Collagen hydrogel, its preparation method and use

Abstract

This application relates to a collagen hydrogel, its preparation method, and use. The preparation method includes the steps of (1) catalyzing the oxidative deamination of collagen using amine oxidase to generate unsaturated aldehyde functional groups, thereby generating intramolecular or intermolecular crosslinks and completing primary crosslinking, and (2) subjecting the primary crosslinked product to secondary crosslinking under the catalysis of a carboxyl activator to obtain the collagen hydrogel. The collagen hydrogel prepared by this method is a pure collagen hydrogel that exhibits a fast setting rate, good biocompatibility (excellent biomimetics), tunable mechanical strength, tunable structural size, good stability, and a wide range of applications.

Description

[Technical Field]

[0001] The present application belongs to the technical field of collagen hydrogels, and specifically relates to collagen hydrogels, their preparation methods and uses. [Background technology]

[0002] Hydrogels are polymerized network structures that can absorb and store large amounts of water. These polymer networks contain hydrophilic groups or regions that can hydrate under neutral conditions to form a gel structure. Due to their highly hydrated three-dimensional network, hydrogels provide space for cell adhesion, proliferation, and differentiation. Therefore, hydrogel scaffolds are highly attractive for cell support and tissue development.

[0003] Collagen is the main component of the extracellular matrix and is the most abundant and widely distributed protein in animal bodies. Due to its triple helix structure, collagen not only has good mechanical properties, but also low antigenicity, blood clotting properties, is easily absorbed by the human body, and promotes cell survival and growth, making it widely used in the biomedical field.

[0004] Currently, most collagen hydrogels are prepared by physical crosslinking, such as low-temperature self-assembly, ultraviolet irradiation, thermal crosslinking, freeze-drying, and hydrogen bonding. These methods have problems such as poor mechanical properties and rapid degradation rates. Alternatively, gelation is achieved by forming a composite system with other materials, resulting in complex components and unclear degradation products.

[0005] CN109265705A discloses a mercapto derivative of collagen, its preparation method, and its use. Using collagen as a raw material, a mercapto compound containing both a mercapto group and a carboxyl group or both a mercapto group and an amino group was used as a modifying agent, and mercapto groups were successfully introduced into the collagen molecular chain through the action of a carboxyl activator. The disclosed side chains of the collagen derivative after mercapto modification were flexible and variable, allowing the preparation of various modified mercapto derivatives of collagen.

[0006] CN107513172A discloses a method for preparing a collagen membrane, which uses hydroxycarboxylic acid-N-hydroxysuccinimide ester to activate collagen to form covalent crosslinks, and then uses an oxidase solution to further catalyze secondary crosslinks. This method has problems such as poor gel flexibility, rapid degradation, uncontrollable structure, slow gelation, and easy distortion during the preparation process, which greatly restrict the use of collagen hydrogels as tissue engineering scaffolds, especially in terms of fibrous materials.

[0007] In summary, a method for preparing collagen hydrogels with fast curing speed, good mechanical properties, slow degradation rate, adjustable structure, and rapid and precise production is extremely important. Summary of the Invention [Problem to be solved by the invention]

[0008] The present application provides a collagen hydrogel, its preparation method and use. The collagen hydrogel prepared by the method is a pure collagen hydrogel, which has a fast setting speed, good biocompatibility (excellent biomimetics), adjustable mechanical strength, adjustable structural size, good stability and a wide range of applications. [Means for solving the problem]

[0009] In aspect 1, the present application provides: A method for preparing a collagen hydrogel, comprising: (1) catalyzing the oxidative deamination of collagen using an amine oxidase to generate unsaturated aldehyde functional groups, generating intramolecular or intermolecular crosslinks and completing primary crosslinking; (2) subjecting the product of the primary crosslinking to secondary crosslinking under the catalysis of a carboxyl activator to obtain the collagen hydrogel; A method of preparation is provided.

[0010] In this study, the gelation transition of pure collagen is achieved by two crosslinking methods: primary crosslinking (enzymatic crosslinking reaction) and secondary crosslinking (amidation crosslinking). The mechanical strength and microstructure of the pure collagen hydrogel are adjusted by changing the concentration of the collagen solution, the amount of enzyme used, and the selection of the curing receiving solution.

