A collagen hydrogel and a preparation method and application thereof

Abstract

The present application relates to a kind of collagen hydrogel and its preparation method and application, the preparation method includes the following steps: (1) using amine oxidase catalytic collagen oxidation deamination generates unsaturated aldehyde group functional group, intramolecular or intermolecular crosslinking occurs, completes first crosslinking;(2) the product of first crosslinking occurs secondary crosslinking under the catalysis of carboxyl activating agent, obtains the collagen hydrogel.In the present application, the collagen hydrogel prepared by the method is pure collagen hydrogel, solidification speed is fast, biocompatibility is good (excellent bionics), mechanical strength is adjustable, structure size is adjustable, stability is good, and application range is wide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of collagen hydrogel, and particularly relates to a collagen hydrogel, a preparation method and application thereof. BACKGROUND

[0002] A hydrogel is a polymeric network that can absorb and retain large amounts of water. Within this polymer network are hydrophilic groups or regions that can hydrate under neutral conditions, thus creating a gel structure. Due to the highly hydrated three-dimensional network, hydrogels provide space for cell adhesion, proliferation and differentiation. Therefore, hydrogel scaffolds have great attraction in loading cells and tissue development.

[0003] Collagen is the main component of the extracellular matrix and is the most abundant and widely distributed protein in the animal body. In addition to its excellent mechanical properties, collagen also has low antigenicity, blood clotting, easy absorption by the human body, and the ability to promote cell survival and growth, and has been widely used in the field of biomedicine.

[0004] At present, most collagen hydrogels are prepared by physical crosslinking such as low-temperature self-assembly, ultraviolet irradiation, thermal crosslinking, freeze-drying, hydrogen bonding force and the like, and have the problems of poor mechanical properties and fast degradation rate; or are realized by forming a composite system with other materials, which has the problems of complex composition, unclear degradation products and the like.

[0005] CN 109265705A discloses a collagen thiolated derivative, a preparation method and application thereof. With collagen as raw material, a thiol compound with both thiol and carboxyl groups or both thiol and amino groups is used as a modifier, and under the action of a carboxyl activator, thiol groups are successfully introduced into the collagen molecular chain. The collagen derivative disclosed by the present application has flexible and variable side chains, and different modified collagen thiolated derivatives are prepared.

[0006] A method for preparing a collagen membrane disclosed in CN 107513172A utilizes hydroxyl carboxylic acid-N-hydroxysuccinimide ester to activate collagen to form covalent crosslinking, and further utilizes an oxidase solution to catalyze secondary crosslinking. This method has the problems of poor colloidal flexibility, fast degradation rate, uncontrolled structure, slow gelation speed, easy distortion and the like in the preparation process, which greatly limits the application of collagen hydrogel in tissue engineering scaffolds, especially in fiber materials.

[0007] In summary, it is crucial to develop a preparation method of collagen hydrogel with fast solidification speed, good colloidal mechanical properties, slow degradation rate, controllable structure and fast and accurate shaping. SUMMARY

[0008] In view of the deficiencies of the prior art, the purpose of the present application is to provide a collagen hydrogel and a preparation method and application thereof, wherein the collagen hydrogel prepared by the method is a pure collagen hydrogel, has a fast solidification speed, good biocompatibility (excellent bionics), adjustable mechanical strength, adjustable structure size, good stability, and a wide application range.

[0009] To achieve the purpose, the present application adopts the following technical solutions:

[0010] In a first aspect, the present application provides a preparation method of a collagen hydrogel, which comprises the following steps:

[0011] (1) collagen is oxidatively deaminated by using amine oxidase to generate unsaturated aldehyde functional groups, intramolecular or intermolecular crosslinking occurs, and the first crosslinking is completed;

[0012] (2) the product of the first crosslinking is subjected to secondary crosslinking under the catalysis of a carboxyl activator to obtain the collagen hydrogel.

[0013] In the present application, the gelation transformation of pure collagen is realized by two crosslinking methods, i.e., the first crosslinking (enzyme crosslinking reaction) and the second crosslinking (amidation crosslinking), and the mechanical strength and microstructure of the pure collagen hydrogel are adjusted by changing the concentration of the collagen solution and the amount of the enzyme, and selecting the solidification receiving liquid.

