Irradiation cross-linked collagen fiber material and its preparation method and application

Abstract

The present invention provides a radiation-crosslinked collagen fiber material and its preparation method and application, which relate to the field of organic polymer compound processing and also to the field of medical material technology. Among them, in the preparation method of the radiation-crosslinked collagen fiber material, the problem of excessive and rapid crosslinking of proteins caused by the irradiation process is solved by changing the solvent system of the collagen fiber, introducing some organic solvents, and reducing the moisture content to control the excitation rate of the electron beam on water molecule free radicals, thereby controlling the degree of crosslinking of the collagen material by irradiation; further, by introducing a dispersant and controlling the irradiation dose, irradiation temperature, material thickness and other processes, uniform crosslinking of the collagen fibers is achieved; after irradiation, the collagen fibers are homogenized in a homogenization process, achieving the injectability of the material, and significantly improving the physical and chemical properties and enzymatic resistance of the obtained radiation-crosslinked collagen fiber material.

Description

Technical Field

[0001] The present invention relates to the field of organic polymer compound processing and the technical field of medical materials, and in particular to a radiation cross-linked collagen fiber material and a preparation method and application thereof. Background Art

[0002] Collagen is a natural biopolymer with excellent biocompatibility and biodegradability, making it widely used in the biomedical field. Its unique molecular structure and biological properties give it excellent cell affinity in terms of cell adhesion, proliferation, and differentiation, helping to promote tissue regeneration and repair. These properties make collagen a particularly suitable base material for tissue engineering scaffolds, providing an ideal microenvironment for cell growth and demonstrating significant potential for application in the repair and regeneration of tissues such as cartilage, skin, and bone.

[0003] However, due to the presence of specific enzymes targeting collagen in the human body, pure collagen materials have poor resistance to enzymatic hydrolysis, which limits their long-term stability in biomedical applications. In order to improve the mechanical properties and resistance to enzymatic hydrolysis of collagen, it is usually modified by cross-linking. However, commonly used chemical cross-linking agents often bring about residue problems and pose potential safety risks. Therefore, physical cross-linking methods are widely used as an alternative, but there are still some shortcomings. Conventional material cross-linking methods, such as ultraviolet irradiation, high temperature, freeze-drying and other methods, have problems such as limited cross-linking strength, uneven cross-linking, and destruction of collagen structure.

[0004] During irradiation, high-energy radiation (such as gamma rays or electron beams) reacts with water molecules, generating a large number of free radicals (such as hydroxyl radicals and hydrogen radicals). These free radicals can attack amino acid residues in collagen molecules, particularly charged groups such as lysine, arginine, and glutamate. These reactions trigger the reorganization of collagen's chemical bonds and promote covalent cross-linking between proteins. Under the catalytic action of free radicals, cross-links are formed between collagen molecules through covalent bonds, creating a larger protein network. This cross-linking significantly increases the strength and elasticity of collagen, making it particularly suitable for biomaterials that require long-term structural retention, such as tissue engineering scaffolds.

[0005] Currently, irradiation is primarily used for protein sterilization. Conventional irradiation, due to the rapid formation of free radicals by electron beams, causes rapid localized cross-linking of collagen, leading to uneven cross-linking, hardening and brittleness, and protein denaturation, making the material unusable. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing a collagen fiber material without introducing exogenous substances for irradiation and effectively preventing local rapid cross-linking of collagen caused by irradiation; another object of the present invention is to provide a collagen fiber material prepared based on the aforementioned method and having good uniformity within a specific cross-linking degree range; another object of the present invention is to provide the use of the aforementioned collagen fiber material in the preparation of medical injectable filling materials and / or tissue engineering scaffolds.

[0007] In a first aspect, the present application provides a method for preparing an irradiated cross-linked collagen fiber material:

[0008] A method for preparing an irradiated cross-linked collagen fiber material comprises the following steps:

[0009] (1) Preparation of collagen fiber intermediates: concentrated collagen fibers are dispersed in an irradiation solvent system, and after centrifugation, a collagen fiber intermediate having a concentration of 35-45 mg / ml is obtained; the irradiation solvent system comprises physiological saline and an organic solvent in a volume ratio of 1:(1-2.4), and a dispersant in an amount of 0.1-2 w / v% of the total amount of the physiological saline and organic solvent;

[0010] (2) Irradiation cross-linking: irradiating the collagen fiber intermediate at a temperature of 20-30°C and an irradiation dose of 10-20 kGy to obtain cross-linked collagen fibers;

[0011] (3) removing the dispersant from the cross-linked collagen fibers and sterilizing them by low-temperature irradiation to obtain a finished collagen fiber material.

[0012] Furthermore, in the preparation of the collagen fiber intermediate, the liquid ratio of the concentrated collagen fiber to the irradiated solvent system is 1:20-1:50.

