063] (2) Spray granulate the above recombinant collagen solution with a pH < 7 using a spray dryer. The spray temperature is 150 °C, the feeding rate is 300 ml / h, and the nozzle diameter is 1 mm to obtain 80 g of the first recombinant collagen microspheres. The spray drying time is 2 h.

[0064] (3) Take 2 g of the first recombinant collagen microspheres for physical crosslinking, which is semi-crosslinking once. The crosslinking conditions are as follows: temperature: 140 °C in a vacuum drying oven, time: 3 h, vacuum degree: -0.1 MPa, to obtain 2 g of the second recombinant collagen microspheres.

[0065] (4) Add 2 g of the second recombinant collagen microspheres to 98 g of water for injection, mix and stir at a rotation speed of 100 r / min for 30 min, and then swell in a 4 °C refrigerator for 6 h to obtain a white dilute solution with suspended particles.

[0066] (5) Fill the white dilute solution with suspended particles into vials and place them in a foam box equipped with ice packs (for maintaining 4 °C) for irradiation sterilization, which is secondary low-temperature irradiation crosslinking; the irradiation crosslinking uses high-energy electron beams, and the irradiation dose is 10 kGy, thereby obtaining the colorless and transparent jelly-like recombinant collagen gel material of Example 1 of the present invention.

[0067] Examples 2 - 5

[0068] Examples 2 - 5 provide a preparation method for a recombinant collagen gel material:

[0069] Other conditions are the same as those in Example 1, and the differences are as follows: the temperature of physical crosslinking of the first recombinant collagen microspheres, the concentration of the second recombinant collagen microspheres, the swelling conditions, and the irradiation crosslinking conditions are shown in Table 1. The colorless and transparent jelly-like recombinant collagen gel materials of Examples 2 - 5 are prepared.

[0070] Comparative Examples 1 - 5

[0071] Comparative Examples 1 - 5 provide a preparation method for a recombinant collagen gel material:

[0072] Other conditions were the same as those in Example 1, except that: the physical cross-linking temperature of the first recombinant collagen microspheres, the concentration of the second recombinant collagen microspheres, the swelling conditions, and the irradiation cross-linking conditions were as shown in Table 1, and the recombinant collagen gel materials of Comparative Examples 1-5 were prepared. Among them, the recombinant collagen in Comparative Example 1 was recombinant type III humanized collagen purchased from the market (purchased from Xi'an Denohis Medical Technology Co., Ltd., batch number: 2308B01, model: RHC-III-Lel).

[0073] Comparative Example 6

[0074] Comparative Example 6 provides a method for preparing a recombinant collagen gel material:

[0075] Other conditions were the same as those in Example 1, except that: 1. The recombinant collagen solution was freeze-dried into a recombinant collagen sponge, then physically cross-linked, frozen with liquid nitrogen for 3-5 min and then ground with a mortar to obtain recombinant collagen powder, which was mixed with injection water to obtain a white dilute solution with suspended particles.

[0076] Comparative Example 7

[0077] Comparative Example 7 provides a method for preparing a recombinant collagen gel material:

[0078] Other conditions were the same as those in Example 1, except that: the material prepared in Comparative Example 7 was prepared according to the current conventional method (high concentration), in which the physical cross-linking temperature was high, the concentration of the recombinant collagen microspheres was relatively high, and the swelling time was long. The prepared material was terminally sterilized by irradiation sterilization or moist heat sterilization, and no secondary cross-linking occurred during this process. The physical cross-linking temperature of the first recombinant collagen microspheres, the concentration of the second recombinant collagen microspheres, the swelling conditions, and the irradiation cross-linking conditions were as shown in Table 1, and the recombinant collagen gel material of Comparative Example 7 was prepared.

