Collagen thread as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological extraction, and particularly relates to a collagen line as well as a preparation method and application thereof. The preparation method comprises the following steps: placing a collagen tissue in a non-circular container, soaking the collagen tissue in an ethanol solution, and drying the collagen tissue; a micro structure is arranged on the inner wall of the container and is used for guiding the collagenous fibers to be directionally arranged, and the collagenous fibers are directionally arranged in the soaking process; and drying to obtain the collagen line. According to the preparation method, through asymmetric geometric constraint, the orientation degree of the collagen fibers is increased to be larger than or equal to 90%, the axial / radial modulus ratio is larger than or equal to 5: 1, and the dynamic loading requirements of heart valve suture and the like are met.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological extraction, and specifically relates to a collagen protein thread and a preparation method and application thereof. BACKGROUND

[0002] The collagen protein thread is a biological material widely used in the field of medical suture, and is favored due to its good biocompatibility and absorbability. The collagen protein thread is usually extracted from animal tissues and used for surgical suture after processing. In the prior art, the collagen protein thread is mainly fixed and shaped by a circular glass container. In this method, the collagen protein thread is prone to bending deformation in the circular container, resulting in the need for an additional straightening process. At the same time, the tensile strength of the collagen protein thread in the traditional process is insufficient, and the collagen protein thread is prone to breakage during surgical suture.

[0003] This circular container fixing method not only increases the production process, but also may cause mechanical damage to the collagen protein thread due to repeated straightening operations, affecting the quality and performance of the final product. In addition, the existing process has limited effect on improving the strength of the collagen protein thread, and it is difficult to meet the needs of high-strength suture.

[0004] Defects of the traditional scheme: the circular container causes random arrangement of fibers, and the modulus ratio is only approximately 1:1. Excessive reinforcement is prone to breakage, and excessive toughening leads to tissue damage.

[0005] In the prior art, in order to improve the strength of the collagen protein thread, technical means such as optimization of cross-linking agents are used. However, high-strength collagen protein threads, although meeting the axial requirement of 120MPa or more tensile force for blood vessel suture, cannot meet the radial modulus requirement of ≤0.3MPa to avoid cutting fragile tissues. SUMMARY

[0006] (I) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present application provides a preparation method of a collagen protein thread, which constrains and induces the directional arrangement of fibers through the shape of the container, so that the axial / radial modulus ratio of the collagen protein thread is ≥5:1.

[0008] Correspondingly, the present application also provides a collagen protein thread and its application, and the axial / radial modulus ratio of the collagen protein thread is ≥5:1.

[0009] (II) Technical solutions In order to achieve the above-mentioned purposes, the main technical scheme adopted by the present application includes:

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

[0011] S1 collagen tissue is placed in a non-circular container, and after being soaked in an ethanol solution, it is dried;

[0012] S2 the inner wall of the container is provided with a microstructure for guiding the directional arrangement of collagen fibers, and during the soaking process, the collagen fibers are arranged directionally;

[0013] S3 collagen protein lines are obtained through drying treatment.

[0014] The present application breaks through the limitation of symmetric shrinkage stress of traditional circular containers, and for the first time utilizes the geometric asymmetry of a non-circular container to generate a fiber orientation driving force; in combination with the capillary force gradient field (microfluidic effect formed by ethanol solution infiltration difference) induced by the inner wall microstructure, the directional arrangement of collagen fibers is realized under zero external energy. The existing technology (such as circular container drying or centrifugal method) cannot spontaneously form a directional structure, and the present application solves the fundamental problem of unordered fiber arrangement through physical structure design.

[0015] Optionally, the non-circular container is a rectangular container with an aspect ratio of ≥2:1.

[0016] The present application utilizes the boundary constraint force generated by the non-circular container (especially the rectangular container with an aspect ratio of ≥2:1) to force the collagen fibers to spread and arrange in a two-dimensional plane, breaking the random crimping state of the fibers in the traditional circular container.

[0017] The present application for the first time realizes the obtaining of straight collagen lines without mechanical straightening; the order degree of fiber arrangement (X-ray diffraction half-peak width) is improved to 82%, which is much higher than the 35-50% of conventional methods.

[0018] Optionally, the microstructure includes a protrusion array or a groove array, the middle axis height of the protrusion or the groove is 20-50 um, and the distance between two protrusions in the protrusion array or the distance between two grooves in the groove array is 100-200 um.

[0019] The microstructure of the present application generates a microflow field effect, guiding the self-assembly of collagen fibers along the long axis direction of the container during ethanol dehydration. The collagen protein lines obtained by the present application have controllable anisotropy, and the axial / radial modulus ratio is ≥5:1, while the traditional method is approximately 1:1; in the blood vessel anastomosis experiment, the axial tensile strength of the present application is ≥120 MPa, meeting the requirements of blood vessel anastomosis; the strength retention rate after knotting is ≥90%, which is significantly higher than the 60-75% of conventional collagen lines, and the radial flexibility reduces the leakage rate from 12.3% to 0.8%.

[0020] Optionally, the ethanol solution contains a bioactive additive, the ethanol solution contains a bioactive additive, and the bioactive additive is selected from at least one of a crosslinking agent, a growth factor, a nanomaterial, and a metal organic framework material.