[0013] The irradiation solvent system contains a relatively high proportion of organic solvents, which can effectively disperse collagen fibers and reduce the concentration of water molecules in the material, thereby reducing the excitation rate of free radicals during irradiation, thereby slowing down the cross-linking of collagen fibers by irradiation. The addition of a dispersant can prevent excessive cross-linking between monomers during subsequent irradiation cross-linking, thereby preventing the formation of a high-strength scaffold structure in the shape of a body, which is beneficial for subsequent homogeneous dispersion. The collagen concentration is controlled between 35-45 mg / ml. Too low a concentration will result in insufficient cross-linking of the material after irradiation, while too high a concentration will result in excessive cross-linking.

[0014] Irradiation at room temperature (20-30°C) can achieve short-range localized crosslinking between collagen fibers while maintaining a slightly fluid state, improving their mechanical properties and resistance to enzymatic degradation. Irradiation doses of 10, 15, and 20 kGy achieve in vivo degradation times of 6, 9, and 15 months, respectively. If the irradiation temperature is too low during crosslinking, insufficient crosslinking may occur, reducing enzymatic resistance. If the irradiation intensity is too high, excessive crosslinking may occur.

[0015] The free radicals generated during the radiation cross-linking process covalently cross-link the amino acids of the collagen side chains, thereby improving the fiber performance; the prepared radiation cross-linked collagen fiber material does not contain any cross-linking agent and is a pure collagen material; the post-irradiation homogenization process uniformly plasmidizes the collagen fibers, has high mechanical strength, and greatly increases the physical and chemical properties and resistance to enzymatic hydrolysis, and is suitable for application as a tissue engineering scaffold material. The material is injectable and can still be evenly and smoothly injected with a 27G syringe needle after cross-linking, and the in vivo degradation cycle is controllable; the radiation cross-linked collagen fiber material prepared by the present invention can also be freeze-dried. The freeze-dried radiation cross-linked collagen fiber material has excellent hemostatic properties and the effect of inducing cell proliferation, and is suitable for use in the field of tissue regeneration and repair.

[0016] The concentrated collagen fibers include any one or more combinations of type I collagen fibers, type II collagen fibers, and type III collagen fibers.

[0017] Furthermore, concentrated collagen fibers can be obtained from the collagen self-assembly precipitation process;

[0018] The specific operation of the collagen self-assembly precipitation process is to regulate the collagen solution to the physiological state in vitro for self-assembly, pH 6.4-7.8, temperature 35-38 ° C, sodium chloride concentration 0.1-0.2 M, and standing time 2-3 hours to achieve the precipitation of collagen fibers; then the precipitated collagen fibers are homogenized and dispersed, and then centrifuged.

[0019] Furthermore, the radiation crosslinking is electron beam irradiation or gamma ray irradiation.

[0020] Furthermore, electron beam irradiation is used, and the thickness of the collagen fiber intermediate does not exceed 10 mm during irradiation.

[0021] Furthermore, the organic solvent includes but is not limited to any one or more of dimethyl sulfoxide, isopropyl alcohol, acetone, ethyl acetate, and methyl tert-butyl ether. As a preferred embodiment of the present invention, the organic solvent includes dimethyl sulfoxide.

[0022] Dimethyl sulfoxide, a commonly used organic solvent, is miscible with physiological saline and the added dispersant in any proportion. Collagen fibers can be stably present in the configured organic solvent system without destroying the triple helical structure of the collagen molecule.

[0023] Furthermore, the dispersant includes any one or more combinations of Triton X-100, Triton X-114, Triton X-405, and TritonGR-7M.

[0024] Triton X-100, as a nonionic surfactant, can effectively improve the dispersion performance of collagen fibers in the solution system and avoid excessive cross-linking of collagen fibers during irradiation.

[0025] Furthermore, the method for removing the dispersant in step (3) includes: redissolution, homogenization and / or ultrasonic treatment, and centrifugation.

[0026] Specifically, the method for removing the dispersant in step (3) includes: adding physiological saline to the cross-linked collagen fibers, controlling the liquid ratio to be 1:20-1:50, homogenizing and dispersing for 30 minutes, ultrasonically extracting for 30-60 minutes, and centrifuging; repeating the operation multiple times.

[0027] Furthermore, the cross-linked collagen fibers are first subjected to ultra-low temperature quick freezing treatment, and then subjected to low-temperature irradiation sterilization treatment at a temperature range of -40--80°C; the irradiation dose of the low-temperature irradiation sterilization treatment is 25-30 kGy.

[0028] In low-temperature irradiation sterilization, the irradiation process keeps the material in a low-temperature frozen state, preventing further cross-linking between collagen fibers during subsequent irradiation, which may lead to changes in fiber particle size. At the same time, the irradiation dose can achieve sterilization of the material without changing its physical and chemical properties.

[0029] Furthermore, in the preparation of the cross-linked collagen fibers, the thickness of the collagen fiber intermediate is 5-8 mm; in the low-temperature irradiation sterilization, the thickness of the cross-linked collagen fibers is 5-8 mm.