[0011] Specifically, collagen (COL) is the most abundant protein in the human and animal bodies. To date, 28 types of collagen have been discovered in vertebrates, of which type I collagen is the most numerous and most versatile. All collagen molecules are composed of three polypeptide chains, i.e., a triple helix. This structure confers several special properties to collagen, such as circular dichroism, low antigenicity, and good clotting activity. Collagen is an amphoteric electrolyte, and the basic amino acids in its molecule (e.g., lysine, histidine, etc.) contain reactive groups, such as ε-amino and imino groups, which can react with many chemical reagents.

[0012] In this application, amine oxidase can be used to enzymatically oxidize lysyl or hydroxylysyl in the peptide chain of collagen to obtain aldehyde lysyl or aldehyde hydroxylysyl, which can then generate chemical crosslinks between aldehyde groups or between aldehyde groups and active amino groups, thereby achieving the goal of rapid hardening.

[0013] Since the method of the present application mimics the mechanism of collagen and elastin maturation in the body, it is expected that the method will also be applicable to the preparation of elastin hydrogels and gelatin hydrogels.

[0014] In the present application, the collagen in the collagen solution is pure collagen, and is preferably one or a combination of at least two of bovine hide-derived type I collagen, bovine hide-derived type II collagen, or mouse tail-derived type I collagen, among which typical but non-limiting combinations include a combination of bovine hide-derived type I collagen and bovine hide-derived type II collagen, a combination of bovine hide-derived type II collagen and mouse tail-derived type I collagen, and a combination of bovine hide-derived type I collagen, bovine hide-derived type II collagen, and mouse tail-derived type I collagen, etc.

[0015] Preferably, the chemical crosslinking reaction according to the present invention is carried out under weakly alkaline conditions, which is a Schiff base reaction and a pH-responsive chemical bond, thereby expanding the scope of application.

[0016] Preferably, the concentration of the collagen solution is 20 to 60 mg / mL, for example, 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, or 58 mg / mL.

[0017] Preferably, the pH of the collagen solution is <7, such as 6.5, 6, 5.5, or 5.

[0018] Preferably, the solvent for the collagen solution comprises an organic acid solution.

[0019] Preferably, the solvent for the collagen solution comprises an acetic acid solution.

[0020] Preferably, the mass fraction of the acetic acid solution is 0.01% to 30%, such as 1%, 2%, 5%, 10%, 15%, 20%, or 25%.

[0021] Preferably, the concentration of the amine oxidase solution is 5 to 7 U / mL, for example, 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, or 6.8 U / mL.

[0022] In this application, by adjusting the collagen concentration and enzyme concentration, the elastic modulus and internal pore structure of the pure collagen hydrogel can be adjusted, thereby expanding the application range of the material.

[0023] Preferably, the pH of the amine oxidase solution is 6.5 to 7.5, such as 6.6, 6.8, 7.0, 7.2, or 7.4.

[0024] Preferably, the amine oxidase comprises one or a combination of at least two of plasma amine oxidase, monoamine oxidase, diamine oxidase, and lysyl oxidase. Typical but non-limiting combinations include a combination of plasma amine oxidase and monoamine oxidase, a combination of monoamine oxidase, diamine oxidase, and lysyl oxidase, and a combination of plasma amine oxidase, monoamine oxidase, diamine oxidase, and lysyl oxidase, with plasma amine oxidase being more preferred.

[0025] In the present application, plasma amine oxidase, monoamine oxidase, diamine oxidase, or lysyl oxidase plays a similar role in the formation of collagen fibers in vivo, and plasma amine oxidase is preferred because it is expected that in situ hardening can be achieved using the patient's own plasma in such studies.

[0026] Preferably, the secondary crosslinking is carried out in a cure-receiving solution that includes a carboxyl activator.

[0027] Preferably, the pH of the hardened receiving solution is 7 to 9, such as 7.2, 7.4, 7.6, 7.8, 8, 8.2, 8.4, 8.6, or 8.8.

[0028] Preferably, the hardened receiving solution comprises a weak alkaline solution.

