High concentration collagen-based hydrogel, preparation method and application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a high-concentration collagen-based hydrogel, its preparation method, and its applications. The high-concentration collagen-based hydrogel provided by this invention is obtained by first modifying natural collagen with a carboxylating agent, and then adding a carbodiimide crosslinking agent for crosslinking. The beneficial effects of this invention are: modifying natural collagen with a carboxylating agent improves its solubility, increasing its solubility in neutral and alkaline aqueous solutions; simultaneously, crosslinking the carboxylated collagen with a carbodiimide crosslinking agent achieves the construction of a carboxylated collagen spatial network. The resulting collagen-based hydrogel is colorless and transparent, possessing a dry matter content and mechanical properties far exceeding those of natural collagen gels, and can be applied in fields such as bio-3D printing, in-situ filling, drug loading, and delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a high-concentration collagen-based hydrogel, its preparation method, and its application. Background Technology

[0002] Collagen, due to its excellent biological properties, has become a key component in the construction of biomedical materials and is widely used in clinical medicine, medical aesthetics, tissue engineering and other fields. Hydrogel is the main form of collagen application. Hydrogel is a three-dimensional spatial network structure with a unique extracellular matrix-like structure formed by physical or chemical cross-linking. It is a soft solid polymer material rich in water.

[0003] Currently, the preparation of collagen-based hydrogels is mainly based on the self-assembly behavior of collagen and physical or chemical cross-linking techniques. Among them, collagen self-assembly requires a long incubation period at a specific temperature to form a hydrogel. Physical cross-linking techniques based on ultraviolet light irradiation are highly dependent on equipment, while chemical cross-linking techniques are the most widely used due to their advantages such as not requiring special equipment and rapid gel formation.

[0004] However, the mechanical properties of natural collagen-based hydrogels are often unsatisfactory and cannot fully meet practical needs. The key factors affecting the mechanical properties of hydrogels are the dry matter content and the network structure formed by the components in the system. This undoubtedly points the way to improving the mechanical properties of collagen-based hydrogels.

[0005] As a high-molecular-weight protein, natural collagen has a molecular weight of approximately 300 kDa. Its solubility, especially in neutral or alkaline aqueous solutions, is very poor, which is a key reason for the insufficient mechanical properties and cumbersome preparation process of collagen-based hydrogels. The physiological environment of living organisms is generally neutral or slightly alkaline. Under acidic conditions, cell proliferation and migration are often greatly inhibited. Therefore, the preparation of collagen-based hydrogels for biomedical applications typically requires first dissolving natural collagen in an acidic solution, such as acetic acid or hydrochloric acid, and then dialysis with a neutral or alkaline PBS buffer solution to remove acidic substances before gel formation.

[0006] To address the problem of poor solubility of natural collagen under neutral and alkaline conditions, a technical solution can be found by adjusting the types and quantities of functional groups on the surface of natural collagen through carboxylation modification, thereby altering the isoelectric point.

[0007] In fact, the applicant had previously achieved succinylation of amino groups on the surface of natural collagen and demonstrated that succinylation grafting modification can regulate the isoelectric point of natural collagen, thereby improving its solubility under neutral or alkaline conditions. Cell experiments also showed that succinylated collagen has good biocompatibility. Furthermore, it is particularly noteworthy that grafting modification of natural collagen does not affect its triple helix structure, a typical structural feature of natural collagen and crucial for its biological functions. For example, the cell adhesion, cell proliferation and migration guidance, and physiological hemostatic properties of collagen denaturation products (collagen peptides or gelatin) are all weakened or lost due to the loss of the triple helix structure.

[0008] Although carboxylation modification can improve the solubility of natural collagen under neutral or alkaline conditions, it also leads to the loss of collagen's self-assembly properties, thus preventing the formation of hydrogel systems with spatial network structures.