[0030] As an optional embodiment, the collagen fiber intermediates are packaged in aluminum foil bags with a thickness of 5-8 mm and irradiated, so that the radiation dose received by the collagen is uniform and the cross-linking degree of the collagen fibers on the upper and lower surfaces of the aluminum foil bags is consistent.

[0031] Furthermore, in the ultra-low temperature quick freezing, the ultra-low temperature is -80°C.

[0032] Furthermore, in the preparation of the irradiated cross-linked collagen fiber material, the thawing process is as follows:

[0033] First place it in a 25 ℃ constant temperature box and let it stand for 20 minutes, then shake it for 1 minute, and then place it in a 25 ℃ constant temperature box and let it stand for 20 minutes. Repeat at least 2 times until it is completely thawed.

[0034] Direct thawing will cause the physiological saline in the material to precipitate first. The increase in local collagen fiber concentration will cause the particle size of the material to change. Through repeated shaking during the thawing process, the thawed and precipitated physiological saline will quickly return to the system, making the material thawed more evenly.

[0035] The present application provides an irradiation-crosslinked collagen fiber material and a preparation method thereof. To address the problem of excessively rapid cross-linking of proteins caused by the irradiation process, the solvent system of the collagen fibers is changed, some organic solvents are introduced, and the moisture content is reduced to control the excitation rate of the electron beam on water molecule free radicals, thereby controlling the degree of cross-linking of the collagen material by irradiation; further, by introducing a dispersant and controlling processes such as irradiation dose, irradiation temperature, and material thickness, uniform cross-linking of the collagen fibers is achieved.

[0036] In a second aspect, the present application provides a collagen fiber material:

[0037] A collagen fiber material prepared by the above method has the following characteristics:

[0038] The average particle size of the collagen fibers is in the range of 100-150 μm.

[0039] The material was filled into a 27 G syringe, and the maximum pushing force was between 11.5 N and 25 N at a pushing speed of 30 mm / min.

[0040] The collagen fiber material prepared in the present application has good mechanical properties and resistance to enzymatic degradation. The complete degradation period in vivo can be adjusted by the irradiation dose, and the complete degradation time in vivo can be controlled between 6-15 months. Furthermore, the collagen fibers are not cross-linked using any chemical cross-linking agents. The prepared material is pure collagen fibers and physiological saline, without any other substances, and has high biological safety.

[0041] In a third aspect, the present application provides an application of a collagen fiber material:

[0042] The collagen fibers obtained by the present invention can be directly thawed and used as gel injection filling. Their excellent biocompatibility and resistance to enzymatic hydrolysis can be used in the fields of skin tissue filling, soft tissue repair, joint injection, drug carrier, etc.

[0043] The collagen fibers obtained by the present invention can be used as tissue engineering scaffolds after being freeze-dried, and can be used in the fields of burn dressing, tissue hemostasis, soft tissue reconstruction, etc.

[0044] Beneficial effects: 1. The present invention adopts a special irradiation solvent. The organic solvent and dispersant in the special irradiation solvent can effectively disperse the collagen fibers, and at the same time reduce the concentration of water molecules in the material, so that the excitation rate of free radicals in the irradiation process is reduced, thereby slowing down the cross-linking of collagen fibers by irradiation, and thus obtaining more uniform cross-linked collagen fibers; further controlling the material concentration before irradiation to 35-45 mg / ml, and irradiating at room temperature (20-30 ℃) and under the conditions of preferred irradiation dose, thereby achieving uniform cross-linking of collagen fibers, making the collagen material have a slightly fluid state for cross-linking, and achieving relatively uniform local cross-linking between collagen fibers in a short range, so that the obtained irradiated cross-linked collagen fiber material has a high degree of cross-linking, enzymatic resistance and uniform and smooth injection performance with a 27 G syringe needle.

[0045] 2. This application uses low-temperature irradiation sterilization treatment at -40-80°C to achieve complete sterilization of cross-linked collagen fibers. At the same time, deep low temperature can prevent further cross-linking of collagen fibers and affect their physical and chemical properties.

[0046] 3. The collagen fibers obtained by the present invention can be directly thawed and used as gel injection filling. Their excellent biocompatibility and resistance to enzymatic hydrolysis can be used in the fields of skin tissue filling, soft tissue repair, joint injection, drug carrier, etc.; the collagen fibers obtained by the present invention can be used as tissue engineering scaffolds after freeze-drying, and can be used in the fields of burn dressing, tissue hemostasis, soft tissue reconstruction, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a photograph of the state of the irradiated cross-linked collagen fiber material obtained by Example 1 of the present invention;

[0048] Figure 2 This is a photograph of the freeze-dried irradiated cross-linked collagen fiber material obtained in Example 3 of the invention;

[0049] Figure 3 This is a comparison chart of the remaining collagen fiber ratios of the irradiated cross-linked collagen fiber materials obtained by Examples 1 to 3 and Comparative Examples 1 to 10 after different enzymatic hydrolysis times. DETAILED DESCRIPTION

[0050] In order to make the technical solution of the present invention clearer, Figure 1 、 Figure 2 、 Figure 3 The present invention is further described in detail with reference to the accompanying drawings and specific embodiments.

