Implantable recombinant collagen microparticles and methods of making the same

Abstract

The application discloses an implantable recombinant collagen micro-particle and a preparation method thereof. Although the materials used in the field of soft tissue filling, such as animal collagen and sodium hyaluronate, have good effects, there are still problems of low biological safety, crosslinking agent residue and short maintenance time. In order to solve the above problems of the existing medical and cosmetic materials, the application adopts a method combining self-assembly and physical crosslinking to prepare an implantable recombinant collagen micro-particle. The micro-particle is completely swelled after adding an aqueous solvent to form an injectable milky white opaque or colorless transparent gel. Compared with traditional medical and cosmetic fillers, the gel prepared by the application has the characteristics of good biocompatibility, a long degradation period, a simple preparation process and easy scale production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to an implantable recombinant collagen microparticle and its preparation method. Background Technology

[0002] Hydrogels are polymeric materials with a three-dimensional network structure and water as the dispersion medium. They possess excellent biocompatibility, porous structure, permeability, and hydrophilicity, and can mimic the natural extracellular matrix, providing a suitable microenvironment for cells. Their applications in tissue repair and regeneration are increasingly widespread. Hydrogels used in the biomedical field can be classified into natural hydrogels and synthetic hydrogels based on their origin. Natural hydrogels, including collagen, gelatin, hyaluronic acid, and chitosan, have good biocompatibility and biodegradability, but poor mechanical properties and are limited by potential immunogenicity in the exogenous host. Synthetic hydrogels, such as polyethylene glycol derivatives, polycaprolactone, and polyvinyl alcohol, are characterized by stable composition, controllable structure, and low immunogenicity, and can withstand strong mechanical loads, thus becoming a research hotspot. Hydrogels can serve as carrier scaffolds providing regenerative templates or matrices. On the one hand, cells can adhere and proliferate on them, coordinating their responses to regenerate damaged tissue after injury; on the other hand, the additional stimulation from drugs, cytokines, and stem cells carried on them can promote tissue regeneration. Currently, the natural polymer materials used to prepare hydrogels are mainly animal collagen and sodium hyaluronate.

[0003] Collagen is a biological macromolecule and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein. Due to its good biocompatibility, biodegradability, and bioactivity, collagen is widely used in medicine, tissue engineering, cosmetics, and other fields. However, commonly used collagen is derived from animal tissues such as pig skin, cow skin, and beef tendon. Animal collagen may contain viral residues and cause immune rejection. Therefore, some users still experience rejection issues such as redness, swelling, allergies, and chronic inflammation at the application site.

[0004] Hydrogels prepared by chemical cross-linking exhibit better stability and higher flexibility. However, because the cross-linking process requires the use of chemical cross-linking agents such as 1,4-butanediol diglycidyl ether and sodium β-glycerophosphate, incomplete removal can cause harm to the human body. Enzymes, as biological macromolecules, are widely present in living organisms. They are safe, non-toxic, highly efficient at catalysis, and can form stable chemical bonds. Compared to traditional cross-linking agents, enzymes are more suitable for use in the human body. However, enzymes are often expensive, which may be a limiting factor for large-scale applications. Furthermore, the poor stability of enzymes may affect the efficiency of the cross-linking reaction and the consistency of the product.

[0005] To avoid the cytotoxic effects associated with chemical cross-linking agents and due to cost issues, physical cross-linking methods can be used. These methods can prevent exogenous substances from entering the collagen, but they provide a lower degree of cross-linking. Summary of the Invention

[0006] The purpose of this invention is to prepare implantable recombinant collagen microparticles to supplement the deficiencies of existing hydrogels and soft tissue filler materials. These implantable recombinant collagen microparticles use recombinant collagen as a raw material and are obtained through a combination of self-assembly and physical cross-linking. Furthermore, by changing the pH of the recombinant collagen solution, rod-shaped or spherical recombinant collagen microparticles can be obtained. They are safe, non-toxic, biodegradable, have excellent biocompatibility, and pose no viral risks. The method of this invention not only avoids the introduction of new chemical substances and eliminates any chemical reagent residues, resulting in better biosafety, but also utilizes the unique self-assembly capability of recombinant collagen raw material for self-assembly, combined with physical cross-linking, to achieve a higher degree of cross-linking. The specific technical solution adopted in this invention is as follows:

[0007] The first aspect of this invention provides a method for preparing implantable recombinant collagen microparticles, comprising:

[0008] (1) Dissolve recombinant collagen in water to obtain a recombinant collagen solution;

[0009] (2) The recombinant collagen solution is dried and granulated to obtain the first recombinant collagen microparticles;

[0010] (3) The first recombinant collagen microparticles are subjected to dry heat crosslinking to obtain the second recombinant collagen microparticles.

[0011] In some implementations, in step (1), a certain amount of recombinant collagen is dissolved in water and stirred until completely dissolved, resulting in a transparent solution and an aqueous solution of recombinant collagen. This recombinant collagen can be rapidly dispersed into nanoscale particles in water and has super hydrophilicity. The collagen molecules have regular hydrophilic groups on the outside, which have super aggregation ability, and hydrophobic groups on the inside, which can form microscaffolds.

[0012] In some implementations, step (2) involves using a spray dryer to dry and granulate the recombinant collagen solution.

[0013] In some implementations, step (2) further includes adjusting the pH of the recombinant collagen solution to less than 7 or greater than 7.

[0014] In some embodiments, the first recombinant collagen microparticles are rod-shaped or spherical.

[0015] In some embodiments, the pH of the recombinant collagen solution is adjusted to less than 7, and the first recombinant collagen microparticles have a spherical structure.

[0016] In some embodiments, the diameter of the spherical structure is 2 to 10 μm, and the interior is a porous mesh structure.

[0017] In some embodiments, the pH of the recombinant collagen solution is adjusted to be greater than 7, and the first recombinant collagen microparticles have a rod-shaped structure.

[0018] In some embodiments, the diameter of the rod-shaped structure is 2 to 5 μm.

[0019] This invention uses a spray dryer for drying. During the drying process, due to the special structure of the recombinant collagen raw material, it can spontaneously assemble into a microscaffold and self-aggregate to form a microparticle structure. Therefore, during the heating and drying process, the protein chains rapidly lose water, move closer to each other, and form a large number of hydrogen bonds, which enhances the self-assembly effect of the recombinant collagen. This process completes the self-assembly of the recombinant collagen. At this time, the recombinant collagen microparticles are dissolved in water and are in a sol state.

[0020] In some embodiments, the drying temperature of the spray dryer is 100-200°C, for example, it can be 120°C, 130°C, 140°C, 150°C, 170°C, 180°C, etc. More preferably, the drying temperature of the spray dryer is 120-190°C.

[0021] In some implementations, dry thermal crosslinking is performed in a vacuum drying oven in step (3).

[0022] This invention involves dry heat crosslinking of first recombinant collagen microparticles in a vacuum drying oven to increase the unwinding temperature, i.e., denaturation temperature, of collagen. After dehydration, crosslinking occurs between collagen molecules, which can significantly improve the mechanical properties of collagen, resulting in second recombinant collagen microparticles with high strength and degradation resistance.

[0023] In some embodiments, the temperature for dry heat crosslinking in the vacuum drying oven is 90–250°C, for example, 100°C, 140°C, 180°C, 200°C, 240°C, etc. More preferably, the temperature of the vacuum drying oven is 100–240°C.

[0024] In some implementations, the dry heat crosslinking time in the vacuum drying oven is 1 to 10 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 9 hours, etc. More preferably, the vacuum drying time is 2 to 9 hours.

[0025] The recombinant collagen of this invention is the recombinant human collagen disclosed in CN108070032B [A purification method for recombinant human collagen].