[0014] Specifically, collagen (COL) is the most abundant protein in human and animal bodies. So far, 28 types of collagen have been found in vertebrate animals. Among them, type I collagen is the most abundant and widely used collagen. All collagen molecules are composed of three polypeptide chains, i.e., a triple helix structure. This structure endows collagen with some special properties, such as circular dichroism, low antigenicity, excellent blood clotting, etc. Collagen is an amphoteric electrolyte, and the basic amino acids (such as lysine, histidine, etc.) in the molecule contain active groups such as ε-amino groups and imino groups, which can react with many chemical reagents.

[0015] In the present application, lysyl or hydroxylysyl on the collagen peptide chain is subjected to enzyme catalysis oxidation by using amine oxidase, and aldehyde lysyl or aldehyde hydroxylysyl is obtained. Further, chemical crosslinking occurs between aldehyde groups or between aldehyde groups and active amino groups, which can achieve the purpose of fast solidification.

[0016] The method of the present application simulates the maturation mechanism of collagen and elastin in vivo, and therefore, the method is also expected to be applicable to the preparation of elastin hydrogel and gelatin hydrogel.

[0017] In the present application, the collagen in the collagen solution is pure collagen, preferably any one of bovine skin-derived type I collagen, bovine skin-derived type II collagen or mouse tail-derived type I collagen or a combination of at least two of them, wherein a typical but non-limiting combination includes a combination of bovine skin-derived type I collagen and bovine skin-derived type II collagen, a combination of bovine skin-derived type II collagen and mouse tail-derived type I collagen, a combination of bovine skin-derived type I collagen, bovine skin-derived type II collagen and mouse tail-derived type I collagen, etc.

[0018] Preferably, the chemical cross-linking reaction in the present application is carried out under weak alkaline conditions, belongs to Schiff base reaction and belongs to pH-responsive chemical bonds, thereby expanding the application scenarios.

[0019] Preferably, the concentration of the collagen solution is 20-60 mg / mL, such as 24 mg / mL, 26 mg / mL, 28 mg / mL, 30 mg / mL, 32 mg / mL, 34 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 48 mg / mL, 50 mg / mL, 52 mg / mL, 54 mg / mL, 56 mg / mL, 58 mg / mL, etc.

[0020] Preferably, the pH of the collagen solution is less than 7, such as 6.5, 6, 5.5, 5, etc.

[0021] Preferably, the solvent of the collagen solution includes an organic acid solution.

[0022] Preferably, the solvent of the collagen solution includes an acetic acid solution.

[0023] Preferably, the mass fraction of the acetic acid solution is 0.01%-30%, such as 1%, 2%, 5%, 10%, 15%, 20%, 25%, etc.

[0024] Preferably, the concentration of the amine oxidase solution is 5-7 U / mL, such as 5.2 U / mL, 5.4 U / mL, 5.6 U / mL, 5.8 U / mL, 6 U / mL, 6.2 U / mL, 6.4 U / mL, 6.6 U / mL, 6.8 U / mL, etc.

[0025] In the present application, by adjusting the concentration of collagen and the concentration of enzyme, the elasticity modulus of pure collagen hydrogel and the internal pore structure can be adjusted, thereby expanding the application range of the material.

[0026] Preferably, the pH of the amine oxidase solution is 6.5-7.5, such as 6.6, 6.8, 7.0, 7.2, 7.4, etc.

[0027] Preferably, the amine oxidase includes any one or a combination of at least two of plasma amine oxidase, monoamine oxidase, diamine oxidase or lysyl oxidase, wherein a typical but non-limiting combination includes a combination of plasma amine oxidase and monoamine oxidase, a combination of monoamine oxidase, diamine oxidase and lysyl oxidase, a combination of plasma amine oxidase, monoamine oxidase, diamine oxidase and lysyl oxidase, etc., and further preferably plasma amine oxidase.

[0028] In the present application, plasma amine oxidase, monoamine oxidase, diamine oxidase or lysyl oxidase plays a similar role in the process of collagen fiber formation in the body, and the reason for the preference of plasma amine oxidase is that such research is expected to achieve in-situ solidification using patient's autologous plasma.

[0029] Preferably, the secondary cross-linking is performed in a solidification receiving solution containing a carboxyl activator.

[0030] Preferably, the solidification receiving solution has a pH value of 7-9, such as 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, 8.8, etc.