[0051] Example 1, a method for preparing an irradiated cross-linked collagen fiber material, comprising the following steps:

[0052] Collagen self-assembly precipitation: An acidic collagen solution extracted from animal sources was adjusted to pH 7.4, a sodium chloride concentration of 0.15 M, and a temperature of 37°C. The solution was allowed to stand for 2.5 hours to simulate the in vitro self-assembly process of collagen. The precipitated collagen fibers were then homogenized and dispersed, followed by centrifugation to obtain concentrated collagen fibers.

[0053] Preparation of cross-linked collagen fibers:

[0054] Redisperse the concentrated collagen fibers: Mix physiological saline and dimethyl sulfoxide in a 1:1 (v / v) ratio. Add 0.1% (w / v) Triton X-100 to the blended solution to form an irradiation solvent system. Disperse the concentrated collagen fibers homogeneously in the irradiation solvent system at a liquid-to-liquid ratio of 1:40. (The liquid-to-liquid ratio refers to the mass ratio of concentrated collagen fibers to irradiation solvent system. For example, 100 g of collagen fibers can be dispersed in 4000 g of irradiation solvent system.) Stir for 30 minutes, then centrifuge to obtain the collagen fiber intermediate. Control the collagen concentration to 35 mg / ml.

[0055] Cross-linking of collagen fiber intermediates: The obtained collagen fiber intermediates were packed into aluminum foil bags with a single bag thickness of 5 mm, and sealed by flattening. Subsequently, electron beam irradiation cross-linking was performed at 20° C. with an irradiation dose of 20 kGy to obtain cross-linked collagen fibers.

[0056] Preparation of irradiated cross-linked collagen fiber materials:

[0057] Removal of dispersant: The cross-linked collagen fibers were redispersed in physiological saline at a liquid ratio of 1:20 and dispersed by homogenization for 30 minutes. After ultrasonic extraction for 60 minutes, the collagen fibers were obtained by centrifugation. The ultrasonic cleaning was repeated three times, and no dispersant was detected.

[0058] The cross-linked collagen fibers after the dispersant was removed were packed into aluminum foil bags with a thickness of 8 mm per bag, and then ultra-low temperature quick-frozen in a -80°C refrigerator.

[0059] Low-temperature irradiation sterilization: The ultra-low-temperature quick-frozen cross-linked collagen fibers were spread flat on dry ice for low-temperature irradiation sterilization with an irradiation dose of 25 kGy.

[0060] Thawing: Place the cross-linked collagen fibers sterilized by low-temperature irradiation in a 25°C constant temperature box and thaw for 20 minutes, then shake for 1 minute and continue thawing for 20 minutes. Repeat 3 times until completely thawed to obtain a slightly fluid irradiation cross-linked collagen fiber gel and obtain an irradiation cross-linked collagen fiber material (appearance photo as shown in the figure). Figure 1 ).

[0061] Example 2, a method for preparing an irradiation-crosslinked collagen fiber material, comprising the following preparation steps:

[0062] Collagen self-assembly precipitation: An acidic collagen solution extracted from animal sources was adjusted to pH 7.4, a sodium chloride concentration of 0.15 M, and a temperature of 37°C. The solution was allowed to stand for 2.5 hours to simulate the in vitro self-assembly process of collagen. The precipitated collagen fibers were then homogenized and dispersed, followed by centrifugation to obtain concentrated collagen fibers.

[0063] Preparation of cross-linked collagen fibers:

[0064] Re-dispersion of concentrated collagen fibers: Physiological saline and acetone were mixed in a ratio of 3:7 (v / v). 0.5% (w / v) Triton X-114 was added to the mixed solution to prepare an irradiation solvent system. The concentrated collagen fibers were homogeneously dispersed in the above irradiation solvent system at a liquid ratio of 1:40. After stirring for 30 min, the collagen fiber intermediates were centrifuged and the concentration was controlled at 45 mg / ml.

[0065] Cross-linking of collagen fiber intermediates: The obtained collagen fiber intermediates were packaged in aluminum foil bags with a single bag thickness of 8 mm, flattened and sealed; then electron beam irradiation cross-linking was performed at 30°C with an irradiation dose of 10 kGy to obtain cross-linked collagen fibers.

[0066] Preparation of irradiated cross-linked collagen fiber materials:

[0067] Removal of dispersant: The cross-linked collagen fibers were redispersed in physiological saline at a liquid ratio of 1:50 and dispersed by homogenization for 30 min. After ultrasonic extraction for 30 min, the collagen fibers were obtained by centrifugation. The ultrasonic cleaning was repeated three times. No dispersant was detected.