[0029] In this application, the setting-receiving solution is a weak alkaline solution containing bicarbonate ions or carbonate ions. The use of a weak alkaline solution as the setting-receiving solution provides the reaction conditions for enzymatic crosslinking, rapidly realizing the gelation transition of the raw material and allowing the Schiff base reaction to occur, forming a reversible covalent crosslinked network of the first layer. At the same time, the setting solution reacts with glacial acetic acid, the solvent of the collagen solution, to generate carbon dioxide bubbles, which then cause gas-liquid displacement within the gel, forming pores.

[0030] Preferably, the weak alkaline solution contains one or a combination of at least two of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium carbonate, or potassium carbonate, among which typical but non-limiting combinations include a combination of sodium bicarbonate and potassium bicarbonate, a combination of potassium bicarbonate, sodium carbonate, and potassium carbonate, and a combination of sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate, etc., with sodium bicarbonate (NaHCO3) being more preferred.

[0031] In this application, the weak alkaline solution is a sodium bicarbonate solution, which avoids collagen denaturation caused by the use of a strong alkaline solution and provides the weak alkaline pH conditions necessary for enzymatic cross-linking. Furthermore, glacial acetic acid, the solvent of the collagen solution, rapidly reacts with the sodium bicarbonate solution to generate CO2 gas inside the hydrogel, which then collects into uniformly sized bubbles. After gas-liquid exchange, abundant pores are formed inside the hydrogel.

[0032] Preferably, the reaction temperature for the primary crosslinking is 4 to 37°C, for example, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C.

[0033] In the present invention, the amine oxidase is allowed to fully exert its catalytic oxidation activity by uniformly mixing the amine oxidase with the collagen solution at room temperature.

[0034] Preferably, the reaction time of the primary crosslinking is 1 s to 12 h, for example, 1 s, 1 h, 2 h, 4 h, 6 h, 8 h, or 10 h.

[0035] In addition, in the present application, before adding the reaction product of the enzyme-catalyzed reaction to the setting liquid to perform gelation, the preparation method may further include centrifuging the reaction product of the enzyme-catalyzed reaction to remove bubbles caused by external factors, thereby preventing the formation of excessively large, non-uniform bubble voids in the gel, which may affect the internal structure of the gel.

[0036] Preferably, the carboxyl activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS).

[0037] In this application, EDC is a water-soluble carbodiimide used as a carboxyl group activation reagent in amide synthesis, as well as for phosphate group activation, cross-linking of proteins and nucleic acids, and preparation of immunoconjugates. It is often used in combination with N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide to enhance coupling efficiency. NHS favors amide bond formation by activating carboxyl groups.

[0038] The acidic amino acids (e.g., glutamic acid and aspartic acid) in the collagen peptide chains provide many side chain carboxyl groups, which have strong reactive activity, and can react with the remaining amino groups in the collagen peptide chains under the action of carboxyl activator EDC / NHS to further form a covalently crosslinked reversible polymer network structure, thus forming pure collagen hydrogel.

[0039] Preferably, the mass ratio of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is (1-10):1, where 1-10 can be 2, 3, 4, 5, 6, 7, 8, or 9, etc.

[0040] Preferably, the concentration of the carboxyl activator is 0.5 to 1 mg / mL, such as 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, or 0.9 mg / mL.

[0041] In the present application, the carboxyl activator is more preferably a combination of EDC and NHC, the mass ratio of the two being (1-10):1, and the concentration being 0.5-1 mg / mL. Under these conditions, the remaining amino groups and carboxyl groups in the collagen hydrogel undergo an amidation reaction to form a second layer of covalently crosslinked network.

[0042] In the present application, preferably, the preparation method further includes gelling the collagen solution using a microinjection technique to produce collagen fibers with different diameters, which can be achieved by changing parameters such as the needle tip diameter, flow rate, and voltage of the injection extrusion.

[0043] In a preferred embodiment, the collagen hydrogel obtained by covalent crosslinking is washed with clean water at least three times (e.g., four, five, or six times) to remove residual compounds, and then freeze-dried for 2 to 3 days (e.g., 2.2 days, 2.4 days, 2.6 days, or 2.8 days) to obtain a dry, pure collagen hydrogel.

[0044] In the above preparation process, amine oxidase was first used to catalytically oxidize collagen, which led to the enzymatic cross-linking reaction, achieving rapid hardening of the pure collagen hydrogel. Subsequently, the carboxylic acid activator EDC / NHS was used to carry out amidation cross-linking, further enhancing the mechanical properties of the pure collagen hydrogel.