[0009] In addition, carboxylation-modified collagen causes the active amino groups on its surface to be replaced by carboxyl groups, making it impossible to construct a hydrogel system using glutaraldehyde as a crosslinking agent. Although carbodiimide crosslinking agents are commonly used, their effectiveness depends on the presence of free amino and carboxyl groups in the crosslinking system. They can be used as crosslinking agents to promote amidation reactions between amino and carboxyl groups. There are currently no reports on the crosslinking of biomacromolecules such as fully carboxylated collagen after carboxylation modification. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a high-concentration collagen-based hydrogel, its preparation method, and its application.

[0011] The high-concentration collagen-based hydrogel provided by this invention is designed based on the correlation between dry matter content and the mechanical properties of hydrogels, combined with the novel discovery that "carbodiimide crosslinking agents promote the gelation of carboxylated collagen." Specifically, by modifying natural collagen with carboxylation, its solubility in physiological pH environments, i.e., neutral or alkaline aqueous solutions, is improved. Furthermore, a spatial network structure is constructed using biocompatible carbodiimide crosslinking agents to achieve the construction of the gel system. The resulting hydrogel has advantages such as high collagen content, simple preparation process, and good mechanical properties.

[0012] The present invention provides a method for preparing a high-concentration collagen-based hydrogel, comprising the following steps:

[0013] (1) Carboxylation modification of collagen: Under conditions below 20°C, natural collagen was grafted with carboxylation reagent to obtain carboxylated collagen with high grafting density.

[0014] (2) Preparation of carboxylated collagen solution: Dissolve the carboxylated collagen obtained in (1) in deionized water or buffer solution to prepare carboxylated collagen solution;

[0015] (3) Formation of collagen-based hydrogel: Weigh a carbodiimide crosslinking agent and add it to the carboxylated collagen solution obtained in (2), stir evenly, and let stand to form a hydrogel; or squeeze the carboxylated collagen solution obtained in (2) into a solution containing a carbodiimide crosslinking agent and let stand to form a hydrogel.

[0016] In the steps involved in the above-described method provided by this invention:

[0017] The carboxylating agent mentioned in (1) is any one of the following: a compound having an lactone ring structure or a dicarboxylic acid compound protected by N-hydroxysuccinimide (NHS).

[0018] Preferably, the carboxylating agent is any one of succinic anhydride and its derivatives, dimethylmaleic anhydride and its derivatives, itaconic anhydride and its derivatives, and N-hydroxysuccinimide monoprotected dicarboxylic acid compounds.

[0019] The carboxylating reagent used in this invention is a reagent that can react directly with amino groups under alkaline conditions and form terminal carboxyl groups. The carboxylating reagent is used in sufficient or excessive amounts to ensure that the active amino groups present in natural collagen can be converted into carboxyl groups.

[0020] The natural collagen described in (1) is a natural type I collagen with a complete triple helix structure that is extracted, isolated and purified from the tissues of vertebrates.

[0021] Preferably, the vertebrate tissues are skin, tendons, and Achilles tendons.

[0022] Preferably, the reaction temperature in (1) is ≤10℃.

[0023] The invention employs a relatively low reaction temperature primarily to avoid collagen denaturation caused by heating or high temperatures, which would cause it to lose its original complete triple helix structure. Furthermore, experiments in this invention have shown that heating collagen at 37°C for a period of time can indeed damage the collagen structure, making it difficult for the collagen to crosslink under the action of the crosslinking agent to obtain a hydrogel.

[0024] Furthermore, the carboxylation modification of collagen in (1) is carried out as follows:

[0025] Natural collagen was dissolved in acetic acid solution to obtain a collagen solution. The obtained collagen solution was dialyzed with phosphate buffer solution until the pH of the dialysate no longer decreased. An excess of carboxylating reagent was added, mixed well, and reacted. The unreacted reagent was removed by the aforementioned dialysis operation to obtain carboxylated collagen, which was then lyophilized for later use.

[0026] Preferably, in the carboxylation modification of collagen in (1), the concentration of collagen solution is 1-8 mg / mL, the pH of phosphate buffer solution is 8-10, the dialysis time of collagen solution with phosphate buffer solution is 12-48 h, the molecular weight cutoff of dialysis bag is 12000-15000 Da, the dialysis solution is changed every 3-4 h, the added carboxylation reagent is in excess, and the carboxylation reaction lasts 12-48 h.

