Implantable recombinant collagen microparticles and method for producing the same

Abstract

This invention provides implantable recombinant collagen microparticles for use in soft tissue filling applications, which have characteristics such as good biocompatibility, a long degradation cycle, a simple manufacturing process, and ease of large-scale mass production, as well as a method for producing the same. [Solution] The method for producing implantable recombinant collagen microparticles comprises (1) dissolving recombinant collagen in water to obtain a recombinant collagen solution, (2) drying and granulating the recombinant collagen solution to obtain first recombinant collagen microparticles, and (3) dry-heat crosslinking the first recombinant collagen microparticles to obtain second recombinant collagen microparticles, wherein the amino acid sequence of the recombinant collagen is shown in a specific sequence.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and specifically relates to an implantable recombinant collagen microparticle and a method for producing the same. Background Art

[0002] Hydrogel is a polymer material using water as a dispersion medium and having a three-dimensional network structure. It has good biocompatibility, a porous structure, permeability and hydrophilicity, can simulate a natural extracellular matrix, provides a suitable microenvironment for cells, and has been increasingly widely applied in tissue repair and regeneration. Hydrogels used in the biomedical field can be divided into natural hydrogels and artificially synthesized hydrogels according to their origins. Natural hydrogels include collagen, gelatin, hyaluronic acid, chitosan and the like, which have good biocompatibility and biodegradability, but suffer from poor mechanical properties and are limited by potential immunogenicity in exogenous hosts. Artificially synthesized hydrogels such as polyethylene glycol derivatives, polycaprolactone and polyvinyl alcohol have the characteristics of stable components, controllable structure, low immunogenicity, and can withstand strong mechanical loads, thus becoming the focus of research. Hydrogels can be used as carrier stents that provide regeneration templates or substrates. On one hand, cells adhere and proliferate on the hydrogel, and can regenerate damaged tissue after injury by coordinating the relevant reactions; on the other hand, exogenous stimuli carried therein such as drugs, cytokines and stem cells can promote tissue regeneration. Currently, natural polymer materials used for preparing hydrogels are mainly animal collagen, sodium hyaluronate and the like.

[0003] Collagen is a biomolecular-weight functional protein that is the most abundant and widely distributed protein in mammals, accounting for 25% to 30% of the total protein content. Due to its good biocompatibility, biodegradability, and bioactivity, collagen is widely used in fields such as pharmaceuticals, tissue engineering, and cosmetics. However, ordinary collagen is derived from animal tissues such as pigskin, cowhide, and beef tendons, and animal collagen may contain residual viruses and potentially cause immune rejection. Therefore, some users still experience rejection reactions such as redness at the application site, allergies, and chronic inflammation.

[0004] Hydrogels produced by chemical crosslinking methods exhibit better stability and higher flexibility. However, the crosslinking process requires the use of chemical crosslinking agents, such as 1,4-butanediol diglycidyl ether and β-glycerin sodium phosphate, and incomplete removal of these agents can cause harm to the human body. Enzymes, as biomacromolecules, are widely present in living organisms, are safe and non-toxic, possess high catalytic efficiency, and can form stable chemical bonds. Compared to conventional crosslinking agents, enzymes are more suitable for use in the human body. However, enzymes are often expensive, which can be a limiting factor for large-scale applications, and their poor stability can affect the efficiency of the crosslinking reaction and the consistency of the products.

[0005] To avoid the cytotoxic effects and cost issues associated with chemical crosslinking agents, a physical crosslinking method can be employed. This method prevents exogenous substances from entering the collagen, but the degree of crosslinking provided is low. [Overview of the project]

[0006] The present invention aims to compensate for the deficiencies of conventional hydrogels and soft tissue fillers by producing implantable recombinant collagen microparticles. These implantable recombinant collagen microparticles are obtained using recombinant collagen as a raw material and a method that combines self-assembly and physical cross-linking. By changing the pH value of the recombinant collagen solution, recombinant collagen microparticles with rod-shaped or spherical structures can be obtained. They are safe, non-toxic, biodegradable, highly biocompatible, and free from the hidden risk of viruses. The method of the present invention does not introduce new chemical substances, avoids the residue of any chemical reagents, and has better biosecurity. Furthermore, by utilizing the self-assembly ability unique to recombinant collagen raw materials and combining it with physical cross-linking, a higher degree of cross-linking can be achieved. The present invention specifically employs the following technical proposals.