[0031] Preferably, the solidification receiving solution includes a weak alkali solution.

[0032] In the present application, the solidification receiving solution is a weak alkali solution containing bicarbonate or carbonate ions. Using a weak alkali solution as the solidification receiving solution can provide the reaction conditions for enzyme cross-linking, rapidly achieve the rapid gelation transformation of raw materials to occur Schiff base reaction to form a first layer of reversible covalent cross-linking network. At the same time, the solidification solution can react with the solvent glacial acetic acid of the collagen solution to generate carbon dioxide gas bubbles to form pores by gas-liquid displacement in the gel interior.

[0033] Preferably, the weak alkali solution includes any one or a combination of at least two of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium carbonate or potassium carbonate, wherein a typical but non-limiting combination includes a combination of sodium bicarbonate and potassium bicarbonate, a combination of potassium bicarbonate, sodium carbonate and potassium carbonate, a combination of sodium bicarbonate, potassium bicarbonate, sodium carbonate and potassium carbonate, etc., and further preferably sodium bicarbonate (NaHCO3).

[0034] In the present application, the weak alkali solution is a sodium bicarbonate solution, which avoids the denaturation of collagen protein caused by the use of a strong alkali solution. It provides the required weak alkali pH conditions for enzyme cross-linking. Moreover, the solvent glacial acetic acid of the collagen solution can rapidly react with the sodium bicarbonate solution to generate CO2 gas in the hydrogel interior, and then gather into uniform-sized bubbles to form abundant pores in the hydrogel interior after gas-liquid displacement.

[0035] Preferably, the reaction temperature of the primary cross-linking is 4-37℃, such as 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, etc.

[0036] In the present application, the amine oxidase is mixed with the collagen solution uniformly at room temperature, so that the amine oxidase fully exerts catalytic oxidation activity.

[0037] Preferably, the reaction time of the primary cross-linking is 1s-12h, such as 1s, 1h, 2h, 4h, 6h, 8h, 10h, etc.

[0038] It should be noted that, in the present application, before the reaction product of the enzyme catalytic reaction is added to the solidification liquid for gelation, the preparation method can further include centrifuging the reaction product of the enzyme catalytic reaction to remove air bubbles caused by external factors, so as to avoid the formation of additional large and uneven air bubble holes in the gel, which affects the internal structure of the gel.

[0039] Preferably, the carboxyl activator includes 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS).

[0040] In the present application, EDC is a carbodiimide soluble in water, which is used as an activating agent for carboxyl groups in amide synthesis, and is also used for activating phosphate groups, cross-linking of proteins and nucleic acids, and preparation of immunoconjugates. EDC is often used in combination with N-hydroxysuccinimide (NHS) or N-hydroxylthiosuccinimide to improve coupling efficiency. NHS is N-hydroxysuccinimide, which activates carboxyl groups to facilitate the formation of amide bonds.

[0041] The acidic amino acids (such as glutamic acid and aspartic acid) in the collagen peptide chain provide more side chain carboxyl groups, which have strong reactivity. Under the action of the carboxyl activator EDC / NHS, the remaining amino groups on the collagen peptide chain can be reacted to further form a covalently cross-linked reversible polymer network structure, forming a pure collagen hydrogel.

[0042] Preferably, the mass ratio of the 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and the N-hydroxysuccinimide is (1-10):1, where 1-10 can be 2, 3, 4, 5, 6, 7, 8, 9, etc.

[0043] Preferably, the concentration of the carboxyl activator is 0.5-1mg / mL, such as 0.6mg / mL, 0.7mg / mL, 0.8mg / mL, 0.9mg / mL, etc.

[0044] In the present application, the carboxyl activator is further preferably a combination of EDC and NHC, and the mass ratio of the two is (1-10):1, and the concentration is 0.5-1mg / mL. Under this condition, the residual amino groups and carboxyl groups in the collagen hydrogel undergo amidation reaction to form a second layer of covalently cross-linked network.

[0045] Optionally, the preparation method further comprises gelating the collagen solution into collagen fibers with different diameters by micro-injection technology. Specifically, the diameter of the needle for injection extrusion and the parameters such as flow rate, voltage, etc. can be changed to achieve the above-mentioned purpose.