[0027] Preferably, the carboxylation modification of collagen in (1) is carried out as follows:

[0028] Natural collagen was dissolved in acetic acid to obtain a collagen solution with a concentration of 1–8 mg / mL. The obtained collagen solution was dialyzed with a phosphate buffer solution with a pH of 8.0–10.0 for 12–48 h. The molecular weight cutoff of the dialysis bag was 12,000–15,000 Da. The dialysate was changed every 3–4 h until the pH of the dialysate no longer decreased. An excess carboxylation reagent was added, mixed well, and reacted for 12–48 h. Unreacted reagent was removed by the aforementioned dialysis operation to obtain carboxylated collagen, which was then lyophilized for later use.

[0029] In the above steps, the buffer solution mentioned in (2) is a buffer solution that does not contain components that react with carboxyl groups.

[0030] Preferably, the buffer solution described in (2) is a phosphate buffer solution.

[0031] Preferably, the concentration of the carboxylated collagen solution in (2) is ≤60mg / mL.

[0032] In the above steps, the carbodiimide crosslinking agent mentioned in (3) is any one of dicyclohexylcarbodiimide, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0033] Preferably, the mass ratio of the crosslinking agent to carboxylated collagen in (3) is (1-5):(5-1).

[0034] Preferably, the operation of extruding the succinylated collagen solution into the carbodiimide crosslinking reagent solution as described in (3) uses any one of the following as the extrusion device: the extrusion device of the 3D printing instrument, a simple syringe, or other commonly used extrusion devices.

[0035] During the research process, the inventors discovered that succinylated collagen obtained after succinic anhydride carboxylation modification can form a hydrogel system in the presence of carbodiimide crosslinking agents, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Furthermore, by adjusting the concentrations of the raw materials and EDC reagent, hydrogels with mechanical properties far superior to those of natural collagen can be obtained. Moreover, EDC is a water-soluble, biocompatible carbodiimide crosslinking agent that is easily removed after the reaction.

[0036] Furthermore, while existing technologies disclose the use of carbodiimide crosslinking agents to promote the formation of carboxyl and amino-m-amide bonds to construct the spatial network structure of hydrogels, the succinylated collagen prepared in this invention can still be crosslinked into gels via EDC even without almost any active amino groups. This new discovery undoubtedly lays the foundation for the gelation behavior of carboxylated modified collagen.

[0037] The beneficial effects of this invention are as follows:

[0038] Firstly, this invention adjusts the isoelectric point of natural collagen through carboxylation modification technology, thereby improving the solubility of collagen in neutral and alkaline environments and obtaining a high-concentration collagen solution.

[0039] Secondly, in view of the defect that collagen loses its self-assembly performance due to carboxylation modification, this invention introduces carbodiimide crosslinking agents to construct hydrogels without containing amino groups, and successfully realizes the construction of carboxylated collagen spatial networks.

[0040] Third, the collagen-based hydrogel obtained by this invention has a dry matter content and mechanical properties that are far higher than those of conventional natural collagen gels. Furthermore, the collagen-based hydrogel is colorless and transparent, and the resulting gel can be applied in fields such as bio-3D printing, in-situ filling, drug loading, and delivery. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the preparation principle of the collagen-based hydrogel in Example 1 of the present invention;

[0042] Figure 2 In Example 1 of the present invention, the grafting rate of succinylated collagen was determined by the 2,4,6-trinitrobenzenesulfonic acid colorimetric method.

[0043] Figure 3 In Example 1 of this invention, the inversion method is used to characterize the gelation behavior of succinylated collagen in the presence of EDC;

[0044] Figure 4 In Example 2 of the present invention, circular dichroism spectroscopy was used to characterize the changes in the triple helix structure of succinylated collagen with heating temperature and heating time.

[0045] Figure 5 The following are examples of succinylated collagen after different heat treatments in Example 2 of the present invention;

[0046] Among them, (a) is succinylated collagen without heat treatment, (b) is succinylated collagen incubated at 37℃ for 10 min, (c) is succinylated collagen incubated at 37℃ for 50 min, and (d) is succinylated collagen incubated at 40℃ for 30 min.

