A recombinant collagen gel material and a preparation method and application thereof

Abstract

The application provides a recombinant collagen gel material and a preparation method and application thereof. The preparation method of the material comprises the following steps: mixing recombinant collagen with a first aqueous phase to obtain a recombinant collagen solution, 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 with a second aqueous phase to swell, to obtain a white dilute solution with suspended particles; and performing low-temperature irradiation crosslinking on the white dilute solution with suspended particles, to obtain the recombinant collagen gel material. The preparation method shortens the production cycle, greatly reduces the production cost, and the gel material prepared by the method has high biological safety, good mechanical properties and degradation performance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically the field of injectable fillers for medical aesthetics, and specifically relates to a recombinant collagen gel material, its preparation method, and its application. Background Technology

[0002] Hydrogels are three-dimensional network biomaterials composed of hydrophilic polymer chains. They swell in water but do not dissolve, exhibiting strong water absorption and retention properties, as well as biocompatibility. They have been extensively studied in fields such as biology and medical engineering. Applications include their use as biomedical materials in implantable medical devices, medical wound dressings, and drug delivery carriers in the biopharmaceutical industry. Furthermore, hydrogels serve as soft tissue fillers, repairing cavities and defects caused by soft tissue injuries or lesions, and are used in cosmetic surgery to remove wrinkles or improve facial contours.

[0003] Recombinant collagen is collagen produced through genetic engineering. Compared to naturally extracted collagen, it boasts higher purity, better biocompatibility, and a more controllable production process. The preparation methods for recombinant collagen hydrogels mainly include physical cross-linking, chemical cross-linking, and enzymatic cross-linking. Physical cross-linking achieves physical cross-linking through repeated freeze-thaw cycles or by utilizing non-covalent bonds (such as hydrogen bonds and hydrophobic interactions). Hydrogels prepared in this way exhibit good mechanical properties, but suffer from drawbacks such as a cumbersome process and difficulty in controlling the degree of cross-linking. Hydrogels prepared by chemical cross-linking often retain residual cross-linking agents and initiators, posing a risk of causing inflammation or cytotoxicity. Enzymatic cross-linking utilizes specific enzyme-catalyzed reactions to achieve cross-linking between polymer or protein molecules. While a green and efficient method, its production cost is high, making large-scale production challenging.

[0004] In summary, existing methods for preparing recombinant collagen hydrogels suffer from problems such as cumbersome processes, difficulty in controlling the degree of cross-linking, residual chemical cross-linking agents, and high costs. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a recombinant collagen gel material, its preparation method, and its applications. The preparation method of this invention significantly reduces production costs, shortens the production cycle, offers high production efficiency, is highly operable, and is easily scalable for mass production. The resulting material exhibits excellent mechanical properties, is safe and non-toxic, biodegradable, has excellent biocompatibility, and poses no viral risks.

[0006] To achieve the above objectives, in one aspect, the present invention provides a method for preparing a recombinant collagen gel material, comprising:

[0007] Recombinant collagen was mixed with the first aqueous phase to obtain a recombinant collagen solution, which was then dried and granulated to obtain the first recombinant collagen microspheres.

[0008] The first recombinant collagen microspheres were physically cross-linked to obtain the second recombinant collagen microspheres;

[0009] The second recombinant collagen microspheres were mixed and stirred with the second aqueous phase and swollen to obtain a white dilute solution with suspended particles.

[0010] A white dilute solution containing suspended particles was subjected to low-temperature irradiation crosslinking 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, transparent, jelly-like gel.

[0012] The recombinant collagen described in this invention is the recombinant human collagen disclosed in CN108070032B [A purification method for recombinant human collagen].

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

[0014] The recombinant collagen raw materials described in the specific embodiments of the present invention 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, the recombinant collagen solution contains 10%-20% recombinant collagen protein.

[0016] According to a specific embodiment of the present invention, the recombinant collagen solution has a pH < 7, preferably a pH of 4-6. According to a specific embodiment of the present invention, in the white dilute solution containing 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 white dilute solution containing suspended particles is the content of 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 are carried out using a spray dryer with a spray temperature of 140-260℃, a feed rate of 200-500ml / h, and a nozzle diameter of 0.75-1.5mm.

