High-orientation collagen fiber as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to a high-orientation collagen fiber as well as a preparation method and application thereof. The preparation method of the high-orientation collagen fiber comprises the following steps: injecting a collagen solution into a buffer solution through an injection needle, and depositing in the buffer solution to form the collagen fiber; the inner diameter D of the injection needle head is 0.05 to 4.0 mm, and the length L of the injection needle head is 1.5 to 15 cm; and the extrusion speed V1 of the injection needle is 5-500 cm / min. According to the method, the injection needle is adopted to extrude the collagen solution, and in the process that the collagen solution passes through the injection needle, pre-collagen molecules can be oriented under the action of extrusion of the inner wall of the injection needle, pressure in the injection direction and the like, so that high-orientation collagen fibers are formed when the pre-collagen molecules enter a buffer solution to be self-assembled; and the preparation method is simple and convenient to operate and high in production efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a highly oriented collagen fiber and its preparation method and application. Background Art

[0002] Collagen is the most abundant and widely distributed protein in mammals, accounting for about 25% - 30% of the total. It is formed by three α - peptide chains with approximately equal molecular weights coiled around each other into a right - hand helix, about 300 nm in length and about 1.0 - 1.5 nm in diameter, and each α - peptide chain contains about 1050 amino acid residues. Due to its good biocompatibility, biodegradability and wide source channels, collagen has been widely used in the fields of food, cosmetics and medicine.

[0003] In the human body, collagen in tendons, ligaments and corneas aggregates together to form large collagen fibers. In tendons, collagen fibers are highly oriented along the long axis of the tendon. This arrangement enables tendons to efficiently transmit the force generated by muscles to bones, thus realizing limb movement. Collagen fibers in ligaments are highly oriented, connecting adjacent bones together and playing a role in stabilizing joints. Collagen fibers in the cornea are mainly type I collagen, and these fibers are arranged in layers. The fibers in each layer are parallel to each other and parallel to the corneal surface, showing a high degree of orientation. Highly oriented collagen fibers have strong mechanical properties. The higher the orientation, the better the tensile properties. Therefore, collagen fiber materials with high orientation are good materials for repairing tissues such as tendons, ligaments and corneas. However, during the extraction and preparation of collagen, the highly oriented aggregated collagen fibers are dissolved into procollagen in an acid solution, destroying the orientation of the collagen fibers. However, due to the properties of collagen and the influence of its low thermal denaturation temperature on its processability, it is difficult to prepare non - oriented collagen into highly oriented collagen fibers.

[0004] Currently, the main methods for processing collagen into highly oriented collagen fibers include electrospinning, electrochemical orientation, twin - screw extrusion, etc. However, in the electrospinning process, solvents such as hexafluoroisopropanol or tetrafluoroisopropanol are usually used. These solvents are highly toxic, expensive, and may also cause the destruction of the triple - helix structure of collagen, resulting in the loss of some biological activities. Electrochemical orientation is that collagen molecules aggregate to form fibers at the isoelectric point in a collagen solution under the action of an electrostatic field. However, this method has low preparation efficiency and is not suitable for industrial mass production. The thermal denaturation temperature of collagen is 42 ± 2 °C. During twin - screw extrusion, a large amount of heat is generated, leading to the denaturation of collagen.

[0005] In view of this, the present invention is specifically proposed. SUMMARY OF THE INVENTION

[0006] The object of the present invention is to provide highly oriented collagen fibers, a preparation method thereof and an application thereof. Collagen is introduced by means of an injection needle, and self-assembled to form highly oriented collagen fibers, while ensuring or even improving the mechanical properties of the collagen fibers.

[0007] The first aspect of the present invention provides a method for preparing highly oriented collagen fibers, comprising the following steps: injecting a collagen solution into a deposition solution through an injection needle, and self-assembling and depositing in the deposition solution to form highly oriented collagen fibers;

[0008] The inner diameter D of the injection needle is 0.083 - 3.810 mm, and the length L of the injection needle is 1.5 - 15 cm; the extrusion speed V1 of the injection needle is 5 - 500 cm / min.

[0009] The inner diameter D, the length L and the extrusion speed V1 satisfy

[0010] wherein, the unit of D is mm, the unit of L is cm, and the unit of V1 is cm / min.

[0011] In a specific embodiment of the present invention, the deposition solution is flowing relative to the injection needle. Further, the flow speed V2 of the deposition solution is 5 - 500 cm / min.