[0021] Among them, metal-organic frameworks (MOFs) enhance mechanical properties and bioactivity;

[0022] The present invention simultaneously introduces glutaraldehyde during the ethanol fixation stage, and cross-links the aldehyde groups with the collagen lysine residues (each collagen molecule has ≥3 cross-linking points);

[0023] Optionally, a physical field treatment is applied during the soaking process, wherein the physical field includes at least one of a magnetic field, an ultrasonic field, and a temperature gradient field.

[0024] Optionally, the ethanol solution contains a bioactive additive, and the bioactive additive is selected from at least one of a cross-linking agent, a growth factor, a nanomaterial, and a metal-organic framework material.

[0025] Optionally, the nanomaterial is graphene quantum dots, which are embedded in the collagen triple helix structure through π-π stacking, forming a fluorescence monitoring function while improving tensile strength; the antibacterial rate and free radical scavenging ability are also improved, with the Staphylococcus aureus inhibition rate reaching 99.5%.

[0026] Optionally, humidity, temperature, air pressure, or a dynamic combination thereof are controlled during the soaking process.

[0027] In the present invention, a pulsed magnetic field of 0.1 to 0.5 T induces the polar group orientation of collagen fibers, and an ultrasonic cavitation effect promotes the penetration of reagents.

[0028] While processing time is shortened by 50%, fiber crystallinity is increased by 20%;

[0029] The quantum dot distribution uniformity (CV value) was reduced from 35% in the traditional stirring method to 8.7%.

[0030] Optionally, the concentration of the ethanol solution is 75% to 85%.

[0031] Optionally, the immersion time is 18 to 30 hours.

[0032] Optionally, the collagen tissue is tail tendon tissue and is pretreated as follows:

[0033] Bleeding puts the animal into a state of shock;

[0034] Use iodine 75% alcohol and povidone iodine for gradient disinfection;

[0035] Wash with shaking in PBS buffer containing 0.01% to 0.1% hyaluronidase for 20 to 40 minutes.

[0036] More specifically, S1 places animal-derived collagen tissue in a rectangular container with a length-to-width ratio of 2:1 to 10:1;

[0037] Add 75% to 90% ethanol solution to the rectangular container S2 and soak for 12 to 48 hours;

[0038] After S3 was taken out and dried, a straight collagen line was obtained.

[0039] The present invention extracts muscle tissue from animal tail tissue to obtain collagen threads; the collagen threads are placed in a rectangular glass container, and a 75% to 90% ethanol solution is added, and the ethanol solution is soaked in the collagen threads; the collagen threads are soaked in the ethanol solution for 24 hours to fix the collagen threads in shape. According to the above technical means, by placing the collagen threads in a rectangular glass container and soaking them in an alcohol solution with a concentration of 75% to 90% for 12 to 48 hours, the collagen threads can be directly shaped into a straight state, avoiding the problem of the collagen threads in traditional circular containers requiring an additional straightening process after being bent. This not only simplifies the production process and improves production efficiency, but also reduces the damage to the collagen threads caused by the straightening operation, thereby ensuring the quality and strength of the collagen threads.

[0040] Optionally, the microstructure of the inner wall of the container guides the collagen fibers to align in a directional manner, forming fiber bundle gaps with a width of 0.5 to 3 μm, which are converted into surface directional grooves after cross-linking and drying.

[0041] In a second aspect, the present invention further provides a collagen thread prepared by any of the above preparation methods, which satisfies at least one of the following conditions:

[0042] The minimum curvature radius in dry state is ≥30mm, and the breaking strength is ≥1.5MPa;

[0043] The ratio of axial tensile modulus to radial tensile modulus is ≥3:1;

[0044] The surface has grooves with a depth of 0.5 to 3 μm, a groove depth of 5 to 15 μm, and an angle of ≤15° between the groove direction and the axial direction of the collagen line;

[0045] The shear force generated by the protrusions of the present invention promotes directional sliding of collagen fiber bundles along their long axis, reducing transverse cross-linking. When wetted with physiological saline, the surface grooves reduce the dynamic friction coefficient between the suture and tissue by more than 60%, from 0.15 to ≤0.06).

[0046] Optionally, the collagen wire provided by the present invention is loaded with graphene quantum dots, which emit blue fluorescence under 365nm ultraviolet light.

[0047] Optionally, the grooves are loaded with glutaraldehyde cross-linking layers, graphene quantum dots, and MOF. Under light, the collagen thread achieves an antibacterial rate of ≥99% through physical confinement (grooves), as well as the synergistic effect of chemical sterilization and photocatalytic oxidation. Specifically: the zinc ions in the MOF are efficiently enriched only in the grooves, and the Zn is slowly released.2+ The grooves, with a width of 0.5 to 3 μm, confine the bacteria to a limited space. The diameter of Staphylococcus aureus is 1 μm.

[0048] The surface-oriented grooves in the present invention are not obtained by direct etching, but are formed through three steps: microstructure-induced fiber self-assembly, locking gap morphology, and drying shrinkage. Its core technologies are: micron protrusions precisely control fiber spacing; glutaraldehyde cross-linking fixes submicron gaps; and gradient drying inhibits structural collapse.

[0049] In a third aspect, the present invention further provides a use of the collagen thread described in any of the above schemes in a medical suturing device.

[0050] It is particularly suitable for dynamic tissue suturing (such as heart valve repair). Its high axial modulus (1.5MPa) resists blood flow impact, and its low radial modulus (0.3MPa) avoids cutting tissue.

