A recombinant collagen-hyaluronic acid complex and a method for preparing the same

Abstract

This invention relates to the field of bioengineering technology and provides a recombinant collagen-hyaluronic acid complex and its preparation method, aiming to solve the problems of easy degradation, short in vivo retention time, and biotoxicity of hyaluronic acid and collagen as natural materials. The recombinant collagen has a triple helix structure, and the hyaluronic acid is covalently coupled to the recombinant collagen to form a complex; the recombinant collagen is an optimized recombinant human type III collagen. The recombinant collagen-hyaluronic acid complex prepared by this invention can significantly improve skin hydration, repair damaged tissue, and enhance the skin's natural elasticity. This complex not only optimizes its biocompatibility and stability and possesses excellent mechanical properties, but also significantly slows down its in vivo degradation rate, thereby prolonging its effective action time in the skin, making it suitable as a support material in the field of medical aesthetics.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a recombinant collagen-hyaluronic acid complex and its preparation method. Background Technology

[0002] Dermal fillers are a common treatment in cosmetic medicine, aiming to improve skin appearance, reduce wrinkles and fine lines, restore facial volume, or improve facial contours. Commonly used filler materials include hyaluronic acid (HA), calcium hydroxyapatite, poly-L-lactic acid, and collagen. Among them, collagen, as a natural facial filler, is an abundant structural protein in the human body, renowned for its excellent biocompatibility, safety, and crucial role in tissue health and function. Collagen is divided into animal-derived and recombinant expression. Traditional animal or human-derived collagen has the risks of animal-derived pathogens and immune responses, as well as issues of low purity and unstable quality. The basic structural unit of collagen is a triple helix structure formed by three polypeptide chains intertwined. Each chain contains a repeating Gly-XY amino acid sequence. These chains form a stable triple helix structure through hydrogen bonds during synthesis, where Gly is glycine, and X and Y are usually proline and hydroxyproline, respectively. While recombinant collagen has high biocompatibility and safety, it also faces high costs and technical challenges in purification and expression, and cannot completely mimic the function of natural collagen.

[0003] Hyaluronic acid (HA), a natural facial filler, is a linear polysaccharide composed of repeating N-acetylglucosamine and glucuronic acid units. It possesses high hydration and biocompatibility, but is easily degraded by hyaluronidase in vivo, requiring frequent replenishment to maintain its effectiveness. Furthermore, hyaluronic acid, when used as a facial filler, exhibits poor support, affecting long-term results. To address these issues, researchers have explored cross-linking techniques between collagen and hyaluronic acid, aiming to combine the advantages of both. However, while chemical cross-linking agents such as glutaraldehyde can effectively cross-link, they may introduce potential toxicity, leading to cytotoxicity and inflammatory responses. Physical cross-linking methods, although avoiding chemical toxicity, typically result in weaker cross-linked structures, leading to poor in vivo stability and requiring further optimization to meet clinical needs. Therefore, this invention proposes a recombinant collagen-hyaluronic acid complex and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant collagen-hyaluronic acid complex and its preparation method, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A recombinant collagen-hyaluronic acid complex, wherein the recombinant collagen has a triple helix structure, and the hyaluronic acid is covalently coupled to the recombinant collagen to form the complex.

[0007] Furthermore, the structural formula of the recombinant collagen-hyaluronic acid complex is as follows:

[0008]

[0009] Furthermore, the recombinant collagen is an optimized recombinant human type III collagen, and its amino acid sequence, as shown in SEQ ID No. 1, was obtained using algorithm design and optimized screening. The molecular weight of the recombinant collagen is selected to be 50–200 kDa, preferably 50–150 kDa, and more preferably 100 kDa.

[0010] Furthermore, the recombinant collagen-hyaluronic acid complex contains γ-polyglutamic acid (γ-PGA) with a molecular weight of 700 kDa.

[0011] The carboxyl groups (-COOH) of recombinant collagen undergo esterification with the hydroxyl groups (-OH) of hyaluronic acid, while γ-PGA, acting as a bioscaffold material, provides the carboxyl groups (-COOH) to undergo amidation with the amino groups (-NH2) of recombinant collagen, forming amide bonds (-CONH) that are highly resistant to enzymatic degradation in the human body, thereby slowing down the degradation rate. This results in a recombinant collagen-hyaluronic acid complex with excellent biocompatibility, high stability, and mechanical properties.