[0045] Preferably, the reaction temperature for the secondary crosslinking is 4 to 37°C, for example, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C.

[0046] Preferably, the reaction time for the secondary crosslinking is 0.1 to 12 hours, such as 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, or 10 hours.

[0047] As a preferred technical solution, the preparation method comprises: (1) A step of performing an enzyme-catalyzed oxidative deamination reaction between a collagen solution having a concentration of 20 to 60 mg / mL and a pH of <7 and an amine oxidase solution having a concentration of 5 to 7 U / mL and a pH of 6.5 to 7.5 at 4 to 37°C for 1 s to 12 h to complete primary crosslinking; (2) Secondary crosslinking of the reaction product of the enzyme-catalyzed reaction in a setting-receiving solution containing a carboxyl activator at pH 7 to 9 at 4 to 37°C for 1 s to 12 h to obtain a collagen hydrogel.

[0048] In this application, the method comprises two steps: Step 1 is a Schiff base reaction mediated by amine oxidase, which allows the hydrogel to harden rapidly within a few seconds, meeting the requirements for precise manufacturing and rapid molding, and expanding the use of the hydrogel in fields such as dry-wet spinning and 3D bioprinting; however, since the Schiff base increases the fluidity of the molecular chains in the hydrogel and is pH-sensitive, Step 2 is an amidation coupling reaction mediated by a carboxyl activator to enhance the stability of the hydrogel.

[0049] In a second aspect, the present application provides a collagen hydrogel prepared by the preparation method according to the first aspect.

[0050] In a third aspect, the present application provides a use of the collagen hydrogel according to the second aspect in a biomedical material or a tissue engineering scaffold.

[0051] In the present application, the collagen hydrogel mimics the mechanism of collagen fiber formation in the body and has excellent biomimetic and biocompatible properties. [Effects of the Invention]

[0052] Compared to the prior art, the present invention has the following beneficial effects:

[0053] (1) In this application, the collagen hydrogel prepared by the above method is a pure collagen hydrogel, which has a fast curing rate, good biocompatibility (excellent biomimetics), adjustable mechanical strength, adjustable structural size, good stability, and a wide range of applications.

[0054] The collagen hydrogel fibers of the present application have a maximum breaking elongation of 79.13±2.54% or more, a maximum tensile stress of 118.60±2.3 kPa or more, and a fiber diameter of 199.59±8.45 to 860.91±15.44 μm. The collagen hydrogel fibers of the present application have good mechanical strength and elastic properties, and a small fiber diameter.

[0055] (2) In this application, the collagen hydrogel prepared by the above method has a faster hardening speed (the gelation time of this application can be achieved within 12 seconds, while conventional technologies require more than 6 minutes) compared to current pure physical collagen hydrogels and multi-component collagen hydrogels, and also improves the mechanical performance and stability of the collagen hydrogel. The gel components are simple and clear, making it suitable for 3D bioprinting and additive manufacturing.

[0056] (3) Compared with the previously developed pure collagen hydrogel crosslinked with EDC / NHS carboxyl activator and then enzymatically, the present invention not only accelerates the curing rate but also generates a large number of densely packed CO2 bubbles inside the collagen hydrogel through a solvent reaction, creating abundant pores. Furthermore, the enzymatic crosslinking process reversibly changes the conformation of the collagen molecular chains, increasing their fluidity and ultimately resulting in collagen hydrogel fibers with good flexibility. Furthermore, the size of the hydrogel fibers can be adjusted, enabling the construction of various types of material scaffolds. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of an actual collagen hydrogel mass prepared by the method described in Example 1. [Figure 2] FIG. 1 is a schematic diagram of an actual collagen hydrogel fiber prepared by the method described in Example 1. [Figure 3] FIG. 1 shows the results of infrared measurement of collagen, an enzymatic cross-linking reaction product, and collagen hydrogel fibers according to the method described in Example 1. [Figure 4] FIG. 1 is a micro-sectional view of a collagen hydrogel fiber prepared by the method described in Example 1. [Figure 5a] FIG. 1 is a diagram showing the surface shape of collagen hydrogel fibers prepared by the method described in Example 1. [Figure 5b]FIG. 1 is a diagram showing the surface shape of collagen hydrogel fibers prepared by the method described in Example 2. [Figure 5c] FIG. 1 is a diagram showing the surface shape of collagen hydrogel fibers prepared by the method described in Example 3. [Figure 5d] FIG. 1 is a diagram showing the surface shape of collagen hydrogel fibers prepared by the method described in Example 4. [Figure 6] FIG. 1 is a stress-strain diagram of collagen hydrogel fibers prepared by the methods described in Examples 1, 5, and 6. [Figure 7] FIG. 1 is a stress-strain diagram of collagen hydrogel fibers prepared by the methods described in Examples 1, 7, and 8. [Figure 8] FIG. 10 is a time scan measurement diagram of the gelation process in the method described in Example 13. [Figure 9] FIG. 1 is a time scanning measurement diagram of the gelation process in the method described in Comparative Example 1. [Figure 10] FIG. 1 illustrates the use of collagen hydrogel fibers prepared by the method described in Example 1 in tissue engineering scaffolds. DETAILED DESCRIPTION OF THE INVENTION