[0026] In some embodiments, the amino acid sequence of the recombinant collagen is shown in SEQ ID No:1.

[0027] The recombinant collagen raw materials described in the specific embodiments of the present invention are all proteins having the amino acid residue sequence shown in SEQ ID No:1 in the sequence listing.

[0028] In some implementations, recombinant human collagen is obtained through plasmid preparation, mixing and transforming the plasmid with Pichia pastoris, screening for multi-copy insertion recombinants, fermentation, and purification. This type of collagen exhibits good biocompatibility and a purity exceeding 95%. Because its structure and function are extremely similar to human collagen, its use can effectively address the viral risks associated with extracting collagen from animal tissues. Furthermore, this recombinant collagen can rapidly disperse into nanoscale particles in water, exhibiting superior hydrophilicity. The collagen molecules have regular hydrophilic groups on the outside, providing strong aggregation ability, and hydrophobic groups on the inside, enabling the formation of microscaffolds. Therefore, this recombinant collagen possesses excellent self-assembly capabilities.

[0029] In some embodiments, the concentration of the recombinant collagen solution is 5-20% (wt), for example, it can be 5% (wt), 7% (wt), 10% (wt), 12% (wt), 15% (wt), 18% (wt), 20% (wt), etc., and more preferably, the concentration of the recombinant collagen solution is 8-15% (wt).

[0030] A second aspect of the present invention provides an implantable recombinant collagen microparticle, wherein the amino acid sequence of the recombinant collagen in the recombinant collagen microparticle is shown in SEQ ID No:1.

[0031] In some embodiments, the implantable recombinant collagen microparticles are prepared by the preparation method described in the first aspect of the present invention.

[0032] The third aspect of the present invention provides a method for preparing recombinant collagen gel, which includes adding an aqueous phase to the second recombinant collagen microparticles (implantable recombinant collagen microparticles) described in the first aspect of the present invention, and obtaining the recombinant collagen gel after swelling.

[0033] In some embodiments, the recombinant collagen gel is a milky white, opaque gel or a colorless, transparent, jelly-like gel.

[0034] In some embodiments, the pH of the recombinant collagen solution is adjusted to less than 7, the first recombinant collagen microparticles are spherical, and an aqueous phase is added to the second recombinant collagen microparticles (implantable recombinant collagen microparticles). After swelling, the recombinant collagen gel is a milky white, opaque gel.

[0035] In some embodiments, the pH of the recombinant collagen solution is adjusted to be greater than 7, the first recombinant collagen microparticles are rod-shaped, and an aqueous phase is added to the second recombinant collagen microparticles (implantable recombinant collagen microparticles). After swelling, the recombinant collagen gel is a colorless, transparent, jelly-like gel.

[0036] In some embodiments, the second recombinant collagen microparticles described in the first aspect of the invention need to be fully swollen after the aqueous phase is added.

[0037] In some implementations, the swelling time required for complete swelling is 10 to 15 hours.

[0038] In some embodiments, the aqueous phase is selected from one or more of ultrapure water, water for injection, and phosphate buffer, preferably water for injection.

[0039] Recombinant collagen microparticles obtained through two processes—self-assembly and dry-heat cross-linking—are water-insoluble implantable recombinant collagen microparticles. When a certain amount of water solvent is added, after complete swelling, they form a milky white, opaque gel or a colorless, transparent, jelly-like gel, both referred to as recombinant collagen gel. In some embodiments, the concentration of the recombinant collagen gel is 5–15% (wt), for example, 5% (wt), 8% (wt), 10% (wt), 12% (wt), 15% (wt), etc. More preferably, the concentration of the recombinant collagen gel is 8–13% (wt).

[0040] In some embodiments, the method for preparing the recombinant collagen gel includes:

[0041] An aqueous phase is added to the second recombinant collagen microparticles (implantable recombinant collagen microparticles) described in the first aspect of the present invention, and the mixture is stirred at a speed of 100-150 r / min for 1-4 h, and then swollen at a temperature of 2-8°C for 10-15 h to obtain a recombinant collagen gel.