[0007] The first embodiment of the present invention is (1) Dissolve recombinant collagen in water to obtain a recombinant collagen solution. (2) The recombinant collagen solution is dried and granulated to obtain first recombinant collagen fine particles. (3) Dry heat crosslinking of the first recombinant collagen microparticles to obtain the second recombinant collagen microparticles, The amino acid sequence of the recombinant collagen is shown in SEQ ID No:1. The present invention provides a method for producing implantable recombinant collagen microparticles characterized by the following features.

[0008] In some embodiments, in step (1), a predetermined amount of recombinant collagen is dissolved in water and stirred until completely dissolved, and the solution is clear, thereby obtaining an aqueous recombinant collagen solution. The recombinant collagen can be rapidly dispersed in water to nanoscale particle sizes, has extremely strong hydrophilicity, and the outside of the collagen molecule has regular hydrophilic groups, has extremely strong cohesive ability, and the inside has hydrophobic groups, allowing it to form microstents.

[0009] In some embodiments, in step (2), the recombinant collagen solution is dried and granulated using a spray dryer.

[0010] In some embodiments, step (2) further includes adjusting the pH of the recombinant collagen solution to less than 7 or greater than 7.

[0011] In some embodiments, the first recombinant collagen microparticles have a rod-like or spherical structure.

[0012] 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.

[0013] In some embodiments, the spherical structure has a diameter of 2 to 10 μm and an internal porous network structure.

[0014] In some embodiments, the pH of the recombinant collagen solution is adjusted to greater than 7, and the first recombinant collagen microparticles have a rod-like structure.

[0015] In some embodiments, the diameter of the rod-shaped structure is 2 to 5 μm.

[0016] In this invention, drying is performed using a spray dryer, and during the drying process, due to the special structure of the recombinant collagen raw material used, it spontaneously assembles into microstents and self-aggregates to form a fine particle structure. Therefore, during the heating drying process, the protein chains rapidly lose water, approach each other and form a large number of hydrogen bonds, strengthening the self-assembly effect of the recombinant collagen. In this process, the self-assembly of the recombinant collagen is completed, and at this time, the recombinant collagen fine particles dissolve in water and become a sol.

[0017] In some embodiments, the drying temperature of the spray dryer is 100 to 200°C, and may be, for example, 120°C, 130°C, 140°C, 150°C, 170°C, 180°C, and more preferably, the drying temperature of the spray dryer is 120 to 190°C.

[0018] In some embodiments, dry heat crosslinking is performed in a vacuum drying box in step (3).

[0019] In this invention, first recombinant collagen microparticles are dry-heat crosslinked in a vacuum drying box to increase the temperature of the helical decomposition structure of collagen, i.e., the denaturation temperature. After dehydrating the collagen, crosslinking occurs between collagen molecules, significantly improving the mechanical properties of the collagen and yielding second recombinant collagen microparticles with high strength and degradation resistance.

[0020] In some embodiments, the temperature at which dry heat crosslinking is performed in a vacuum drying box is 90 to 250°C, and may be, for example, 100°C, 140°C, 180°C, 200°C, 240°C, etc., and more preferably the temperature of the vacuum drying box is 100 to 240°C.

[0021] In some embodiments, the time for dry heat crosslinking in a vacuum drying box is 1 to 10 hours, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 9 hours, etc., and more preferably, the time in the vacuum drying box is 2 to 9 hours.

[0022] The recombinant collagen of the present invention is the recombinant human collagen disclosed in CN108070032B [(Method for purifying recombinant human collagen)].

[0023] The recombinant collagen raw materials described in the specific examples of the present invention are all proteins having the amino acid residue sequence shown as SEQ ID No:1 in the sequence listing.