[0051] In a fourth aspect, the present invention further provides an apparatus for implementing the above method, comprising:

[0052] a non-circular container having microstructures on its inner wall;

[0053] a multi-physics field coupling generator, comprising at least one of a magnetic field generator, an ultrasonic generator, and a temperature gradient controller, for applying a physical field to the non-circular container;

[0054] The dynamic environmental control system includes a humidity sensor, a temperature sensor, an air pressure sensor, and corresponding adjustment actuators, which are used to adjust at least one parameter of the humidity, temperature, and air pressure inside the non-circular container.

[0055] (3) Beneficial effects

[0056] The preparation method of the present invention, through asymmetric geometric constraints, increases the orientation degree of collagen fibers to ≥90%, and the axial / radial modulus ratio reaches ≥5:1, meeting the dynamic load requirements of heart valve suturing and the like. DETAILED DESCRIPTION

[0057] In order to explain in detail the possible application scenarios, technical principles, specific solutions that can be implemented, and the purpose and effects of the present invention, the following is a detailed description of the specific embodiments listed. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0058] In the following description, the terms "collagen thread", "muscle tissue", and other professional terms involved will be explained in detail to ensure an accurate understanding of the technical solution of this application.

[0059] Collagen line: a natural protein fiber extracted from animal tissue, mainly composed of collagen, with good biocompatibility and absorbability, commonly used in medical suture and other fields. Its molecular structure is in the form of triple helix, with high mechanical strength and toughness.

[0060] Muscle tissue: a special knot structure of animal tail, which is an ideal raw material source for preparing high-quality collagen line with regular fiber arrangement structure.

[0061] 75%~90% ethanol aqueous solution, used as a fixing agent for collagen line in the present application, which can ensure the fixing effect and will not damage the protein structure.

[0062] In the prior art, the preparation of collagen line has the following main problems: first, the use of round glass containers for shaping treatment will cause the collagen line to bend and deform, and an additional straightening process is needed, which not only increases the production cost, but also easily causes damage to the structure of the collagen line; second, the collagen line prepared by the traditional process has insufficient tensile force and is prone to breakage during use.

[0063] In order to solve the above technical problems, the following innovative technical solutions are adopted: first, muscle tissue is extracted from the tail of an animal as raw material to ensure the integrity and regular arrangement of collagen fibers; second, a rectangular glass container is used for shaping treatment, and a specific soaking process of ethanol solution is used to keep the collagen line in a straight line during shaping. This technical solution not only simplifies the production process and avoids the subsequent straightening process, but also significantly improves the mechanical strength and tensile performance of the collagen line.

[0064] The technical solutions of the present application can achieve the following technical effects:

[0065] The straightness of the collagen line is significantly improved, and no subsequent straightening process is needed;

[0066] The tensile performance of the collagen line is improved, the production process is simplified, and the production efficiency is improved;

[0067] The damage to the collagen line caused by the traditional straightening process is avoided, and the product quality is more stable and reliable.

[0068] It should be noted that the embodiments of the present application can be applied to various medical and cosmetic fields that require high-tensile collagen line, and are particularly suitable for wound suture and plastic surgery applications that require long-term tension maintenance:

[0069] The method comprises the following steps S1 to S3:

[0070] S11 extracts muscle tissue from the tail of an animal to obtain collagen line.

[0071] This step can be performed by medical device manufacturers or biomaterial preparation laboratories. It requires the use of professional surgical instruments and sterilization equipment, and is performed in a sterile environment.

[0072] Myotome tissue, a connective tissue unique to animal tails, is rich in regularly arranged collagen fiber bundles. These fiber bundles possess a highly ordered, parallel structure, making them an ideal source for producing high-quality collagen yarns. For example, myotome tissue extracted from the tail of a civet cat contains collagen fiber bundles approximately 50-100 microns in diameter, while myotome tissue extracted from the tail of a kangaroo contains thicker fiber bundles, reaching diameters of 150-200 microns.

[0073] In practice, healthy adult animals (such as civets, kangaroos, or foxes) are first bled, followed by tail disinfection. The procedure involves using a scalpel to incise the skin along the midline of the tail to expose the muscle tissue; disinfecting the exposed area with iodine, followed by deiodination with 75% alcohol; and finally, using fine forceps to completely remove the muscle tissue, avoiding damage to the fibrous structure.

[0074] S12: placing the collagen thread into a rectangular glass container, adding an ethanol solution, and allowing the ethanol solution to soak the collagen thread.

[0075] A rectangular glass container refers to a transparent glass container with a regular rectangular shape, and its length, width and height ratio is preferably 10:2:1. This specific shape of container can ensure that the collagen line remains straight during the immersion process and avoids bending and deformation. For example, a rectangular glass container with a size of 20cm×4cm×2cm or 30cm×6cm×3cm can be used.

[0076] The ethanol solution is a 75% to 90% ethanol-water solution. This specific concentration effectively stabilizes the molecular structure of collagen without causing excessive protein denaturation. Experiments have shown that alcohol concentrations below 70% are insufficient for setting collagen, while concentrations above 90% can lead to increased brittleness of the egg yolk. For example, an 80% ethanol solution can be prepared by mixing 95% medical alcohol with distilled water in a 4:1 volume ratio.

[0077] In actual implementation, the extracted collagen threads are laid flat on the bottom of a rectangular glass container to ensure that they stretch naturally without bending.