[0046] In an optional embodiment, the collagen hydrogel obtained by covalent cross-linking is washed with clean water for at least 3 times (for example, 4 times, 5 times, 6 times, etc.), and freeze-dried for 2-3 days (for example, 2.2 days, 2.4 days, 2.6 days, 2.8 days, etc.) to obtain dried pure collagen hydrogel.

[0047] In the above preparation process, first, the rapid solidification of the pure collagen hydrogel is achieved by catalytic oxidation of collagen by amine oxidase to induce enzyme cross-linking reaction; second, the mechanical properties of the pure collagen hydrogel are further enhanced by amide cross-linking using carboxyl activator EDC / NHS.

[0048] Preferably, the reaction temperature of the secondary cross-linking is 4-37℃, for example, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, etc.

[0049] Preferably, the reaction time of the secondary cross-linking is 0.1-12h, for example, 1h, 2h, 4h, 6h, 8h, 10h, etc.

[0050] As a preferred technical solution, the preparation method comprises the following steps:

[0051] (1) collagen solution with a concentration of 20-60mg / mL and pH<7 is mixed with amine oxidase solution with a concentration of 5-7U / mL and pH of 6.5-7.5, and enzyme catalytic oxidation deamination reaction is carried out at 4-37℃ for 1s-12h to complete the first cross-linking;

[0052] (2) the reaction product of the enzyme catalytic reaction is placed in a solidification receiving liquid containing a carboxyl activator with a pH value of 7-9, and secondary cross-linking is carried out at 4-37℃ for 1s-12h to obtain collagen hydrogel.

[0053] In the present application, the method comprises two steps in time: the first step is the Schiff base reaction mediated by amine oxidase, which enables the hydrogel to be rapidly solidified within seconds, meeting the requirements of precision manufacturing and rapid prototyping, and expanding the application of the hydrogel in the fields of dry-wet spinning and 3D biological printing, etc. However, since the Schiff base improves the flowability of the molecular chains in the hydrogel while being sensitive to pH, in order to increase the stability of the hydrogel, the second step of amide coupling reaction mediated by carboxyl activator is carried out to enhance the stability.

[0054] In a second aspect, the present application provides a collagen hydrogel prepared by the preparation method of the first aspect.

[0055] In a third aspect, the present application provides a use of the collagen hydrogel of the second aspect in a biomedical material or a tissue engineering scaffold.

[0056] In the present application, the collagen hydrogel simulates the mechanism of collagen fiber formation in vivo, and has excellent bionics and biocompatibility.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] (1) In the present application, the collagen hydrogel prepared by the method is a pure collagen hydrogel, has a fast solidification speed, good biocompatibility (excellent bionics), adjustable mechanical strength, adjustable structure size, good stability, and a wide application range.

[0059] The maximum tensile elongation of the collagen hydrogel fiber is above 79.13±2.54%, the maximum tensile stress is above 118.60±2.3kPa, and the fiber diameter is between 199.59±8.45-860.91±15.44μm. The collagen hydrogel fiber has good mechanical strength and elastic properties, and a small fiber diameter.

[0060] (2) In the present application, compared with the current pure physical collagen hydrogel, multi-component blended collagen hydrogel, etc., the collagen hydrogel prepared by the method not only improves the solidification speed (the gelation time of the present application can be realized within 12s, and the prior art needs more than 6min), but also improves the mechanical properties and stability of the collagen hydrogel, and the gel composition is simple and clear, and can be applied to 3D biological printing and additive manufacturing.

[0061] (3) In the present application, compared with the existing pure collagen hydrogel crosslinked by the first EDC / NHS carboxyl activator and then crosslinked by an enzyme, the solidification speed is improved, and a large number of dense CO2 bubbles are generated in the collagen hydrogel by means of the reaction between solvents to form rich pores. At the same time, enzyme crosslinking causes the conformation of collagen molecular chains to reversibly change, increases the fluidity of the molecular chains, and finally makes the collagen hydrogel fiber have good flexibility. In addition, the size of the hydrogel fiber is also controlled, and on this basis, a variety of material scaffold structures can be constructed. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 is a schematic diagram of the block-shaped collagen hydrogel prepared by the method of Example 1;

[0063] Figure 2 is a schematic diagram of the collagen hydrogel fiber prepared by the method of Example 1;

[0064] Figure 3are infrared test results of collagen, enzyme cross-linking reaction product and collagen hydrogel fiber involved in the method described in Example 1;