[0042] In some embodiments, the method for preparing the recombinant collagen gel further includes sterilizing the recombinant collagen gel.

[0043] In some implementations, the sterilization conditions are: 120°C moist heat sterilization for 30 minutes in a high-temperature, high-pressure steam sterilizer.

[0044] A fourth aspect of the present invention provides a recombinant collagen gel, wherein the amino acid sequence of the recombinant collagen in the recombinant collagen gel is shown in SEQ ID No:1.

[0045] In some embodiments, the recombinant collagen gel is prepared by the preparation method described in the third aspect of the present invention.

[0046] In some embodiments, the pH value of the recombinant collagen gel is 5 to 7.

[0047] In this invention, the implantable recombinant collagen microparticles (spherical structure) obtained by the preparation method described herein, after complete swelling, form a milky white, opaque gel that can maintain its shape in vivo for more than 6 months, exhibiting a long degradation period. Furthermore, when injected into tissues, it is not prone to displacement and possesses strong support properties. The colorless, transparent, jelly-like gel obtained by the method of this application, after complete swelling, can also be used in cosmetic procedures such as mesotherapy.

[0048] The fifth aspect of this invention provides the application of the implantable recombinant collagen microparticles described in the second aspect of this invention or the recombinant collagen gel described in the fourth aspect of this invention in the preparation of medical materials;

[0049] In some implementations, the medical material is used for cosmetic filling, cosmetic surgery, soft tissue repair, or anti-adhesion.

[0050] Beneficial effects:

[0051] The implantable recombinant collagen microparticles prepared using the method described in this application are non-toxic, residue-free, and contain only recombinant collagen as a raw material, resulting in high safety compared to traditional chemical cross-linking methods. Compared to traditional enzymatic cross-linking methods (such as TG enzyme), they exhibit a higher cross-linking rate and lower cost. The combination of self-assembly and physical cross-linking achieves a higher degree of cross-linking, overcoming the drawback of low cross-linking degree in physical cross-linking. The implantable recombinant collagen microparticles (spherical structure) prepared using this method, after complete swelling, form a milky white, opaque gel. When injected into the target location, they provide strong support and are less prone to displacement, maintaining their position in vivo for over 6 months, exhibiting a long degradation cycle and good biocompatibility. The implantable recombinant collagen microparticles (rod-shaped structure) prepared using this method, after complete swelling, can form a colorless, transparent, jelly-like gel, showing good application potential in aesthetic procedures such as mesotherapy. The preparation process is simple, easy to scale up for mass production, and highly suitable for applications in aesthetic filling, aesthetic surgery, soft tissue repair, and anti-adhesion. Attached Figure Description

[0052] Figure 1 This is an image of the recombinant collagen gel.

[0053] Figure 2 This is a transmission electron microscope image of recombinant collagen raw material. Figure 2 (a) Scanning electron micrograph of implanted recombinant collagen microparticles (spherical structure) Figure 2 (b) Scanning electron micrograph of implanted recombinant collagen microparticles (rod-like structure). Figure 2 c);

[0054] Figure 3 This is a hematoxylin-eosin staining image of subcutaneous tissue on the back of a rat;

[0055] Figure 4 This is a schematic diagram illustrating the swelling properties of implantable recombinant collagen microparticles;

[0056] Figure 5 This is a schematic diagram of the injection force of implanted recombinant collagen microparticles;

[0057] Figure 6 This is a schematic diagram of the in vivo degradation of implanted recombinant collagen microparticles. Detailed Implementation

[0058] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0059] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0060] Unless otherwise stated, the experimental methods, detection methods, and preparation methods not described in detail in this invention all adopt conventional techniques in this technical field.