[0058] In order to facilitate understanding of the present application, the present application provides the following examples. Those skilled in the art should understand that the above examples are only for understanding the present application and should not be considered as specifically limiting the present application.

[0059] In the present application, each example used large molecular collagen extracted from bovine hide collagen, and the main component was type 1 collagen. [Example]

[0060] Example 1 This example provides a method for preparing collagen hydrogel, including the following steps:

[0061] (1) 40 mg of pure collagen was dissolved in 1 mL of 1% acetic acid aqueous solution by mass fraction, and completely dissolved in an ultrasonic oscillator and a 37°C water bath until a transparent solution was obtained, forming a collagen solution.

[0062] (2) 20 mg of plasma amine oxidase was accurately weighed, and 1 mL of double-distilled water was added to dissolve it thoroughly. The solution was dispensed into aliquots and stored at -20°C, carefully shielded from light, to form an amine oxidase solution with a pH of 7 and a concentration of 6.8 U / mL.

[0063] (3) 10 μL of the above plasma amine oxidase solution was added to 1 mL of the dissolved collagen solution, mixed thoroughly and homogeneously, and the enzyme-catalyzed primary crosslinking reaction was carried out at 25°C for 30 minutes. After centrifugation to remove air bubbles, the mixture was sucked into a 1 mL syringe (syringe size 25G) and prepared for extrusion.

[0064] (4) The product of the enzymatic cross-linking reaction was extruded into a pre-cooled aqueous NaHCO3 solution containing 50% wt EDC:NHS (4:1) and cured at 25 °C for 30 min to complete the secondary cross-linking, yielding doubly cross-linked pure collagen hydrogel fibers.

[0065] (5) The collagen hydrogel obtained by the above crosslinking was immersed in double distilled water and washed three times to obtain collagen hydrogel fibers.

[0066] Examples 2 to 4 The difference between Examples 2 to 4 and Example 1 was that the syringe sizes were 27G (Example 2), 22G (Example 3), and 18G (Example 4), respectively; the rest were the same as Example 1.

[0067] Example 5 This example provides a method for preparing collagen hydrogel, including the following steps:

[0068] (1) 30 mg of pure collagen was dissolved in 1 mL of 1% acetic acid solution by mass fraction, and completely dissolved under the action of an ultrasonic oscillator and a 37°C water bath until a transparent solution was obtained, forming a collagen solution.

[0069] (2) 20 mg of plasma amine oxidase was accurately weighed, and 1 mL of double-distilled water was added to dissolve it thoroughly. The solution was dispensed into aliquots and stored at -20°C, carefully shielded from light, to form an amine oxidase solution with a pH of 7 and a concentration of 6.8 U / mL.

[0070] (3) 10 μL of the above plasma amine oxidase solution was added to 1 mL of the dissolved collagen solution, mixed thoroughly and homogenously, and subjected to enzyme-catalyzed primary crosslinking at 25°C for 30 minutes. After centrifugation to remove air bubbles, the mixture was sucked into a 1 mL syringe and prepared for extrusion.

[0071] (4) The above collagen solution was 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 crosslinking, yielding doubly crosslinked pure collagen hydrogel fibers.

[0072] (5) The collagen hydrogel obtained by the above crosslinking was immersed in double distilled water and washed three times to obtain collagen hydrogel fibers.