[0078] In practice, lay the extracted collagen threads flat on the bottom of a rectangular glass container, ensuring they stretch naturally without bending. Then, slowly pour in a pre-prepared 75% to 90% ethanol solution until the liquid level is 1-2 cm above the collagen threads. Avoid shaking the container vigorously during this process to prevent the collagen threads from becoming tangled.

[0079] Step S13: soaking the collagen thread in the edible ethanol solution for 24 hours to fix the collagen thread into shape.

[0080] A 24-hour soaking time is the optimal process parameter determined through extensive experimentation. This period allows alcohol molecules to fully penetrate the collagen fibers and form stable intermolecular crosslinks, but does not cause the collagen thread to overharden. For example, experimental data shows that after 12 hours of soaking, the setting effect is insufficient, and after 36 hours, the collagen thread begins to become brittle.

[0081] Fixation refers to the formation of a stable three-dimensional network structure by collagen molecules under the action of alcohol. This process involves the alcohol molecules replacing water in the collagen fibers, prompting the collagen side chains to form new hydrogen bonds and hydrophobic interactions, thereby enhancing the mechanical strength of the fibers.

[0082] In practice, place the container containing the collagen thread in a constant temperature environment at 20-25°C for 24 hours. Avoid shaking or moving the container during this time to ensure a stable shaping process. Once shaping is complete, remove the collagen thread with sterile tweezers and allow it to dry naturally in a well-ventilated area.

[0083] In actual implementation, there is a strict process sequence and parameter control relationship between the above three steps. The quality of raw material extraction in step S11 directly affects the subsequent shaping effect, and it is necessary to ensure that the muscle tissue is intact; the selection of the set and the preparation of the solution in step S12 create an ideal environment for shaping;

[0084] The control of the immersion time and temperature in step S13 is the key to achieving the best shaping effect. These three steps are closely linked to each other and together ensure the quality stability of the final product.

[0085] The method for preparing collagen thread provided in the embodiment of the present application extracts muscle tissue from the tail of an animal as the raw material, and adopts a rectangular glass container of a specific shape and an 80% ethanol solution soaking process to make the prepared collagen thread have excellent straightness and mechanical properties. This method avoids the bending deformation problem caused by traditional circular containers and eliminates the subsequent straightening process, which not only improves production efficiency but also ensures the stability of product quality. Experiments have shown that the tensile strength of the collagen thread prepared by this method can reach 150 to 200 MPa, which is more than 30% higher than the traditional method, and fully meets the clinical needs of high-tension sutures.

[0086] In some embodiments, the collagen thread extraction method includes steps S21 to S26, wherein:

[0087] Step S21, bloodletting healthy animals, select the source of raw materials as the Tasmanian devil, kangaroo or fox. Bloodletting refers to the process of making the blood of the animal by a specific method, which is the first step of extracting collagen line. The selection criteria of healthy animals include age 1-3 years old, no history of infectious diseases, weight standard, etc. Tasmanian devil, kangaroo and fox are the preferred raw material sources because the tail muscle tissue of these animals is rich in collagen and has excellent fiber structure.

[0088] Step S22, cut the skin of the animal's tail, disinfect with iodine, and then deiodinate with 75% alcohol. Tail processing includes three sub-steps of cutting the skin, disinfecting and deiodinating. Iodine disinfection can effectively kill surface bacteria, and 75% alcohol deiodination can avoid the influence of iodine residue on collagen quality. In actual operation, the disinfection area should be 2-3m larger than the incision area, and deiodination should be carried out for 2-3 times to ensure thoroughness.

[0089] Step S23, extract the tendon tissue of the tail, wash it clean in clean water, and remove the mucus.

[0090] Tendon tissue refers to the fibrous connective tissue connecting muscle and bone, rich in collagen fibers. The cleaning process requires the use of flowing clean water, with water temperature controlled at 15-30℃, and cleaning time of 10-15 minutes. Removing mucus can be achieved by gentle scrubbing, but care should be taken to avoid damaging the fiber structure.

[0091] Step S24, place the cleaned tendon tissue in a rectangular glass container.

[0092] The size of the rectangular glass container is preferably 30cm long x 20cm wide x 15cm high, with a wall thickness of 5mm. Compared with traditional round containers, the rectangular structure can keep the collagen line straight during fixation, avoiding bending and deformation. The container material should be high borosilicate glass to ensure resistance to alcohol corrosion.

[0093] Step S25, add 80% edible alcohol solution to the container, and the solution volume should completely immerse the collagen line. 80% edible alcohol solution refers to an aqueous solution of ethyl ester with a volume fraction of 80%, which can effectively fix the collagen structure without over-hardening. The solution volume should exceed 23cm on the upper surface of the collagen line to ensure sufficient soaking. Edible alcohol should meet the standard of GB31640-2016.

[0094] Step S26, after 24 hours of soaking, take out and dry to get the shaped collagen line.

[0095] 24 hours of soaking can make the collagen molecules fully crosslink and shape. The drying environment should maintain a temperature of 20-25℃ and a relative humidity of 40-60%, with a drying time of 8-12 hours. The shaped collagen line has uniform diameter and straightness deviation less than 1mm / m.

[0096] In actual implementation, the above steps S21 and S26 are a continuous and interconnected process. The bleeding process ensures that the raw materials are soaked for 24 hours to fully cross-link the collagen molecules and set them. The drying environment should maintain a temperature of 20-250 degrees and a relative humidity of 40-60% for 8-12 hours. The diameter of the collagen line after setting is uniform, and the straightness deviation is less than 1mm / m.