[0068] The cross-linked collagen fibers from which the dispersant had been removed were packaged in aluminum foil bags with a thickness of 5 mm per bag, and then ultra-low temperature quick-frozen in a -80°C refrigerator.

[0069] Low-temperature irradiation sterilization: The ultra-low-temperature quick-frozen cross-linked collagen fibers were spread flat on dry ice for low-temperature irradiation sterilization at a dose of 30 kGy.

[0070] Thawing: Place the low-temperature irradiated cross-linked collagen fibers in a 25°C incubator for 20 minutes to thaw, then shake for 1 minute and continue thawing for 20 minutes. Repeat this process three times until completely thawed to obtain irradiated cross-linked collagen fiber material.

[0071] Example 3, a method for preparing an irradiation-crosslinked collagen fiber material, comprising the following preparation steps:

[0072] Collagen self-assembly precipitation: An acidic collagen solution extracted from animal sources was adjusted to pH 7.4, a sodium chloride concentration of 0.15 M, and a temperature of 37°C. The solution was allowed to stand for 2.5 hours to simulate the in vitro self-assembly process of collagen. The precipitated collagen fibers were then homogenized and dispersed, followed by centrifugation to obtain concentrated collagen fibers.

[0073] Preparation of cross-linked collagen fibers:

[0074] Re-dispersion of concentrated collagen fibers: Physiological saline and isopropanol were mixed in a ratio of 2:3 (v / v). 2% (w / v) Triton GR-7M was added to the mixed solution to obtain an irradiation solvent system. The concentrated collagen fibers were homogeneously dispersed in the irradiation solvent system at a liquid ratio of 1:40. After stirring for 30 min, the collagen fiber intermediates were obtained by centrifugation and the concentration was controlled at 40 mg / ml.

[0075] Cross-linking of collagen fiber intermediates: The obtained collagen fiber intermediates were packaged in aluminum foil bags with a single bag thickness of 7 mm, flattened and sealed; then electron beam irradiation cross-linking was performed at 25°C with an irradiation dose of 15 kGy to obtain cross-linked collagen fibers.

[0076] Preparation of irradiated cross-linked collagen fiber materials:

[0077] Removal of dispersant: The cross-linked collagen fibers were redispersed in physiological saline at a liquid ratio of 1:40 and dispersed by homogenization for 30 min. After ultrasonic extraction for 35 min, the collagen fibers were obtained by centrifugation. The ultrasonic cleaning was repeated three times. No dispersant was detected.

[0078] The cross-linked collagen fibers after removing the dispersant were packed into aluminum foil bags with a thickness of 6 mm per bag and quickly frozen in a -80°C refrigerator.

[0079] Low-temperature irradiation sterilization: The ultra-low-temperature quick-frozen cross-linked collagen fibers were spread flat on dry ice for low-temperature irradiation sterilization at a dose of 28 kGy.

[0080] Freeze-drying: The collagen fibers sterilized by low-temperature irradiation are directly freeze-dried to obtain irradiation cross-linked collagen fiber materials. The state of the irradiation cross-linked collagen fiber materials is as follows: Figure 2 .

[0081] Comparative Example 1 is a method for preparing an irradiated cross-linked collagen fiber material. The difference from Example 1 is that, in the preparation of the cross-linked collagen fibers, an equal amount of physiological saline is used to replace the irradiation solvent system.

[0082] Comparative Example 2 is a method for preparing a radiation-crosslinked collagen fiber material. The difference from Example 1 is that in the collagen fiber intermediate crosslinking step, the thickness of a single aluminum foil bag is 10 mm.

[0083] Comparative Example 3, a method for preparing an irradiated cross-linked collagen fiber material, differs from Example 1 in that the collagen concentration of the collagen fiber intermediate in the concentrated collagen fiber redispersion step is 55 mg / ml.

[0084] Comparative Example 4 is a method for preparing a radiation-crosslinked collagen fiber material. The difference from Example 2 is that in the collagen fiber intermediate crosslinking step, the radiation crosslinking control dosage is 25 kGy.

[0085] Comparative Example 5 is a method for preparing a radiation-crosslinked collagen fiber material. The difference from Example 2 is that in the collagen fiber intermediate crosslinking step, the radiation crosslinking is controlled at a dose of 5 kGy.

[0086] Comparative Example 6 is a method for preparing a radiation-crosslinked collagen fiber material. The difference from Example 2 is that in the collagen fiber intermediate crosslinking step, the radiation crosslinking is performed in a frozen state at -20°C.

[0087] Comparative Example 7, a method for preparing an irradiated cross-linked collagen fiber material, differs from Example 1 in that, in the step of removing the dispersant, three homogenization and ultrasonic extraction and cleaning are not used. Specifically, the cross-linked collagen fibers are redispersed in physiological saline at a liquid ratio of 1:20, the collagen fibers are obtained by centrifugation, and then sterilized by low-temperature irradiation.