[0024] In some embodiments, recombinant human collagen is obtained by plasmid preparation, transformation via mixing plasmids with Pichia pastoris, screening of multi-copy insertion recombinants, fermentation, and purification. Such collagen has good biocompatibility and a purity of 95% or more, and its structure and function are extremely similar to the collagen native to the human body. Therefore, the use of such collagen can effectively eliminate the hidden risk of viruses associated with extracting collagen from animal tissues. In addition, the recombinant collagen can be rapidly dispersed to a nano-level particle size in water and has super strong hydrophilicity; the collagen molecules have regularly arranged hydrophilic groups on the outside and possess super strong aggregation ability, and have hydrophobic groups on the inside that can form micro-stents, so the recombinant collagen has excellent self-assembly ability.

[0025] In some embodiments, the concentration of the recombinant collagen solution is 5~20% (wt), which may be, for example, 5% (wt), 7% (wt), 10% (wt), 12% (wt), 15% (wt), 18% (wt), or 20% (wt), more preferably, the concentration of the recombinant collagen solution is 8~15% (wt).

[0026] In a second aspect of the present invention, there is provided an implantable recombinant collagen microparticle, wherein the amino acid sequence of the recombinant collagen in the implantable recombinant collagen microparticle is set forth in SEQ ID No: 1, and the implantable recombinant collagen microparticle is produced by the production method described in the first aspect of the present invention.

[0027] In a third aspect of the present invention, there is provided a method for producing a recombinant collagen gel, comprising adding an aqueous phase to the second recombinant collagen microparticles (implantable recombinant collagen microparticles) according to the first aspect of the present invention, allowing the microparticles to swell, and then obtaining the recombinant collagen gel.

[0028] In some embodiments, the recombinant collagen gel is a milky white opaque gel or a colorless transparent jelly-like gel.

[0029] In some embodiments, the pH of said recombinant collagen solution is adjusted to less than 7, said first recombinant collagen microparticles have a spherical structure, and the recombinant collagen gel obtained after adding an aqueous phase to second recombinant collagen microparticles (implant-type recombinant collagen microparticles) and allowing the microparticles to swell is a milky white opaque gel.

[0030] In some embodiments, the pH of said recombinant collagen solution is adjusted to higher than 7, said first recombinant collagen microparticles have a rod-shaped structure, and the recombinant collagen gel obtained after adding an aqueous phase to second recombinant collagen microparticles (implant-type recombinant collagen microparticles) and allowing the microparticles to swell is a colorless and transparent jelly-like gel.

[0031] In some embodiments, after adding an aqueous phase to the second recombinant collagen microparticles according to the first aspect of the present invention, it is necessary to allow complete swelling.

[0032] In some embodiments, the swelling time required for complete swelling is 10 to 15 hours.

[0033] In some embodiments, the aqueous phase is one or more selected from the group consisting of ultrapure water, water for injection, and phosphate buffer, preferably water for injection.

[0034] Recombinant collagen microparticles obtained through two processes of self-assembly and dry heat crosslinking of recombinant collagen are water-insoluble implant-type recombinant collagen microparticles. After adding a predetermined amount of aqueous solvent and allowing complete swelling, the product is a milky white opaque gel or a colorless transparent jelly-like gel, both of which are referred to as recombinant collagen gels. In some embodiments, the concentration of said recombinant collagen gel is 5 to 15% (wt), which may be, for example, 5% (wt), 8% (wt), 10% (wt), 12% (wt), or 15% (wt), and more preferably, the concentration of said recombinant collagen gel is 8 to 13% (wt).

[0035] In some embodiments, the method for producing the recombinant collagen gel includes the following steps.

[0036] A aqueous phase was added to the second recombinant collagen microparticle (implantable recombinant collagen microparticle) described in the first embodiment of the present invention, stirred at a rotation speed of 100-150 r / min for 1-4 hours, and then swelled for 10-15 hours under a temperature of 2-8°C to obtain a recombinant collagen gel.

[0037] In some embodiments, the method for producing the recombinant collagen gel further includes sterilizing the recombinant collagen gel.