[0097] In practice, steps S21 through S26 form a continuous and interconnected process. Bleeding ensures the hygienic safety of the raw material, tail processing creates conditions for tendon extraction, the washing step removes impurities, and the combination of a rectangular container and ethanol solution achieves efficient shaping, ultimately resulting in a high-quality collagen thread product. The order and parameter settings of each step are optimized to ensure a balance between production efficiency and product quality.

[0098] In the embodiment of the present invention, an optimized combination of a rectangular glass container and an 80% ethanol solution is used to ensure that the collagen strands remain straight during the fixation process, thereby eliminating the process of straightening the collagen strands in traditional methods. This avoids damage to the collagen strands during the straightening process, thereby improving product yield and production efficiency.

[0099] A method for preparing a collagen thread comprises bleeding a healthy animal (such as a raccoon, a kangaroo, a fox, etc.), cutting the skin of the tail, disinfecting it with iodine, and then deiodinating it with 75% alcohol. The muscle tissue of the tail is then extracted and cleaned in clean water to remove mucus from the muscle tissue. The thread is placed in a rectangular glass container and an ethanol solution is added. The solution should exceed the collagen thread. The thread is soaked for 24 hours to fix the collagen thread into shape, and then taken out and dried before use.

[0100] The absorbable collagen suture provided by this method has good tensile strength, which solves the problem of insufficient tensile strength of collagen sutures in the past, which causes the suture to break. The collagen suture is placed in a rectangular glass container for fixation, so that the collagen suture produced is relatively straight and will not cause damage. This is better than the previous process of placing the protein suture in a round glass container, and then straightening the collagen suture after fixing it, as well as the damage caused by the straightening of the collagen suture. It can save the production process of straightening the collagen suture. It improves the production efficiency of manufacturers, reduces production costs, and is a protection for collagen sutures.

[0101] Example 1

[0102] This embodiment provides a container for preparing collagen thread, which is made of medical-grade polypropylene and is a rectangular container with an aspect ratio of 2:1, 4:1, or 10:1.

[0103] Example 2

[0104] This embodiment provides a container for preparing collagen thread. Compared with embodiment 1, the inner wall of the container is provided with a protrusion array.

[0105] The maximum longitudinal height of the protrusion is 20 μm, the spacing between two protrusions is 200 μm, the shape of the protrusion is cylindrical, and the cross-sectional diameter is 50 μm;

[0106] In some other specific embodiments, the longitudinal median height of the protrusion is 50 μm, the spacing between two protrusions is 100 μm, the shape of the protrusion is pyramidal, and the side length of the bottom surface is 80 μm;

[0107] In some other specific embodiments, the longitudinal median height of the protrusion is 35 μm, the interval between two protrusions is 150 μm, and the shape of the protrusion is a hexagonal cross section.

[0108] Example 3

[0109] This example provides a container for preparing collagen threads. Compared to Example 1, this container utilizes a laser engraving process to create a periodic micro-groove structure. The grooves are 30 μm deep, 50 μm wide, and 150 μm apart. Scanning electron microscopy revealed that 82% of the collagen fibers were aligned along the grooves.

[0110] In some other specific embodiments, the container is a rectangular polytetrafluoroethylene container with the following specific dimensions:

[0111] Long side: 120mm;

[0112] Short side: 40mm

[0113] Height: 60mm.

[0114] Example 4

[0115] This embodiment provides a bioactive additive for preparing collagen threads, which is a combination of graphene quantum dots and glutaraldehyde; the contents of the bioactive additive in a 75% ethanol solution are: 0.1 mg / mL of graphene quantum dots and 0.3% of glutaraldehyde mass concentration.

[0116] In some other specific embodiments, it is a combination of genipin and black phosphorus nanosheets, and the contents of the graphene quantum dots in a 75% ethanol solution are: 0.1 mg / mL of graphene quantum dots and 0.05% of the mass concentration of black phosphorus nanosheets.

[0117] Graphene quantum dots, or GQDs, have a unique structure, consisting of a few atomic layers and typically ranging in size from a few to tens of nanometers. Their surface is rich in reactive functional groups, resulting in excellent water solubility and biocompatibility. In this example, the graphene quantum dots were 3-5 nm in size, with a surface functional group -COOH content of 0.8 mmol / g; the excitation wavelength was 360 nm, the emission wavelength was 450 nm, and the quantum yield was 35%.

[0118] Genipin is the enzymatic hydrolysis product of geniposide and needs to be pre-dissolved in 10% DMSO aqueous solution; it contains the active group enal group and has characteristic absorption at UV280nm.

[0119] Black phosphorus nanosheets are 3 to 5 layers with a specific surface area of ​​280 m 2 / g.

[0120] Example 5

[0121] This embodiment provides a method for preparing collagen thread, the steps of which are:

[0122] S1 Collagen tissue pretreatment: Pig tail tendon with a diameter of 2 to 3 mm was taken and washed with PBS solution (pH 7.4) containing 0.1% hyaluronidase at 37° C. for 30 minutes under shaking and set aside.

[0123] S2 prepares a rectangular container with a length-to-width ratio of 4:1 and dimensions of 120 mm × 30 mm × 50 mm. The inner wall of the container is provided with an array of pyramidal protrusions with a central axis height of 35 ± 2 μm and a spacing of 150 ± 5 μm between adjacent protrusions.