[0012] The above-described use of the recombinant collagen-hyaluronic acid complex in the preparation of tissue engineering materials.

[0013] A method for preparing the recombinant collagen-hyaluronic acid complex according to the above-described method includes the following steps:

[0014] Step 1: Dissolve recombinant collagen in 10-100 mM MES buffer prepared with water for injection, and add γ-PGA to the solution to form the first complex system;

[0015] Step 2: Mix the first composite system with the first crosslinking agent to form the first crosslinking system;

[0016] Step 3: Mix the first crosslinking system with the second crosslinking agent to obtain the collagen-scaffold crosslinking system; the second crosslinking agent is different from the first crosslinking agent;

[0017] Step 4: Hyaluronic acid is mixed with the collagen-scaffold crosslinking system to obtain a recombinant collagen-hyaluronic acid complex. Then, the recombinant collagen-hyaluronic acid complex is mixed in 2-(N-methylamino)ethanesulfonic acid (MES) buffer.

[0018] Furthermore, the bioscaffold material can be dimethyl glutarate, polylysine, chitosan, alginate, γ-PGA, polyethylene glycol, or uronic acid. γ-PGA with a molecular weight of 700 kDa is preferred.

[0019] Furthermore, the hyaluronic acid can be low molecular weight hyaluronic acid, medium molecular weight hyaluronic acid, high molecular weight hyaluronic acid, ultra-high molecular weight hyaluronic acid, cross-linked hyaluronic acid, hydrolyzed hyaluronic acid, acetylated hyaluronic acid, or sodium hyaluronic acid. After cross-linking with recombinant collagen, hyaluronic acid can form a stable three-dimensional network structure, providing better mechanical strength and elasticity. Low molecular weight hyaluronic acid can effectively fill the gaps between collagen fibers during the cross-linking process, enhancing the overall stability and durability of the material. Hyaluronic acid also has good sustained-release properties; after cross-linking with collagen, low molecular weight hyaluronic acid can gradually release the active ingredients, prolonging its duration of action in the skin. Therefore, low molecular weight hyaluronic acid is preferred, specifically 5–200 kDa, more preferably 20–100 kDa, and even more preferably 50 kDa or 70 kDa.

[0020] Furthermore, the first crosslinking agent can be an aldehyde crosslinking agent, an alcohol crosslinking agent, an imine crosslinking agent, or an alkyl crosslinking agent. It is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1,2-ethylenediamine, 4,4'-diaminodiphenyl ether, and dimethyl glutarate (DMS). Preferably, it is EDC, with a volume percentage concentration of 2-30%. More preferably, it is 5-20%, and even more preferably, it is 8-15%.

[0021] Furthermore, the second crosslinking agent can be an aldehyde crosslinking agent, an alcohol crosslinking agent, an imine crosslinking agent, or an alkyl crosslinking agent. Preferably, it is N-hydroxysuccinic acid imide (NHS), which forms an imine crosslinking agent mixture with the EDC added in the previous step. This imine crosslinking agent mixture acts as an amide reaction between the amine and carboxyl groups. The molar ratio of EDC to NHS is not particularly limited, but 1:1, 2:1, 6:1, and 5:2 are preferred, 2:1, 6:1, and 5:2 are more preferred, and 6:1 is ultimately chosen. There are also no particular limitations on the reaction concentration, as long as it allows for the crosslinking reaction between amine and carboxyl groups to occur between and / or within collagen molecules.

[0022] Furthermore, the imine crosslinking agent used has a covalent crosslinking reaction between amine and carboxyl groups. Therefore, the imine crosslinking agent can enable the amine and carboxyl groups between and / or within collagen molecules to crosslink.

[0023] Furthermore, in step 1, the concentration of recombinant collagen is 20–40 mg / mL.

[0024] Furthermore, the pH of the overall reaction system in the preparation method is 3-6, preferably 3-4, and more preferably 3 or 3.5. The reaction temperature is 4-30°C, preferably 4-25°C, and more preferably 18-23°C.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention provides a recombinant collagen-hyaluronic acid complex with a triple helix structure and its preparation method, aiming to solve the problems of easy degradation, short in vivo retention time, and biotoxicity of hyaluronic acid and collagen as natural materials. The recombinant collagen-hyaluronic acid complex prepared by this invention can significantly improve skin hydration, repair damaged tissue, and enhance the skin's natural elasticity. This complex not only optimizes its biocompatibility and stability and possesses excellent mechanical properties, but also significantly slows down its in vivo degradation rate, thereby prolonging its effective action time in the skin, making it suitable as a support material in the field of medical aesthetics. Attached Figure Description

[0027] Figure 1 Electrophoresis results for the purification of recombinant collagen.