[0061] The recombinant collagen used in the following examples is from Shaanxi Huikang Biotechnology Co., Ltd., and its amino acid sequence is as follows: GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAG NQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP(SEQ ID No:1), and other materials and formulations used, unless otherwise specified, are commercially available.

[0062] Example 1

[0063] Step 1: Weigh 50g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 450g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to acidic (pH=5.24) to obtain a 10% (wt) recombinant collagen solution.

[0064] Step 2: The above solution is dried and granulated using a spray dryer at 120°C to obtain spherical recombinant collagen microparticles with a diameter of 2-10 μm.

[0065] Step 3: The first recombinant collagen microparticles with spherical structure are subjected to dry heat cross-linking in a vacuum drying oven at 100°C for 2 hours to obtain the second recombinant collagen microparticles with spherical structure (implantable recombinant collagen microparticles) with high strength.

[0066] Step 4: Weigh 1g of the spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 12.5g of water for injection, swell in a 4℃ refrigerator for 12h, and stir at 120r / min for 2h. The resulting milky white opaque recombinant collagen gel has a concentration of 8% (wt) and a pH of 6.7.

[0067] Example 2

[0068] Step 1: Weigh 60g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 440g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to acidic (pH=4.85) to obtain a 12% (wt) recombinant collagen solution.

[0069] Step 2: The above solution is dried and granulated using a spray dryer at 140°C to obtain spherical recombinant collagen microparticles with a diameter of 2-10 μm.

[0070] Step 3: The first recombinant collagen microparticles with spherical structure are subjected to dry heat cross-linking in a vacuum drying oven at 120°C for 3 hours to obtain the second recombinant collagen microparticles with spherical structure (implantable recombinant collagen microparticles) with high strength.

[0071] Step 4: Weigh 1g of the spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 10g of water for injection, swell in a 4℃ refrigerator for 12h, and stir at 120r / min for 2h. The resulting milky white opaque recombinant collagen gel has a concentration of 10% (wt) and a pH of 6.9.

[0072] Example 3

[0073] Step 1: Weigh 65g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 435g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to acidic (pH=5.32) to obtain a 13% (wt) recombinant collagen solution.

[0074] Step 2: The above solution is dried and granulated using a spray dryer at 165°C to obtain spherical recombinant collagen microparticles with a diameter of 2-10 μm.

[0075] Step 3: The first recombinant collagen microparticles with spherical structure are subjected to dry heat cross-linking in a vacuum drying oven at 135℃ for 4 hours to obtain the second recombinant collagen microparticles with spherical structure (implantable recombinant collagen microparticles) with high strength.

[0076] Step 4: Weigh 1.1g of the spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 9.9g of water for injection, swell in a refrigerator at 4℃ for 12h, and stir at 120r / min for 2h. The resulting milky white opaque recombinant collagen gel has a concentration of 11% (wt) and a pH of 6.6.

[0077] Example 4

[0078] Step 1: Weigh 70g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 430g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to acidic (pH=4.62) to obtain a 14% (wt) recombinant collagen solution.

[0079] Step 2: The above solution is dried and granulated using a spray dryer at 175°C to obtain spherical recombinant collagen microparticles with a diameter of 2-10 μm.

[0080] Step 3: The first recombinant collagen microparticles with spherical structure are subjected to dry heat cross-linking in a vacuum drying oven at 170℃ for 3 hours to obtain the second recombinant collagen microparticles with spherical structure (implantable recombinant collagen microparticles) with high strength.

[0081] Step 4: Weigh 1.2g of the spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 9.8g of water for injection, swell in a refrigerator at 4℃ for 12h, and stir at 120r / min for 2h. The resulting milky white opaque recombinant collagen gel has a concentration of 12% (wt) and a pH of 6.8.

[0082] Example 5

[0083] Step 1: Weigh 75g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 425g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to acidic (pH=4.287) to obtain a 15% (wt) recombinant collagen solution.

[0084] Step 2: The above solution is dried and granulated using a spray dryer at 180°C to obtain spherical recombinant collagen microparticles with a diameter of 2-10 μm.

