Method for preparing cross-linked implant

Abstract

The invention discloses a method for preparing a cross-linked implant. According to the embodiment of the invention, the method comprises the following steps: performing cross-linking treatment on collagen in the presence of a cross-linking agent so as to obtain the cross-linked implant, wherein the cross-linking agent is N-hydroxy succinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and the cross-linking agent is a mixture of N-hydroxy succinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide; the collagen is selected from at least one of natural collagen and recombinant collagen; the recombinant collagen is selected from at least one of recombinant I-type human collagen and recombinant III-type human collagen. The method can be used for large-scale production, is simple and easy to implement, can be used for preparing cross-linking implants with various concentrations, and has the characteristics of good viscoelasticity, enzymolysis resistance, high cross-linking degree, high melting point, no cross-linking agent residue and the like.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, specifically to a method for preparing a cross-linked implant, and more specifically, to a cross-linked implant. Background Technology

[0002] Collagen is the most abundant protein in the human body. Due to its abundance and mechanical strength, it has been widely used in the pharmaceutical, cosmetic, and research industries. After being synthesized in cells, collagen is transferred to different tissues. Under the action of proline hydroxylase, proline (Pro) is catalyzed to form hydroxyproline (HyP), forming a triple helix structure. Through self-assembly, it forms collagen microfibrils, which further polymerize into collagen fibers and collagen bundles.

[0003] The market demand for collagen is substantial. Animal collagen extracted from animal tissues has extremely high requirements for its source, resulting in low purity, high immunogenicity, and the presence of animal viruses, posing certain safety risks and incurring high production costs. While recombinant humanized collagen possesses a triple helix structure, its functionality and stability after implantation in vivo require further investigation. Collagen implantation primarily provides support to local tissues, increases the utilization of implanted collagen fibers by local tissues, and promotes the regeneration of collagen fibers and cells within the local tissues.

[0004] Uncrosslinked collagen is easily degraded after implantation, resulting in loss of function. Frequent injections also increase patient discomfort. Therefore, further crosslinking of recombinant collagen to extend its lifespan in the body is essential. However, the addition of crosslinking agents, if not completely removed, can cause local inflammatory reactions. Furthermore, crosslinking agents such as glutaraldehyde and epoxides are biotoxic. Additionally, crosslinked implants, due to changes in covalent bonds and structure, may not be metabolized and removed from the body. Therefore, the selection of both collagen and crosslinking agents presents significant challenges.

[0005] Existing cross-linking processes mostly involve dialysis, lyophilization, and washing to remove the cross-linking agent. This requires large dialysis volumes and the cross-linking process takes at least three days, which is detrimental to large-scale production. Furthermore, considering the different implantation characteristics of various tissues, cross-linking implants with varying degrees of cross-linking should be considered to meet the needs of different users for different implantation sites. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a method for preparing a cross-linked implant. The cross-linked implant obtained according to the embodiments of this application possesses good viscoelasticity, resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, and no cross-linking agent residue. Furthermore, cross-linked implants with different degrees of cross-linking can be obtained through two-step cross-linking, meeting the needs of different users for different implantation sites.

[0007] This application is based on the following discoveries of the inventors:

[0008] Currently, most recombinant collagen on the market is humanoid or humanized collagen, mainly in fragment form, and lacks the complete biological activity of collagen. Alternatively, collagen is obtained from bovine and porcine hides, which has extremely high requirements for its source, limits production scale, and also carries risks of high immunogenicity and animal-derived viruses. Uncrosslinked collagen is easily degraded after implantation, losing its function, and frequent injections increase patient discomfort. When crosslinking collagen, toxicity or inflammatory reactions introduced by the crosslinking agent should be avoided, and the crosslinking process should facilitate industrial production. Furthermore, considering the different implantation characteristics of various tissues, too low a degree of crosslinking leads to easy degradation in vivo, while too high a degree of crosslinking results in different degradation rates in different tissues, potentially preventing degradation for extended periods. Therefore, crosslinked implants with varying degrees of crosslinking and melting points should be prepared to meet the needs of different users. Because crosslinked collagen is a solid or semi-solid gel and insoluble, conventional testing methods cannot evaluate its performance.

[0009] Based on this, the collagen raw materials selected in this invention are recombinant type I human collagen or recombinant type III human collagen, which are completely identical to the full-length sequence of natural human collagen, possessing the complete biological activity of natural human collagen, without immunogenicity, without viral risks, and can be degraded by the human body. The cross-linking agents used are N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, avoiding the use of biotoxic glutaraldehyde, epoxides, etc. The cross-linking process is simple to operate, has a short production time, and is conducive to industrial production. Simultaneously, through two-step cross-linking, cross-linked implants with different degrees of cross-linking and melting points are obtained to meet the needs of different users for different implantation sites. Regarding the detection methods, the detection methods for collagen content, enzymatic hydrolysis resistance, residual N-hydroxysuccinimide, and residual 1-ethyl-(3-dimethylaminopropyl)carbodiimide have been optimized, which is more conducive to the evaluation of the characteristics of collagen cross-linked implants.

[0010] In a first aspect, the present invention provides a method for preparing a cross-linked implant. According to an embodiment of the invention, the method comprises: cross-linking collagen in the presence of a cross-linking agent to obtain the cross-linked implant; wherein the cross-linking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the collagen is selected from at least one of natural collagen and recombinant collagen; the recombinant collagen is selected from at least one of recombinant type I human collagen and recombinant type III human collagen; the recombinant type I human collagen has an amino acid sequence that is 80% homologous to or has the same amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the recombinant type III human collagen has an amino acid sequence that is 80% homologous to or has the same amino acid sequence as shown in SEQ ID NO: 3. The method according to the embodiment of the invention can prepare a cross-linked implant with good viscoelasticity, resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, and no cross-linking agent residue.

[0011] In a second aspect, the present invention provides a crosslinking implant. According to an embodiment of the present invention, the crosslinking implant is prepared by the method described in the first aspect of the present invention. The crosslinking implant according to an embodiment of the present invention exhibits good viscoelasticity, good resistance to enzymatic hydrolysis, high degree of crosslinking, high melting point, and no crosslinking agent residue.