[0065] Figure 4 are micro cross-section images of collagen hydrogel fiber prepared by the method described in Example 1;

[0066] Figure 5a are surface topography images of collagen hydrogel fiber prepared by the method described in Example 1;

[0067] Figure 5b are surface topography images of collagen hydrogel fiber prepared by the method described in Example 2;

[0068] Figure 5c are surface topography images of collagen hydrogel fiber prepared by the method described in Example 3;

[0069] Figure 5d are surface topography images of collagen hydrogel fiber prepared by the method described in Example 4;

[0070] Figure 6 are stress-strain images of collagen hydrogel fiber prepared by the method described in Example 1, Example 5 and Example 6;

[0071] Figure 7 are stress-strain images of collagen hydrogel fiber prepared by the method described in Example 1, Example 7 and Example 8;

[0072] Figure 8 are time scanning test images of the gelation process in the method described in Example 13;

[0073] Figure 9 are time scanning test images of the gelation process in the method described in Comparative Example 1;

[0074] Figure 10 are application images of collagen hydrogel fiber prepared by the method described in Example 1 in tissue engineering scaffolds. DETAILED DESCRIPTION

[0075] For the purpose of understanding the present application, the present application is illustrated by the following examples. It should be apparent to those skilled in the art that the examples are merely used to help understand the present application and should not be regarded as specific limitation to the present application.

[0076] In the present application, the macromolecular collagen extracted from bovine skin collagen is used in each example, and the main component is collagen type 1.

[0077] Example 1

[0078] The present embodiment provides a preparation method of collagen hydrogel, which comprises the following steps:

[0079] (1) 40 mg of pure collagen was dissolved in 1 mL of 1% acetic acid aqueous solution, and completely dissolved into a transparent solution under the action of an ultrasonic oscillator and a 37°C water bath to form a collagen solution;

[0080] (2) 20 mg of plasma amine oxidase was accurately weighed, added into 1 mL of double distilled water, and fully dissolved, and then stored at -20°C in a dark place to form an amine oxidase solution, with a pH of 7 and a concentration of 6.8 U / mL;

[0081] (3) 10 μL of the above plasma amine oxidase solution was added into 1 mL of the dissolved collagen solution, fully mixed, and subjected to a 30 min enzyme catalysis for a first crosslinking reaction at 25°C, and then centrifuged to remove bubbles, and then sucked into a 1 mL syringe (a syringe with a size of 25G) for extrusion;

[0082] (4) The product of the enzyme crosslinking reaction was extruded into a pre-cooled NaHCO3 aqueous solution containing 50% wt of EDC:NHS (4:1), and then solidified for 30 min at 25°C to complete a second crosslinking, thereby obtaining a double-crosslinked pure collagen hydrogel fiber;

[0083] (5) The collagen hydrogel obtained by crosslinking was immersed in double distilled water, and washed for 3 times to obtain a collagen hydrogel fiber.

[0084] Examples 2-4

[0085] Examples 2-4 are different from Example 1 in that the sizes of the syringes are 27G (Example 2), 22G (Example 3), and 18G (Example 4), respectively, and the rest are the same as those of Example 1.

[0086] Example 5

[0087] The present example provides a preparation method of a collagen hydrogel, which comprises the following steps:

[0088] (1) 30 mg of pure collagen was dissolved in 1 mL of 1% acetic acid aqueous solution, and completely dissolved into a transparent solution under the action of an ultrasonic oscillator and a 37°C water bath to form a collagen solution;

[0089] (2) 20 mg of plasma amine oxidase was accurately weighed, added into 1 mL of double distilled water, and fully dissolved, and then stored at -20°C in a dark place to form an amine oxidase solution, with a pH of 7 and a concentration of 6.8 U / mL;

[0090] (3) 10 μL of the above plasma amine oxidase solution was added into 1 mL of the dissolved collagen solution, fully mixed, and subjected to a 30 min enzyme catalysis for a first crosslinking reaction at 25°C, and then centrifuged to remove bubbles, and then sucked into a 1 mL syringe (a syringe with a size of 25G) for extrusion;

[0091] (4) The collagen solution is extruded into a pre-cooled NaHCO3 solution containing 50%wt EDC:NHS (4:1), and cured at 25°C for 30 min to complete the secondary cross-linking, obtaining a double-cross-linked pure collagen hydrogel fiber;

[0092] (5) The collagen hydrogel obtained by cross-linking is immersed in double-distilled water and washed 3 times, obtaining a collagen hydrogel fiber.