[0085] Step 3: The first recombinant collagen microparticles with spherical structure are subjected to dry heat cross-linking in a vacuum drying oven at 190℃ for 2 hours to obtain the second recombinant collagen microparticles with spherical structure (implantable recombinant collagen microparticles) with high strength.

[0086] Step 4: Weigh 1.3g of the spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 9.7g of water for injection, swell in a refrigerator at 4℃ for 12h, and stir at 120r / min for 2h. The resulting milky white opaque recombinant collagen gel has a concentration of 13% (wt) and a pH of 6.5.

[0087] Example 6

[0088] Step 1: Weigh 50g of recombinant collagen raw material (protein with the amino acid residue sequence shown in SEQ ID No:1), dissolve it in 450g of water for injection, stir at room temperature for 4 hours until completely dissolved, and form a colorless and transparent solution. Adjust the pH to alkaline (pH=8.87) to obtain a 10% (wt) recombinant collagen solution.

[0089] Step 2: The above solution is dried and granulated using a spray dryer at 120°C to obtain first recombinant collagen microparticles with rod-shaped structures of approximately 2-5 μm in diameter and varying lengths.

[0090] Step 3: The first recombinant collagen microparticles with rod-shaped structure are subjected to dry heat cross-linking in a vacuum drying oven at 100°C for 2 hours to obtain the second recombinant collagen microparticles with cross-linked rod-shaped structure (implantable recombinant collagen microparticles).

[0091] Step 4: Weigh 1g of the cross-linked rod-shaped second recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through the two processes of self-assembly and dry heat cross-linking, add 12.5g of water for injection, swell in a refrigerator at 4℃ for 12h, and stir at 120r / min for 2h. The resulting colorless and transparent jelly-like recombinant collagen gel has a concentration of 8% (wt) and a pH of 7.

[0092] Comparative Example 1

[0093] Except for omitting the self-assembly step, it is basically the same as in Example 1.

[0094] Comparative Example 2

[0095] Except for omitting the dry heat crosslinking step, it is basically the same as in Example 1.

[0096] Comparative Example 3

[0097] Except that the recombinant collagen was replaced with commercially available recombinant collagen (purchased from Xi'an Denuo Haisi Medical Technology Co., Ltd., batch number: 2308B01), the rest was basically the same as in Example 1.

[0098] Effect Experiment

[0099] 1. Comparative test of the appearance of implanted recombinant collagen microparticles after they have fully swollen into a gel.

[0100] (1) After the implantable recombinant collagen microparticles were completely swollen into a gel with water, 2 ml of the gel was placed in a transparent glass bottle, ensuring that the sample was uniform and free of air bubbles. Under standard fluorescent lighting, the appearance of the material was photographed using a digital camera, and the transparency, color, and flowability of the material were compared. The gels prepared in Examples 1-6 and Comparative Examples 1-3 after complete swelling of the implantable recombinant collagen microparticles were compared, and their appearance and state were observed. The experimental results are shown in Table 1.

[0101] Table 1. Comparison of appearance after implanted recombinant collagen microparticles have fully swollen into gel.

[0102]

[0103] The above experimental results show that the implantable recombinant collagen microparticles in Examples 1-5, after complete swelling with water, all exhibited an opaque, milky-white gel state with low fluidity. The implantable recombinant collagen microparticles in Example 6, after complete swelling, formed a colorless, transparent, jelly-like gel. Comparative Examples 1 and 3 also exhibited a milky-white gel state with low fluidity, while Comparative Example 2 formed a transparent, colorless sol state with high fluidity. The experimental results are as follows: Figure 1 As shown.

[0104] Recombinant collagen raw materials are rapidly dispersed into nanoscale particles in water, as shown in transmission electron microscopy. Figure 2 In Example a, scanning electron microscope (SEM) images of the surface and internal structure of the implantable recombinant collagen microparticles (spherical structures) obtained in step 1 are shown below. Figure 2 In Figure b, a scanning electron microscope image of the implantable recombinant collagen microparticles (rod-shaped structure) obtained in step 6 of Example 6 is shown below. Figure 2 c.