[0012] In a specific embodiment of the present invention, the flow direction of the deposition solution is the same as the extrusion direction of the injection needle. Further, the ratio V1 / V2 of the extrusion speed V1 to the flow speed V2 is 0.8 - 1.2.

[0013] In a specific embodiment of the present invention, the collagen solution is mainly prepared from collagen and an acid solution. Further, in the collagen solution, the concentration W / V of collagen is 0.1% - 20%.

[0014] In a specific embodiment of the present invention, the pH of the acid solution is 0.5 - 5.

[0015] In a specific embodiment of the present invention, the collagen solution further comprises a photo-crosslinking agent. Further, the concentration W / V of the photo-crosslinking agent is 0.005% - 5%.

[0016] In a specific embodiment of the present invention, the photo-crosslinking agent comprises at least one of riboflavin, sodium riboflavin phosphate, camphorquinone, curcumin and eosin Y.

[0017] In a specific embodiment of the present invention, when the collagen solution contains a photo-crosslinking agent, the preparation method further includes: performing a light treatment on the collagen fibers.

[0018] In a specific embodiment of the present invention, the pH of the deposition solution is 6-9. Further, the concentration of the deposition solution is 1X-10X.

[0019] In a specific embodiment of the present invention, the deposition solution includes at least one of PBS buffer solution, DPBS buffer solution, TBE buffer solution, Tris-HCl buffer solution, HEPES buffer solution, PIPEs buffer solution, MOPS buffer solution, TEA buffer solution, Tricine buffer solution, sodium carbonate-sodium bicarbonate buffer solution, and NaCl solution.

[0020] In a specific embodiment of the present invention, the temperature of the deposition solution is 4-40 °C.

[0021] In a specific embodiment of the present invention, the preparation method further includes: collecting the collagen fibers and performing a modification treatment; the modification treatment includes at least one of the following:

[0022] (1) Crosslinking the collagen fibers in a solution containing a chemical crosslinking agent;

[0023] (2) Enzymatically crosslinking the collagen fibers in a solution containing an enzyme;

[0024] (3) Performing a thermal crosslinking treatment on the collagen fibers.

[0025] In a specific embodiment of the present invention, in the solution containing a chemical crosslinking agent, the concentration of the chemical crosslinking agent W / V is 0.02%-3%. Further, the chemical crosslinking agent includes at least one of glutaraldehyde, formaldehyde, EDC-NHS, genipin, catechol, and maleimide-based crosslinking agents.

[0026] In a specific embodiment of the present invention, in the solution containing an enzyme, the concentration of the enzyme W / V is 0.1%-2%. Further, the enzyme includes at least one of transglutaminase, lysyl oxidase, and horseradish peroxidase.

[0027] In a specific embodiment of the present invention, in the thermal crosslinking treatment, the temperature of the collagen fibers in a vacuum state is 100-140 °C, and the treatment time is 12-96 h.

[0028] The second aspect of the present invention provides a highly oriented collagen fiber prepared by the preparation method of the first aspect.

[0029] The third aspect of the present invention provides an application of the highly oriented collagen fibers of the second aspect in the preparation of tendon, ligament, cornea and / or blood vessel repair products.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) In the present invention, a collagen solution is extruded by an injection needle. During the process of the collagen solution passing through the injection needle, procollagen molecules will be oriented under the action of shear force, and highly oriented collagen fibers are formed during self-assembly in the deposition solution; moreover, the preparation method of the present invention is simple to operate and has high production efficiency.

[0032] (2) The present invention can further regulate the inner diameter, length, extrusion speed, etc. of the injection needle to adjust the orientation of procollagen forming collagen fibers, so as to balance the improvement of the orientation and mechanical properties of collagen fibers.

[0033] (3) The collagen fibers of the present invention have high orientation and can be used in the preparation of products such as tendons, ligaments, surgical sutures, beauty lifting threads, corneas and / or blood vessel repairs. Description of the Drawings

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 It is a photograph of the highly oriented collagen fibers prepared in Example 4 of the present invention;

[0036] Figure 2 It is the test results of the mechanical properties of the collagen fibers prepared in different embodiments of the present invention. Detailed Embodiments