[0073] Example 6 This example provides a method for preparing collagen hydrogel, including the following steps:

[0074] (1) 50 mg of pure collagen was dissolved in 1 mL of 1% acetic acid solution by mass fraction, and completely dissolved under the action of an ultrasonic oscillator and a 37°C water bath until a transparent solution was obtained, forming a collagen solution.

[0075] (2) 20 mg of plasma amine oxidase was accurately weighed, and 1 mL of double-distilled water was added to dissolve it thoroughly. The solution was dispensed into aliquots and stored at -20°C, carefully shielded from light, to form an amine oxidase solution with a pH of 7 and a concentration of 6.8 U / mL.

[0076] (3) 10 μL of the above plasma amine oxidase solution was added to 1 mL of the dissolved collagen solution, mixed thoroughly and homogenously, and the enzyme-catalyzed primary crosslinking reaction was carried out at 25°C for 30 minutes. After centrifugation to remove air bubbles, the mixture was sucked into a 1 mL syringe and prepared.

[0077] (4) The above collagen solution was extruded into a pre-cooled NaHCO3 solution containing 50% wt EDC:NHS (4:1) and cured at 25 °C for 30 min to complete gelation and secondary crosslinking, yielding doubly crosslinked pure collagen hydrogel fibers.

[0078] (5) The collagen hydrogel obtained by the above crosslinking was immersed in double distilled water and washed three times to obtain collagen hydrogel fibers.

[0079] Examples 7-8 The differences between Examples 7 and 8 and Example 1 were that the mass of pure collagen was 20 mg (Example 7) and 60 mg (Example 8), and the mass concentrations of the formed collagen solutions were 20 mg / mL and 60 mg / mL, respectively; the rest were the same as in Example 1.

[0080] Examples 9-10 Examples 9 and 10 differ from Example 1 in that the concentrations of plasma amine oxidase were 4 U / mL (Example 9) and 8 U / mL (Example 10), respectively; the rest were the same as Example 1.

[0081] Example 11 The difference between this example and Example 1 is that in step (4), the product of the enzymatic cross-linking reaction was 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 cross-linking, obtaining cross-linked pure collagen hydrogel fibers; the rest were the same as in Example 1.

[0082] Example 12 The difference between this example and Example 1 is that NaHCO3 was not used in step (4), and the rest was the same as Example 1.

[0083] Example 13 The difference between this example and Example 1 is that NaHCO3 and EDC / NHS were not used in step (4), and the rest were the same as Example 1.

[0084] Comparative Example 1 The difference between this comparative example and Example 1 is that secondary crosslinking was not carried out, and the rest was the same as Example 1.

[0085] Comparative Example 2 The difference between this comparative example and Example 1 is that only secondary crosslinking was carried out, and the rest was the same as Example 1.

[0086] Comparative Example 3 The difference between this comparative example and Example 1 is that the order of the two crosslinking reactions was reversed, and the rest was the same as Example 1.

[0087] Performance Test 1. Using the collagen hydrogel fibers described in Example 1 or Examples 1 to 4 as an example, the following tests were carried out.

[0088] (1) Macrostructure Figure 1 shows the actual collagen hydrogel described in Example 1. Figure 1 shows that a large number of densely packed, uniformly distributed air bubbles are present inside the hydrogel mass, forming a void structure.

[0089] The test sample used in Figure 1 was obtained by centrifugation in step (3) to remove air bubbles, i.e., by immersing the product after the enzyme-catalyzed reaction in the NaHCO3 solution without extruding it with a syringe. This preparation method was used to easily observe the external shape of the hydrogel aggregates.

[0090] Figure 2 shows the actual collagen hydrogel fibers described in Example 1. Figure 2 shows that the collagen hydrogel fibers have a uniform linear structure and can be collected and prepared to obtain a collagen regular fiber scaffold, which has a shape similar to that of natural spinal cord.