[0105] 2. Biocompatibility testing of implantable recombinant collagen microparticles

[0106] (1) Twenty-four healthy adult SD rats were randomly divided into eight groups of three. The rats' backs were dehaired and prepared with a depilatory agent. After cleaning the skin with water, the prepared skin was disinfected with iodine and then deiodized with 75% alcohol. The implantable recombinant collagen microparticles prepared in Examples 1-5 and 1-3 were completely dissolved in water to form a gel and then injected into the rats in each group. 0.2 mL was injected subcutaneously into each rat. After being housed in the same environment for one week, the injected rats were euthanized by cervical dislocation. The injected epidermis on their backs was collected for pathological analysis to observe the inflammatory response. The experimental results are shown in Table 2.

[0107] Table 2 Results of Inflammation Response Test

[0108]

[0109] The above experimental results show that rats injected with the gel after complete swelling of implantable recombinant collagen microparticles in Examples 1-5 basically did not produce an inflammatory response. The inflammatory response after injection with the gel after complete swelling of implantable recombinant collagen microparticles in Comparative Examples 1 and 2 was also relatively mild, while the inflammatory response after injection with the gel after complete swelling of implantable recombinant collagen microparticles in Comparative Example 3 was more severe.

[0110] After sampling, the skin in which the experimental material was injected was fixed in neutral formaldehyde. Then, it was stained using the hematoxylin-eosin staining method, photographed under a regular optical microscope, and subjected to pathological tissue analysis. The experimental results are as follows: Figure 3 As shown.

[0111] Experimental results showed that one week after subcutaneous injection into rats, the rats in the example group showed no obvious inflammatory response, while the commercially available group showed a more severe inflammatory response. This indicates that the implantable recombinant collagen microparticles prepared in this application are a safe, non-toxic, and biocompatible biomaterial that can effectively solve the problems of poor biocompatibility and viral risks associated with traditional biomaterials.

[0112] 3. Swelling performance test of implantable recombinant collagen microparticles

[0113] Experimental Method: The gel obtained from the implantable recombinant collagen microparticles in Example 1, after complete swelling with water, was lyophilized. 5 mg of the lyophilized gel was immersed in purified water and then swollen in a 37°C incubator. The gel was removed at 15 min, 30 min, 60 min, 120 min, and 180 min, and excess surface moisture was absorbed. The weight of the gel (w1) was measured and compared with its initial weight (w0). Swelling rate = (w1 / w0) × 100%.

[0114] Experimental results: The lyophilized gel reached a swelling rate of 1400% after immersion in water for 60 minutes, then slowly decreased and stabilized at around 1300%. The experimental results are as follows... Figure 4 As shown.

[0115] Experimental results show that implantable recombinant collagen microparticles have a high swelling ratio. Since the material can absorb growth factors through water absorption and swelling, a higher swelling ratio allows more aqueous solution to enter the material, which helps the material adsorb and retain growth factors.

[0116] 4. Injection force test of gel after complete swelling of implanted recombinant collagen microparticles

[0117] Experimental Method: The gel obtained in Example 1 after complete swelling of the implantable recombinant collagen microparticles was loaded into a threaded 1mL syringe. Ensure the material evenly fills the syringe, avoiding air bubbles or voids. Install a 26G needle, ensuring a tight connection between the needle and the syringe. Fix the syringe to the clamp of an electronic universal testing machine, ensuring the syringe is vertical and stable. Set the loading rate of the electronic universal testing machine to 10mm / min, and completely eject the material from the syringe, obtaining a force-displacement graph.

[0118] Experimental results show that the injection force of the gel is around 15N, indicating that the material has good injection performance. The low injection force also reflects that the viscosity of the material is moderate, neither too thin to be difficult to control, nor too viscous to increase the difficulty of the doctor's operation. The experimental results are as follows... Figure 5 As shown.