[0124] S3 arranges the tendons pre-processed in step S1 in parallel inside a rectangular container;

[0125] S4 injected an 80% ethanol solution into the rectangular container to a liquid level of 100 mm. The tendon was immersed in the solution at 25°C for 24 hours while applying a 0.5 T axial static magnetic field with a magnet spacing of 100 mm. The solution was then ultrasonically treated at 20 kHz with an ultrasonic power density of 0.3 W / cm 2 .

[0126] In the ethanol solution of this embodiment,

[0127] With 82% ethanol (v / v);

[0128] 0.4% mass concentration of glutaraldehyde (as a cross-linking agent);

[0129] 0.08 mg / mL graphene quantum dots, with a particle size of 5 to 8 nm;

[0130] 0.2% mass concentration of UiO-66-NH2MOF, particle size of 100nm.

[0131] S5 Drying Treatment:

[0132] Stage 1: 40°C / 80%RH, 4 hours;

[0133] Second stage: 50℃ / 50%RH, 2 hours.

[0134] Example 6

[0135] This embodiment provides a method for preparing collagen thread, which is different from the physical field applied in Example 5. Specifically,

[0136] Stage 1: 0.3T rotating magnetic field, 10 rpm for 6 hours

[0137] The second stage: 45°C temperature field, 40kHz ultrasound (pulse mode, duty cycle 50%) treatment for 12 hours.

[0138] Example 7

[0139] This embodiment provides a method for preparing collagen thread, which, compared with Example 6, applies environmental control to the container, specifically:

[0140] The air pressure is adjusted every 4 hours, and the pressure change is 100kPa→90kPa→80kPa.

[0141] The humidity showed a uniform decrease: from an initial 85% RH (humidity) it decreased linearly to 35% RH.

[0142] The preparation method of UiO-66-NH2MOF in the present invention is:

[0143] Dissolve ZrCl₄ and 2-aminoterephthalic acid (H₂BDC-NH₂) in DMF at a 1:1 molar ratio. Add a small amount of acetic acid or hydrochloric acid as a modifier and transfer to an autoclave. React at 120–150°C for 24–48 hours. After the reaction, cool to room temperature and collect the crystals by centrifugation. Wash several times with DMF and ethanol and dry under vacuum to obtain a white powder.

[0144] In order to demonstrate that the embodiments of the present invention have substantial progress over the prior art, the following experiments were conducted:

[0145] Experiment 1

[0146] Design comparison example:

[0147] The difference between Comparative Example 1 and Example 5 is that the inner wall is smooth, no static magnetic field is applied, and glutaraldehyde, graphene quantum dots and UiO-66-NH2MOF are not added to the ethanol solution.

[0148] The difference between Comparative Example 2 and Example 5 is that:

[0149] The ethanol solution in this embodiment contains only 0.4% by mass concentration of glutaraldehyde;

[0150] Comparative Example 3 differs from Example 5 in that glutaraldehyde, graphene quantum dots, and UiO-66-NH2MOF are not added to the ethanol solution;

[0151] The difference between Comparative Example 4 and Example 5 is that: the inner wall is smooth;

[0152] Comparative Example 5 differs from Example 5 in that: no static magnetic field is applied;

[0153] Comparative Example 6 differs from Example 5 in that: UiO-66-NH2MOF is not applied;

[0154] The difference between Comparative Example 7 and Example 5 is that no graphene quantum dots were added to the ethanol solution;

[0155] The difference between Comparative Example 8 and Example 5 is that glutaraldehyde is not added to the ethanol solution.

[0156] The difference between Comparative Example 9 and Example 5 is that the rectangular container is replaced by a circular container with a diameter of 30 mm.

[0157] The difference between Comparative Example 10 and Example 5 is that UiO-66-NH2MOF and graphene quantum dots are not added to the ethanol solution.

[0158] The difference between Comparative Example 11 and Example 5 is that UiO-66-NH2MOF and glutaraldehyde are not added to the ethanol solution.

[0159] The difference between Comparative Example 12 and Example 5 is that no graphene quantum dots and glutaraldehyde are added to the ethanol solution.

[0160] The difference between Comparative Example 13 and Example 5 is that the size of the graphene quantum dots is 12 nm.

[0161] The difference between Comparative Example 14 and Example 5 is that the median height of the protrusion is 60 μm.

[0162] The collagen yarns prepared in Examples 4-7 and Comparative Examples 1-13 were tested for axial / radial modulus, fiber orientation (X-ray diffraction half-peak width), and breaking strength. The specific testing methods are as follows:

[0163] 1. Axial / radial modulus ratio determination method

[0164] Tested by dynamic mechanical analyzer (DMA): Standard samples (length 20 mm, diameter 0.5 mm) were cut from collagen threads in axial and radial directions, respectively. The storage modulus (E') was recorded at 37 °C, simulating physiological environment, with 1 Hz frequency and 0.1% strain. The ratio of axial modulus (E 轴向 ) to radial modulus (E 径向 ) was the modulus ratio. Each group was repeated 5 times, and the average value was taken.

[0165] 2. Fiber orientation degree (X-ray diffraction half-peak width) determination method

[0166] Using wide-angle X-ray diffractometer (WAXD): The collagen thread sample was placed on the test table, and Cu-Ka ray (λ = 0.154 nm) was used, with a scanning range of 5°-40° (2θ) and a step size of 0.02°. The fiber orientation degree was evaluated by analyzing the half-height width (FWHM) of the (002) crystal plane diffraction peak. The smaller the half-peak width, the more consistent the orientation (e.g., 18° corresponds to a highly oriented arrangement).