[0012] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0013] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0014] Figure 1 The image shows the appearance of the cross-linked implant in Example 1 of this application;

[0015] Figure 2 The melting point detection diagram of the cross-linked implant in Example 1 of this application is shown;

[0016] Figure 3 The diagram shows the amino acid composition detection of the cross-linked implant in Example 1 of this application;

[0017] Figure 4 The following are standard curves showing the detection of residual N-hydroxysuccinimide (NHS) crosslinking agent in Examples 1 and 2 of this application;

[0018] Figure 5The diagram shows the standard curves for detecting the residual amount of the crosslinking agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in Examples 1 and 2 of this application. Detailed Implementation

[0019] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0020] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0024] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0025] This application discloses a method for preparing a cross-linked implant and the cross-linked implant itself, which are described in detail below.

[0026] Methods for preparing cross-linked implants

[0027] In a first aspect, the present invention provides a method for preparing a cross-linked implant. According to an embodiment of the present invention, the method includes: cross-linking collagen in the presence of a cross-linking agent to obtain the cross-linked implant; wherein the cross-linking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the collagen is selected from at least one of natural collagen and recombinant collagen; the recombinant collagen is selected from at least one of recombinant type I human collagen and recombinant type III human collagen; the recombinant type I human collagen has an amino acid sequence that is 80% homologous to or has the same amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the recombinant type III human collagen has an amino acid sequence that is 80% homologous to or has the same amino acid sequence as shown in SEQ ID NO: 3. It should be noted that the cross-linking agent of the present invention avoids the use of biotoxic cross-linking agents such as glutaraldehyde and epoxides; the cross-linking process is simple to operate, has a short production time, and is conducive to industrial production. According to the method of the present invention, the preparation time is short, and can be completed within 2 days, which is at least half the time of conventional preparation methods. It can prepare cross-linked implants with good viscoelasticity, resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, no cross-linking agent residue, and good biocompatibility and stability.

[0028] According to an embodiment of the present invention, the collagen is selected from at least one of natural collagen and recombinant collagen.

[0029] According to an embodiment of the present invention, the recombinant collagen is selected from at least one of recombinant type I human collagen and recombinant type III human collagen.

[0030] According to embodiments of the present invention, the recombinant type I human collagen has an amino acid sequence that is identical to or has 80% homology with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. According to embodiments of the present invention, the preparation method of the recombinant type I human collagen is described in CN119954936A. According to embodiments of the present invention, the recombinant type I human collagen has a triple helix structure composed of two COL1A1 amino acid sequences and one COL1A2 sequence (molecular weight approximately 281 kDa).

[0031] According to an embodiment of the present invention, the amino acid sequence encoding COL1A1 after enzymatic digestion with N-terminal and C-terminal propeptides removed is shown in SEQ ID NO:4, and the amino acid sequence encoding COL1A2 after enzymatic digestion with N-terminal and C-terminal propeptides removed is shown in SEQ ID NO:5.

[0032] According to an embodiment of the present invention, the recombinant type III human collagen has an amino acid sequence that is identical to or has 80% homology with the amino acid sequence shown in SEQ ID NO: 3. According to an embodiment of the present invention, the recombinant type III human collagen has a triple helix structure composed of three COL3A1 amino acid sequences (molecular weight approximately 283 kDa). According to an embodiment of the present invention, the preparation method of the recombinant type III human collagen is described in CN119874880A. According to an embodiment of the present invention, the amino acid sequence encoding COL3A1 after enzymatic digestion with the N-terminal and C-terminal propeptides removed is shown in SEQ ID NO: 6.

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] It should be noted that the cross-linked implant prepared in this invention uses recombinant type I human collagen or recombinant type III human collagen as raw material, which is completely consistent with the full-length sequence of natural human collagen, possesses the complete biological activity of natural human collagen, has no immunogenicity, no viral risks, and can be degraded by the human body.

[0040] According to an embodiment of the present invention, the crosslinking process is a one-step crosslinking.

[0041] According to an embodiment of the present invention, the concentration of the crosslinking agent in the crosslinking system is 1-5 mmol / L, for example, it can be 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L and 5 mmol / L or a range between the two, such as 2-5 mmol / L, 1-4 mmol / L, preferably 2-3 mmol / L.

[0042] According to an embodiment of the present invention, the crosslinking treatment is carried out at a temperature of 2 to 40°C, for example, 2°C, 3°C, 4°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, or a range between the two, 3 to 40°C, 4 to 40°C, preferably 15 to 38°C, more preferably 25 to 35°C, for 4 to 48 hours, preferably 5 to 30 hours.

[0043] According to an embodiment of the present invention, the crosslinking treatment is carried out at a stirring speed of 50 to 200 rpm, for example, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, or a range between the two, 60 to 200 rpm, 70 to 200 rpm, preferably 100 to 150 rpm.

[0044] According to an embodiment of the present invention, the crosslinking treatment includes at least two crosslinking steps.

[0045] According to an embodiment of the present invention, the crosslinking treatment is performed at least twice.

[0046] According to an embodiment of the present invention, the crosslinking treatment is performed 1 to 3 times, for example, 1 time, 2 times, or 3 times, preferably 1 to 2 times, and most preferably 2 times.

[0047] According to an embodiment of the present invention, the crosslinking treatment includes a first crosslinking treatment and a second crosslinking treatment.

[0048] According to an embodiment of the present invention, the first crosslinking treatment and the second crosslinking treatment are carried out under conditions in the presence of at least one of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0049] According to an embodiment of the present invention, the crosslinking agents used in the first crosslinking treatment and the second crosslinking treatment are different.

[0050] According to an embodiment of the present invention, the first crosslinking treatment is carried out in the presence of N-hydroxysuccinimide, and the second crosslinking treatment is carried out in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0051] According to an embodiment of the present invention, the first crosslinking treatment is carried out in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the second crosslinking treatment is carried out in the presence of N-hydroxysuccinimide.

[0052] According to an embodiment of the present invention, the first crosslinking treatment is carried out in the presence of a crosslinking agent at a final concentration of 0.1-0.5 mmol / L, for example, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L and 0.5 mmol / L, or a range between the two of 0.2-0.5 mmol / L, 0.1-0.4 mmol / L, preferably 0.1-0.3 mmol / L, for a first incubation of 10-60 min, for example, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, or a range between the two of 20-60 min, 10-50 min, preferably 15-30 min.