[0119] 5. Degradation test of gel after complete swelling of implanted recombinant collagen microparticles

[0120] In Experiment 1 above, rats in Example 1 and Comparative Examples 1-3 were sacrificed at 2, 8, and 24 weeks after feeding, and epidermal tissue from the injection sites on their backs was collected to observe the size of the material. The experimental results are as follows: Figure 6 As shown.

[0121] Experimental results showed that in Group 1, the gel injected subcutaneously into rats underwent some degradation over time, with a small amount of material remaining at 24 weeks. In Group 1 (Comparative Example), no material residue was observed when the gel was injected subcutaneously into rats at week 2. Similarly, in Groups 2 and 3 (Comparative Examples), no material residue was observed when the gel was injected subcutaneously into rats at week 8. Therefore, the method for preparing implantable recombinant collagen microparticles provided in this application yields a gel with a suitable degradation rate after complete swelling, effectively solving the problems of traditional gel fillers failing to degrade or degrading too quickly, and also effectively addressing the issue of poor biocompatibility in traditional biomaterials.

[0122] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing implantable recombinant collagen microparticles, comprising: (1) Dissolve recombinant collagen in water to obtain a recombinant collagen solution; (2) The pH of the recombinant collagen solution was adjusted to less than 7, and it was dried and granulated by spray dryer to obtain the first recombinant collagen microparticles; (3) The first recombinant collagen microparticles were subjected to dry heat crosslinking to obtain the second recombinant collagen microparticles; The first recombinant collagen microparticle has a spherical structure with a diameter of 2~10μm and an internal porous mesh structure. The drying temperature of the spray dryer is 120~190℃; The amino acid sequence of the recombinant collagen is shown in SEQ ID No:1; The temperature for dry heat crosslinking is 100~240℃.

2. The preparation method according to claim 1, wherein, In step (3), dry heat crosslinking is performed in a vacuum drying oven.

3. The preparation method according to claim 2, wherein, The dry heat crosslinking time in the vacuum drying oven is 1~10h.

4. The preparation method according to claim 3, wherein, The dry heat crosslinking time in the vacuum drying oven is 2~9 hours.

5. The preparation method according to claim 1, wherein, The concentration of the recombinant collagen solution is 5-20% (wt).

6. The preparation method according to claim 5, wherein, The concentration of the recombinant collagen solution is 8-15% (wt).

7. An implantable recombinant collagen microparticle, wherein, The amino acid sequence of the recombinant collagen in the recombinant collagen microparticles is shown in SEQ ID No:

1. The implantable recombinant collagen microparticles are prepared by the preparation method according to any one of claims 1-6.

8. A method for preparing a recombinant collagen gel, comprising adding an aqueous phase to the second recombinant collagen microparticles as described in any one of claims 1-6, and obtaining a recombinant collagen gel after swelling.

9. The preparation method according to claim 8, wherein, The second recombinant collagen microparticles need to be fully swollen after being added to the aqueous phase.

10. The preparation method according to claim 9, wherein, The swelling time required for complete swelling is 10-15 hours.

11. The preparation method according to claim 8, wherein, The aqueous phase is selected from one or more of ultrapure water, water for injection, and phosphate buffer.

12. The preparation method according to claim 8, wherein, The recombinant collagen gel is a milky white, opaque gel or a colorless, transparent, jelly-like gel.

13. A recombinant collagen gel, wherein, The amino acid sequence of the recombinant collagen in the recombinant collagen gel is shown in SEQ ID No:

1. The recombinant collagen gel is prepared by the preparation method according to any one of claims 8-12.

14. The use of the implantable recombinant collagen microparticles of claim 7 or the recombinant collagen gel of claim 13 in the preparation of medical materials for cosmetic filling, soft tissue repair or anti-adhesion.

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