[0038] In some embodiments, the sterilization conditions involve moist heat sterilization at 120°C for 30 minutes in a high-temperature, high-pressure steam sterilizer.

[0039] A fourth embodiment 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, and the recombinant collagen gel is manufactured by the manufacturing method described in the third embodiment of the present invention.

[0040] In some embodiments, the pH of the recombinant collagen gel is 5 to 7.

[0041] In the present invention, the opaque, milky-white gel obtained after the implantable recombinant collagen microparticles (spherical structure) have fully swollen by the method for producing implantable recombinant collagen microparticles described in the present invention can be maintained in the body for more than 6 months after injection and has a long degradation cycle. Furthermore, when injected into tissue, it is resistant to displacement and has strong support properties. The colorless, transparent, jelly-like gel obtained after the implantable recombinant collagen microparticles (rod-shaped structure) have fully swollen by the method of the present invention may be used in cosmetic medical procedures such as mesotherapy.

[0042] A fifth embodiment of the present invention provides the use of implantable recombinant collagen microparticles according to the second embodiment of the present invention or recombinant collagen gel according to the fourth embodiment of the present invention in the manufacture of medical materials.

[0043] In some embodiments, the medical material is used for cosmetic medical fillings, cosmetic medical surgery, soft tissue repair, or adhesion prevention.

[0044] Beneficial effects:

[0045] The implantable recombinant collagen microparticles produced by the method described herein are highly safe compared to conventional chemical crosslinking methods, as the produced material is non-toxic, leaves no residue, and contains only recombinant collagen as a raw material. Compared to conventional enzymatic crosslinking methods (such as TG enzymes), it has a higher crosslinking rate and lower cost. By using a method that combines self-assembly and physical crosslinking, a higher degree of crosslinking is achieved, compensating for the disadvantage of low crosslinking with physical crosslinking. The implantable recombinant collagen microparticles (spherical structure) produced by the method described herein, after complete swelling, are a milky white opaque gel. When injected into the target location, they provide strong support, are resistant to displacement, can be maintained in the body for more than 6 months, and have a long degradation cycle and good biocompatibility. The implantable recombinant collagen microparticles (rod-shaped structure) produced by the method described herein may be a colorless, transparent jelly-like gel after complete swelling, and have excellent applicability in cosmetic surgery such as mesotherapy. The manufacturing process is simple, making it easy to mass-produce on a large scale, and is highly suitable for fields such as cosmetic medical fillers, cosmetic surgery, soft tissue repair, and adhesion prevention. [Brief explanation of the drawing]

[0046] [Figure 1] This is an external view of recombinant collagen gel. [Figure 2]These are transmission electron microscope images of recombinant collagen raw materials (Figure 2a); scanning electron microscope images of implantable recombinant collagen microparticles (spherical structure) (Figure 2b); and scanning electron microscope images of implantable recombinant collagen microparticles (rod-shaped structure) (Figure 2c). [Figure 3] This is a hematoxylin-eosin stained image of rat dorsal subcutaneous tissue. [Figure 4] This is a schematic diagram illustrating the swelling performance of implantable recombinant collagen microparticles. [Figure 5] This is a schematic diagram illustrating the injection force of implantable recombinant collagen microparticles. [Figure 6] This is a schematic diagram illustrating the in vivo degradation of implantable recombinant collagen microparticles. [Modes for carrying out the invention]

[0047] 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 following specific embodiments, and that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments and not to limit the scope of protection of the present invention.

[0048] Where numerical ranges are indicated in the examples, it should be understood that, unless otherwise specified in the present invention, any numerical value between the two endpoints of each range and any numerical value between the two endpoints are selectable. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, the present invention can also be realized using any prior art methods, equipment, and materials that are similar or equivalent to those described in the examples of the present invention, based on the prior art understanding of those skilled in the art and the description of the present invention.