[0053] According to an embodiment of the present invention, the second crosslinking treatment is carried out in the presence of a crosslinking agent at a final concentration of 5 to 100 mmol / L, for example, 5 mmol / L, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 100 mmol / L, preferably 20 to 50 mmol / L, for a second incubation of 0.5 to 30 h, for example, 0.5 h, 1 h, 2 h, 5 h, 7 h, 9 h, 11 h, 13 h, 15 h, 17 h, 19 h, 21 h, 23 h, 25 h, 27 h, 29 h, 30 h, or a range between the two, 1 to 30 h, 2 to 30 h, preferably 5 to 25 h.

[0054] According to an embodiment of the present invention, the collagen is first mixed with a phosphate buffer solution.

[0055] According to an embodiment of the present invention, the collagen exists in liquid form.

[0056] According to embodiments of the present invention, the concentration of collagen is 1 to 6 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL or a range between the two, 2 to 6 mg / mL, 1 to 5 mg / mL, preferably 2 to 4 mg / mL.

[0057] According to an embodiment of the present invention, the phosphate buffer solution comprises sodium monohydrogen phosphate and sodium dihydrogen phosphate.

[0058] According to embodiments of the present invention, the concentration of the phosphate buffer solution is 10–200 mmol / L, for example, it can be 10 mmol / L, 20 mmol / L, 30 mmol / L, 50 mmol / L, 70 mmol / L, 90 mmol / L, 110 mmol / L, 130 mmol / L, 150 mmol / L, 170 mmol / L, 190 mmol / L, 200 mmol / L, or a range between the two, 20–200 mmol / L, 10–190 mmol / L, preferably 50–100 mmol / L.

[0059] According to an embodiment of the present invention, the pH value of the phosphate buffer solution is 11.0 to 13.0, preferably 11.5 to 12.5.

[0060] According to an embodiment of the present invention, the volume ratio of the collagen to the phosphate buffer solution is (5-30):1, preferably (8-15):1.

[0061] According to an embodiment of the present invention, the pH value of the first mixed treatment product is 6.0 to 8.0, for example, it can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 or a range between the two, 6.2 to 8.0, 6.0 to 7.8, preferably 6.8 to 7.6.

[0062] According to an embodiment of the present invention, the process further includes incubating the first mixed product.

[0063] According to an embodiment of the present invention, the incubation treatment is carried out at a temperature of 4 to 35°C, for example, 4°C, 5°C, 6°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 33°C, 34°C, 35°C, or a range between the two, 5 to 35°C, 6 to 35°C, preferably 15 to 30°C, for 1 to 20 hours, preferably 3 to 10 hours.

[0064] According to an embodiment of the present invention, the process further includes a first centrifugation treatment of the crosslinked product.

[0065] According to an embodiment of the present invention, the first centrifugation is performed at a temperature of 2 to 10°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or a range between the two, 3 to 10°C, 4 to 10°C, and a rotation speed of 12000 to 15000g, for example, 12000g, 13000g, 14000g, 15000g or a range between the two, 13000 to 15000g, for 20 to 50 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or a range between the two, 25 to 50 minutes, 30 to 50 minutes.

[0066] According to an embodiment of the present invention, the process further includes purifying the first centrifuged product.

[0067] According to an embodiment of the present invention, the purification process includes a first purification process and a second purification process.

[0068] According to an embodiment of the present invention, the first purification process is achieved by: subjecting the first centrifuged product to a second mixing process with a glycine solution; subjecting the second mixed product to a third incubation process; and subjecting the third incubation product to a second centrifugation process.

[0069] According to an embodiment of the present invention, the second purification process is achieved by: subjecting the second centrifugation product to a third mixing process with phosphate buffer; subjecting the third mixing product to a fourth incubation process; and subjecting the fourth incubation product to a third centrifugation process.

[0070] According to embodiments of the present invention, the concentration of the glycine solution is 0.05 to 0.5 mol / L, for example, it can be 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, or a range between the two, such as 0.1 to 0.5 mol / L or 0.05 to 0.4 mol / L, preferably 0.1 to 0.3 mol / L.

[0071] According to an embodiment of the present invention, the first purification process is performed 1 to 3 times, preferably 1 to 2 times.

[0072] According to embodiments of the present invention, the concentration of the phosphate buffer is 5–20 mmol / L, for example, it can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, or a range between the two, 6–20 mmol / L, 7–20 mmol / L, preferably 8–15 mmol / L.

[0073] According to an embodiment of the present invention, the second purification process is performed 1 to 5 times, for example, 1, 2, 3, 4, or 5 times, preferably 2 to 4 times.

[0074] According to an embodiment of the present invention, the third incubation treatment is carried out at a temperature of 2 to 30°C, for example, 2°C, 3°C, 4°C, 5°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, or a range between the two, 3 to 30°C, 4 to 30°C, preferably 2 to 10°C, for 20 to 120 minutes, for example, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or a range between the two, 30 to 120 minutes, 20 to 110 minutes, preferably 30 to 60 minutes.

[0075] According to an embodiment of the present invention, the fourth incubation treatment is carried out at a temperature of 2 to 30°C, for example, 2°C, 3°C, 4°C, 5°C, 8°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, or a range between the two, 3 to 30°C, 4 to 30°C, preferably 2 to 10°C, for 20 to 120 minutes, for example, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, or a range between the two, 30 to 120 minutes, 20 to 110 minutes, preferably 30 to 60 minutes.

[0076] According to an embodiment of the present invention, the second centrifugation is performed at a temperature of 2 to 10°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or a range between the two, 3 to 10°C, 4 to 10°C, and a rotation speed of 12000 to 15000g, for example, 12000g, 13000g, 14000g, 15000g or a range between the two, 13000 to 15000g, for 20 to 50 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or a range between the two, 25 to 50 minutes, 30 to 50 minutes.

[0077] According to an embodiment of the present invention, the third centrifugation treatment is carried out at a temperature of 2 to 10°C, for example, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C or a range between the two, 3 to 10°C, 4 to 10°C, and a rotation speed of 12000 to 15000g, for example, 12000g, 13000g, 14000g, 15000g or a range between the two, 13000 to 15000g, for 20 to 50 minutes, for example, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes or a range between the two, 25 to 50 minutes, 30 to 50 minutes.

[0078] According to an embodiment of the present invention, the method further includes adding excipients to the purified product.

[0079] According to an embodiment of the present invention, the excipients include at least one selected from lidocaine hydrochloride, physiological saline, and phosphate buffer.