[0079] Table 1 Preparation conditions of Examples 1-5 and Comparative Examples 1-7

[0080]

[0081] Effect Example

[0082] The following performance tests were carried out on the recombinant collagen gel materials prepared in the above examples and comparative examples:

[0083] 1. Scanning electron microscopy: After the recombinant collagen microspheres obtained by spray granulation in Example 1, the prepared white dilute solution with suspended particles, and the recombinant collagen gel material were freeze-dried, they were placed in liquid nitrogen for 3-5 min and then broken apart, and the cross-section was observed for microscopic morphology using a scanning electron microscope. The results were as Figure 1As shown: A: After spray granulation, the recombinant collagen forms a microsphere structure with a diameter of about 10 μm; B: The interior of the microsphere is a porous network structure; C: Crosslinking points are formed between some collagen molecular chains in the dilute white solution with suspended particles (as shown in the box); D: The cross-section of the recombinant collagen gel material is a honeycomb-like porous network structure, which is dense. The porous network structure is conducive to the diffusion of nutrients and promotes cell proliferation.

[0084] 2. Appearance test method: Compare the appearances of the products prepared before and after the second crosslinking of Examples 1-5 and Comparative Examples 1-7. The comparison results are shown in Table 2. The appearance comparison of the products prepared before and after the second crosslinking of Example 1 is shown in Figure 2 , where Figure 2 a in Figure 2 is the state after the first physical semi-crosslinking preparation solution and swelling,

[0085] 3. Morphology after sample injection: Fill the gel materials prepared in Examples 1-5 and Comparative Examples 4 and 6 into syringes and inject them into glass dishes. Observe the morphology of the samples. The results are shown in Table 2. The morphology of the sample of Example 1 after injection is as shown in Figure 3 A in Figure 3 . The morphologies of the samples of the remaining Examples 2-5 after injection are basically the same as that of Example 1; the morphology of the sample of Comparative Example 4 after injection is as shown in Figure 3 B in

[0086] Table 2 Appearance test results

[0087]

[0088] Note: " / " represents that the sample is in a dilute solution state and cannot be formed after injection, and is in a flowing liquid state.

[0089] 4. The injection force is tested on a universal mechanical testing machine. For the 2 ml sample after filling in Example 1, install a 27G needle, fix it on the mechanical testing machine, and inject it at a constant speed of 10 mm / min until the sample is pushed out with a constant force value to end the experiment. Record the curve of the injection force changing with time during the injection process of the sample, and record the maximum force value during the injection process as the injection force. The results are shown in Table 3. Figure 4 For the test of the injection force of the recombinant collagen filler for injection in Example 1, it can be obtained from Figure 4 that the injection force of the recombinant collagen gel material is approximately around 16 N.

[0090] 5. Osmotic pressure: Take 300 μl of the sample and perform the test according to the General Principles (0632 Determination of Osmolality - 1 Determination of Osmolality) in the Fourth Part of the Chinese Pharmacopoeia (2020 Edition). The results are shown in Table 3: The osmotic pressure of the recombinant collagen gel materials prepared in Examples 1 - 5 is in the range of 270 - 310 mOsm / L, approaching the ideal state of human physiology and activities.

[0091] 6. Rotational viscosity: Take 200 ml of the sample and perform viscosity detection at 25 °C using a No. 5 rotor of a rotational viscometer at a rotational speed of 25 rpm. The results are shown in Table 3: The rotational viscosity of the recombinant collagen gel materials prepared in Examples 1 - 5 is in the range of 6000 - 9000 cp.

[0092] Table 3 Performance test results of the materials prepared in examples and comparative examples

[0093] Osmotic pressure (mOsm / L) Bolus injection force (N) Rotational viscosity (cp) Example 1 296.0 16 6500 Example 2 298.4 17 7920 Example 3 304.8 18 8335 Example 4 297.3 16 6755 Example 5 299.2 16 7026 Comparative Example 1 279.6 / 1006 Comparative Example 2 275.5 / 1504 Comparative Example 3 209.4 / 307 Comparative Example 4 304.5 20N 9086 Comparative Example 5 275.5 / 1050 Comparative Example 6 270.0 / 400 Comparative Example 7 308.2 25N 12360

[0094] 7. Biological evaluation: Test according to GB / T 16886: Perform a cytotoxicity experiment on the samples prepared in Examples 1 - 5 and Comparative Examples 1 - 7. The results are shown in Table 4: When the extraction stock solution of the recombinant collagen gel materials prepared in the comparative examples and examples acts on L929 cells for 24 h, the cell viability is above 85%, and there is no obvious cytotoxicity.