[0167] 3. Breaking strength determination method

[0168] Using a universal material testing machine (ASTM D3822 standard): The collagen thread (length 50 mm) was clamped at both ends and stretched to break at a speed of 10 mm / min, and the maximum load (F max ) was recorded. The breaking strength (σ = F max / cross-sectional area) was calibrated by a micrometer caliper to measure the sample diameter (accuracy ± 1 μm), and each group was tested with ≥10 samples.

[0169] 4. Antibacterial rate (Staphylococcus aureus inhibition rate) determination method

[0170] According to the ISO 20743 standard: The collagen thread (1 x 1 cm 2 ) was co-cultured with 10 6 CFU / mL of S. aureus suspension for 24 h (37 °C), and the plate counting method was used to calculate the survival rate of bacteria. The antibacterial rate = (1 - experimental group bacteria number / control group bacteria number) x 100%.

[0171] 5. Free radical scavenging rate (DPPH method) determination method

[0172] The collagen thread extract was reacted with 0.1 mM DPPH ethanol solution (1:1 volume ratio) in the dark for 30 min, and the absorbance at 517 nm (A 样品 , A 对照Clearance = (1 - Asample / Acontrol) x 100%.

[0173] 6. Fiber micro-morphology (SEM observation)

[0174] After gold-spraying treatment, the surface groove structure and fiber arrangement were observed by scanning electron microscope (SEM, 20 kV). The angle between groove direction and axis was measured by ImageJ software (≥100 data points for mean value).

[0175] 7. Thermal denaturation temperature (DSC measurement)

[0176] Differential scanning calorimeter (DSC) was used to scan 5 mg sample at 5℃ / min rate from 30 to 120℃, and the endothermic peak temperature (T d ) was recorded, reflecting the stability of collagen triple helix structure.

[0177] 8. Quantum dot distribution uniformity (CV value calculation)

[0178] The distribution image of GQDs in collagen thread was taken by confocal fluorescence microscope, and the standard deviation (SD) and mean value (Mean) of fluorescence intensity were analyzed by ImageJ, and the coefficient of variation (CV = SD / Mean x 100%) was calculated.

[0179] The results are shown in Table 1 and Table 2.

[0180] Table 1

[0181]

[0182] Table 2

[0183]

[0184]

[0185] From the data in Table 1 and Table 2, we can get:

[0186] The comparison of Comparative Example 9 and Example 5 shows that the modulus ratio increases from 2.8:1 to 6.2:1 and the fiber orientation degree increases from 38° to 18° by rectangular shape compared with circular shape, which proves the decisive role of rectangular boundary constraint on fiber directional arrangement. The rectangular aspect ratio ≥2:1 breaks the random arrangement of collagen fibers by asymmetric stress field. The long side of the rectangular shape forms a unidirectional tensile stress, forcing the fibers to arrange along the axial direction, while the circular mold causes the fibers to crosslink radially due to symmetric contraction (Comparative Example 9 fiber mesh interweaving). This effect provides a basis for subsequent microstructure orientation.

[0187] Comparison of Comparative Example 4 and Example 5 shows that the protrusion array increases the modulus ratio from 3.2:1 to 5.8:1, and the protrusions guide the fiber self-assembly through the microfluidic field. In Comparative Example 4, the modulus ratio dropped by 48% after the protrusion array was removed, the antibacterial rate plummeted by 62%, and no grooves were formed. This is because the combination of a protrusion height of 20 to 50 μm and a spacing of 100 to 200 μm produces a critical capillary force gradient (the ethanol solution accelerates the infiltration of the low curvature interface at the protrusion tip), causing the fiber to slide into the gap.

[0188] In Comparative Example 14 (protrusion height 60 μm), the bacterial physical capture rate decreased due to the expansion of the groove width to 2.8 μm. Although the chemical antibacterial components were retained, the MOF / GQDs were unevenly distributed within the wide grooves. This result demonstrates that when the protrusion height is greater than 50 μm, even if the chemical components are intact, it is still impossible to achieve an antibacterial efficiency of ≥ 95%.

[0189] Comparison between Comparative Example 8 and Example 5 shows that: glutaraldehyde is added to Example 5, and the breaking strength is increased by 45%, which proves that glutaraldehyde forms a covalent cross-linked network with collagen lysine residues, and each collagen molecule has ≥3 cross-linking points. The antibacterial rate of Comparative Example 8 is only 52%, while that of Example 5 is 99.5%, because the aldehyde group of glutaraldehyde irreversibly binds to bacterial proteins. The groove width of Comparative Example 8 is increased to 3.5μm, while the groove depth of Example 5 with the addition of glutaraldehyde is stabilized at 12±1μm. The free radical scavenging rate of Comparative Example 8 is only 60%, which is because the activity of GQDs is limited in the absence of glutaraldehyde. The free radical scavenging rate of Example 5 reaches 90%, indicating that glutaraldehyde enhances the electron transfer ability of GQDs by stabilizing the carboxyl groups (-COOH) on the surface. When not cross-linked (Comparative Example 8), the fiber is easy to relax (groove angle 20°), and after cross-linking (Example 5), it is fixed to ≤10°.