[0047] Figure 6 The dissolution of succinylated collagen in Example 3 of the present invention;

[0048] Figure 7 The mechanical properties of succinylated collagen hydrogel and natural collagen hydrogel were characterized by rheological methods in Example 3 and Comparative Example 4 of the present invention.

[0049] Figure 8 This is a product image obtained by 3D printing using a 30 mg / mL succinylated collagen aqueous solution in Example 4 of the present invention;

[0050] Figure 9 The inverted method was used in Comparative Example 1 of the present invention to characterize the gelation of succinylated collagen in the presence of glutaraldehyde.

[0051] Figure 10 The inverted method in Comparative Example 2 of this invention is used to characterize the gelation of natural collagen in the presence of EDC.

[0052] Figure 11 The inversion method in Comparative Example 3 of this invention is used to characterize the gelation of natural collagen in the presence of glutaraldehyde.

[0053] Figure 12 The dissolution of natural collagen in Comparative Example 4 of the present invention is shown. Detailed Implementation

[0054] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0055] Example 1

[0056] The preparation principle of collagen-based hydrogels in this embodiment is as follows: Figure 1 As shown.

[0057] (1) Preparation of high grafting density succinylated collagen

[0058] Under conditions below 10°C, 500 mg of bovine Achilles tendon type I collagen was weighed, dissolved in 0.5 mol / L acetic acid solution, and then transferred into a dialysis bag with a molecular weight cutoff of 14000 Da. After dialysis with phosphate buffer solution at pH 9.0 for 48 h, the collagen solution was transferred to a beaker.

[0059] 100 mg of succinic anhydride was dissolved in a small amount of dimethyl sulfoxide and added dropwise to the above solution. While adding the solution, the pH of the system was adjusted with 0.5 mol / L NaOH and maintained at 8.0–9.0. The reaction was stirred for 24 h. The reaction system was then transferred to a dialysis bag and dialyzed with deionized water for 48 h. The dialysis fluid was replaced every 3 h. After that, the solution was freeze-dried to obtain succinylated collagen.

[0060] The grafting rate of succinylated collagen was determined by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) colorimetric method, such as... Figure 2 As shown.

[0061] (2) Preparation of succinylated collagen solution

[0062] The succinylated collagen prepared in (1) was dissolved in phosphate buffer solutions with pH values ​​of 5.0, 7.0 and 9.0, respectively, to obtain 2 mL of 8 mg / mL succinylated collagen solution.

[0063] (3) Gel formation of succinylated collagen

[0064] The succinylated collagen solution prepared in (2) was transferred to a transparent glass bottle, EDC was added and quickly mixed evenly, and allowed to stand to form a hydrogel; wherein the mass ratio of succinylated collagen to EDC was 1.6:1.

[0065] The gelation of succinylated collagen under EDC was characterized by an inversion experiment, as shown in the attached figure. Figure 3 As shown.

[0066] from Figure 2 As can be seen, the more free amino acids in the collagen sample, the stronger the absorption peak at 346nm. Taking the number of free amino acids in natural collagen as 100%, the grafting rate of free amino acids in grafted modified collagen can be calculated. Therefore, the amino grafting rate of succinylated collagen is 98.2%.

[0067] Figure 3 The results show that the succinylated collagen prepared in Example 1 can form a colorless and transparent gel system under the cross-linking effect of EDC, even under different pH conditions, i.e., acidic or alkaline environments.

[0068] Example 2

[0069] The succinylated collagen prepared in Example 1 was subjected to heating to disrupt its triple helix structure, resulting in denatured succinylated collagen. The specific procedure is as follows:

[0070] The succinylated collagen prepared in Example 1 was dissolved in a phosphate buffer solution with a pH of 7.0 to prepare a solution with a concentration of 8 mg / mL. The solution was then incubated at 37°C for 10 min and 50 min, or at 40°C for 30 min, to obtain three types of denatured succinylated collagen with different degrees of triple helix structure destruction.