[0093] Example 6

[0094] The embodiment provides a preparation method of a collagen hydrogel, comprising the following steps:

[0095] (1) 50 mg of pure collagen is dissolved in 1 mL of 1% acetic acid solution, and completely dissolved into a transparent solution under the action of an ultrasonic oscillator and a 37°C water bath, forming a collagen solution;

[0096] (2) 20 mg of plasma amine oxidase is accurately weighed, added into 1 mL of double-distilled water, and fully dissolved, and stored at -20°C in a light-proof manner, forming an amine oxidase solution with a pH of 7 and a concentration of 6.8 U / mL;

[0097] (3) 10 μL of the above-mentioned plasma amine oxidase solution is added into 1 mL of the dissolved collagen solution, fully mixed, and subjected to a 30 min enzyme catalysis primary cross-linking reaction at 25°C, and after centrifugation to remove bubbles, the collagen solution is sucked into a 1 mL syringe;

[0098] (4) The collagen solution is extruded into a pre-cooled NaHCO3 solution containing 50%wt EDC:NHS (4:1), and cured at 25°C for 30 min to complete the secondary cross-linking, obtaining a double-cross-linked pure collagen hydrogel fiber;

[0099] (5) The collagen hydrogel obtained by cross-linking is immersed in double-distilled water and washed 3 times, obtaining a collagen hydrogel fiber.

[0100] Examples 7-8

[0101] Examples 7-8 are different from Example 1 in that the mass of the pure collagen is 20 mg (Example 7) and 60 mg (Example 8), the mass concentration of the formed collagen solution is 20 mg / mL and 60 mg / mL respectively, and the rest is the same as Example 1.

[0102] Examples 9-10

[0103] Examples 9-10 are different from Example 1 in that the concentration of the plasma amine oxidase is 4 U / mL (Example 9) and 8 U / mL (Example 10) respectively, and the rest is the same as Example 1.

[0104] Example 11

[0105] The difference between this embodiment and Example 1 is that step (4) is as follows: the product of the above enzyme crosslinking reaction is extruded into a pre-cooled NaHCO3 solution containing 50% wt% EDC:NHS (4:1), and cured at 4°C for 30 min to complete the gelation and secondary crosslinking, and crosslinked pure collagen hydrogel fiber is obtained. The rest is the same as in Example 1.

[0106] Example 12

[0107] The difference between this embodiment and embodiment 1 is that step (4) does not include NaHCO3, while the rest is the same as in embodiment 1.

[0108] Example 13

[0109] The difference between this embodiment and embodiment 1 is that step (4) does not include NaHCO3 and EDC / NHS, while the rest is the same as in embodiment 1.

[0110] Comparative Example 1

[0111] The difference between this comparative example and Example 1 is that no secondary crosslinking is performed; otherwise, they are the same as in Example 1.

[0112] Comparative Example 2

[0113] The difference between this comparative example and Example 1 is that only two crosslinking processes were performed; otherwise, they are the same as in Example 1.

[0114] Comparative Example 3

[0115] The difference between this comparative example and Example 1 is that the order of the two crosslinking reactions is reversed; otherwise, they are the same as in Example 1.

[0116] Performance testing

[0117] 1. Taking the collagen hydrogel fiber described in Example 1 or Examples 1-4 as an example, the following tests were conducted:

[0118] (1) Macro Structure

[0119] Figure 1 This is a physical demonstration of the collagen hydrogel described in Example 1, by... Figure 1 As can be seen, the blocky hydrogel contains a large number of dense and uniform air bubbles, forming a porous structure.

[0120] in, Figure 1 The test sample used was the product after centrifugation to remove air bubbles in step (3), that is, the product after the enzyme-catalyzed reaction was not squeezed out with a syringe, but soaked in NaHCO3 solution. The preparation method is to facilitate the observation of the appearance morphology of the block hydrogel.

[0121] Figure 2is a physical demonstration of the collagen hydrogel fiber described in Example 1, which is prepared by Figure 2 It can be seen that the collagen hydrogel fiber has a uniform linear structure, and the collagen ordered fiber scaffold is prepared by collection.