[0049] Unless otherwise specified, experimental methods, detection methods, and manufacturing methods not described in detail in this invention all employ prior art in the field of this technology. [Examples]

[0050] The recombinant collagen used in the following examples is derived from Shaanxi Huikang Biotechnology Co., Ltd., and the amino acid sequence of said recombinant collagen is: GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPG KPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSN GPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKPGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGKP (SEQ ID No: 1). Unless otherwise specified, other materials and formulations used can all be obtained through commercial channels.

[0051] Example 1

[0052] Step 1: Weigh 50 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 450 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to acidic (pH=5.24) to obtain a 10% (wt) recombinant collagen solution.

[0053] Step 2: The above solution was dried and granulated at 120°C using a spray dryer to obtain first recombinant collagen microparticles with a spherical structure and a diameter of 2-10 μm.

[0054] Step 3: The first recombinant collagen microparticles with a spherical structure were dry-heat crosslinked in a vacuum drying chamber at 100°C for 2 hours to obtain a second recombinant collagen microparticle (implantable recombinant collagen microparticle) with a high-strength spherical structure.

[0055] Step 4: 1 g of the second type of recombinant collagen microparticles (implantable recombinant collagen microparticles) with a spherical structure obtained through two processes, self-assembly and dry heat crosslinking, was weighed, 12.5 g of sterile water for injection was added, and the mixture was swelled in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting milky white opaque recombinant collagen gel had a concentration of 8% (wt) and a pH of 6.7.

[0056] Example 2

[0057] Step 1: Weigh 60 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 440 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to acidic (pH=4.85) to obtain a 12% (wt) recombinant collagen solution.

[0058] Step 2: The above solution was dried and granulated at 140°C using a spray dryer to obtain first recombinant collagen microparticles with a spherical structure and a diameter of 2-10 μm.

[0059] Step 3: The first recombinant collagen microparticles with a spherical structure were dry-heat crosslinked in a vacuum drying chamber at 120°C for 3 hours to obtain a second recombinant collagen microparticle (implantable recombinant collagen microparticle) with a high-strength spherical structure.

[0060] Step 4: 1 g of the second type of recombinant collagen microparticles (implantable recombinant collagen microparticles) with a spherical structure obtained through two processes, self-assembly and dry heat crosslinking, was weighed, 10 g of sterile water for injection was added, and the mixture was swelled in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting milky white opaque recombinant collagen gel had a concentration of 10% (wt) and a pH of 6.9.

[0061] Example 3

[0062] Step 1: Weigh 65 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 435 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to acidic (pH=5.32) to obtain a 13% (wt) recombinant collagen solution.

[0063] Step 2: The above solution was dried and granulated at 165°C using a spray dryer to obtain first recombinant collagen microparticles with a spherical structure and a diameter of 2-10 μm.

[0064] Step 3: The first recombinant collagen microparticles with a spherical structure were dry-heat crosslinked in a vacuum drying chamber at 135°C for 4 hours to obtain a second recombinant collagen microparticle (implantable recombinant collagen microparticle) with a high-strength spherical structure.

[0065] Step 4: 1.1 g of the second set of spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through two processes, self-assembly and dry heat crosslinking, was weighed, 9.9 g of sterile water for injection was added, and the mixture was allowed to swell in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting milky white, opaque recombinant collagen gel had a concentration of 11% (wt) and a pH of 6.6.

[0066] Example 4

[0067] Step 1: Weigh 70 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 430 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to acidic (pH=4.62) to obtain a 14% (wt) recombinant collagen solution.

[0068] Step 2: The above solution was dried and granulated at 175°C using a spray dryer to obtain first recombinant collagen microparticles with a spherical structure and a diameter of 2-10 μm.

[0069] Step 3: The first recombinant collagen microparticles with a spherical structure were dry-heat crosslinked in a vacuum drying chamber at 170°C for 3 hours to obtain a second recombinant collagen microparticle (implant-type recombinant collagen microparticle) with a high-strength spherical structure.

[0070] Step 4: 1.2 g of the second set of spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through two processes, self-assembly and dry heat crosslinking, was weighed, 9.8 g of sterile water for injection was added, and the mixture was allowed to swell in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting milky white, opaque recombinant collagen gel had a concentration of 12% (wt) and a pH of 6.8.