[0080] Cross-linking implant

[0081] In a second aspect, the present invention provides a crosslinking implant. According to an embodiment of the present invention, the crosslinking implant is prepared by the method described in the first aspect of the present invention. The crosslinking implant according to an embodiment of the present invention exhibits good viscoelasticity, good resistance to enzymatic hydrolysis, high degree of crosslinking, high melting point, and no crosslinking agent residue.

[0082] According to an embodiment of the present invention, the elastic modulus G' of the crosslinking implant is 2000-6000 Pa, and the viscous modulus G” is 100-2000 Pa.

[0083] According to an embodiment of the present invention, the enzymatic resistance of the cross-linked implant is 30 to 100 times that of non-cross-linked collagen, for example, it can be 30, 40, 50, 60, 70, 80, 90, 100 times or a range between the two, such as 40 to 100 times or 30 to 90 times.

[0084] According to embodiments of the present invention, the degree of crosslinking of the collagen crosslinkers in the crosslinking implant is 30% to 85%, for example, it can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or a range between the two, such as 35% to 85% or 30% to 80%.

[0085] According to an embodiment of the present invention, the melting point of the crosslinking implant is 50-75°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 73°C, 74°C, 75°C or a range between the two, such as 51-75°C or 52-75°C.

[0086] Cross-linking implant

[0087] In a third aspect, the present invention provides a cross-linking implant. According to embodiments of the present invention, the cross-linking implant comprises a collagen cross-linker. The cross-linking implant according to embodiments of the present invention exhibits good viscoelasticity, good resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, and no cross-linking agent residue.

[0088] According to an embodiment of the present invention, the elastic modulus G' of the crosslinking implant is 2000-6000 Pa, and the viscous modulus G” is 100-2000 Pa.

[0089] According to an embodiment of the present invention, the enzymatic resistance of the cross-linked implant is 30 to 100 times that of non-cross-linked collagen, for example, it can be 30, 40, 50, 60, 70, 80, 90, 100 times or a range between the two, such as 40 to 100 times or 30 to 90 times.

[0090] According to embodiments of the present invention, the degree of crosslinking of the collagen crosslinkers in the crosslinking implant is 30% to 85%, for example, it can be 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or a range between the two, such as 35% to 85% or 30% to 80%.

[0091] According to an embodiment of the present invention, the melting point of the crosslinking implant is 50-75°C, for example, it can be 50°C, 51°C, 52°C, 53°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 73°C, 74°C, 75°C or a range between the two, such as 51-75°C or 52-75°C.

[0092] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0093] Example 1: Preparation of cross-linking implant

[0094] The collagen raw materials used in this embodiment are recombinant type I human collagen stock solutions (the amino acid sequences of the recombinant type I human collagen after enzymatic digestion are shown in SEQ ID NO: 4 or SEQ ID NO: 5, and the amino acid sequences of the recombinant type I human collagen after enzymatic digestion are shown in SEQ ID NO: 1 or SEQ ID NO: 2) or recombinant type III human collagen stock solutions (the amino acid sequences of the recombinant type III human collagen after enzymatic digestion are shown in SEQ ID NO: 6, and the amino acid sequences of the recombinant type I human collagen after enzymatic digestion are shown in SEQ ID NO: 3) obtained by our company based on technologies such as genetic engineering, molecular biology, and fermentation engineering. By simultaneously adding N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) for cross-linking, the obtained cross-linked implant has the characteristics of good viscoelasticity, resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, and no cross-linking agent residue.

[0095] (1) Assembly

[0096] Take 50mM phosphate buffer solution with pH 12 and add it to recombinant collagen stock solution at a volume ratio of 1:10 for the first mixing treatment. The pH of the mixed solution (the product of the first mixing treatment) is around 7.0. Incubate at 20℃ for 5h to obtain collagen fiber precipitation, i.e. assembled fibers.

[0097] (2) Crosslinking

[0098] NHS and EDC, both with a final concentration of 2 mmol / L, were slowly added to the assembled collagen fiber solution in a single step, accompanied by stirring at 50 rpm. The solution was then incubated at 20°C for 20 h with stirring at 100 rpm. After incubation, the solution was centrifuged at 14000g for 30 min at 4°C. The supernatant was carefully discarded, and the resulting precipitate was the cross-linked gel.

[0099] (3) Removal of crosslinking agent

[0100] The presence of cross-linking agents NHS and EDC poses a potential safety hazard to the implantation of cross-linked implants and must be removed as much as possible, with residual amounts controlled below 2 ppm. Since the cross-linked gel is in a solid state and NHS and EDC are in a soluble state, the cross-linking agents can be removed by extraction and centrifugation using appropriate buffer solutions.

[0101] First purification treatment: First, add 0.1 mol / L glycine to the crosslinked gel obtained in step (2) for a second mixing treatment. After mixing, perform a third incubation treatment and let it stand for 30 min. Place it in a centrifuge and perform a second centrifugation treatment at 14000g for 30 min at 4℃. After taking it out, carefully discard the supernatant and repeat the operation. Second purification treatment: Further add 10 mmol / L phosphate buffer solution (pH 7.5) for a third mixing treatment. Perform a fourth incubation treatment under the same incubation conditions and a third centrifugation treatment under the same centrifugation conditions. Repeat 2 to 3 times to obtain a crosslinked gel without crosslinking agent.

[0102] (4) Preparation of cross-linked implant final products

[0103] In step (3), lidocaine hydrochloride, physiological saline, or phosphate buffer are added to the crosslinking gel from which the crosslinking agent has been removed to further prepare a crosslinking implant with a concentration of 1-50 mg / mL. The crosslinking implant is then directly filled into the gel or further freeze-dried into a lyophilized powder.

[0104] Example 2: Preparation method of cross-linked implants with different degrees of cross-linking

[0105] Cross-linked implants with different degrees of cross-linking are prepared using a two-stage cross-linking method. The first cross-linking treatment involves incubation with a low concentration of NHS or EDC, causing cross-linked structures to form on the surface of the recombinant collagen while leaving the interior uncross-linked. The second cross-linking treatment involves further cross-linking of the internal collagen by adding an appropriately high concentration of NHS or EDC. By controlling the concentration of NHS or EDC and the cross-linking time, cross-linked implants with different degrees of cross-linking and melting points can be obtained. The cross-linking agent added in the first cross-linking treatment must be different from that added in the second cross-linking treatment.