[0095] Table 4 Cytotoxicity results

[0096]

[0097] 8. Test on the treatment effect of the photoaging model: For the 1 ml samples filled in Example 1 and Comparative Example 1, install a 34G needle, and evenly inject 1 ml of the solution into the dorsal skin of the photoaging model SKH - 1 mice in a dot - like manner. In the blank control group, evenly inject 1 ml of normal saline into the dorsal skin of the photoaging model SKH - 1 mice in a dot - like manner. At 0 week (0w), 1 week (1w), and 2 weeks (2w) respectively, use a digital camera and a digital microscope to record the exposed dorsal skin, wrinkles, pigmentation, and dorsal skin texture of each group of rats after injection. The results are shown in Figure 5 、 Figure 6 ; At the same time, use a skin assistant analyzer (skin analysis system, CBS - 806, Wuhan Boshi Electronics Co., Ltd., Wuhan, China) to detect the skin condition, and take 3D images of the epidermis every week to measure the three main parameters representing the skin condition: skin elasticity, collagen content, and moisture content. Select three measurement points evenly distributed on the dorsal skin, and measure each point three times to reduce measurement errors. Calculate the average value according to the measurement data. The results are shown in Figures 5 to 8 。

[0098] 9. Histological results of the treatment effect of the photoaging model: After 2 weeks of treatment, the tissues in the injection areas of the blank control group and the experimental group were cut, about 1 cm * 1 cm, rinsed with normal saline, flattened and placed in an embedding cassette, fixed in 4% formaldehyde for at least 24 hours, and then embedded in paraffin and sectioned into 5 μm. The HE method (hematoxylin-eosin staining) was used to observe the distribution of tissue cells and matrix in the injection area and the presence of inflammation, etc. The results are shown in Figure 9 , where Figure 9 a in Figure 9 is the blank control, and

[0099] Experimental results

[0100] 1. Test results of the treatment effect of the photoaging model:

[0101] Figure 5 and Figure 6 are the effect diagrams of the treatment of the photoaging model with the gel materials prepared in Example 1 and Comparative Example 1, respectively.

[0102] As Figure 5 shown in the results, after 2 weeks of injection of the gel material prepared in Example 1, compared with the blank control group (normal saline group): the wrinkles are reduced, the skin texture is delicate and shiny, and the skin is relatively flat, indicating that the recombinant collagen gel material prepared in Example 1 can significantly improve the macroscopic manifestations of skin photoaging.

[0103] As Figure 6 shown in the results, after 1 week of injection of the gel material prepared in Comparative Example 1, ulceration occurred at the injection site of the skin of the model mice. After 2 weeks of injection, compared with the control group (normal saline group): the skin texture is rough and dull, indicating that the gel material prepared in Comparative Example 1 is relatively difficult to significantly improve the macroscopic manifestations of skin photoaging compared with the material in Example 1.

[0104] Table 5, Figure 7 is the statistical results of skin elasticity, collagen content, and moisture of the photoaging model mice treated with the recombinant collagen gel material prepared in Example 1 at 0w, 1w, and 2w.

[0105] Table 5 Statistical results of skin elasticity, collagen content, and moisture in Example 1

[0106]

[0107] As shown in Table 5, Figure 7 the results show that the increments of collagen content, moisture content, and skin elasticity in the experimental group (material in Example 1) are significantly higher than those in the control group at 0w, 1w, and 2w, indicating that the recombinant collagen gel material prepared in Example 1 can significantly improve the skin state of skin photoaging.

[0108] Table 6 Figure 8 Statistical results of skin elasticity, collagen content, and moisture of the gel material prepared in Comparative Example 1 for the photoaged mice at 0w, 1w, and 2w.