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the drawings and specific embodiments. However, those skilled in the art will understand that the following described embodiments are some embodiments of the present invention, not all embodiments, and are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. Those not specified in the embodiments are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0038] Currently, the main methods for preparing collagen fibers include electrospinning, electrochemical orientation, twin-screw extrusion, etc. Since collagen is almost insoluble in neutral water and most organic solvents, and can only be dissolved in acidic aqueous solutions and most organic solvents, when using these solvents for electrospinning of collagen, the fiber formation effect is poor, and usually it is necessary to blend with other polymer materials to form fibers with good morphology. During the blending process, hexafluoroisopropanol or tetrafluoroisopropanol is usually used as a solvent, which is highly toxic, expensive, and may also cause the destruction of the triple helix structure of collagen, resulting in the loss of some biological activities. Limited by the equipment and preparation efficiency of electrochemical orientation, the method of preparing collagen fibers by electrochemical orientation is currently not suitable for industrial mass production. The twin-screw extrusion method also has limitations for collagen with a low thermal denaturation temperature. To solve these problems, the present invention realizes the preparation of highly oriented collagen fibers by using an injection needle in an aqueous phase system, avoiding costly and toxic organic solvents, and avoiding high-temperature steps. At the same time, the operation is simple and suitable for industrial mass production.

[0039] Based on this, in the first aspect of the present invention, a method for preparing highly oriented collagen fibers is provided, including the following steps: injecting a collagen solution into a deposition solution through an injection needle, and self-assembling and depositing in the deposition solution to form highly oriented collagen fibers;

[0040] The inner diameter D of the injection needle is 0.083 - 3.810 mm, and the length L of the injection needle is 1.5 - 15 cm; the extrusion speed V1 of the injection needle is 5 - 500 cm / min;

[0041] The inner diameter D, length L, and extrusion speed V1 satisfy:

[0042]

[0043] In the preparation method of the present invention, an injection needle is used to inject a collagen solution into a deposition solution, and the shear force during the injection process of the collagen solution aligns the procollagen molecules in the solution, and highly oriented collagen fibers are formed when the procollagen molecules self-assemble after entering the deposition solution.

[0044] The present invention regulates the inner diameter, length, and corresponding extrusion speed of the injection needle within the above ranges to ensure the high orientation of collagen fibers, ensure the mechanical properties of collagen fibers, etc., and improve production efficiency. For example, in different embodiments, the inner diameter D of the injection needle can be any range formed by two of 0.083 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 3.810 mm, for example, 0.1 - 1.8 mm; the length L of the injection needle can be any range formed by two of 1.5 cm, 2 cm, 4 cm, 5 cm, 8 cm, 10 cm, 15 cm, for example, 2 - 8 cm; the extrusion speed V1 of the injection needle can be any range formed by two of 5 cm / min, 10 cm / min, 20 cm / min, 40 cm / min, 60 cm / min, 80 cm / min, 100 cm / min, 150 cm / min, 200 cm / min, 250 cm / min, 300 cm / min, 500 cm / min, for example, 20 - 200 cm / min.

[0045] In actual operation, the collagen solution can be filled into a syringe. The syringe is connected to an injection needle and an injection pump, and the extrusion speed of the injection needle is regulated by the injection pump. Here, the extrusion speed refers to the distance that the collagen solution advances in the injection needle per minute when flowing through the injection needle.

[0046] The inner diameter D, length L, and extrusion speed V1 satisfy:

[0047] , for example, it can be 2, 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or any range formed by two of them, such as 2 - 10, 10 - 30, 30 - 50, 50 - 70, 70 - 90, 90 - 100, etc. The present invention has found through research that regulating within the above ranges helps to improve the orientation, fiber diameter, and mechanical properties of collagen fibers at the microscopic level. It should be noted that when calculating the results corresponding to the above relationships, the units represented by D, L, and V1 are not considered, and only the numerical values corresponding to when the unit of D is mm, the unit of L is cm, and the unit of V1 is cm / min are used to calculate the results corresponding to the relevant relationships.

[0048] In a specific embodiment of the present invention, the deposition liquid is flowing relative to the injection needle. Further, the flow rate V2 of the deposition liquid is 5 to 500 cm / min, for example, it can be 5 cm / min, 10 cm / min, 15 cm / min, 20 cm / min, 25 cm / min, 30 cm / min, 35 cm / min, 40 cm / min, 45 cm / min, 50 cm / min, 60 cm / min, 80 cm / min, 100 cm / min, 120 cm / min, 150 cm / min, 200 cm / min, 250 cm / min, 300 cm / min, 350 cm / min, 400 cm / min, 450 cm / min, 500 cm / min or a range composed of any two of them. Injecting the collagen solution into the flowing deposition liquid can avoid the aggregation of collagen fibers at the needle tip. Further regulating the flow rate within the above range helps to ensure that the collagen solution forms linear collagen fibers by self-assembly during the flow process of the deposition liquid as it is injected into the deposition liquid, which is more conducive to subsequent collection and processing, etc. In actual operation, the flowing deposition liquid can be achieved and regulated by a circulation pump, but it is not limited thereto.