[0028] Figure 2 Examples of recombinant collagen-hyaluronic acid complexes obtained in Examples 2-5 are shown in the diagram. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0030] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0031] Example 1: Preparation of recombinant collagen using a CHO cell expression system;

[0032] 1-1. Construction of Recombinant Collagen Gene Vector: The recombinant collagen is an optimized recombinant human type III collagen. The amino acid sequence of the recombinant collagen was obtained using algorithm design and optimization screening, as shown below:

[0033]

[0034] 1-2. Construction of engineered cells: Plasmids containing the target gene are transfected into host cells (CHO-K1) by electroporation. After culturing in an incubator, cell pool screening is performed to obtain candidate pool cells for subsequent collagen preparation.

[0035] 1-3. Preparation of collagen: Recombinant CHO cells were cultured in shake flasks for propagation, and collagen was expressed in large quantities in a fermenter. The expression level of protein in the fermentation supernatant was determined by SDS-PAGE electrophoresis.

[0036] (1) Stop culturing when the cells in the fermenter reach the expected density. Collect the cell culture medium and centrifuge to collect the supernatant.

[0037] (2) SDS-PAGE electrophoresis was used to detect the purity and impurities of the centrifuged samples.

[0038] (3) The supernatant of the fermentation broth was filtered through a 0.22 μm filter membrane and purified by MMC composite column chromatography and 4FF molecular sieve chromatography. The sample was concentrated using a concentration tube and quantitatively analyzed by BCA quantification.

[0039] (4) Sample freeze-drying and storage: The concentrated and quantified recombinant collagen was freeze-dried. The sample was placed in a freeze-drying bottle and frozen at 40°C for 4 hours. The pre-frozen sample was then placed in a freeze dryer at -50 to -80°C and a vacuum of 10 to 20 Pa for preliminary drying for 24 to 48 hours. After preliminary drying, the temperature of the freeze-drying chamber was gradually increased to 20 to 25°C while maintaining the vacuum for 12 to 24 hours to remove residual moisture. After completion, the pressure of the freeze-drying chamber was slowly restored, the freeze-drying bottle was removed, and it was immediately sealed with a sealing cap to prevent the sample from absorbing moisture. The freeze-dried sample was placed in a dry, sealed container and stored at a low temperature of -20°C or -80°C to ensure the long-term stability and activity of the sample.

[0040] 1-4. Characterization of recombinant collagen: Denaturing non-reducing SDS-PAGE was performed on the recombinant collagen samples to detect trimer assembly. The results are as follows: Figure 1 As shown, lanes 1-5 are the eluted samples during the purification process, and lane 6 is the purified recombinant collagen. It can be seen that the recombinant collagen in lane 6 has a high purity, and most of the other impurities have been eluted and recombined.

[0041] Example 2: Preparation of recombinant collagen-hyaluronic acid complex;

[0042] 2-1. Preparation of reaction system buffer: Dissolve MES at 100mM in water for injection, pH 3.5, filter sterilize through a 0.22μm filter membrane, and then use it for subsequent applications.

[0043] 2-2. Preparation of recombinant collagen: Dissolve the above recombinant collagen at 35 mg / mL in 100 mM, pH 3.5 MES buffer until the protein is completely dissolved and forms a homogeneous and clear solution.

[0044] 2-3. First cross-linking reaction: Dissolve EDC at 400mM in 100mM, pH 3.5 MES solution as the first cross-linking agent and cross-link it with the 35mg / mL recombinant collagen solution in step 2-2. The cross-linking time is not less than 30 minutes. After the cross-linking reaction is completed, centrifuge and collect the cross-linked recombinant type III collagen fibers formed by this first cross-linking reaction.

[0045] 2-4. Second cross-linking reaction: NHS is added to the solution in step 2-3 to form an imine cross-linking agent mixture with EDC (the molar ratio of EDC to NHS is 6:1), which activates the carboxyl groups in the reaction. After the cross-linking reaction is completed, the cross-linked recombinant type III collagen fibers formed by this second cross-linking reaction are collected by centrifugation.