[0091] (2) Infrared measurement Infrared measurements were performed on the pure collagen (COL) used in step (1) of Example 1, the enzymatic cross-linking reaction product (COL-PAO) in step (3), and the collagen hydrogel fibers (COL-PAO-E / N) obtained in step (5). As shown in Figure 3, a comparative analysis of the infrared spectra of collagen and the enzymatic cross-linked collagen hydrogel revealed a peak at 1900 cm -1 The C=O that appeared near the aldehyde group indicates that an aldehyde group was formed after the reaction, proving that a Schiff base reaction occurred. -1 From the amide I and amide II bands in the vicinity, it was found that both the amide bands of the enzymatically cross-linked collagen hydrogel and the amide bands of the secondary cross-linked collagen hydrogel were strengthened, and the amide bands at 2100 cm -1 Nearby -NH 3+ From the absorption band of -NH, after the collagen cross-linking reaction, the free amino groups decreased, the content of protonated amino groups decreased, and the -NH 3+ It was found that the peak intensity of the absorption band of 1 was significantly reduced, and the above phenomenon indicates that the collagen underwent an enzymatic cross-linking reaction and a secondary cross-linking reaction.

[0092] (3) Micro surface structure The cross section of the collagen hydrogel fiber described in Example 1 was measured with a scanning electron microscope to observe its surface shape, and the results are shown in FIG.

[0093] The results of SEM analysis of the structure and shape of the collagen hydrogel fibers described in Examples 1 to 4, i.e., the collagen hydrogel fibers of different sizes obtained, are shown in Figures 5a, 5b, 5c, and 5d.

[0094] SEM analysis showed that many post-crosslinking voids appeared on the surface of the cured collagen hydrogel, indicating that the crosslinking reaction was complete.

[0095] (4) Time scanning measurement The results of time-scanning measurements of the primary crosslinking reaction of the collagen hydrogel fibers described in Example 1 using a rotational rheometer are shown in Figure 8. The results show that at approximately 12 s, the elastic modulus (G') exceeded the viscous modulus (G"), and then G' stabilized and remained greater than G", indicating that the gelation reaction of the collagen solution occurred at approximately 12 s, allowing it to harden rapidly.

[0096] (5) Biological properties: The collagen hydrogel fibers were assembled into a fibrous scaffold (PCFS) with a diameter of approximately 2 mm and a length of approximately 4 cm. Its injury repair function was examined in a rat model of complete spinal cord injury. Three months after treatment, immunofluorescence labeling of Tuj-1-positive neurons in the tissue of the injured area was performed. The results are shown in Figure 10. It was found that PCFS can effectively promote the development of endogenous neurons, which is beneficial for injury repair.

[0097] 2. The collagen hydrogel fibers described in Examples 1 to 12 and Comparative Examples 1 and 2 were subjected to the following tests.

[0098] (1) Mechanical performance: A 10N sensor was used, placed on the tensile jig of the sensor, and the test parameters were set according to the size of the sample. When a deviation appeared in the test curve, the tensile test was stopped, and the system automatically obtained the mechanical performance value.

[0099] Test parameters: length 10 mm, diameter 260 μm (Examples 1, 5, 6, 7, 8, 9, 10, 11), diameter 210 μm (Example 2), diameter 420 μm (Example 3), diameter 860 μm (Example 4), compression speed 0.5 mm / min.

[0100] (2) Fiber diameter: The diameter of the fibers was observed using a scanning electron microscope and statistically analyzed.

[0101] The test results are summarized in Table 1 and Figures 6 and 7.

[0102] [Table 1]

[0103] Analysis of the data in Table 1 revealed that the maximum breaking elongation of the collagen hydrogel fibers according to the present application was 45.53±16.03% to 98.66±2.56%, the maximum tensile stress was 118.60±2.3 kPa to 328.42±10.64 kPa, and the fiber diameter was between 199.59±8.45 and 860.91±15.44 μm, indicating that the collagen hydrogel fibers according to the present application have good mechanical strength and elastic properties, and the fiber diameter is adjustable.

[0104] Examples 1, 5, and 6 were analyzed, and the results, as shown in Figure 6, indicate that the collagen hydrogel fibers of the present invention have good mechanical strength and elastic properties, and within a certain range, the mechanical strength of the collagen hydrogel gradually increases with increasing initial substrate concentration.

[0105] Analysis of Examples 1 and 7-8 showed that the performance of Examples 7-8 was worse than that of Example 1. The results, as shown in Figure 7, indicate that the mechanical performance of the obtained collagen hydrogel changes with the change in substrate enzyme concentration within a certain range, and that the optimal mechanical performance can be achieved by optimizing the enzyme substrate concentration. In this application, it is more advantageous for the concentration of the collagen solution to be 30-50 mg / mL to prepare high-performance collagen hydrogels.