[0071] Take 2 mL of the sample solution obtained above, add 10 mg of EDC to crosslink into a gel. Simultaneously, circular dichroism spectroscopy is used to characterize the changes in the triple helix structure of succinylated collagen with heating temperature and time. The results are shown in the appendix. Figure 4 Furthermore, the gelation behavior and stability of the resulting gel were characterized by an inversion experiment, as shown in the attached figure. Figure 5 As shown.

[0072] Figure 4 The study showed that succinylated collagen has a triple helix structure with similar integrity to natural collagen, but the triple helix integrity of succinylated collagen decreased significantly with prolonged heating time or increased heating temperature.

[0073] Figure 5 In the figures, (a) is succinylated collagen without heat treatment, (b) is succinylated collagen incubated at 37°C for 10 min, (c) is succinylated collagen incubated at 37°C for 50 min, and (d) is succinylated collagen incubated at 40°C for 30 min.

[0074] Figure 5 The results show that untreated succinylated collagen can form a stable gel;

[0075] Although the systems obtained by incubating succinylated collagen at 37°C for 10 min and 50 min could form gels, they degraded after 3 h and 15 min, respectively; while the system obtained by incubating succinylated collagen at 40°C for 30 min could not form gels.

[0076] The above results indicate that the triple helix structure of succinylated collagen is a key factor affecting its EDC crosslinking and gelation properties. If the triple helix structure is destroyed, natural collagen can hardly gel again.

[0077] Example 3

[0078] The mechanical properties of the succinylated collagen hydrogel prepared in Example 1 were determined.

[0079] The succinylated collagen prepared in Example 1 was dissolved in a beaker containing a phosphate buffer solution with a pH of 7.0 to prepare a 10 mg / mL succinylated collagen phosphate buffer solution. The flowability of the succinylated collagen phosphate buffer solution was observed by tilting the beaker.

[0080] Based on the above steps, the concentration of the succinylated collagen phosphate buffer solution was increased to 30 mg / mL, and the pH was set to 7.0. The beaker was then tilted further, and the fluidity of the collagen solution was observed, as shown in the attached figure. Figure 6 As shown.

[0081] The elastic modulus (G') and viscous modulus (G”) of succinylated collagen were further measured. The results are shown in Table 1 below and appendix. Figure 7 As shown by the blue and black lines in the diagram.

[0082] Table 1. Elastic modulus (G') and viscous modulus (G”) of succinylated collagen

[0083] Sample Name Elastic modulus (G') (Pa) Viscous modulus (G”) (Pa) Succinylated collagen 311 13

[0084] The results show that the 30 mg / mL succinylated collagen phosphate buffer solution still exhibits a fluid dynamic, and the process does not require acetic acid dissolution / phosphate buffer dialysis. The mechanical properties of the 30 mg / mL succinylated collagen hydrogel obtained by the above system are 311 Pa.

[0085] Example 4

[0086] The succinylated collagen prepared in Example 1 was dissolved in deionized water to prepare a 30 mg / mL succinylated collagen aqueous solution. This solution was then printed using a 3D printing device onto a 10 mg / mL EDC aqueous solution. The resulting sample is shown below. Figure 8 As shown.

[0087] As can be seen, the collagen-based hydrogel prepared by this invention is colorless and transparent, has good mechanical properties, and can exhibit good technical effects when applied to 3D printing.

[0088] Example 5

[0089] Based on Example 1, the carboxylating agent was changed and replaced with itaconic anhydride, while all other conditions remained the same as in Example 1.

[0090] Example 6

[0091] Based on Example 1, the carboxylating agent was changed and replaced with dimethylmaleic anhydride, while all other conditions remained the same as in Example 1.

[0092] Example 7

[0093] Based on Example 1, the carboxylating agent was changed and replaced with N-hydroxysuccinimide monoprotected adipic acid, while all other conditions remained the same as in Example 1.

[0094] Example 8

[0095] Based on Example 1, only in step (3) the crosslinking agent is replaced with 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, and the rest of the operation is the same as in Example 1.