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

[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 mixing and stirring speed is 80-120 r / min, and the time is 30-60 min.

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

[0023] According to a specific embodiment of the present invention, the irradiation crosslinking is performed using an electron beam or 60 Co-γ rays, with an irradiation dose of 10-25 kGy.

[0024] According to a specific embodiment of the present invention, when the white dilute solution containing suspended particles is subjected to irradiation crosslinking, the temperature of the white dilute solution containing 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 more combinations of ultrapure water, water for injection, and phosphate buffer.

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

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

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

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

[0030] After freeze-drying, the recombinant collagen gel material of the present invention has a honeycomb-like porous structure in its cross-section, which is dense and facilitates 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 more combinations of ultrapure water, water for injection, and phosphate buffer;

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

[0033] In this invention, the injection force measurement conditions are as follows: 2 mL of recombinant collagen gel material sample is filled, a 27G needle is attached, and the sample is injected at a constant speed of 10 mm / min until the sample is completely ejected with a constant force value. During the experiment, the maximum force value is measured as 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 this invention, the rotational viscosity measurement conditions are as follows: take 200 ml of the sample, use a No. 5 rotor, rotate at 25 rpm, and perform viscosity measurement using a rotational viscometer at 25°C.

[0037] On the other hand, the present invention also provides the above-described preparation method or the recombinant collagen gel material prepared by the above-described preparation method or the application of the above-described 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 medical aesthetic water light therapy, medical aesthetic filling, soft tissue repair, or anti-adhesion.

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

[0040] Firstly, this invention employs a two-stage crosslinking process. The first physical crosslinking is a semi-crosslinking, which raises the unwinding temperature (denaturation temperature) of some collagen molecules. After dehydration, only some collagen molecular chains form crosslinking points. The second crosslinking is achieved through low-temperature irradiation, which generates free radicals from the amino acid residues in the recombinant collagen, causing them to recombine in a very short time. The low temperature slows down the diffusion rate of free radicals and promotes the formation of the crosslinked network, thus achieving secondary crosslinking and preparing the recombinant collagen gel material. The production process does not require the introduction of additional chemical crosslinking agents, resulting in high biosafety, non-toxicity, and excellent biocompatibility.

[0041] Secondly, achieving low-concentration recombinant collagen microspheres can achieve the same effect as high-concentration raw material preparation, greatly reducing production costs, making it highly operable and easy to scale up production.

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

[0043] Fourth, low-temperature irradiation cross-linking can effectively prevent bacteria, extend storage time, and ensure the quality of hydrogels.

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

[0045] Sixth, it has a simple composition, containing only one type of recombinant collagen, and has high biosafety. Attached Figure Description

[0046] Figure 1 The images shown are SEM images of the material prepared in Example 1, where (A) is a scanning electron microscope image (5000x) of the first recombinant collagen microspheres, and (B) is a scanning electron microscope image (100000x) of the internal porous structure of the first recombinant collagen microspheres; and (C) and (D) are scanning electron microscope images (200x) of the cross-sections of the recombinant collagen gel material prepared in Example 1 after one cross-linking (C) and two cross-linkings (D) and after freeze-drying.

[0047] Figure 2 Comparison of the appearance of samples prepared after primary and secondary crosslinking of the recombinant collagen gel material prepared in Example 1;

[0048] Figure 3 The prepared recombinant collagen gel material samples were loaded into a syringe and injected into a glass dish. The morphology of the samples after injection was observed. Among them, A is the morphology of the sample after injection of Example 1, B is the morphology of the sample after injection of Comparative Example 4, and C is the morphology of the sample after injection of Comparative Example 7.

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

[0050] Figure 5 The image shows the effect of photoaging model treatment using the recombinant collagen gel material prepared in Example 1.

[0051] Figure 6 The image shows the effect of photoaging model treatment using the recombinant collagen gel material prepared in Comparative Example 1.

[0052] Figure 7 Statistical results of skin elasticity, collagen content, and moisture content in mice with a photoaging model treated with collagen gel material prepared in Example 1 at 0w, 1w, and 2w.

[0053] Figure 8 Statistical results of skin elasticity, collagen content, and moisture content in mice with a photoaging model treated with recombinant collagen gel material prepared for Comparative Example 1 at 0w, 1w, and 2w.