[0106] Example 13 and Comparative Example 1 were analyzed, and Example 13 was unable to gel under conditions that did not contain NaHCO. The results, as shown in Figure 8 (Example 13) and Figure 9 (Comparative Example 1), demonstrated that NaHCO provides the necessary conditions for enzymatic cross-linking.

[0107] Analysis of Comparative Example 3 and Example 1 demonstrated that prior PAO enzyme-catalyzed crosslinking increased the reversible conformational changes of the internal molecular chains of the collagen hydrogel, enhancing its fluidity, increasing the elasticity of the hydrogel fibers, and broadening the application range of the fibers, as shown in Table 1.

[0108] Although the present application has described the detailed method of the present application through the above examples, the applicant declares that the present application is not limited to the above examples, that is, it does not mean that the present application must be carried out depending on the above detailed methods. Those skilled in the art should understand that any improvements to the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific forms, etc. are all within the protection scope and disclosure scope of the present application.

Claims

1. A method for preparing a collagen hydrogel, comprising: (1) catalyzing the oxidative deamination of collagen using an amine oxidase to generate unsaturated aldehyde functional groups, generating intramolecular or intermolecular crosslinks and completing primary crosslinks; (2) subjecting the product of the primary crosslinking to secondary crosslinking under the catalysis of a carboxyl activator to obtain the collagen hydrogel; Preparation method.

2. In step (1), the collagen and the amine oxidase are separately prepared as a collagen solution and an amine oxidase solution, respectively, and then the catalytic reaction is carried out; Preferably, the concentration of the collagen solution is 20 to 60 mg / mL; Preferably, the pH of the collagen solution is <7, Preferably, the solvent for the collagen solution comprises an organic acid solution; Preferably, the solvent for the collagen solution comprises an acetic acid solution; Preferably, the mass fraction of the acetic acid solution is 0.01% to 30%. The preparation method according to claim 1.

3. the concentration of the amine oxidase solution is 5-7 U / mL; Preferably, the pH of the amine oxidase solution is between 6.5 and 7.5; Preferably, the amine oxidase comprises any one or a combination of at least two of plasma amine oxidase, monoamine oxidase, diamine oxidase, or lysyl oxidase. The preparation method according to claim 2.

4. The secondary crosslinking is carried out in a cure-receiving solution containing a carboxyl activator; Preferably, the pH of the hardened receiving solution is 7 to 9; Preferably, the hardened receiving solution comprises a weak alkaline solution; Preferably, the weak alkaline solution contains any one or a combination of at least two of sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, sodium carbonate, or potassium carbonate. The preparation method according to any one of claims 1 to 3.

5. The reaction temperature of the primary crosslinking is 4 to 37°C, Preferably, the reaction time of the primary crosslinking is 1 s to 12 h. The preparation method according to any one of claims 1 to 4.

6. the carboxyl activator comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and / or N-hydroxysuccinimide; Preferably, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is (1 to 10):1; Preferably, the concentration of the carboxyl activator in the cured receiving solution is 0.5 to 1 mg / mL. The preparation method according to any one of claims 1 to 5.

7. The reaction temperature of the secondary crosslinking is 4 to 37°C, Preferably, the reaction time of the secondary crosslinking is 0.1 to 12 h. The preparation method according to claim 6.

8. (1) performing an enzyme-catalyzed oxidative deamination reaction between a collagen solution having a concentration of 20-60 mg / mL and a pH of <7 and an amine oxidase solution having a concentration of 5-7 U / mL and a pH of 6.5-7.5 at 4-37°C for 1 s-12 h to complete primary crosslinking; (2) subjecting the reaction product of the primary crosslinking reaction to gelation and secondary crosslinking in a hardening-receiving solution containing a carboxyl activator and having a pH of 7 to 9 at 4 to 37°C for 1 s to 12 h to obtain a collagen hydrogel; The preparation method according to any one of claims 1 to 7.

9. Prepared by the method according to any one of claims 1 to 8. Collagen hydrogel.

10. Use of the collagen hydrogel according to claim 9 in a biomedical material or a tissue engineering scaffold.

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