[0049] In a specific embodiment of the present invention, the flow direction of the deposition liquid is the same as the extrusion direction of the injection needle. Further, the ratio V1 / V2 of the extrusion speed V1 to the flow rate V2 is 0.8 to 1.2, for example, it can be 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2 or a range composed of any two of them. This can avoid the situation where the extruded collagen solution is not carried away by the deposition liquid and accumulates at the outlet due to the deposition liquid flow rate being too slow compared to the extrusion speed, and the situation where the deposition liquid flow rate is too fast and pulls the extruded collagen solution, resulting in the breakage of the formed collagen fibers, thereby ensuring the continuity and stability of the formation of linear collagen fibers.

[0050] In a specific embodiment of the present invention, the collagen solution is mainly prepared from collagen and an acid solution. Further, in the collagen solution, the concentration W / V of collagen is 0.1% to 20%, for example, it can be 0.1%, 1%, 3%, 5%, 8%, 10%, 12%, 15%, 18%, 20% or a range composed of any two of them, and preferably 1.0% to 8%.

[0051] It is understood that the collagen solution referred to in the present invention does not limit the form of collagen in the solution, but refers to a solution made of collagen. In the collagen solution of the present invention, collagen mainly exists in the form of procollagen molecules. Among them, the collagen can be a collagen material with a complete triple helix structure extracted from animal tissues, and the animal tissues include but are not limited to pig skin, cowhide, pig bone, cow bone, mouse tail, pig articular cartilage, cow articular cartilage, fish skin, etc. Further, the concentration W / V involved in the present invention refers to the ratio of the mass of collagen in the solution to the volume of the solvent.

[0052] In a specific embodiment of the present invention, the temperature of the collagen solution is regulated to 1-8 °C, such as 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C or the range composed of any two of them. It has been found that at a lower temperature, the fluidity of the collagen solution is better, which is more conducive to injection, and the self-assembly ratio of procollagen molecules in the solution is lower at a lower temperature. When it enters the deposition solution, with the change of the environment, it has a faster self-assembly speed in the deposition solution, ensuring high orientation.

[0053] In a specific embodiment of the present invention, the pH of the acid solution is 0.5-5, preferably 2-4. Further, in the acid solution, the acid includes at least one of hydrochloric acid, phosphoric acid, sulfuric acid, citric acid and acetic acid.

[0054] In a specific embodiment of the present invention, the collagen solution further includes a photo-crosslinking agent. Further, the concentration W / V of the photo-crosslinking agent is 0.005%-5%, for example, it can be 0.005%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5% or the range composed of any two of them. By further adding a photo-crosslinking agent to the collagen solution, after self-assembly to form collagen fibers, light treatment can be carried out to improve the crosslinking degree of the collagen fibers, and further improve the mechanical properties and stability of the collagen fibers.

[0055] In a specific embodiment of the present invention, the photo-crosslinking agent includes at least one of riboflavin, sodium riboflavin phosphate, camphorquinone, curcumin and eosin Y.

[0056] In a specific embodiment of the present invention, when the collagen solution includes a photo-crosslinking agent, the preparation method further includes: performing light treatment on the collagen fibers. Further, the light treatment includes: a wavelength of 300-600 nm, a light intensity of 100-10000 mW / cm 2 , and a light irradiation time of 3-300 min.

[0057] In actual operation, when the collagen solution contains a photo-crosslinking agent, after light treatment, it can be treated by conventional freeze-drying, vacuum drying, supercritical carbon dioxide drying or air drying, etc.

[0058] In a specific embodiment of the present invention, the pH of the deposition solution is 6 to 9. Further, the concentration of the deposition solution is 1X to 10X.

[0059] In a specific embodiment of the present invention, the deposition solution includes at least one of PBS buffer (phosphate buffer), DPBS buffer (phosphate buffer without calcium and magnesium ions), TBE buffer (tris(hydroxymethyl)aminomethane-borate buffer), Tris-HCl buffer (tris(hydroxymethyl)aminomethane-hydrochloric acid buffer), HEPES buffer (2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid buffer), PIPEs buffer (piperazine-1,4-bis(2-ethanesulfonic acid) buffer), MOPS buffer (3-(N-morpholino)propanesulfonic acid buffer), TEA buffer (triethanolamine buffer), Tricine buffer (tris(hydroxymethyl)methylglycine buffer), sodium carbonate-sodium bicarbonate buffer, and NaCl solution, wherein the concentration of the NaCl solution can be 2 to 4M.