[0054] Figure 9 These are images of hematoxylin-eosin stained mice 2 weeks after aging with the recombinant collagen gel material prepared in Example 1. Detailed Implementation

[0055] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0056] In actual production, those skilled in the art will understand that, provided that qualified products can be obtained, some related process steps can be adjusted or added or subtracted.

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0059] GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPG KPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSN GPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGS QGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP(SEQ ID No:1).

[0060] Example 1

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

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

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

[0064] (3) Take 2g of the first recombinant collagen microspheres for physical cross-linking, which is a single half cross-linking. The cross-linking conditions are: temperature: vacuum drying oven 140℃, time: 3h, vacuum degree: -0.1MPa, to obtain 2g of the second recombinant collagen microspheres.

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

[0066] (5) The white dilute solution with suspended particles was filled into vials and placed in a foam box containing ice packs (to maintain 4°C) for irradiation sterilization, which is a secondary low-temperature irradiation crosslinking. The irradiation crosslinking uses a high-energy electron beam with an irradiation dose of 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 method for preparing recombinant collagen gel material:

[0069] Other conditions were the same as in Example 1, except that the temperature for physical cross-linking of the first recombinant collagen microspheres, the concentration of the second recombinant collagen microspheres, the swelling conditions, and the irradiation cross-linking conditions are shown in Table 1. Colorless, transparent, jelly-like recombinant collagen gel materials of Examples 2-5 were prepared.

[0070] Comparative Examples 1-5

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

[0072] Other conditions were the same as in Example 1, except that the temperature for physical cross-linking 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, thus preparing recombinant collagen gel materials for Comparative Examples 1-5. The recombinant collagen in Comparative Example 1 was commercially available recombinant type III humanized collagen (purchased from Xi'an Denuo Haisi 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 are the same as in Example 1, except that: 1. The recombinant collagen solution is freeze-dried into a recombinant collagen sponge, then physically cross-linked, frozen with liquid nitrogen for 3-5 minutes, and then ground with a mortar to obtain recombinant collagen powder. After mixing with water for injection, a white dilute solution with suspended particles is obtained.

[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 in Example 1, except that the material prepared in Comparative Example 7 was prepared using conventional methods (high concentration), which involved a high physical cross-linking temperature, a high concentration of recombinant collagen microspheres, and a long swelling time. The prepared material was terminally sterilized by irradiation sterilization or moist heat sterilization, without secondary cross-linking. 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 are shown in Table 1. The recombinant collagen gel material of Comparative Example 7 was thus prepared.

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

[0080]

[0081] Example of effect

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

[0083] 1. Scanning Electron Microscopy: The recombinant collagen microspheres granulated by spraying in Example 1, the prepared white dilute solution containing suspended particles, and the recombinant collagen gel material were freeze-dried, placed in liquid nitrogen for 3-5 minutes, and then broken apart. The cross-sections were observed using a scanning electron microscope. The results are as follows: Figure 1As shown: A: After spray granulation, the recombinant collagen forms microspheres with a diameter of approximately 10 μm; B: The microspheres have a porous network structure inside; C: In the white dilute solution with suspended particles, some collagen molecular chains form cross-linking points (as shown in the box); D: The cross-section of the recombinant collagen gel material is porous and honeycomb-like, dense; the porous network structure is conducive to the diffusion of nutrients and promotes cell proliferation.

[0084] 2. Appearance Testing Method: The appearance of the products prepared before and after secondary crosslinking in Examples 1-5 and Comparative Examples 1-7 was compared. The comparison results are shown in Table 2. The appearance comparison of the products prepared before and after secondary crosslinking in Example 1 is shown in Table 2. Figure 2 ,in, Figure 2 In this context, 'a' represents the solution after the first physical semi-crosslinking preparation and the state after swelling. Figure 2 In this context, 'b' represents the state after low-temperature irradiation crosslinking.

[0085] 3. Morphology after sample injection: The gel materials prepared in Examples 1-5 and Comparative Examples 4 and 6 were injected into glass dishes using syringes, and the morphology of the samples was observed. The results are shown in Table 2. The morphology of the sample after injection in Example 1 is as follows: Figure 3 As shown in A, the post-injection morphology of the samples in Examples 2-5 is basically the same as that in Example 1; the post-injection morphology of the sample in Comparative Example 4 is as shown in Figure 1. Figure 3 As shown in B; the morphology of the sample in Comparative Example 7 after injection is as shown in Figure B. Figure 3 As shown in C.