[0088] Comparative Example 8, a method for preparing an irradiated cross-linked collagen fiber material, differs from Example 1 in that dry ice is not used in the low-temperature irradiation sterilization step, and irradiation is not performed at a low temperature. The specific operation is as follows:

[0089] The ultra-low temperature quick-frozen cross-linked collagen fibers were packed in aluminum foil bags with a thickness of 8 mm per bag, and then sterilized by irradiation at a dose of 25 kGy at room temperature of 20°C.

[0090] Comparative Example 9: A method for preparing an irradiated cross-linked collagen fiber material. The difference from Example 1 is that in the preparation of the cross-linked collagen fibers, the solvent is only a mixture of physiological saline and dimethyl sulfoxide in a ratio of 1:1 (v / v), and no dispersant Triton X-100 is added.

[0091] Comparative Example 10 is a method for preparing an irradiated cross-linked collagen fiber material. The difference from Example 1 is that in the preparation of the cross-linked collagen fibers, the irradiation solvent system used is not mixed with dimethyl sulfoxide, but contains equal amounts of physiological saline and Triton X-100.

[0092] Performance testing:

[0093] Experimental samples: The irradiated cross-linked collagen fiber materials prepared by the preparation methods of the irradiated cross-linked collagen fiber materials of Examples 1 to 3 and Comparative Examples 1 to 10 were used as experimental samples. The injection performance, enzymatic hydrolysis resistance, cross-linking degree, and solvent residue of the materials were tested. The specific experimental process and experimental results are as follows:

[0094] 1. Material injection performance

[0095] The experimental samples were dispensed into syringes and tested for sample extrusion and injection using a universal tensile testing machine using a 27 G syringe needle. The extrusion speed was set at 30 mm / min, and the maximum extrusion force during the extrusion process was set at 25 N. If the extrusion force during the entire extrusion process was less than 25 N, the sample was considered qualified. If the extrusion force was greater than 25 N or needle blockage occurred during the measurement, the sample was considered unqualified. The specific experimental results are shown in Table 1.

[0096] Table 1. Injectability of radiation-crosslinked collagen fiber materials obtained using Examples 1 to 3 and Comparative Examples 1 to 10

[0097]

[0098] According to the results in the above table, the collagen fiber materials prepared in Examples 1 and 2 can pass smoothly through 27 G syringe needles inject evenly without needle blockage; Comparative Example 1 has needle blockage, which is because no solvent and dispersant solvent system are used before the radiation cross-linking process, resulting in collagen aggregation during the irradiation process and local excessive cross-linking, and subsequent homogenization cannot thoroughly disperse the collagen evenly; Comparative Example 2 has a high pushing force and is unqualified, which is due to the high stacking thickness of the material and uneven electron beam irradiation during radiation cross-linking; Comparative Example 3 has an excessively high pushing force and is unqualified, which is due to the excessively high collagen concentration before radiation cross-linking, resulting in uneven cross-linking and local excessive cross-linking; Comparative Example 4 has a needle blockage, which is due to the excessively high dose of radiation cross-linking, resulting in local rapid cross-linking of collagen fibers and uneven material; Comparative Examples 5 and 6 have qualified pushing forces; Comparative Example 7 has an excessively high pushing force, which is due to the lack of homogenized ultrasonic treatment, resulting in local cross-linking of the irradiated collagen fibers and inability to be smoothly injected; Comparative Example 8 has a needle blockage, which is due to radiation cross-linking at room temperature, resulting in secondary cross-linking and fiber entanglement. Comparative Examples 9 and 10 experienced needle blocking. This was because no dispersant or organic solvent was used in the solvent system during the radiation cross-linking process, which resulted in excessive cross-linking of the collagen fibers and needle blocking.

[0099] 2. Particle size determination

[0100] The sample was diluted 50 times with normal saline, and the average particle size of the collagen fibers in the solution was tested by dynamic light scattering. All sample solutions were added to polystyrene cuvettes for detection. The average particle size distribution range of collagen fibers in normal saline can be analyzed by dynamic light scattering. The collagen fibers in the solution will scatter the light passing through, and the mutual superposition of scattered light will cause the superposition or reduction of the light intensity in the dark and bright areas of the particles, so that the light intensity appears in the form of fluctuations. Under the irradiation of laser, the scattered light intensity of the moving particles will also fluctuate. The frequency of the fluctuation is related to the size of the particles. The changes in the speed of ion fluctuations are recorded by dynamic light scattering to obtain the particle size distribution information of the particles in the solution. As shown in the following table, the average particle size distribution results of the samples are shown in Table 2.