[0060] The collagen solution is in an acidic condition. After being injected into the deposition solution, the pH and ionic strength of the deposition solution change significantly compared with the collagen solution, and the collagen solution self-assembles into collagen fibers in the deposition solution.

[0061] In a specific embodiment of the present invention, the temperature of the deposition solution is 25°C to 40°C, for example, it can be 25°C, 30°C, 35°C, 40°C, or a range composed of any two of them. The rate of self-assembly of the collagen solution into collagen fibers in the deposition solution is adjusted by the temperature of the deposition solution.

[0062] In a specific embodiment of the present invention, the preparation method further includes: collecting the collagen fibers and performing a modification treatment; the modification treatment includes at least one of the following:

[0063] (1) Crosslinking the collagen fibers in a solution containing a chemical crosslinking agent;

[0064] (2) Enzymatically crosslinking the collagen fibers in a solution containing an enzyme;

[0065] (3) Thermally crosslinking the collagen fibers.

[0066] The above modification treatment is to perform chemical crosslinking, thermal crosslinking, or enzymatic crosslinking on the obtained highly oriented collagen fibers. Through the above modification treatment, the mechanical properties of the collagen fibers can be further improved. Specifically, one of the above three modification treatment methods can be used, or multiple modification treatments can be performed. When multiple modification treatments are used, the order of the various modification treatments is not limited.

[0067] In a specific embodiment of the present invention, in the solution containing a chemical crosslinking agent, the concentration of the chemical crosslinking agent W / V is 0.02% to 3%, for example, it can be 0.02%, 0.1%, 0.5%, 1%, 2%, 3% or the range composed of any two of them.

[0068] In a specific embodiment of the present invention, the chemical crosslinking agent includes at least one of glutaraldehyde, formaldehyde, EDC-NHS, genipin, catechol, and maleimide-based crosslinking agents. Among them, the solvent used in the solution containing the chemical crosslinking agent may include but is not limited to at least one of the above deposition solutions for depositing collagen fibers.

[0069] In a specific embodiment of the present invention, the crosslinking treatment in the solution containing the chemical crosslinking agent includes soaking the collagen fibers in the solution containing the chemical crosslinking agent, and the soaking time is 1 min to 24 h, for example, it can be 1 min, 20 min, 40 min, 1 h, 8 h, 12 h, 20 h, 24 h or the range composed of any two of them.

[0070] In a specific embodiment of the present invention, in the solution containing an enzyme, the concentration of the enzyme W / V is 0.1% to 2%, for example, it can be 0.1%, 0.5%, 0.8%, 1%, 1.5%, 2% or the range composed of any two of them. Further, the enzyme includes at least one of transglutaminase, lysyl oxidase, and horseradish peroxidase. Among them, the solvent used in the solution containing the enzyme may include but is not limited to water.

[0071] In a specific embodiment of the present invention, in the enzyme crosslinking treatment, the pH of the solution is 6 to 8, the temperature is 30 to 40 °C, and the time is 1 to 48 h, for example, it can be 1 h, 4 h, 8 h, 12 h, 16 h, 24 h, 36 h, 48 h or the range composed of any two of them.

[0072] In a specific embodiment of the present invention, in the thermal crosslinking treatment, the temperature is 100 to 140 °C under a vacuum state, for example, it can be 100 °C, 110 °C, 120 °C, 130 °C, 140 °C or the range composed of any two of them; the time is 12 to 96 h, for example, it can be 12 h, 24 h, 36 h, 48 h, 72 h, 96 h or the range composed of any two of them. In actual operation, before the thermal crosslinking treatment, the collagen fibers are pre-treated by conventional drying to remove the moisture in the collagen fibers, and then placed in a vacuum environment for the above thermal crosslinking treatment.

[0073] After the above modification treatment, the collagen fibers can be conventionally dried according to actual needs, including but not limited to freeze-drying, vacuum drying, supercritical carbon dioxide drying, or air-drying, etc., and then can be collected by means such as drum collection.

[0074] The second aspect of the present invention provides a highly oriented collagen fiber prepared by the preparation method of the first aspect.

[0075] The collagen fiber of the present invention not only has high orientation but also has excellent mechanical properties.