[0106] Example 2-1: The specific preparation process is shown below:

[0107] Assembly: Take 50mM phosphate buffer solution with pH 12 and add recombinant collagen stock solution at a volume ratio of 1:10 for the first mixing treatment. The pH of the mixed solution (the product of the first mixing treatment) is around 7.0. Incubate at 20℃ for 5h to obtain collagen fiber precipitation, i.e. assembled fiber.

[0108] First crosslinking treatment: NHS with a final concentration of 0.2 mmol / L was added to the assembled fibers and stirred and incubated at 25°C for 20 min.

[0109] Second crosslinking treatment: In the first crosslinking treatment system, EDC with final concentrations of 20 mmol / L, 30 mmol / L, 40 mmol / L and 50 mmol / L were added, and the mixture was stirred and incubated at 25°C for 5 h, 15 h and 25 h respectively.

[0110] Removal of crosslinking agent: First purification treatment: First, add 0.1 mol / L glycine to the obtained crosslinked gel for a second mixing treatment. After mixing, perform a third incubation treatment and let it stand for 30 min. Place it in a centrifuge and perform a second centrifugation treatment at 14000g for 30 min at 4℃. After taking it out, carefully discard the supernatant and repeat the operation. Second purification treatment: Further add 10 mmol / L phosphate buffer solution (pH 7.5) for a third mixing treatment. Perform a fourth incubation treatment under the same incubation conditions. Perform a third centrifugation treatment under the same centrifugation conditions. Repeat 2-3 times to obtain a crosslinked gel without crosslinking agent.

[0111] The preparation of the cross-linked implant final product is the same as step (4) in Example 1.

[0112] Example 2-2:

[0113] Assembly: Take 50mM phosphate buffer solution with pH 12 and add recombinant collagen stock solution at a volume ratio of 1:10 for the first mixing treatment. The pH of the mixed solution (the product of the first mixing treatment) is around 7.0. Incubate at 20℃ for 5h to obtain collagen fiber precipitation, i.e. assembled fiber.

[0114] First crosslinking treatment: Add EDC to the assembled fiber at a final concentration of 0.2 mmol / L and stir and incubate at 25°C for 20 min.

[0115] Second crosslinking treatment: NHS with final concentrations of 20 mmol / L, 30 mmol / L, 40 mmol / L, and 50 mmol / L was added to the first crosslinking treatment system, and the mixture was stirred and incubated at 25°C for 5 h, 15 h, and 25 h, respectively.

[0116] Removal of crosslinking agent: First purification treatment: First, add 0.1 mol / L glycine to the obtained crosslinked gel for a second mixing treatment. After mixing, perform a third incubation treatment and let it stand for 30 min. Place it in a centrifuge and perform a second centrifugation treatment at 14000g for 30 min at 4℃. After taking it out, carefully discard the supernatant and repeat the operation. Second purification treatment: Further add 10 mmol / L phosphate buffer solution (pH 7.5) for a third mixing treatment. Perform a fourth incubation treatment under the same incubation conditions. Perform a third centrifugation treatment under the same centrifugation conditions. Repeat 2-3 times to obtain a crosslinked gel without crosslinking agent.

[0117] The preparation of the cross-linked implant final product is the same as the preparation of the cross-linked implant final product in step (4) of Example 1.

[0118] Example 3: This example is basically the same as Example 2-1, except that the crosslinking is performed three times. The third crosslinking treatment is the same as the second crosslinking treatment, except that the final concentration of the crosslinking agent is different: In the second crosslinking treatment system, EDC with a final concentration of 20-50 mmol / L is added, and the mixture is stirred and incubated at 25°C for 5-25 hours. The other processes are basically the same as in Example 2-1. In the third crosslinking treatment system, 50-100 mmol / L of EDC is added, and the other treatment methods are the same as in the second crosslinking.

[0119] Comparative Example 1: The experimental procedure was the same as in Example 1, except that the collagen used was recombinant type I or type III humanized collagen (molecular weight approximately 40 kDa) expressed by our Pichia pastoris expression system. It was cultured in BMGY medium in a 500 mL shake flask for 72 h to obtain cell precipitate. The precipitate was homogenized, centrifuged at 12000 rpm for 30 min, and the supernatant was discarded to obtain the precipitate. After reconstitution, the precipitate was salted out with 1 mol / L sodium chloride solution for 20 h, impurities were removed by ion exchange chromatography, and the solution was concentrated by ultrafiltration through a 30 kDa membrane to obtain the recombinant type I or type III humanized collagen stock solution. The preparation process of the cross-linking implant was basically the same as in Example 1.

[0120] Test Example 1: Performance Evaluation Method of Cross-linked Implants

[0121] Protein content:

[0122] Since cross-linked recombinant human collagen implants are insoluble, conventional detection methods are ineffective, with only the Kjeldahl method being suitable. However, this method cannot remove nitrogen from non-protein sources and has poor accuracy. Therefore, this application establishes the following detection method. The hydroxyproline content coefficient in recombinant human collagen is stable. By first detecting the hydroxyproline content in the cross-linked implant, the content of recombinant human collagen is calculated using the hydroxyproline content coefficient. Furthermore, it was found that the results of the relative quantification method are consistent with those of the absolute quantification method, and the operation and calculation are simpler. The specific steps of the detection method are shown below.

[0123] Solution preparation:

[0124] pH 6.0 buffer: Weigh 57g sodium acetate trihydrate, 37.5g trisodium citrate, 5.5g citric acid monohydrate, 385mL isopropanol, add 500mL water, adjust the pH to 6.0 with citric acid monohydrate, and dilute with water to 1000mL.

[0125] Chloramine T solution: Weigh 3.5g of chloramine T and dilute with water to 50mL.

[0126] Oxidizing agent solution: Mix chloramine T solution and pH 6.0 buffer solution at a ratio of 1:4.

[0127] 60% perchloric acid solution: Measure 43 mL of perchloric acid and dilute with water to 50 mL.

[0128] p-Dimethylaminobenzaldehyde solution: Weigh 10g of p-dimethylaminobenzaldehyde and dissolve it in 15mL of 60% perchloric acid solution.

[0129] Colorimetric reagent: Measure 15 mL of p-dimethylaminobenzaldehyde solution and dissolve it in 65 mL of isopropanol.