[0096] Example 9

[0097] Based on Example 2, only in step (3) the crosslinking agent was replaced with 2-(7-azobenzotriazole)-N,N,N′,N′-tetramethylurea hexafluorophosphate, and the rest of the operation was the same as in Example 1.

[0098] Example 10

[0099] Based on Example 1, grass carp skin collagen was used as raw material, succinic anhydride was used as carboxylation reagent, and dicyclohexylcarbodiimide was used as crosslinking agent. The ratio of crosslinking agent to collagen was 1:1.

[0100] The properties of the collagen-based hydrogels prepared in Examples 5-10 are shown in Table 2 below.

[0101] Table 2 shows the mechanical properties of the hydrogels obtained in Examples 5-10.

[0102]

[0103]

[0104] Example 11

[0105] Based on Example 1, only the ratio of carboxylated collagen to crosslinking agent was changed, with the ratio set to 1:5, 1:1, and 5:1 respectively; and the properties of the prepared collagen were characterized. The properties of the prepared collagen-based hydrogels are shown in Table 3 below.

[0106] Table 3. Mechanical properties of the hydrogel obtained in Example 11

[0107] The ratio of carboxylated collagen to crosslinking agent Elastic modulus (G') (Pa) Viscous modulus (G”) (Pa) 1:5 121 9 1:1 289 12 5:1 463 21

[0108] Comparative Example 1

[0109] Steps (1) to (2) are the same as in Example 1. In step (3), glutaraldehyde, commonly used in the preparation of collagen raw materials, is used instead of EDC as a crosslinking agent to repeat the gelation process of the succinylated collagen system under different pH conditions. The gelation of the succinylated collagen is shown in the attached figure. Figure 9 As shown.

[0110] In fact, when glutaraldehyde is used as a crosslinking agent, it forms a Schiff base structure by promoting the reaction between the aldehyde group and the amino group in the molecule. The crosslinking mechanism of EDC is to promote the amidation reaction between the amino and carboxyl groups in the molecule. Succinylated collagen modified by carboxylation cannot form a gel when glutaraldehyde is used as a crosslinking agent. That is, there is no active amino group in the succinylated collagen prepared in this invention.

[0111] Comparative Example 2

[0112] (1) Take the bovine Achilles tendon type I collagen used in Example 1 directly, dissolve it in phosphate buffer solution, and prepare bovine Achilles tendon type I collagen phosphate buffer solutions of 2 mL, 8 mg / mL, and pH of 5.0, 7.0 and 9.0 respectively.

[0113] (2) The bovine Achilles tendon type I collagen phosphate buffer solution prepared in (1) was transferred to a transparent glass bottle. EDC was added and the mixture was quickly mixed evenly. The solution was allowed to stand to form a hydrogel, and its gelation behavior was characterized by an inversion experiment, as shown in the attached figure. Figure 10 As shown.

[0114] Comparative Example 3

[0115] (1) Take the bovine Achilles tendon type I collagen used in Example 1 directly, dissolve it in PBS buffer, and prepare bovine Achilles tendon type I collagen phosphate buffer solutions of 2 mL, 8 mg / mL, and pH 5.0, 7.0 and 9.0 respectively;

[0116] (2) The bovine Achilles tendon type I collagen phosphate buffer solution prepared in (1) was transferred to a transparent glass bottle, glutaraldehyde was added, and the mixture was quickly mixed evenly. The solution was allowed to stand to form a hydrogel, and its gelation behavior was characterized by an inverted inversion experiment, as shown in the attached figure. Figure 11 As shown.

[0117] The results of Comparative Examples 2 and 3 show that bovine Achilles tendon type I collagen is a natural collagen containing a large number of amino and carboxyl groups. Therefore, it can be cross-linked into a gel regardless of whether EDC or glutaraldehyde is used as a cross-linking agent. However, at a concentration of 8 mg / mL, the resulting gel system is milky white and opaque. The poor light transmittance of this type of gel system makes it difficult to directly apply to biomedical materials such as artificial corneas that require light transmittance.

