Preparation method of collagen and hyaluronic acid co-crosslinked gel

Abstract

The invention belongs to the technical field of biomedical treatment, and particularly relates to a preparation method of collagen and hyaluronic acid co-crosslinked gel. Comprising the following steps: (1) mixing sodium hyaluronate with a first part of cross-linking agent solution and a first part of alkali liquor, and carrying out pre-crosslinking reaction to obtain pre-crosslinked hyaluronic acid gel; mixing collagen with a second part of the cross-linking agent solution and a second part of the alkali liquor, and carrying out a pre-crosslinking reaction to obtain pre-crosslinked collagen gel; and (2) stirring and blending the pre-crosslinked hyaluronic acid gel and the pre-crosslinked collagen gel, adding the residual alkali liquor in the stirring and blending process, carrying out a co-crosslinking reaction, and purifying to obtain the collagen and hyaluronic acid co-crosslinked gel. A multi-dimensional network structure is constructed through a synergistic cross-linking technology of sodium hyaluronate and collagen, so that the mechanical strength and the structural stability of the gel are remarkably enhanced, the problem of phase separation easily occurring in a traditional material is effectively solved, and the gel is suitable for the field of medical cosmetology.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a method for preparing a collagen hyaluronic acid co-crosslinked gel. Background Art

[0002] With the rapid development of medical cosmetic technology, injectable fillers have attracted widespread attention due to their minimal invasiveness and immediate effects. Collagen and hyaluronic acid (HA) are currently among the most widely used biomaterials. As the main structural protein of human tissue, collagen has good biocompatibility and tissue support, but it degrades quickly and the filling effect is not durable enough when used alone. Hyaluronic acid can provide an immediate filling effect due to its excellent water retention and viscoelasticity, but cross-linked HA fillers are easily degraded by hyaluronidase in the body, with limited long-term maintenance effects and may cause local inflammatory reactions.

[0003] In the prior art, although the fillers of physically mixing collagen and hyaluronic acid can partially combine the advantages of both, due to the lack of chemical cross-linking, phase separation is prone to occur, resulting in unstable mechanical properties. In addition, some co-cross-linking processes do not fully realize the three-dimensional cross-linked network between molecules, and insufficient cross-linking degree or cross-linking agent residue problems may affect the safety and effectiveness of the product. For example, the synchronous cross-linking of collagen and hyaluronic acid in traditional processes may lead to uneven reactions, and the amount of cross-linking agent is difficult to accurately control, which ultimately affects the swelling degree, degradation rate and biocompatibility of the gel. Therefore, there is an urgent need for a new filling material and a preparation method thereof that can combine the advantages of long-term regeneration of collagen and instant filling of hyaluronic acid, while having a stable cross-linked structure and controllable degradation performance. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing a collagen hyaluronic acid co-cross-linked gel, constructing a multidimensional network structure through the synergistic cross-linking technology of sodium hyaluronate and collagen, and through two-step pre-cross-linking, significantly enhancing the mechanical strength and structural stability of the gel, effectively solving the phase separation problem that is prone to occur in traditional materials, and is suitable for the field of medical cosmetology.

[0005] The technical solution adopted by the present invention is as follows: The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing sodium hyaluronate with the first part of the cross-linking agent solution and the first part of the alkali solution to perform a pre-cross-linking reaction to obtain a pre-cross-linked hyaluronic acid gel; Mixing the collagen with the second part of the cross-linking agent solution and the second part of the alkali solution to perform a pre-cross-linking reaction to obtain a pre-cross-linked collagen gel; (2) adding the pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel into a double planetary mixer for stirring and blending. During the stirring and blending process, adding the remaining alkali solution to carry out a co-cross-linking reaction, and obtaining the collagen hyaluronic acid co-cross-linked gel after purification; The cross-linking agent solution is prepared by mixing the cross-linking agent with water at a mass ratio of 0.5: (25-50), and the alkali solution is prepared by mixing sodium hydroxide with water at a mass ratio of 0.5: (30-50).

[0006] The weight average molecular weight of the sodium hyaluronate is 200,000 to 2,000,000 Da.

[0007] The collagen is recombinant collagen.

[0008] The mass ratio of sodium hyaluronate to collagen is (10-30):15.

[0009] The crosslinking agent is 1,4-butanediol glycidyl ether or polyethylene glycol diglycidyl ether, preferably 1,4-butanediol glycidyl ether. Under alkaline conditions, 1,4-butanediol glycidyl ether can react with hydroxyl groups on hyaluronic acid and recombinant collagen to form stable ether bonds.