[0109] Table 6 Statistical results of skin elasticity, collagen content, and moisture of Comparative Example 1

[0110]

[0111] Table 6, Figure 8 The results showed that the increments of collagen, moisture, and elasticity in the experimental group (the material of Comparative Example 1) were not significantly higher than those in the control group at 0w, 1w, and 2w, indicating that the gel material prepared in Comparative Example 1 could not significantly improve the skin state of photoaged skin.

[0112] 2. Histological results of the treatment effect of the photoaging model:

[0113] Figure 9 Hematoxylin-eosin staining pictures of the photoaged mice at 2w with the recombinant collagen gel material prepared in Example 1. Among them, Figure 9 a in is the blank control, Figure 9 b in is the experimental group (the material of Example 1). As Figure 9 The histological results showed that the implanted recombinant collagen material prepared in Example 1 had good biocompatibility, without obvious inflammation and granuloma, and the collagen fibers in the experimental group were significantly more closely arranged than those in the blank control group, indicating that the recombinant collagen gel material prepared by the technical solution of the present invention could significantly improve the skin state of photoaged skin.

Claims

1. A preparation method of a recombinant collagen gel material, which comprises: Mixing recombinant collagen with a first aqueous phase to obtain a recombinant collagen solution, and drying and granulating to obtain first recombinant collagen microspheres; Physically crosslinking the first recombinant collagen microspheres to obtain second recombinant collagen microspheres; Mixing and stirring the second recombinant collagen microspheres with a second aqueous phase and swelling to obtain a dilute solution with white suspended particles; Performing low-temperature irradiation crosslinking on the dilute solution with white suspended particles to obtain a recombinant collagen gel material.

2. The preparation method according to claim 1, wherein, The amino acid sequence of the recombinant collagen is as shown in SEQ ID No: 1; Preferably, the pH of the recombinant collagen solution is < 7, and more preferably, the pH is 4 - 6; Preferably, in the recombinant collagen solution, the mass fraction of recombinant collagen is 10% - 20%; Preferably, in the dilute solution with white suspended particles, the mass fraction of the second recombinant collagen microspheres is 2% - 4%.

3. The preparation method according to claim 1, wherein The drying and granulating is carried out by a spray dryer, the spray temperature is 140 - 260 °C, the feeding speed is 200 - 500 ml / h, and the nozzle diameter is 0.75 - 1.5 mm.

4. The preparation method according to claim 1, wherein, The temperature of the physical crosslinking is 120 - 160 °C, and the time is 2 - 5 h.

5. The preparation method according to claim 1, wherein, The rotation speed of the mixing and stirring is 80 - 120 r / min, and the time is 30 - 60 min: Preferably, the temperature of the swelling is 2 - 8 °C, and the time is 6 - 10 h.

6. The preparation method according to claim 1, wherein, The irradiation crosslinking is carried out by using electron beam or 60 Co-γ ray, and the irradiation dose is 10 - 25 kGy; Preferably, when performing irradiation crosslinking, the temperature of the dilute solution with white suspended particles is 2 - 8 °C.

7. The preparation method according to claim 1, wherein, The first aqueous phase or the second aqueous phase is selected from one or a combination of two or more of ultrapure water, water for injection, and phosphate buffer solution.

8. A recombinant collagen gel material prepared by the preparation method according to any one of claims 1 - 7.

9. A recombinant collagen gel material, wherein, Calculated based on the total mass of the recombinant collagen gel material being 100%, the content of the recombinant collagen is 2% - 4%, and the content of the aqueous phase is 96% - 98%; The amino acid sequence of the recombinant collagen is as shown in SEQ ID No: 1; The recombinant collagen gel material is a colorless and transparent jelly-like gel with a porous network structure inside.

10. Use of the preparation method according to any one of claims 1 - 7 or the recombinant collagen gel material according to claim 8 or 9 in the preparation of medical materials; Preferably, the medical materials are used for medical aesthetic hydroporation, medical aesthetic filling, soft tissue repair, or anti-adhesion.

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