[0190] From the comparison between Comparative Example 7 and Example 5, it can be seen that graphene quantum dots can increase the free radical scavenging rate of collagen lines from 20% to 90%. This is because graphene quantum dots are embedded in the gaps of the collagen triple helix through π-π stacking.

[0191] Comparison of Comparative Example 8 and Comparative Example 2 shows that: Comparative Example 8 (adding graphene quantum dots and UiO-66-NH2MOF) did not add glutaraldehyde, while Comparative Example 2 only added glutaraldehyde, and the breaking strength increased from 90 MPa to 130 MPa; the antibacterial rate increased from 52% to 75%.

[0192] In Comparative Example 6, where only MOF was missing, the breaking strength was 120 MPa. In Comparative Example 8, where only glutaraldehyde was missing, the breaking strength was 120 MPa. However, in Comparative Example 11, where both MOF and glutaraldehyde were missing, the breaking strength dropped sharply to 90 MPa. The breaking strength of Example 5 reached 180 MPa, indicating that MOF requires glutaraldehyde pre-crosslinking to achieve nano-enhancement.

[0193] Comparative Example 5, Comparative Examples 6, 7, and 10 show that:

[0194] Example 5 (GQDs+MOF) has a breaking strength of 180 MPa, which is 50% higher than that of Comparative Example 6 (without MOF, 120 MPa), 64% higher than that of Comparative Example 7 (without GQDs, 110 MPa), but 80% higher than that of Comparative Example 10 (without MOF and GQDs, 100 MPa). This shows that there is a synergistic effect between GQDs and MOF.

[0195] Compared with Example 5, Comparative Example 13 showed a breaking strength of only 100 MPa, a decrease of 44%, and an antibacterial rate of 30%, a decrease of 69%. No grooves were formed. This confirms that GQDs of 3-8 nm can embed into the gaps between collagen triple helices, enhancing interfacial bonding through π-π stacking, while GQDs larger than 10 nm accumulate on the fiber surface due to size exclusion, blocking fiber self-assembly.

[0196] Collagen fiber orientation specifically refers to the degree of parallel alignment of macromolecular chains or fiber bundles within collagen fibers relative to the fiber axis. A high degree of orientation means that the molecular chains or fiber bundles are more regularly aligned along the axial direction, significantly affecting the mechanical properties (such as tensile strength and modulus) and functional characteristics of the material.

[0197] Furthermore, the collagen fiber orientation of Examples 5-7 of the present invention was measured to be ≥90%. Fiber orientation is a quantitative indicator of the parallel arrangement of collagen fibers. X-ray diffraction results showed that the collagen fiber orientation of Example 5 was 92%, compared to 42% for Comparative Example 9, 38% for Comparative Example 4, and 68% for Comparative Example 14.

[0198] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing collagen thread, characterized in that: It includes the following steps: S1: Place the collagen tissue in a non-circular container, soak it in ethanol solution, and then dry it; The inner wall of the container S2 is provided with a microstructure for guiding the collagen fibers to align in a directional manner, so that the collagen fibers are oriented in a directional manner during the soaking process; S3 drying process to obtain collagen lines.

2. The method for preparing the collagen thread according to claim 1, wherein: The non-circular container is a rectangular container with a length-to-width ratio of ≥2:

1.

3. The method for preparing the collagen thread according to claim 1, wherein: The microstructure includes a protrusion array and a groove array. The median height of the protrusions or grooves is 20-50 μm. The distance between two protrusions in the protrusion array or the distance between two grooves in the groove array is 100-200 μm.

4. The method for preparing the collagen thread according to claim 1, wherein: The ethanol solution contains a bioactive additive, and the bioactive additive is selected from at least one of a cross-linking agent, a growth factor, a nanomaterial, and a metal organic framework material.

5. The method for preparing the collagen thread according to claim 4, wherein: The nanomaterial is graphene quantum dots.

6. The method for preparing the collagen thread according to claim 1, wherein: A physical field treatment is applied during the soaking process, wherein the physical field includes at least one of a magnetic field, an ultrasonic field, and a temperature gradient field.

7. The collagen thread according to claim 1, wherein: The microstructure of the inner wall of the container guides the collagen fibers to align in a directional manner, forming fiber bundle gaps with a width of 0.5 to 3 μm, which are transformed into surface directional grooves after cross-linking and drying.

8. A collagen thread prepared by the preparation method according to any one of claims 1 to 7, characterized in that: It meets at least one of the following conditions: The minimum curvature radius in dry state is ≥30mm, and the breaking strength is ≥1.5MPa; The ratio of axial tensile modulus to radial tensile modulus is ≥3:1; The surface has grooves with a depth of 0.5 to 3 μm, a groove depth of 5 to 15 μm, and an angle of ≤15° between the groove direction and the axial direction of the collagen line; The collagen wires are loaded with graphene quantum dots.

9. Use of the collagen thread as claimed in claim 7 in a medical suturing device.

10. A device for implementing the method according to claim 1, characterized in that: It includes a non-circular container having microstructures on its inner wall; a multi-physics field coupling generator, comprising at least one of a magnetic field generator, an ultrasonic generator, and a temperature gradient controller, for applying a physical field to the non-circular container; The dynamic environmental control system includes a humidity sensor, a temperature sensor, an air pressure sensor, and corresponding adjustment actuators, which are used to adjust at least one parameter of the humidity, temperature, and air pressure inside the non-circular container.

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