[0086] Table 2 Appearance Test Results

[0087]

[0088] Note: " / " indicates that the sample is in a dilute solution state and cannot be molded after injection; it is a flowing liquid state.

[0089] 4. The injection force was tested using a universal mechanical testing machine. A 2ml sample filled in Example 1 was fitted with a 27G needle and fixed on the mechanical testing machine. The sample was injected at a constant speed of 10mm / min until it was ejected with a constant force. The injection force was recorded as a function of time during the injection process. The maximum force during the injection process was recorded 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 Example 1, from Figure 4 It can be obtained that the injection force of the recombinant collagen gel material is approximately 16N.

[0090] 5. Osmotic pressure: Take 300 μl of sample and test it according to the general rules (0632 Osmotic pressure molar concentration determination method-1 Determination of osmotic pressure molar concentration) of the Chinese Pharmacopoeia (2020 edition, Part IV). 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, which is close to the ideal state of human physiology and activity.

[0091] 6. Rotational viscosity: Take 200 ml of sample and use a rotational viscometer with rotor No. 5 at 25 rpm at 25 ℃ to measure the viscosity. 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 materials prepared in the examples and comparative examples

[0093] 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: Tested according to GB / T 16886: The samples prepared in Examples 1-5 and Comparative Examples 1-7 were subjected to cytotoxicity experiments. The results are shown in Table 4. When the extract of the recombinant collagen gel material prepared in the comparative examples and examples was used to treat L929 cells for 24 hours, the cell viability was above 85% and there was no obvious cytotoxicity.

[0095] Table 4 Cytotoxicity Results

[0096]

[0097] 8. Photoaging Model Treatment Effect Test: 1 ml of the sample from Example 1 and Comparative Example 1 was filled into the dorsal skin of SKH-1 photoaging model rats. A 34G needle was attached to the needle, and 1 ml of the solution was injected evenly into the dermal layer. In the blank control group, 1 ml of physiological saline was injected evenly into the dorsal skin of SKH-1 photoaging model rats. At weeks 0 (0w), 1 (1w), and 2 (2w), the exposed dorsal skin, wrinkles, pigmentation, and texture of the rats were recorded weekly using a digital camera and digital microscope. Results are shown in [link to results]. Figure 5 , Figure 6 Simultaneously, a skin-assisted analyzer (skin analysis system, CBS-806, Wuhan Boshi Electronics Co., Ltd., Wuhan, China) was used to detect skin condition. Epidermal 3D images were taken weekly to measure three key parameters representing skin condition: skin elasticity, collagen content, and moisture content. Three measurement points were evenly distributed on the dorsal skin, with three measurements taken at each point to minimize measurement error. The average value was calculated based on the measurement data. See [link to results]. Figures 5 to 8 .

[0098] 9. Histological Results of Photoaging Model Treatment: Two weeks after treatment, tissue samples (approximately 1 cm x 1 cm) from the injection areas of the blank control group and the experimental group were excised, rinsed with physiological saline, flattened, and placed in an embedding cassette. The tissues were fixed in 4% formaldehyde for at least 24 hours, then embedded in paraffin and sectioned into 5 μm sections. The distribution of cells and matrix in the injection area and the presence of inflammation were observed using hematoxylin-eosin staining (HE method). Results are shown in [link to results]. Figure 9 ,in Figure 9 'a' in the text represents the blank control. Figure 9 In this context, b represents the experimental group.

[0099] Experimental results

[0100] 1. Results of treatment efficacy test on photoaging model:

[0101] Figure 5 , Figure 6 The images show the effects of photoaging treatment on the gel materials prepared in Example 1 and Comparative Example 1, respectively.

[0102] like Figure 5 The results showed that, compared with the blank control group (saline group), the gel material prepared in Example 1 had fewer wrinkles, finer skin texture, and more radiance after 2 weeks of injection, and the skin was relatively smooth. This indicates that the recombinant collagen gel material prepared in Example 1 can significantly improve the macroscopic manifestations of skin photoaging.