[0046] 2-5. Recombinant collagen-bioscaffold crosslinking: Add γ-PGA at twice the amount of recombinant collagen to the crosslinking system in step 2-4 as a scaffold to provide carboxyl groups for crosslinking with the solution. Collect the recombinant collagen-bioscaffold crosslinked complex by centrifugation.

[0047] 2-6. Add hyaluronic acid: Add 35 mg / mL of hyaluronic acid to the recombinant collagen-bioscaffold cross-linked complex solution from step 2-5, and cross-link in an acidic buffer solution at pH 3.5 for at least 30 minutes to form a recombinant collagen-hyaluronic acid complex.

[0048] 2-7. Cleaning impurities and unreacted crosslinking agents: Use water for injection or physiological saline at a ratio of 10 times the volume to clean 3 times.

[0049] 2-8. Lyophilization: Place the sample in a lyophilization bottle and freeze at 40°C for 4 hours. Place the pre-frozen sample in a lyophilizer for initial drying for 24–48 hours. After initial drying, gradually increase the temperature of the lyophilization chamber while maintaining a constant vacuum for 12–24 hours to remove residual moisture. After completion, place the lyophilized sample in a dry, sealed container and store it in a low-temperature environment.

[0050] Example 3: Preparation of recombinant collagen-hyaluronic acid complex;

[0051] 3-1. Preparation of reaction system buffer solution: Same as step 2-1 in Example 1.

[0052] 3-2. Preparation of recombinant collagen: Dissolve the above recombinant collagen at 35 mg / mL in 100 mM, pH 3.5 MES buffer until the protein is completely dissolved and forms a homogeneous and clear solution.

[0053] 3-3. First cross-linking reaction: Dissolve EDC at 600mM in 100mM, pH 3.5 MES solution as the first cross-linking agent and cross-link it with the 35mg / mL recombinant collagen solution in 3-2. The cross-linking time is not less than 30 minutes. After the cross-linking reaction is completed, centrifuge and collect the cross-linked recombinant type III collagen fibers formed by this first cross-linking reaction.

[0054] 3-4. Second cross-linking reaction: NHS is added to the solution in step 3-3 to form an imine cross-linking agent mixture with EDC (the molar ratio of EDC to NHS is 2:1), which activates the carboxyl groups in the reaction. After the cross-linking reaction is completed, the cross-linked recombinant type III collagen fibers formed by this second cross-linking reaction are collected by centrifugation.

[0055] 3-5. Recombinant collagen-bioscaffold crosslinking: Add γ-PGA at a concentration ten times that of recombinant collagen to the crosslinking system in step 3-4 as a scaffold to provide carboxyl groups for crosslinking with the solution. Collect the recombinant collagen-bioscaffold crosslinked complex by centrifugation.

[0056] 3-6. Add hyaluronic acid: Add 35 mg / mL of hyaluronic acid to the recombinant collagen-bioscaffold cross-linked complex solution from step 3-5, and cross-link in an acidic buffer solution at pH 3.5 for at least 30 minutes to form a recombinant collagen-hyaluronic acid complex.

[0057] 3-7. Cleaning impurities and unreacted crosslinking agents: Wash 3 times with 10mM phosphate buffer solution at a 10-fold volume ratio.

[0058] 3-8. Lyophilization: Place the sample in a lyophilization bottle and freeze at 40°C for 4 hours. Place the pre-frozen sample in a lyophilizer for preliminary drying for 24–48 hours. After preliminary drying, gradually increase the temperature of the lyophilization chamber while maintaining a constant vacuum for 12–24 hours to remove residual moisture. After completion, place the lyophilized sample in a dry, sealed container and store it in a low-temperature environment.

[0059] Example 4: Preparation of recombinant collagen-hyaluronic acid complex;

[0060] 4-1. Preparation of reaction system buffer solution: Same as step 2-1 in Example 1.

[0061] 4-2. Preparation of recombinant collagen: Dissolve the above recombinant collagen at 35 mg / mL in the buffer solution prepared in step 4-1 until the protein is completely dissolved and forms a homogeneous and clear solution.

[0062] 4-3. Recombinant collagen cross-linking reaction: Glutaraldehyde is used as a cross-linking agent at a reaction concentration of 30-40 ppm and cross-linked with the solution in step 4-2 for a cross-linking time of not less than 30 minutes. The cross-linked recombinant collagen fibers are collected by centrifugation.