[0122] (2) Infrared test

[0123] The infrared test is performed on the pure collagen (COL) used in step (1) of Example 1, the enzyme crosslinking reaction product (COL-PAO) in step (3), and the collagen hydrogel fiber (COL-PAO-E / N) obtained in step (5), and the results are shown in Figure 3 As can be seen from the comparison and analysis of the infrared spectra of collagen and enzyme crosslinked collagen hydrogel, the C=O appearing near 1900 cm -1 indicates that aldehyde groups are generated after the reaction, proving that the Schiff base reaction has occurred; the amide I band and the amide II band near 1500-1650 cm -1 It can be seen that the amide band of both the enzyme crosslinked collagen hydrogel and the secondary crosslinked collagen hydrogel is strengthened; the absorption band of -NH -1 near 2100 cm 3+ , the free amino group content of collagen decreases after crosslinking reaction, and the absorption peak intensity of -NH 3+ significantly decreases, which indicates that the enzyme crosslinking reaction and the secondary crosslinking reaction have occurred.

[0124] (3) Microstructure

[0125] The cross section of the collagen hydrogel fiber described in Example 1 is subjected to scanning electron microscope test, and the surface morphology is observed, and the results are shown in Figure 4 .

[0126] The collagen hydrogel fibers described in Examples 1-4, i.e., the structure and morphology of the collagen hydrogel fibers of different sizes obtained are subjected to SEM analysis, and the results are shown in Figure 5a , Figure 5b , Figure 5c , Figure 5d .

[0127] SEM analysis shows that many crosslinked pores appear on the surface of the solidified collagen hydrogel, indicating that the crosslinking reaction is very complete.

[0128] (4) Time scanning test

[0129] The time scanning test is performed on the primary crosslinking reaction of the collagen hydrogel fiber described in Example 1 by using a rotational rheometer, and the results are shown in Figure 8As shown, the results show that the elastic modulus (G') is greater than the viscous modulus (G") at about 12s, and then G' is stable and greater than G", indicating that the collagen solution forms a gel reaction at about 12s and can be quickly solidified.

[0130] (5) Biological performance: the collagen hydrogel fibers were assembled into fiber bundle scaffolds (PCFS) with a diameter of about 2mm and a length of about 4cm to verify their damage repair function in a complete spinal cord injury rat model, and after 3 months of treatment, the Tuj-1 positive neurons in the damaged area were immunofluorescently labeled. The results are shown in Figure 10 It can be seen that PCFS can effectively promote the occurrence of endogenous neurons and is beneficial to damage repair.

[0131] 2. The collagen hydrogel fibers described in Examples 1-12 and Comparative Examples 1-2 were tested as follows:

[0132] (1) Mechanical properties: using a 10N sensor, placed on the sensor tensile clamp, set the test parameters according to the size of the sample, after the test curve appears mutation, stop stretching, the system automatically obtains the mechanical property value;

[0133] Test parameters: 10mm long, 260μm in diameter (Examples 1, 5, 6, 7, 8, 9, 10, 11), 210μm in diameter (Example 2), 420μm in diameter (Example 3), 860μm in diameter (Example 4), compression rate 0.5mm / min.

[0134] (2) Fiber diameter: the fiber diameter was observed and counted by scanning electron microscopy.

[0135] The test results are summarized in Table 1 and Figures 6-7 .

[0136] Table 1

[0137]

[0138] As can be seen from the analysis of the data in Table 1, the maximum tensile elongation of the collagen hydrogel fibers described in the present application is 45.53±16.03% to 98.66±2.56%, the maximum tensile stress is 118.60±2.3kPa to 328.42±10.64KPa, and the fiber diameter is between 199.59±8.45-860.91±15.44μm. The collagen hydrogel fibers described in the present application have good mechanical strength and elastic properties, and the fiber diameter can be adjusted.

[0139] As can be seen from the analysis of Examples 1, 5 and 6, the results are shown in Figure 6As shown, the collagen hydrogel fiber of the present application has good mechanical strength and elastic properties, and within a certain range, the mechanical strength of the collagen hydrogel gradually increases with the gradual increase of the initial substrate concentration.