[0103] like Figure 6 The results showed that after 1 week of injection, the gel material prepared in Comparative Example 1 caused ulceration at the injection sites on the skin of the model mice. After 2 weeks of injection, compared with the control group (saline group), the skin texture was rough and dull, indicating that the gel material prepared in Comparative Example 1 was less effective than the material in Example 1 in significantly improving the macroscopic manifestations of skin photoaging.

[0104] Table 5 Figure 7 Statistical results of skin elasticity, collagen content, and moisture content in mice with a photoaging model 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 showed that the experimental group (material of Example 1) had significantly higher increases in collagen content, moisture content, and skin elasticity than the control group (0w, 1w, 2w, respectively), indicating that the recombinant collagen gel material prepared in Example 1 can significantly improve the condition of photo-aged skin.

[0108] Table 6 Figure 8 Statistical results of skin elasticity, collagen content, and moisture in mice with photoaging model prepared as Comparative Example 1 at 0w, 1w, and 2w.

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

[0110]

[0111] Table 6, Figure 8 The results showed that the experimental group (material of Comparative Example 1) did not show a significant increase in collagen, moisture and elasticity compared to the control group (0w, 1w and 2w), indicating that the gel material prepared in Comparative Example 1 could not significantly improve the skin condition caused by photoaging.

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

[0113] Figure 9 Images of hematoxylin-eosin stained mice 2 weeks after aging with the recombinant collagen gel material prepared in Example 1. Figure 9 'a' in the text represents the blank control. Figure 9 In this context, 'b' represents the experimental group (materials from Example 1). Figure 9 Histological results showed that the recombinant collagen material prepared in Example 1 had good biocompatibility, with no obvious inflammation or granulomas. Furthermore, the collagen fibers in the experimental group were significantly more tightly arranged than those in the blank control group, indicating that the recombinant collagen gel material prepared by the technical solution of the present invention can significantly improve the condition of photo-aged skin.

Claims

1. A method for preparing a recombinant collagen gel material, comprising: Recombinant collagen was mixed with the first aqueous phase to obtain a recombinant collagen solution, which was then dried and granulated to obtain the first recombinant collagen microspheres. The first recombinant collagen microspheres were physically cross-linked to obtain the second recombinant collagen microspheres; The second recombinant collagen microspheres were mixed and stirred with the second aqueous phase and swollen to obtain a white dilute solution with suspended particles. A white dilute solution containing suspended particles was subjected to low-temperature irradiation crosslinking to obtain a recombinant collagen gel material. The amino acid sequence of the recombinant collagen is shown in SEQ ID No:1; The drying and granulation are carried out using a spray dryer with a spray temperature of 140-260℃, a feed rate of 200-500 ml / h, and a nozzle diameter of 0.75-1.5 mm. The physical cross-linking temperature is 120-160℃, and the time is 2-5 hours; The swelling temperature is 2-8℃, and the time is 6-10h; In the white dilute solution containing suspended particles, the mass fraction of the second recombinant collagen microspheres is 2%-4%. The recombinant collagen gel material is a colorless, transparent, jelly-like gel.

2. The preparation method according to claim 1, wherein, The pH of the recombinant collagen solution is 4-6.

3. The preparation method according to claim 1, wherein, The recombinant collagen solution contains 10%-20% recombinant collagen protein.

4. The preparation method according to claim 1, wherein, The mixing speed is 80-120 r / min, and the time is 30-60 min.

5. The preparation method according to claim 1, wherein, The irradiation crosslinking is performed using an electron beam or... 60 Co-γ rays, with an irradiation dose of 10-25 kGy.

6. The preparation method according to claim 1, wherein, During irradiation crosslinking, the temperature of the white, dilute solution with suspended particles is 2-8 ℃.

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

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

9. A recombinant collagen gel material, wherein, Based on the total mass of the recombinant collagen gel material as 100%, the content of the recombinant collagen is 2%-4%, and the aqueous phase content is 96%-98%. The amino acid sequence of the recombinant collagen is shown in SEQ ID No:1; The recombinant collagen gel material is a colorless, transparent, jelly-like gel with a porous mesh structure inside; The recombinant collagen gel material is injected with a force of 15-20N using a 27G needle.

10. The application 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 for medical aesthetic water light therapy, medical aesthetic filling, soft tissue repair or anti-adhesion.

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