[0071] Example 5

[0072] Step 1: Weigh 75 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 425 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to acidic (pH=4.287) to obtain a 15% (wt) recombinant collagen solution.

[0073] Step 2: The above solution was dried and granulated at 180°C using a spray dryer to obtain first recombinant collagen microparticles with a spherical structure and a diameter of 2-10 μm.

[0074] Step 3: The first recombinant collagen microparticles with a spherical structure were dry-heat crosslinked in a vacuum drying chamber at 190°C for 2 hours to obtain a second recombinant collagen microparticle (implantable recombinant collagen microparticle) with a spherical structure and high strength.

[0075] Step 4: 1.3 g of the second set of spherical recombinant collagen microparticles (implantable recombinant collagen microparticles) obtained through two processes, self-assembly and dry heat crosslinking, was weighed, 9.7 g of sterile water for injection was added, and the mixture was allowed to swell in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting milky white, opaque recombinant collagen gel had a concentration of 13% (wt) and a pH of 6.5.

[0076] Example 6

[0077] Step 1: Weigh 50 g of recombinant collagen raw material (protein having the amino acid residue sequence shown in SEQ ID No: 1), dissolve it in 450 g of sterile water for injection, stir at room temperature for 4 hours until completely dissolved, obtain a colorless and transparent solution, adjust the pH to alkaline (pH=8.87) to obtain a 10% (wt) recombinant collagen solution.

[0078] Step 2: The above solution was dried and granulated at 120°C using a spray dryer to obtain first recombinant collagen microparticles with a rod-like structure, having a diameter of approximately 2-5 μm and varying lengths.

[0079] Step 3: The first rod-shaped recombinant collagen microparticles were dry-heat crosslinked in a vacuum drying chamber at 100°C for 2 hours to obtain the second crosslinked rod-shaped recombinant collagen microparticles (implant-type recombinant collagen microparticles).

[0080] Step 4: 1 g of the second recombinant collagen microparticles (implantable recombinant collagen microparticles) with a cross-linked rod structure obtained through two processes, self-assembly and dry heat cross-linking, was weighed, 12.5 g of sterile water for injection was added, and the mixture was swelled in a refrigerator at 4°C for 12 hours. After stirring at a rotation speed of 120 r / min for 2 hours, the resulting colorless, transparent, jelly-like recombinant collagen gel had a concentration of 8% (wt) and a pH of 7.

[0081] Comparative Example 1

[0082] Except for not performing the self-assembly step, this is basically the same as Example 1.

[0083] Comparative Example 2

[0084] Except for the absence of the dry heat crosslinking step, this is basically the same as Example 1.

[0085] Comparative Example 3

[0086] This example is basically the same as Example 1, except that the recombinant collagen was replaced with commercially available recombinant collagen (purchased from Xi'an Denuohei Medical Technology Co., Ltd., lot number: 2308B01).

[0087] Effect experiment

[0088] 1. Comparative test of appearance after implantable recombinant collagen microparticles have completely swelled into a gel.

[0089] (1) After adding water to the implantable recombinant collagen microparticles and allowing them to swell completely into a gel, 2 ml was taken and placed in a clear glass bottle, ensuring that the sample was uniform and bubble-free. The sample was illuminated with a standard fluorescent lamp, and the appearance of the material was photographed with a digital camera to compare the transparency, color, and fluidity of the materials. The gels produced in Examples 1-6 and Comparative Examples 1-3 after the implantable recombinant collagen microparticles had fully swelled were compared, and their appearance and condition were observed. The experimental results are shown in Table 1.

[0090] [Table 1]

[0091] As can be seen from the experimental results above, after adding water to the implantable recombinant collagen microparticles of Examples 1 to 5 and allowing them to swell completely, they all became an opaque milky white gel state and all had low fluidity. The implantable recombinant collagen microparticles of Example 6 became a colorless transparent jelly-like gel after swelling completely. Comparative Examples 1 and 3 also became a milky white gel state and all had low fluidity. Comparative Example 2 became a transparent colorless sol state and had high fluidity. The experimental results are shown in Figure 1.