[0101] Table 2. Average particle sizes of irradiated cross-linked collagen fiber materials obtained using Examples 1 to 3 and Comparative Examples 1 to 10

[0102]

[0103] According to the results in the above table, the average particle size of the collagen fiber materials prepared in Examples 1 and 2 is between 100-150 microns, which meets the injection requirements; the average particle size of Comparative Example 1 reaches 434.44±109.46 microns, which is too large and uneven. This is because no solvent and dispersant solvent system are used before the irradiation cross-linking process, which causes collagen aggregation during the irradiation process and leads to local excessive cross-linking. The average particle size of Comparative Example 2 was higher than 150 μm due to uneven electron beam irradiation caused by the high material stacking thickness during radiation cross-linking. The average particle size of Comparative Example 3 was 348.11 μm, with large fluctuations, due to uneven cross-linking and localized over-crosslinking caused by excessive collagen concentration before radiation cross-linking. The average particle size of Comparative Example 4 failed to meet the standard due to excessive radiation cross-linking dose, which led to localized rapid cross-linking of collagen fibers. The average particle sizes of Comparative Examples 5 and 6 were both less than 100 μm due to insufficient cross-linking. The average particle size of Comparative Example 7 was greater than 150 μm due to the lack of homogenizing ultrasonic treatment. The average particle size of Comparative Example 8 was 337.65 μm due to secondary cross-linking and fiber entanglement caused by radiation cross-linking at room temperature. The average particle size of Comparative Example 9 was greater than 150 μm due to the lack of a dispersant in the solvent system. The average particle size of Comparative Example 10 was 234.75 ± 67.19 μm due to the lack of an organic solvent in the solvent system, which led to localized over-cross-linking of collagen fibers.

[0104] 3. Material resistance to enzymatic hydrolysis

[0105] Collagen fiber samples prepared in the comparative examples and examples were placed in 10 mL centrifuge tubes (4 g per tube). 100 U / mL of type I collagenase hydrolyzate was added and hydrolyzed in a 37°C waterbath. Six samples were prepared for each group. After different hydrolysis times, the precipitates were centrifuged to obtain the remaining collagen fibers. The precipitates were weighed and the remaining collagen fibers were calculated to determine the material's resistance to enzymatic hydrolysis.

[0106] The results are as follows Figure 3 As shown, Examples 1 and 2 both have high levels of resistance to enzymatic hydrolysis; Comparative Example 1 has slightly poorer enzymatic hydrolysis performance; Comparative Examples 2 and 3 have lower crosslinking levels and poorer enzymatic hydrolysis resistance due to their higher thickness and material concentration during irradiation crosslinking; Comparative Example 4 has higher crosslinking levels and higher enzymatic hydrolysis resistance due to increased irradiation dose; Comparative Example 5 degrades faster due to insufficient crosslinking due to the lower irradiation dose; Comparative Example 6 has the fastest degradation rate due to poor irradiation crosslinking due to the low-temperature crystallization process, which results in a lack of fluidity in the collagen material; Comparative Example 7 does not use ultrasonic cleaning, which has no effect on the degree of irradiation crosslinking; Comparative Example 8 has the strongest enzymatic hydrolysis resistance due to the second irradiation sterilization process at room temperature, resulting in secondary crosslinking. Comparative Examples 9 and 10 have no significant reduction in enzymatic hydrolysis capacity due to excessive local crosslinking caused by the irradiation crosslinking system.

[0107] 4. Cross-linking degree

[0108] The degree of crosslinking of the material was determined by measuring the number of free amino groups in the experimental object. Three replicates were tested for each sample. The results of the crosslinking determination are shown in Table 3.

[0109] The test method for cross-linking degree is as follows:

[0110] Standard curve drawing:

[0111] Accurately weigh 75 mg of glycine and dissolve it in 10 ml of water to obtain a 0.1 M glycine solution. Pipette the 0.1 M glycine solution and dilute it to 10 ml to prepare a 0.25-2.50 mM glycine standard solution for determination of the standard curve.

[0112] To 0.150 ml of glycine standard solution, add 1.2 ml of buffer (0.05 mol / L borax, pH 10) and 1.2 ml of TNBS solution. Place the test tube in a dark, 50°C water bath for 60 min. Add 1.4 ml of deionized water and 1 ml of 0.1 mol / L hydrochloric acid. Cool at room temperature for 30 min and measure absorbance at 340 nm.

[0113] Use water as the blank solution. A quartz cuvette is required.

[0114] A standard curve was drawn with the free amino group concentration (mM) as the horizontal axis and the absorbance as the vertical axis.

[0115] 1. Sample determination

[0116] The sample concentration to be measured is between 0.25 and 2.50 mM, and the free amino group concentration of the sample solution is calculated based on the standard curve.

[0117] 2. Calculation of collagen cross-linking degree

[0118] Free amino content (umol / mg) = free amino concentration / protein concentration

[0119] Cross-linking degree = (free amino content before modification - free amino content after modification) / free amino content before modification * 100%

[0120] The theoretical value of free amino group content before modification: 0.25umol / mg.