[0010] In the step (1), the mass ratio of sodium hyaluronate to the cross-linking agent solution is 18:(30-150); the mass ratio of sodium hyaluronate to the alkali solution is 18:(25-180).

[0011] In the step (1), the mass ratio of collagen to cross-linking agent solution is 15:(25-70); the mass ratio of collagen to alkali solution is 15:(15-80).

[0012] In the step (1), the temperature of the pre-crosslinking reaction is 20-40°C and the time is 20-40 minutes.

[0013] In the step (2), the co-crosslinking reaction temperature is 25-35°C and the time is 15-25h.

[0014] The purification includes soaking and washing with an isotonic PBS buffer solution having a pH value of 6.8 to 7.3.

[0015] The method for preparing the collagen hyaluronic acid co-crosslinked gel also includes the following steps: granulating the purified gel through a sieve, filling the obtained particles into a pre-filled syringe, and performing a wet heat sterilization treatment.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention forms a multi-dimensional cross-linked network of hyaluronic acid-hyaluronic acid, collagen-collagen and hyaluronic acid-collagen through the pre-cross-linking and co-cross-linking process of sodium hyaluronate and collagen, which significantly improves the mechanical strength and stability of the gel and effectively avoids the phase separation phenomenon; at the same time, the long-term regeneration ability of collagen and the instant filling characteristics of hyaluronic acid are used in synergy, which can not only quickly improve the facial contour after injection, but also maintain the long-term effect through the continuous regeneration effect of collagen, prolong the maintenance time of a single injection, and reduce the frequency of repeated injections; in addition, the prepared collagen hyaluronic acid co-cross-linked gel is a milky white gel, which can reduce the visibility of injection marks by scattering light, make the light and shadow transition between the filled part and the surrounding tissue more natural, and avoid the "reflective bright spots" that may appear in transparent gel under strong light, providing a kind of injection material solution for the field of medical cosmetology that has both instant efficacy and continuous regeneration advantages, and the injection effect is naturally invisible, meeting the strict requirements of the field of medical cosmetology for the safety, effectiveness and aesthetics of injection materials. DETAILED DESCRIPTION

[0017] The present invention is further described below with reference to the embodiments, but they do not limit the implementation of the present invention.

[0018] Unless otherwise specified, the raw materials used in the examples and comparative examples are conventional commercially available raw materials, and the process methods used in the examples and comparative examples are conventional methods in the art unless otherwise specified.

[0019] Some of the raw materials used in the examples and comparative examples are described as follows: The PBS buffer is an isotonic PBS buffer with a pH value of 7±0.2, and its preparation method is as follows: accurately weigh sodium dihydrogen phosphate (NaH 2 PO 4 ) 7.5g, disodium hydrogen phosphate (Na 2 HPO 4 ) 33g and sodium chloride (NaCl) 1350g are added into a clean preparation tank. After adding appropriate amount of deionized water for initial dissolution, continue to add deionized water until the total volume of the solution is 150L. Fine-tune the pH value to the target range of 7±0.2 by dropping dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH) solution.

[0020] Example 1 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 55 parts by weight of the first part of the cross-linking agent solution and 35 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 35 parts by weight of the second part of the cross-linking agent solution and 25 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0021] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:40, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:40.

[0022] Example 2 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 30 parts by weight of the first part of the cross-linking agent solution and 25 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 25 parts by weight of the second part of the cross-linking agent solution and 15 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 5 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0023] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water at a mass ratio of 0.5:50, and the alkali solution is prepared by mixing sodium hydroxide and water at a mass ratio of 0.5:50.

[0024] Example 3 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 80 parts by weight of the first part of the cross-linking agent solution and 100 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 60 parts by weight of the second part of the cross-linking agent solution and 80 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 30 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0025] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:25, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:30.

[0026] Example 4 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 10 parts by weight of sodium hyaluronate (weight average molecular weight of 2 million Da), 80 parts by weight of the first part of the cross-linking agent solution and 100 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 70 parts by weight of the second part of the cross-linking agent solution and 80 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0027] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:25, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:25.

[0028] Example 5 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 30 parts by weight of sodium hyaluronate (weight average molecular weight of 200,000 Da), 30 parts by weight of the first part of the cross-linking agent solution and 25 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 25 parts by weight of the second part of the cross-linking agent solution and 15 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 10 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0029] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water at a mass ratio of 0.5:50, and the alkali solution is prepared by mixing sodium hydroxide and water at a mass ratio of 0.5:50.