[0063] 4-4. Recombinant collagen-bioscaffold crosslinking: Add the same amount of γ-PGA as recombinant collagen to the crosslinking system in step 4-3 to provide carboxyl groups for crosslinking with the solution as a scaffold. Collect the recombinant collagen-bioscaffold crosslinked complex by centrifugation.

[0064] 4-5. Add hyaluronic acid: Add 35 mg / mL of hyaluronic acid to the recombinant collagen-bioscaffold cross-linked complex solution from step 4-4, and cross-link in an acidic buffer solution at pH 3.5 for at least 30 minutes to form a recombinant collagen-hyaluronic acid complex.

[0065] 4-6. Cleaning impurities and unreacted crosslinking agents: Use water for injection or physiological saline at a ratio of 10 times the volume to clean 3 times.

[0066] 4-7. Lyophilization: Place the sample in a lyophilization bottle and freeze at 40°C for 4 hours. Place the pre-frozen sample in a lyophilizer for initial drying for 24–48 hours. After initial drying, gradually increase the temperature of the lyophilization chamber while maintaining a constant vacuum for 12–24 hours to remove residual moisture. After completion, place the lyophilized sample in a dry, sealed container and store it in a low-temperature environment.

[0067] Example 5: Preparation of recombinant collagen-hyaluronic acid complex;

[0068] 5-1. Preparation of the reaction system buffer solution: Same as step 2-1 in Example 1.

[0069] 5-2. Preparation of recombinant collagen: Dissolve the above recombinant collagen at 35 mg / mL in the buffer solution prepared in step 5-1 until the protein is completely dissolved and forms a homogeneous and clear solution.

[0070] 5-3. First cross-linking reaction: Glutaraldehyde at a reaction concentration of 35 ppm is used as a cross-linking agent to cross-link with the 35 mg / mL recombinant collagen solution from step 5-2. The cross-linking time is not less than 16 hours to carry out the intermolecular and / or intramolecular amino group covalent cross-linking reaction. After the cross-linking reaction is completed, the first cross-linked collagen fibers formed by this first cross-linking reaction are collected by centrifugation.

[0071] 5-4. Second cross-linking reaction: EDC and NHS are mixed to form an imine cross-linking agent mixture (EDC / NHS) as the second cross-linking agent, which is then added to the collagen fiber solution of the first cross-linking agent in step 5-3.

[0072] 5-5. Add hyaluronic acid: Add 35 mg / mL of hyaluronic acid to the solution in 5-4 and crosslink it in an acidic buffer solution at pH 3.5 for 4 hours.

[0073] 5-6. Cleaning impurities and unreacted crosslinking agents: Wash three times with 10mM phosphate buffer solution at a 10-fold volume ratio to form the recombinant collagen-hyaluronic acid complex of the present invention.

[0074] 5-7. Lyophilization: Place the sample in a lyophilization bottle and freeze at 40°C for 4 hours. Place the pre-frozen sample in a lyophilizer for preliminary drying for 24–48 hours. After preliminary drying, gradually increase the temperature of the lyophilization chamber while maintaining a constant vacuum for 12–24 hours to remove residual moisture. After completion, place the lyophilized sample in a dry, sealed container and store it in a low-temperature environment.

[0075] Example 6: Performance testing of recombinant collagen-hyaluronic acid complex;

[0076] 6-1 Mechanical Property Testing: The elastic modulus and compressive strength of the cross-linked recombinant collagen-hyaluronic acid complex were tested to ensure that its mechanical properties met application requirements and provided structural support. Rheological and viscosity analyses of the recombinant collagen-hyaluronic acid complex were performed using a DHR rheometer. For rheological analysis, the angular frequency (ω) was varied between 100 and 0.1 rad / s, and the initial strain (γ) was set to 0.01%. Figure 2 The examples shown are test samples from Examples 2 to 5. It can be seen that the samples in all four examples are semi-transparent, non-flowing solids. The products using EDC / NHS mixture as a crosslinking agent have the same rheological properties as the products crosslinked with glutaraldehyde, a commonly used crosslinking agent. This proves that crosslinking with EDC / NHS mixture as a crosslinking agent and glutaraldehyde as a crosslinking agent can produce similar results. At the same time, using EDC / NHS mixture as a crosslinking agent does not require washing to remove excess toxic glutaraldehyde and does not introduce other chemicals.