[0130] method:

[0131] Take approximately 50 mg of the cross-linking implant prepared in Examples 1-3 and Comparative Example 1, respectively, and place them in hydrolysis tubes. Add an appropriate amount of 6 mol / L HCl and seal the tubes with nitrogen. Hydrolyze at 105℃ for 16-24 h, cool, and transfer the hydrolysis products of the cross-linking implant prepared in Examples 1-3 and Comparative Example 1 to volumetric flasks. Wash the hydrolysis tubes with water, combine the washings, and transfer them to volumetric flasks. Add 2 drops of phenolphthalein indicator and 6 mol / L NaOH until the solution turns pink. Dilute to 10 mL with water and shake well.

[0132] Take 0.5 mL each of hydroxyproline reference standard (for absolute quantification, the concentration points of the standard curve are 20 μg / mL, 15 μg / mL, 10 μg / mL, 7.5 μg / mL, and 5 μg / mL; or for relative quantification, only the 20 μg / mL concentration point) and the hydrolysis product sample after dilution. Add 1 mL of isopropanol and 0.5 mL of oxidant solution to each, respectively. After mixing, let stand at room temperature for 5 min. Then add 6.5 mL of colorimetric reagent to each, mix, and heat each tube in a 60℃ water bath for 15 min, then cool. Use a blank as a control and measure the absorbance at 560 nm. Calculate the hydroxyproline content in the sample solution based on the hydroxyproline reference standard content, and further calculate the collagen content in the sample.

[0133]

[0134] Where 0.16, or 16%, is the hydroxyproline content coefficient in recombinant human collagen; 10mL is the fixed volume.

[0135] Melting point:

[0136] The cross-linking implants prepared in Examples 1-3 and Comparative Example 1 were diluted to 1.0 mg / mL with 10 mM PB (pH 7.5) and analyzed using Auto MicroCal Capillary-DSC. The operating parameters were set as follows:

[0137]

[0138] The data was analyzed using MicroCal VP-Capillary-DSC Saftware 2.0.

[0139] Amino acid composition:

[0140] Take approximately 30 mg of the crosslinking implant prepared in Examples 1-3 and Comparative Example 1, respectively, into a hydrolysis tube, add 2 mL of 7 mol / L hydrochloric acid solution, purge with nitrogen for 2 min, and acid hydrolyze at 105 °C for 24 h. Transfer to a rotary evaporator flask and evaporate to dryness on an evaporator. Remove the rotary evaporator flask, add 2.5 mL of 0.05 mol / L sodium acetate aqueous solution, dissolve completely, and measure 25 μL into a 1.5 mL centrifuge tube. Add 12.5 μL of 1 mol / L triethylamine acetonitrile solution and vortex to mix. Then add 12.5 μL of 0.1 mol / L PITC acetonitrile solution, vortex to mix, and let stand at room temperature for 1 h. Add 100 μL of n-hexane solution, vortex for 1 min, and let stand for 10 min. Measure 30 μL of the lower layer solution into a 1.5 mL centrifuge tube, add 270 μL of 0.05 mol / L sodium acetate aqueous solution, mix thoroughly, and analyze by instrument.

[0141] Chromatographic conditions:

[0142] Mobile phase A: 0.05 mol / L sodium acetate aqueous solution

[0143] Mobile phase B: Methanol: Acetonitrile: Water (20:60:20)

[0144] Column: Diamonsil AAA, DIKMA or equivalent column

[0145] Detector: Ultraviolet detector

[0146] Wavelength: 254nm

[0147] Column temperature: 35℃

[0148] Injection volume: 50 μL

[0149] Flow rate: 1.0 mL / min

[0150] Gradient elution:

[0151] time min 0 39 40 45 46 60 B％ 5 48 100 100 5 5

[0152] Resistance to enzymatic hydrolysis:

[0153] Uncrosslinked dry powder (obtained by centrifugation, removal of supernatant, and lyophilization following the assembly steps of Example 1) was used as a control. Crosslinked implants (crosslinked samples) prepared in Examples 1-3 and Comparative Example 1, along with the control, were digested with collagenase. The supernatant after digestion was quantified using the BCA method. The digestion rates of the crosslinked samples and the uncrosslinked control were calculated. The ratio of the digestion rate of the crosslinked sample to that of the uncrosslinked control, i.e., the relative digestion rate, was further calculated and used as an indicator of enzyme digestion resistance. This method allows for a comprehensive comparative analysis of digestion rate results from different samples on different days, eliminating the need for simultaneous detection. Analysis can be performed after 1 hour of digestion, and the relative digestion rate after extended digestion time is structurally consistent with the relative digestion rate after 1 hour, significantly reducing detection time. The detection method is as follows:

[0154] Weigh approximately 3 mg of the cross-linking implant sample prepared in Examples 1-3 and Comparative Example 1, and approximately 50 mg of the control. Record the weight. Add collagenase solution to a final concentration of 1 U / mL, and dilute with 50 mmol / L Tris (pH 7.5) buffer to a final volume of 1 mL. Mix well. Incubate the prepared sample and standard simultaneously on a shaker at 37°C and 200 rpm for 1 hour. Centrifuge at 12000 rpm for 10 minutes, collect the supernatant, and perform BCA quantification. Calculate the enzyme digestion rate using the formula:

[0155] Enzyme digestion rate, % = c × 1 / (a ​​× b / 1000) × 100%

[0156] a: Sample weight, mg;

[0157] b: Collagen content in the sample, mg / mL or mg / g;

[0158] c: BCA quantification result, mg / mL;

[0159] 1: Enzyme digestion and incubation volume, mL;

[0160] 1000: Conversion factor, 1mL≈1g=1000mg.

[0161] Relative enzyme digestion rate = Sample enzyme digestion rate / Control enzyme digestion rate.

[0162] 3.5 EDC Residue

[0163] This method employs the carbodiimide residue determination method from the Chinese Pharmacopoeia. However, since the cross-linked implant is insoluble, an extraction method is used for detection. Furthermore, because EDC is easily degraded in conventional aqueous solutions, this application uses 2-morpholinoethanesulfonic acid (MES) solution to extract the cross-linked implants prepared in Examples 1-3 and Comparative Example 1 to prevent EDC degradation, thus maintaining a detection accuracy (recovery rate) between 98% and 102%. The standard curve was optimized to achieve a detection sensitivity of 1 ppm. The detection method is as follows:

[0164] Take about 0.4g of sample and add an equal volume of 100mmol MES (pH 8.0) solution to obtain a 0.5g / mL sample MES suspension. Incubate at 60℃ for 1h, centrifuge at 12000rpm for 20min, and carefully remove the supernatant.