[0030] Example 6 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 55 parts by weight of the first part of the cross-linking agent solution and 35 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 20° C. for 40 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 35 parts by weight of the second part of the cross-linking agent solution and 25 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 25°C for 25 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0031] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:40, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:40.

[0032] Example 7 The method for preparing the collagen hyaluronic acid co-crosslinked gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 55 parts by weight of the first part of the cross-linking agent solution and 35 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 40° C. for 20 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of recombinant collagen with 35 parts by weight of the second part of the cross-linking agent solution and 25 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 35°C for 15 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0033] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:40, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:40.

[0034] Comparative Example 1 The difference from Example 1 is that the cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water at a mass ratio of 0.3:50, and the rest is the same as Example 1.

[0035] Comparative Example 2 The difference from Example 1 is that the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.3:50, and the rest is the same as Example 1.

[0036] Comparative Example 3 The difference from Example 1 is that the preparation method of the gel is: 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 15 parts by weight of recombinant collagen, 90 parts by weight of cross-linking agent solution and 80 parts by weight of alkali solution are directly mixed and stirred, and the cross-linking reaction is carried out at 30° C. for 20.5 hours. The rest is the same as Example 1.

[0037] Comparative Example 4 The preparation method of the gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da), 20 parts by weight of the first part of the cross-linking agent solution and 15 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of the recombinant collagen with 15 parts by weight of the second part of the cross-linking agent solution and 10 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0038] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:140, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:140.

[0039] Comparative Example 5 The preparation method of the gel comprises the following steps: (1) mixing 18 parts by weight of sodium hyaluronate (weight average molecular weight of 1.5 million Da) with 100 parts by weight of the first part of the cross-linking agent solution and 120 parts by weight of the first part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked hyaluronic acid gel; Mixing 15 parts by weight of the recombinant collagen with 80 parts by weight of the second part of the cross-linking agent solution and 100 parts by weight of the second part of the alkali solution, and performing a pre-cross-linking reaction at 30° C. for 30 minutes to obtain a pre-cross-linked collagen gel; (2) The pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel are added to a double planetary mixer and stirred and blended. During the stirring and blending process, the remaining 20 parts by weight of alkali solution are added, and the co-cross-linking reaction is carried out at 30°C for 20 hours. After soaking and rinsing with an isotonic PBS buffer solution with a pH value of 7±0.2, the collagen hyaluronic acid co-cross-linked gel is obtained. The purified gel is granulated through a 60-mesh sieve, and the obtained granules are filled into a prefilled syringe, and subjected to wet heat sterilization (121°C, 8 min).

[0040] The cross-linking agent solution is prepared by mixing 1,4-butanediol glycidyl ether and water in a mass ratio of 0.5:10, and the alkali solution is prepared by mixing sodium hydroxide and water in a mass ratio of 0.5:10.

[0041] The collagen hyaluronic acid co-crosslinked gels prepared in Examples 1-7 and Comparative Examples 1-5 were respectively tested for performance, and the testing method was as follows: Swelling degree (%): squeeze out the gel in the prefilled syringe and take a sample, and test it in accordance with YY / T 0962-2021; react to the degree of cross-linking of the gel, the higher the degree of cross-linking, the greater the swelling degree.

[0042] Stability test: Take 20 portions of the collagen hyaluronic acid co-crosslinked gel prepared in Examples 1-7 and Comparative Examples 1-5, respectively, put them into a stability test box, and place them at 75% humidity and 45°C for 5 months to observe whether the samples have stratification and count the number of stratified samples.

[0043] Allergenicity: Tested in accordance with GB / T 16886.10-2017; In vitro degradation performance (%): Referring to YY / T 0474-2004, freeze-dry the sample to be tested, measure its initial mass, and then immerse the sample in a phosphate buffer with a pH value of 7.4 at 37±1℃ for 26 weeks. Take it out and freeze-dry it again, weigh the mass after drying, and calculate the mass loss rate by (initial mass-mass after drying) / initial mass×100%; Elastic modulus G′ (pa): Rheological measurements are performed using a rheometer at 25°C. The ability of a reactive material to recover its original shape after being subjected to a dynamic force (storage modulus). The higher the G′, the stronger the ability of the gel to recover its original shape after deformation and the less likely it is to collapse and displace in the body.

[0044] The test results are shown in Table 1.