[0077] 6-2. Stability Testing: The stability of the recombinant collagen-hyaluronic acid complex was tested under different temperature and humidity conditions to assess its degradation during storage and use. The recombinant collagen-hyaluronic acid complexes from Examples 2-5 were aliquoted into sterile centrifuge tubes and sealed. Accelerated stability and long-term stability tests were performed at different times and temperatures. 10g of the lyophilized recombinant collagen-hyaluronic acid complex sample was weighed and immersed in 100mL of PBS with a collagenase concentration of 12.5U / mL. The sample was incubated at 37℃ for 7 days. The solution was removed by filtration, and the sample was washed three times with deionized water. After lyophilization, the sample was weighed, and its mechanical properties were measured using a DHR rheometer to calculate the degradation rate. The stability of the protein was determined based on changes in mechanical properties. The results are shown in Tables 1 and 2.

[0078] Table 1. Accelerated stability test results (%) of recombinant collagen-hyaluronic acid complex

[0079] sample High temperature for 6 hours 1 day at room temperature 2 days at room temperature Room temperature for one week Example 2 0.3 0.5 0.9 1.5 Example 3 2.7 4.5 6.1 8.4 Example 4 5.3 7.4 9.5 11.3 Example 5 1.3 2.6 3.4 5.0

[0080] Table 2. Stability test results of recombinant collagen-hyaluronic acid complex (%)

[0081]

[0082]

[0083] As shown in Tables 1 and 2, the recombinant collagen-hyaluronic acid complex prepared in this invention exhibits exceptionally good stability. Test results show that the recombinant collagen-hyaluronic acid complex in Example 2 is stable for at least 6, 12, 18, and 24 months, and maintains a degradation rate of less than or equal to 3.0% at 24 months. In contrast, the other examples all showed varying degrees of degradation, with Examples 3 and 4 showing degradation rates greater than 15.0% at 24 months. These degradation results indicate that the product obtained by cross-linking collagen and hyaluronic acid using suitable bioscaffold materials and an EDC / NHS mixture has better mechanical properties than the glutaraldehyde cross-linked product, while without the addition of new chemical substances, exhibiting higher biocompatibility and no potential toxicity.

[0084] In summary, this invention provides a recombinant collagen-hyaluronic acid complex with a triple helix structure and its preparation method, aiming to solve the problems of hyaluronic acid and collagen being easily degraded, having a short in vivo retention time, and exhibiting biotoxicity as natural materials. The recombinant collagen-hyaluronic acid complex prepared by this invention can significantly improve skin hydration, repair damaged tissue, and enhance the skin's natural elasticity. This complex not only optimizes its biocompatibility and stability and possesses excellent mechanical properties, but also significantly slows down its in vivo degradation rate, thereby prolonging its effective action time in the skin, making it suitable as a support material in the field of medical aesthetics.

[0085] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a recombinant collagen-hyaluronic acid complex, characterized in that, Includes the following steps: Step 1: Dissolve recombinant collagen in 10-100mM MES buffer prepared with water for injection, and add γ-PGA to the solution to form the first complex system; Step 2: Mix the first composite system with the first crosslinking agent to form the first crosslinking system; Step 3: Mix the first crosslinking system with the second crosslinking agent to obtain the collagen-scaffold crosslinking system; The second crosslinking agent is different from the first crosslinking agent; Step 4: Mix hyaluronic acid with the collagen-scaffold cross-linking system to obtain a recombinant collagen-hyaluronic acid complex; The recombinant collagen has a triple helix structure, and the hyaluronic acid is coupled to the recombinant collagen through covalent bonds to form a complex; The recombinant collagen is an optimized recombinant human type III collagen, and the amino acid sequence of the recombinant collagen is shown in SEQ ID No. 1; The recombinant collagen-hyaluronic acid complex contains γ-PGA with a molecular weight of 700 kDa.

2. The preparation method according to claim 1, characterized in that, In step 1, the concentration of recombinant collagen is 20-40 mg / mL.

3. The preparation method according to claim 1, characterized in that, The pH of the overall reaction system in the preparation method is 3-6, and the reaction temperature is 4-30℃.

4. The preparation method according to claim 1, characterized in that, The first crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

5. The preparation method according to claim 4, characterized in that, The second crosslinking agent is N-hydroxy-succinic acid imide, and the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxy-succinic acid imide is 6:

1.

6. The use of a recombinant collagen-hyaluronic acid complex prepared by the method according to any one of claims 1 to 5 in the preparation of tissue engineering materials.

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