[0121] Table 3. List of crosslinking degrees of irradiated crosslinked collagen fiber materials obtained using Examples 1 to 3 and Comparative Examples 1 to 10

[0122]

[0123] The results of the material cross-linking degree are basically consistent with the enzymolysis resistance. The higher the cross-linking degree of the material, the stronger its enzymolysis resistance. The cross-linking degrees of Examples 1-3 are between 87-89%. The cross-linking degree of Comparative Example 1 fluctuates greatly. This is due to the uneven cross-linking caused by the lack of a dispersed solvent system. The cross-linking degree of Comparative Examples 2 and 3 decreases due to the high thickness and high concentration of the material. The cross-linking degree of Comparative Example 4 is very high, which is caused by the high irradiation dose used. The cross-linking degree of Comparative Example 5 is low due to the low irradiation dose. Comparative Example 6 is irradiated and cross-linked at low temperature. The collagen fibers cannot move after freezing, resulting in a significant reduction in their cross-linking degree. Comparative Example 7 does not use ultrasonic cleaning, which has no obvious effect on the cross-linking degree of the material. Comparative Example 8 has a high degree of cross-linking due to secondary irradiation cross-linking. Comparative Examples 9 and 10 do not use dispersants and organic solvents in the irradiation system, resulting in rapid and uneven irradiation cross-linking, so the cross-linking degree is relatively high.

[0124] 5. Solvent residue

[0125] The qualification of the product is determined by measuring the residual organic solvent and dispersant used in the test sample. The residual solvent is determined by measuring the residual concentration in the extract after extraction.

[0126] Table 4. Solvent residue results of radiation cross-linked collagen fiber materials obtained using Examples 1 to 3 and Comparative Examples 1 to 10

[0127]

[0128] From the above results, it can be seen that during the preparation process of collagen fibers, if homogenization and ultrasonic treatment are repeated three times, the organic solvent and dispersant in the final product are washed away and undetectable; only residues are detected in Example 7, which is unqualified.

[0129] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing an irradiated cross-linked collagen fiber material, characterized in that: The steps include: (1) Preparation of collagen fiber intermediates: Dispersing concentrated collagen fibers in an irradiation solvent system, and obtaining a collagen fiber intermediate with a concentration of 35-45 mg / ml after centrifugation; the irradiation solvent system comprises physiological saline and an organic solvent in a volume ratio of 1:1-2.4, and a dispersant in an amount of 0.1-2 w / v% of the total amount of the physiological saline and organic solvent; (2) Irradiation cross-linking: irradiating the collagen fiber intermediate at a temperature of 20-30°C and an irradiation dose of 10-20 kGy to obtain cross-linked collagen fibers; (3) removing the dispersant from the cross-linked collagen fibers and sterilizing them by low-temperature irradiation to obtain a finished collagen fiber material; The method for removing the dispersant in step (3) includes: redissolution, homogenization and / or ultrasonic treatment, and centrifugation; The low-temperature irradiation sterilization process is to firstly subject the cross-linked collagen fibers to ultra-low temperature quick freezing treatment, and then subject them to low-temperature irradiation sterilization treatment at a temperature range of -80°C to -40°C; the irradiation dose of the low-temperature irradiation sterilization treatment is 25-30 kGy; The thickness of the cross-linked collagen fiber intermediate is 5-8 mm; The organic solvent includes any one or more of dimethyl sulfoxide, isopropyl alcohol, acetone, ethyl acetate, and methyl tert-butyl ether; The dispersant includes any one or more combinations of Triton X-100, Triton X-114, Triton X-405, and Triton GR-7M.

2. The method for preparing a radiation cross-linked collagen fiber material according to claim 1, characterized in that: The concentrated collagen fibers include any one or more combinations of type I collagen fibers, type II collagen fibers, and type III collagen fibers.

3. The method for preparing a radiation cross-linked collagen fiber material according to claim 1, characterized in that: The radiation crosslinking is electron beam irradiation or gamma ray irradiation.

4. The method for preparing a radiation cross-linked collagen fiber material according to claim 1, characterized in that: The method for removing the dispersant in step (3) comprises: adding physiological saline to the cross-linked collagen fibers, controlling the liquid ratio to be 1:20-1:50, homogenizing and dispersing for 30 minutes, ultrasonically extracting for 30-60 minutes, and centrifuging; and repeating the operation multiple times.

5. Collagen fiber material prepared by the method according to any one of claims 1 to 4.

6. The collagen fiber material according to claim 5, characterized in that: The degree of cross-linking of the material is between 85% and 90%.

7. The collagen fiber material according to claim 6, characterized in that: The average particle size of the collagen fibers is in the range of 100-150 μm.

8. The collagen fiber material according to claim 6, characterized in that: The material is filled into a 27G syringe, and the maximum pushing force is between 11.5N and 25N at a pushing speed of 30mm / min.

9. Use of the collagen fiber material according to any one of claims 6 to 8 in the preparation of medical injection filling materials and / or tissue engineering scaffolds.

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