[0140] Analyzing Example 1 and Examples 7-8, the performance of Examples 7-8 is not as good as Example 1, and the results are as shown in Table 1. Figure 7 As shown, it is proved that within a certain range, the mechanical properties of the collagen hydrogel change with the change of the substrate enzyme concentration, and the optimal mechanical properties can be obtained after optimizing the enzyme substrate concentration. In the present application, the concentration of the collagen solution is 30-50 mg / mL, which is more conducive to the preparation of high-performance collagen hydrogel.

[0141] Analyzing Example 13 and Comparative Example 1, Example 13 cannot be gelled without NaHCO3, and the results are as shown in Table 1. Figure 8 (Example 13), Figure 9 (Comparative Example 1) shows that NaHCO3 provides the necessary conditions for enzyme crosslinking.

[0142] Analyzing Comparative Example 3 and Example 1, as shown in Table 1, it is proved that the first PAO enzyme catalytic crosslinking will increase the reversible change of the molecular chain conformation in the collagen hydrogel, thereby improving its fluidity, increasing the elasticity of the hydrogel fiber, and expanding the application range of the fiber.

[0143] The applicant declares that the above examples illustrate the detailed method of the present application, but the present application is not limited to the above detailed method, that is, it does not mean that the present application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing a collagen hydrogel, characterized by, The preparation method comprises the following steps: (1) using amine oxidase to catalyze collagen to oxidize and deaminate to generate unsaturated aldehyde functional groups, and to occur intramolecular or intermolecular crosslinking to complete primary crosslinking; (2) using a carboxyl activator to catalyze secondary crosslinking of the product of the primary crosslinking to obtain the collagen hydrogel; The secondary crosslinking is performed in a solidification receiving liquid containing a carboxyl activator; The solidification receiving liquid comprises a sodium bicarbonate solution.

2. The production method according to claim 1, characterized by, In step (1), the collagen and the amine oxidase are independently formed into a collagen solution and an amine oxidase solution, respectively, and then a catalytic reaction is performed.

3. The production method according to claim 2, characterized by, The concentration of the collagen solution is 20-60 mg / mL.

4. The preparation method according to claim 2, characterized in that, The solvent of the collagen solution comprises an organic acid solution.

5. The preparation method according to claim 4, characterized in that, The solvent of the collagen solution comprises an acetic acid solution.

6. The production method according to claim 5, wherein The mass fraction of the acetic acid solution is 0.01%-30%.

7. The preparation method according to claim 2, characterized in that, The concentration of the amine oxidase solution is 5-7 U / mL.

8. The preparation method according to claim 2, characterized in that, The pH of the amine oxidase solution is 6.5-7.

5.

9. The preparation method according to claim 2, characterized in that, The amine oxidase comprises any one or a combination of at least two of plasma amine oxidase, monoamine oxidase, diamine oxidase or lysyl oxidase.

10. The method of claim 1, wherein, The reaction temperature of the primary crosslinking is 4-37℃.

11. The method of claim 1, wherein, The reaction time of the primary crosslinking is 1 s-12 h.

12. The method of claim 1, wherein, The carboxyl activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide.

13. The method of claim 12, wherein, The mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide is (1-10):

1.

14. The method of claim 1, wherein, The concentration of the carboxyl activator in the solidification receiving liquid is 0.5-1 mg / mL.

15. The method of claim 1, wherein, The reaction temperature of the secondary crosslinking is 4-37℃.

16. The method of claim 1, wherein, The reaction time of the secondary crosslinking is 0.1-12 h.

17. The method of claim 1, wherein, The preparation method comprises the following steps: (1) performing an enzymatic catalytic oxidation and deamination reaction at 4-37℃ for 1 s-12 h on a collagen solution with a concentration of 20-60 mg / mL and a pH of less than 7 and an amine oxidase solution with a concentration of 5-7 U / mL and a pH of 6.5-7.5 to complete primary crosslinking; (2) performing gel formation and secondary crosslinking in a solidification receiving liquid containing a carboxyl activator with a pH of 7-9 at 4-37℃ for 1 s-12 h on the reaction product of the primary crosslinking reaction to obtain a collagen hydrogel.

18. A collagen hydrogel, characterized in that, The collagen hydrogel is obtained by the preparation method of any one of claims 1-17.

19. Use of the collagen hydrogel of claim 18 in a biomedical material or a tissue engineering scaffold.

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