[0165] Prepare EDC standard solutions with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, and 16 μg / mL.

[0166] Take 0.2 mL of the sample solution and standard solution, and add 1.8 mL of dimethylbarbituric acid test solution to each. Take 0.2 mL of water as a blank control and perform the same procedure. Mix each tube well, let stand in the dark at room temperature for 30 minutes, add 2.0 mL of pyridine acetate solution to each tube, mix well, and measure the absorbance at a wavelength of 599 nm ± 2 nm.

[0167] Plot a linear relationship between the concentration of the EDC standard and its corresponding absorbance, calculate the EDC concentration according to the equation, and then calculate the residual EDC amount (ppm) in the sample.

[0168] EDC residue (ppm) = EDC concentration / 0.5.

[0169] 3.6NHS residue

[0170] Since the cross-linked implant is insoluble, an extraction method is used for detection. At the same time, the detection method is optimized to achieve a detection sensitivity of 0.25 ppm.

[0171] Weigh approximately 0.2 g of the cross-linking implant sample prepared in Examples 1-3 and Comparative Example 1, dilute it with purified water to a concentration of 0.2 g / mL, mix well, incubate on a shaker at 37°C and 120 rpm for 1 hour, then centrifuge at 12000 rpm for 15 min, and take the supernatant after centrifugation for testing.

[0172] EDC standard solutions were prepared using purified water to obtain concentrations of 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.2 μg / mL, 0.1 μg / mL, and 0.05 μg / mL, respectively.

[0173] Take 15 μL each of the sample solution and EDC standard solution for HPLC detection. The instrumentation conditions are as follows:

[0174] Mobile phase: 0.1% aqueous phosphoric acid solution

[0175] Column: Ultimate AQ-C18 or equivalent column

[0176] Detector: 2489 UV detector; Wavelength: 205nm

[0177] Column temperature: 30℃; Flow rate: 0.6 mL / min

[0178] Washing method: isocratic washing; Washing time: 20 minutes

[0179] The concentration of NHS in the sample was calculated based on the standard curve, and then the concentration of NHS in the sample was further calculated.

[0180] NHS residue (ppm) = NHS concentration / sample mass.

[0181] 3.8 Viscoelasticity

[0182] The mechanical properties of the cross-linked implants were tested using an Anton Paar rheometer with a dynamic frequency scan measurement of 1%. The elastic modulus G' and viscous modulus G" of the cross-linked implants prepared in Examples 1-3 and Comparative Example 1 were analyzed.

[0183] 3.9 Degree of crosslinking

[0184] Uncrosslinked samples (obtained by centrifugation and removal of supernatant following the assembly steps of Example 1) and crosslinked implants obtained in Examples 1-3 and Comparative Example 1 were all prepared into lyophilized powders. 3 mg of each powder was weighed and added to 1 mL of 0.1 mol / L Tris (pH 7.5) buffer. Then, 1 mL of 1% TNBS solution was added, and the mixture was reacted at 60°C for 1 h. 2 mL of 6 mol / L hydrochloric acid was added, and the mixture was reacted at 60°C for 1 h. After cooling to room temperature, 5 mL of purified water was added. 2 mL of this mixture was taken out, and 4 mL of diethyl ether was added. The mixture was mixed thoroughly and allowed to stand for phase separation. The upper layer was discarded. This process was repeated twice. After the diethyl ether had completely evaporated, the absorbance was measured at 345 nm.

[0185] Crosslinking degree calculation:

[0186] Degree of crosslinking (%) = (Absorbance of uncrosslinked sample - Absorbance of crosslinked implant sample) / Absorbance of uncrosslinked sample.

[0187] The cross-linked implants prepared according to the methods of Examples 1-3 and Comparative Example 1 were analyzed according to the method described in Test Example 1. This application exemplarily shows partial results of the cross-linked implants prepared in Examples 1-3 and Comparative Example 1. The results of Example 1 are shown in Table 1 and... Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown in Table 2, the results of Comparative Example 1 are shown in Table 2. The results of detecting residual crosslinking agents - hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in the crosslinking implant prepared in Example 2 are shown in Table 3. Figure 4 and Figure 5 As shown.

[0188] Table 1

[0189]

[0190] Table 2

[0191] Inspection items result Melting point Gel dried at 61℃, then lyophilized at 92℃. Resistance to enzymatic hydrolysis 30 times that of non-crosslinked samples viscoelasticity G' is 1000-2000 Pa, and G" is 500-1500 Pa.

[0192] The results show that the cross-linked implant obtained by simultaneously cross-linking with two cross-linking agents (NHS and EDC) in this application (in one step) exhibits good viscoelasticity, resistance to enzymatic hydrolysis, high degree of cross-linking, high melting point, and no cross-linking agent residue. Comparing the results of Example 1 and Comparative Example 1, it can be seen that the recombinant human collagen cross-linked implant prepared in this application is superior to the recombinant humanized collagen cross-linked implant prepared by the same method in terms of melting point, resistance to enzymatic hydrolysis, and viscoelasticity.

[0193] The results of the crosslinking degree and melting point of the crosslinked implant prepared in Example 2 are shown in Table 3. By controlling the order of addition of NHS and EDC, the concentration of addition, and the crosslinking incubation time, crosslinked implants with different crosslinking degrees and melting points can be obtained to meet the needs of different users for different implantation sites and provide more choices.

[0194] Table 3

[0195]

[0196] The results showed that, under the same concentration and crosslinking time conditions, the method of adding NHS for the first crosslinking and EDC for the second crosslinking resulted in a higher degree of crosslinking and melting point in the crosslinked implant compared to the method of adding EDC for the first crosslinking and NHS for the second crosslinking.

[0197] The cross-linked implant prepared in Example 3 had a high degree of cross-linking and melting point, but its elastic modulus G' was too high (above 8000 Pa), resulting in a very hard state, and its protein concentration was low (not higher than 30 mg / mL). The results of the cross-linked implants prepared in Examples 3 and 2 indicate that cross-linking twice is more beneficial for preparing cross-linked implants with good viscoelasticity, resistance to enzymatic hydrolysis, different degrees of cross-linking, different melting points, and no cross-linking agent residue.