[0045] Table 1 Performance test results

[0046] It can be seen from the test data in Table 1 that the collagen hyaluronic acid co-crosslinked gel prepared in Examples 1-3 has a high degree of swelling, which indicates that the degree of crosslinking of the gel is high. At the same time, all samples did not show stratification, showing good stability. In addition, the gel prepared in Examples 1-3 has a slow degradation rate in vitro and a long maintenance time; its elastic modulus G' is high, which means that the gel has a strong resistance to deformation, which further indicates that it is less likely to collapse and displace in vivo. With the increase in the amount of crosslinking agent, the swelling degree of the gel increases and the degree of crosslinking becomes higher. In order to control the crosslinking agent residue in the actual production process, a lower amount of crosslinking agent is usually selected.

[0047] By comparing Example 1, Example 4 and Example 5, it can be seen that when the molecular weight of sodium hyaluronate is higher and in the reaction system, the amount of cross-linking agent and alkali added is more compared to the amount of sodium hyaluronate added, the cross-linking degree of the gel will be greater, the swelling degree will be higher, and the mass loss rate will be smaller.

[0048] Comparison of Example 1, Example 6 and Example 7 shows that the reaction temperature has a major influence on the mechanical strength of the gel. A lower reaction temperature helps the gel to exhibit a high elastic modulus.

[0049] Compared with Example 1, the amount of crosslinking agent used in Comparative Example 1 was reduced, resulting in poor gelation and low crosslinking degree, and stratification occurred in the stability test. At the same time, the in vitro degradation rate of the gel was fast, the mechanical properties were poor, and the elastic modulus was low.

[0050] Compared with Example 1, in Comparative Example 2, due to the reduced amount of sodium hydroxide, the system failed to reach the ideal pH reaction condition, resulting in poor gelling performance.

[0051] Compared with Example 1, in Comparative Example 3, since the hyaluronic acid and collagen were not pre-cross-linked, the hyaluronic acid and collagen in the gel did not form an interpenetrating network structure with lines forming a network, which made the in vitro degradation rate of the gel faster, the elastic modulus was lower than that of the embodiment, and there was a significant difference in mechanical strength.

[0052] Compared with Example 1, in Comparative Example 4, due to the small amount and low concentration of the crosslinking agent solution and the alkali solution, the degree of crosslinking is incomplete, which makes the mechanical properties and stability of the gel poor.

[0053] Compared with Example 1, the concentration of the alkali solution in Comparative Example 5 was too high, and the hyaluronic acid and collagen were degraded, and a gel could not be formed.

Claims

1. A method for preparing a collagen hyaluronic acid co-crosslinked gel, characterized in that: The following steps are involved: (1) mixing sodium hyaluronate with the first part of the cross-linking agent solution and the first part of the alkali solution to perform a pre-cross-linking reaction to obtain a pre-cross-linked hyaluronic acid gel; Mixing the collagen with the second part of the cross-linking agent solution and the second part of the alkali solution to perform a pre-cross-linking reaction to obtain a pre-cross-linked collagen gel; (2) stirring and blending the pre-cross-linked hyaluronic acid gel and the pre-cross-linked collagen gel, adding the remaining alkali solution during the stirring and blending process to carry out a co-cross-linking reaction, and obtaining the collagen hyaluronic acid co-cross-linked gel after purification; The cross-linking agent solution is prepared by mixing the cross-linking agent with water at a mass ratio of 0.5: (25-50), and the alkali solution is prepared by mixing sodium hydroxide with water at a mass ratio of 0.5: (30-50).

2. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: The weight average molecular weight of the sodium hyaluronate is 200,000 to 2,000,000 Da; and the collagen is recombinant collagen.

3. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: The mass ratio of sodium hyaluronate to collagen is (10-30):

15.

4. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: The cross-linking agent is 1,4-butanediol glycidyl ether or polyethylene glycol diglycidyl ether.

5. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: In the step (1), the mass ratio of sodium hyaluronate to the cross-linking agent solution is 18:(30-150); the mass ratio of sodium hyaluronate to the alkali solution is 18:(25-180).

6. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: In the step (1), the mass ratio of collagen to cross-linking agent solution is 15:(25-70); the mass ratio of collagen to alkali solution is 15:(15-80).

7. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: In the step (1), the temperature of the pre-crosslinking reaction is 20-40°C and the time is 20-40 minutes.

8. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: In the step (2), the co-crosslinking reaction temperature is 25-35°C and the time is 15-25h.

9. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: The purification includes soaking and washing with PBS buffer.

10. The method for preparing the collagen hyaluronic acid co-crosslinked gel according to claim 1, characterized in that: The following steps are also included: The purified gel is granulated through a sieve, and the obtained granules are filled into prefilled syringes and sterilized by wet heat.

Images