[0118] Comparative Example 4

[0119] The mechanical properties of bovine Achilles tendon type I natural collagen used in Example 1 of this invention were measured using the same method as in Example 3. The mechanical properties of the bovine Achilles tendon type I natural collagen are shown in Table 4 and the appendix below. Figure 7 The solid and dashed black lines in the figure illustrate the collagen flow during the measurement process, as shown in the attached figure. Figure 12 As shown.

[0120] Table 4. Mechanical properties of bovine Achilles tendon type I natural collagen.

[0121]

[0122]

[0123] Combined with Table 4 and Appendix Figure 7 It can be seen that the mechanical properties of bovine Achilles tendon type I natural collagen are only 21 Pa, while the mechanical properties of succinylated collagen hydrogel in Example 3 are as high as 311 Pa, which is 15 times that of natural collagen hydrogel. Furthermore, the highest concentration of natural collagen is 10 mg / mL, and the solubility of collagen is limited, making it difficult to obtain a solution system with a higher concentration. The utilization rate and mechanical properties of collagen are far inferior to those of the modified collagen-based hydrogel in this invention.

[0124] Figure 12 The results showed that bovine Achilles tendon type I collagen could not be dissolved in PBS buffer solution at pH 7.0. It had to be dissolved in acetic acid first, and then dialyzed with phosphate buffer solution. The resulting 10 mg / mL system was in a gel state and could not be extruded, 3D printed or other operations.

[0125] However, the succinylated collagen obtained after modification in this invention has good solubility in phosphate buffer solution, and the 10 mg / mL system still has good fluidity.

Claims

1. A method for preparing a high concentration collagen-based hydrogel, characterized by, It comprises the following steps: (1) Carboxylation modification of collagen: under the condition of less than 20℃, grafting modification of natural collagen is carried out by using carboxylation reagent, so as to obtain carboxylated collagen with high grafting density, and the carboxylation reagent is sufficient or excess, so as to ensure that the active amino group in the natural collagen can be converted into carboxyl group; The natural collagen is natural type I collagen with complete triple helix structure, which is extracted and separated and purified from the tissue of vertebrate animals; The carboxylation reagent is any one of succinic anhydride and its derivatives, dimethyl maleic anhydride and its derivatives, itaconic anhydride and its derivatives, and N-hydroxysuccinimide monoprotected dicarboxylic acid compound; (2) Preparation of carboxylated collagen solution: the carboxylated collagen obtained in (1) is dissolved in deionized water or buffer solution to prepare a carboxylated collagen solution; (3) Gelation of collagen-based hydrogel: a carbodiimide cross-linking reagent is weighed and added to the carboxylated collagen solution obtained in (2), stirred uniformly, and left to form a hydrogel; or the carboxylated collagen solution obtained in (2) is extruded into a solution containing a carbodiimide cross-linking reagent, and left to form a hydrogel.

2. A method of preparing a high concentration collagen-based hydrogel according to claim 1, characterized by, The carboxylation modification of collagen in (1) is carried out as follows: The natural collagen is dissolved in acetic acid solution to obtain a collagen solution, and the obtained collagen solution is dialyzed using a phosphate buffer solution until the pH of the dialysate no longer decreases, an excess of carboxylation reagent is added, mixed, and then reacted, and the unreacted reagent is removed by the aforementioned dialysis operation to obtain carboxylated collagen, which is lyophilized for later use.

3. The method for preparing a high-concentration collagen-based hydrogel as described in claim 1, characterized in that, The buffer solution in (2) is a buffer solution containing no carboxyl-reactive components.

4. The method for preparing a high-concentration collagen-based hydrogel as described in claim 1, characterized in that, The carbodiimide cross-linking reagent in (3) is any one of dicyclohexyl carbodiimide, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride.

5. The method for preparing a high-concentration collagen-based hydrogel as described in claim 1, characterized in that, The mass ratio of the cross-linking reagent to the carboxylated collagen in (3) is (1-5):(5-1).

6. A high-concentration collagen-based hydrogel obtained by using the preparation method of the high-concentration collagen-based hydrogel according to claim 1.

7. Application of the hydrogel according to claim 6 in biological 3D printing, in-situ gelation system, drug loading and transportation.

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