[0198] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0199] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for preparing a cross-linked implant, characterized in that, include: Collagen is cross-linked in the presence of a cross-linking agent to obtain the cross-linked implant. The crosslinking agent is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; The collagen is selected from at least one of natural collagen and recombinant collagen; The recombinant collagen is selected from at least one of recombinant type I human collagen and recombinant type III human collagen; The recombinant type I human collagen has an amino acid sequence that is identical to or has 80% homology with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; The recombinant type III human collagen has an amino acid sequence that is identical to or has 80% homology with the amino acid sequence shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that, The crosslinking treatment is a one-step crosslinking process; Optionally, the concentration of the crosslinking agent in the crosslinking system is 1-5 mmol / L, preferably 2-3 mmol / L; Optionally, the crosslinking treatment is carried out at a temperature of 2-40°C, preferably 15-38°C, more preferably 25-35°C for 4-48 hours, preferably 5-30 hours. Optionally, the crosslinking treatment is carried out at a stirring speed of 50 to 200 rpm, preferably 100 to 150 rpm.

3. The method according to claim 1, characterized in that, The crosslinking process includes at least two crosslinking steps.

4. The method according to claim 3, characterized in that, The crosslinking treatment includes a first crosslinking treatment and a second crosslinking treatment; Optionally, the first crosslinking treatment and the second crosslinking treatment are carried out under conditions in the presence of at least one of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; Preferably, the crosslinking agents used in the first crosslinking treatment and the second crosslinking treatment are different; Preferably, the first crosslinking treatment is carried out in the presence of N-hydroxysuccinimide, and the second crosslinking treatment is carried out in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide; Preferably, the first crosslinking treatment is carried out in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and the second crosslinking treatment is carried out in the presence of N-hydroxysuccinimide; Optionally, the first crosslinking treatment is carried out by incubating for 10 to 60 minutes, preferably 15 to 30 minutes, in the presence of a crosslinking agent at a final concentration of 0.1-0.5 mmol / L, preferably 0.1-0.3 mmol / L. Optionally, the second crosslinking treatment is carried out by a second incubation for 0.5 to 30 hours, preferably 5 to 25 hours, in the presence of a crosslinking agent at a final concentration of 5 to 100 mmol / L, preferably 20 to 50 mmol / L.

5. The method according to claim 1, characterized in that, The collagen is pre-mixed with a phosphate buffer solution; Optionally, the collagen is in liquid form; Optionally, the concentration of the collagen is 1–6 mg / mL, preferably 2–4 mg / mL. Optionally, the phosphate buffer solution comprises sodium monohydrogen phosphate and sodium dihydrogen phosphate; Optionally, the concentration of the phosphate buffer solution is 10–200 mmol / L, preferably 50–100 mmol / L; Optionally, the pH value of the phosphate buffer solution is 11.0 to 13.0, preferably 11.5 to 12.5; Optionally, the volume ratio of the collagen to the phosphate buffer solution is (5-30):1, preferably (8-15):1; Optionally, the pH value of the first mixed treatment product is 6.0 to 8.0, preferably 6.8 to 7.6; Optionally, the process further includes incubating the first mixed product; Optionally, the incubation treatment is carried out at a temperature of 4–35°C, preferably 15–30°C, for 1–20 hours, preferably 3–10 hours.

6. The method according to claim 1, characterized in that, This further includes a first centrifugation treatment of the cross-linked product; Optionally, the first centrifugation is carried out for 20 to 50 minutes at a temperature of 2 to 10°C and a rotation speed of 12,000 to 15,000 g.

7. The method according to claim 6, characterized in that, Further, it includes purifying the product from the first centrifugation process.

8. The method according to claim 7, characterized in that, The purification process includes a first purification process and a second purification process; Optionally, the first purification process is implemented in the following manner: The first centrifuged product is then mixed with a glycine solution in a second process. The product of the second mixed treatment is subjected to a third incubation treatment; The product from the third incubation treatment was subjected to a second centrifugation treatment. Optionally, the second purification process is implemented in the following manner: The product from the second centrifugation process is then mixed with phosphate buffer solution in a third process. The product of the third mixing treatment is then subjected to a fourth incubation treatment. The product from the fourth incubation treatment was subjected to a third centrifugation treatment. Optionally, the concentration of the glycine solution is 0.05–0.5 mol / L, preferably 0.1–0.3 mol / L; Optionally, the first purification process is performed 1 to 3 times, preferably 1 to 2 times; Optionally, the concentration of the phosphate buffer solution is 5–20 mmol / L, preferably 8–15 mmol / L; Optionally, the second purification process is performed 1 to 5 times, preferably 2 to 4 times; Optionally, the third incubation treatment is carried out at a temperature of 2-30°C, preferably 2-10°C, for 20-120 minutes, preferably 30-60 minutes. Optionally, the fourth incubation treatment is carried out at a temperature of 2-30°C, preferably 2-10°C, for 20-120 minutes, preferably 30-60 minutes. Optionally, the second centrifugation is carried out for 20 to 50 minutes at a temperature of 2 to 10°C and a rotation speed of 12,000 to 15,000 g. Optionally, the third centrifugation process is carried out for 20 to 50 minutes at a temperature of 2 to 10°C and a rotation speed of 12,000 to 15,000 g.

9. The method according to claim 8, characterized in that, Further, this includes adding excipients to the purified product; Optionally, the excipients include at least one selected from lidocaine hydrochloride, physiological saline, and phosphate buffer.

10. A cross-linking implant, characterized in that, include: Prepared by the method described in any one of claims 1 to 9.

11. A cross-linking implant, characterized in that, include: Collagen cross-linkers.

12. The cross-linking implant according to claim 10 or 11, characterized in that, The elastic modulus G' of the crosslinking implant is 2000-6000 Pa, and the viscous modulus G” is 100-2000 Pa; Optionally, the enzymatic resistance of the cross-linked implant is 30 to 100 times that of non-cross-linked collagen; Optionally, the degree of cross-linking of the collagen cross-linkers in the cross-linking implant is 30% to 85%; Optionally, the melting point of the cross-linking implant is 50–75°C.

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