[0092] The recombinant collagen raw material was rapidly dispersed in water to nanoscale particle size, and its transmission electron microscope image is shown in Figure 2a. Scanning electron microscope images of the surface and internal structure of the implantable recombinant collagen microparticles (spherical structure) obtained in the steps of Example 1 are shown in Figure 2b. Scanning electron microscope images of the implantable recombinant collagen microparticles (rod-shaped structure) obtained in the steps of Example 6 are shown in Figure 2c.

[0093] 2. Biocompatibility testing of implantable recombinant collagen microparticles

[0094] (1) Twenty-four healthy adult SD rats were randomly divided into eight groups of three. Hair removal was performed on the backs of the rats using a hair removal agent, the skin was washed with water, the rat skin was disinfected with iodine tincture after hair removal, and then deiodinated with 75% alcohol. To the eight groups of rats, implantable recombinant collagen microparticles prepared in Examples 1-5 and Comparative Examples 1-3 were added with water and swelled completely into a gel, and then injected. 0.2 mL was injected subcutaneously into each rat. The injected rats were kept in the same environment for one week, then killed by neck amputation, and the injected dorsal epidermis was taken for pathological analysis and observed for inflammatory responses. The experimental results are shown in Table 2.

[0095] [Table 2]

[0096] As can be seen from the experimental results above, rats injected with the gel after the implantable recombinant collagen microparticles of Examples 1 to 5 had fully swollen showed almost no inflammatory response. Similarly, when injected with the gel after the implantable recombinant collagen microparticles of Comparative Examples 1 and 2 had fully swollen, the inflammatory response was mild. However, when injected with the gel after the implantable recombinant collagen microparticles of Comparative Example 3 had fully swollen, the inflammatory response was severe.

[0097] After sampling, the skin injected with the experimental material was fixed in neutral formaldehyde. Then, it was stained using the hematoxylin-eosin staining method, photographed with a standard light microscope, and subjected to histopathological analysis. The experimental results are shown in Figure 3.

[0098] The experimental results showed that one week after subcutaneous injection of the corresponding material into rats, no clear inflammatory response was observed in the rats in the test group, but a relatively severe inflammatory response was observed in the commercially available group. This indicates that the implantable recombinant collagen microparticles manufactured in this invention are a safe, non-toxic, and biocompatible biomaterial that can effectively solve the problems of poor biocompatibility and the hidden risks of viruses associated with conventional biomaterials.

[0099] 3. Swelling performance test of implantable recombinant collagen microparticles

[0100] Experimental method: After adding water to the implantable recombinant collagen microparticles obtained in the steps of Example 1 and allowing them to swell completely, the resulting gel was freeze-dried. 5 mg of the freeze-dried gel was taken and immersed in purified water, then swelled in a 37°C incubator. The gel was removed at 15 min, 30 min, 60 min, 120 min, and 180 min, and excess water on the surface was absorbed and dried. The weight of the gel (w1) was measured and compared to its initial weight (w0). Swelling rate = (w1 / w0) × 100%.

[0101] Experimental results: After immersion in water for 60 minutes, the freeze-dried gel reached a swelling rate of 1400%, which then gradually decreased and stabilized at approximately 1300%. The experimental results are shown in Figure 4.

[0102] Experimental results showed that implantable recombinant collagen microparticles have a high swelling rate. The material can absorb growth factors through water absorption and swelling, and the high swelling ratio allows more aqueous solution to penetrate the material, which contributes to the adsorption and preservation of growth factors.

[0103] 4. Injection strength test of the gel after implantable recombinant collagen microparticles have fully swelled.

[0104] Experimental Method: The gel obtained in Example 1, after the implantable recombinant collagen microparticles had fully swollen, was placed in a 1 mL screw-cap syringe. Efforts were made to ensure that the material was uniformly filled into the syringe, avoiding bubbles and gaps. A 26G needle was attached, and a tight connection between the needle and the syringe was ensured. The syringe was fixed to a jig of an electronic universal testing machine to ensure its vertical position and stability. The loading speed of the electronic universal testing machine was set to 10 mm / min, and all the material was extruded from the syringe to obtain a force-displacement relationship diagram.

[0105] As can be seen from the experimental results, the gel injection force is approximately 15N, indicating that the material has good injection performance. The low injection force also reflects the appropriate viscosity of the material; it is neither too thin to be difficult to control nor too viscous to increase the difficulty of handling by the physician. The experimental results are shown in Figure 5.

[0106] 5. Decomposition test of the gel after implantable recombinant collagen microparticles have fully swelled.

[0107] In the experiment described in Experiment Example 1 above, rats from Example 1 and Comparative Examples 1-3 were killed at 2, 8, and 24 weeks after rearing, respectively. The epidermis from the injection site on their backs was then collected, and the size of the material was observed. The experimental results are shown in Figure 6.

[0108] As can be seen from the experimental results, when the gel of experimental example group 1 was injected subcutaneously into rats, the material was degraded subcutaneously to some extent over time, and a small amount of material remained even after 24 weeks. When the gel of comparative example group 1 was injected subcutaneously into rats, no material residue was observed when the material was collected at 2 weeks. When the gels of comparative examples groups 2 and 3 were injected subcutaneously into rats, no material residue was observed when the material was collected at 8 weeks. As can be seen from this, the present invention provides a gel obtained by a method for producing implantable recombinant collagen microparticles after the implantable recombinant collagen microparticles have fully swollen, has a relatively appropriate degradation rate, can effectively solve the problem that conventional gel fillers cannot be degraded or degrade too quickly, and can also effectively solve the problem that conventional biocompatibility of biomaterials is poor.

[0109] The above-described embodiments are intended to facilitate the understanding and use of the invention by those skilled in the art. Those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring progressive work. Therefore, the present invention is not limited to the embodiments described above, and any improvements or 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 all be within the scope of protection of the present invention.

Claims

1. (1) Dissolve recombinant collagen in water to obtain a recombinant collagen solution. (2) The recombinant collagen solution is dried and granulated to obtain first recombinant collagen fine particles. (3) Dry heat crosslinking of the first recombinant collagen microparticles to obtain the second recombinant collagen microparticles, The amino acid sequence of the recombinant collagen is shown in SEQ ID No:

1. A method for producing implantable recombinant collagen microparticles, characterized by the above.

2. The manufacturing method according to claim 1, characterized in that in step (2), the recombinant collagen solution with a concentration of 5-20% (wt) is dried and granulated at a drying temperature of 100-200°C using a spray dryer.

3. Step (2) further comprises adjusting the pH of the recombinant collagen solution to less than 7 or greater than 7, wherein the first recombinant collagen microparticles have a rod-shaped or spherical structure. The aforementioned spherical structure has a diameter of 2 to 10 μm and an internal porous network structure. The manufacturing method according to claim 1, characterized in that the diameter of the rod-shaped structure is 2 to 5 μm.

4. The manufacturing method according to claim 1, characterized in that in step (3), dry heat crosslinking is performed in a vacuum drying box at a temperature of 90 to 250°C for 1 to 10 hours.

5. 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, and the implantable recombinant collagen microparticle is manufactured by the manufacturing method described in any one of claims 1 to 4.

6. A method for producing recombinant collagen gel, characterized by comprising adding an aqueous phase to a second recombinant collagen microparticle described in any one of claims 1 to 4, swelling it, and then obtaining a recombinant collagen gel.

7. The manufacturing method according to claim 6, characterized in that the recombinant collagen gel is a milky white opaque gel or a colorless, transparent jelly-like gel.

8. 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, and the recombinant collagen gel is manufactured by the manufacturing method described in claim 6.

9. Use of implantable recombinant collagen microparticles according to claim 5 or recombinant collagen gel according to claim 8 in the manufacture of medical materials.

10. The use of the medical material according to claim 9 is characterized in that it is used for cosmetic medical fillings, soft tissue repair, or adhesion prevention.

Images