[0076] The third aspect of the present invention provides an application of the highly oriented collagen fiber of the second aspect in the preparation of tendon, ligament, corneal and / or blood vessel repair products.

[0077] In actual operation, the prepared highly oriented collagen fiber can be processed into a collagen medical surgical suture, or woven into a collagen tendon and ligament repair bundle, or woven into a tubular collagen tube for blood vessel transplantation, or woven into a collagen tissue patch, a lifting thread for medical aesthetics, etc.

[0078] Example 1

[0079] This example provides a preparation method of collagen fiber, which includes the following steps:

[0080] (1) Dissolve the collagen with the triple helix structure intact in a phosphoric acid aqueous solution with pH = 2 at 4°C to make the collagen concentration 3.5% (W / V), and add riboflavin thereto to make its concentration in the solution 0.025% (W / V) to obtain a collagen solution.

[0081] (2) Load the collagen solution prepared in step (1) into an injection pump and connect an injection needle, and then slowly inject the collagen solution at 4°C into the flowing 10X PBS buffer at 40°C. When the injected collagen solution enters the buffer, it quickly self-assembles to form collagen fibers and flows along with the buffer;

[0082] Among them, the inner diameter of the injection needle is 0.337 mm (23G needle specification), the needle length is 5 cm, the pushing speed of the injection pump is adjusted so that the flow rate of the collagen solution in the injection needle is 100 cm / min; the flow rate of the 10X PBS buffer relative to the injection needle is 100 cm / min, and the flow direction is the same as the extrusion and injection direction of the collagen solution.

[0083] (3) Collect the collagen fibers in step (2), lay them flat 10 cm below a blue light lamp (wavelength 450 nm), and irradiate them at an intensity of 500 mW / cm 2 for 1 h. Then collect the collagen fibers with a roller.

[0084] (4) Pack the collagen fibers collected in step (3) into an ethylene oxide sterilization bag, seal it, and place it in a supercritical carbon dioxide extraction kettle. Dry it using the supercritical carbon dioxide method to obtain dry collagen fibers. During supercritical carbon dioxide drying, control the pressure in the extraction kettle to be 35 MPa, the SCCO2 flow rate to be 3 L / min, and maintain the extraction time for 3 h.

[0085] Example 2

[0086] This example provides a method for preparing collagen fibers, which includes the following steps:

[0087] (1) Dissolve the collagen with a complete triple helix structure in an acetic acid aqueous solution with pH = 2.5 at 4°C to make the collagen concentration 5% (W / V) to obtain a collagen solution.

[0088] (2) Load the collagen solution prepared in step (1) into a syringe pump and connect it to a syringe needle. Then slowly inject the collagen solution at 4°C into the flowing 10X sodium carbonate-sodium bicarbonate buffer solution at 40°C. When the injected collagen solution enters the buffer solution, it quickly self-assembles to form collagen fibers and flows along with the buffer solution;

[0089] Among them, the inner diameter of the syringe needle is 0.838 mm (18G specification), the needle length is 5 cm, adjust the pushing speed of the syringe pump to make the flow rate of the collagen solution in the syringe needle 200 cm / min; the flow rate of the 10X sodium carbonate-sodium bicarbonate buffer solution relative to the syringe needle is 220 cm / min, and the flow direction is the same as the extrusion and injection direction of the collagen solution.

[0090] (3) Use a roller to collect the collagen fibers in step (3) and perform freeze-drying to obtain dry collagen fibers.

[0091] Example 3

[0092] This example provides a method for preparing collagen fibers, which includes the following steps:

[0093] (1) Dissolve the collagen with a complete triple helix structure in a hydrochloric acid aqueous solution with pH = 3 at 4°C to make the collagen concentration 3% (W / V) to obtain a collagen solution.

[0094] (2) Load the collagen solution prepared in step (1) into a syringe pump and connect it to a syringe needle. Then slowly inject the collagen solution at 4°C into the flowing 10X DPBS buffer solution at 40°C. When the injected collagen solution enters the buffer solution, it quickly self-assembles to form collagen fibers and flows along with the buffer solution;

[0095] Among them, the inner diameter of the injection needle is 0.413 mm (22G gauge), the needle length is 4 cm, the pushing speed of the regulating injection pump is adjusted to make the flow rate of the collagen solution in the injection needle 400 cm / min; the flow rate of the 10X DPBS buffer solution relative to the injection needle is 360 cm / min, and the flow direction is the same as the extrusion and injection direction of the collagen solution.

[0096] (3) Collect the collagen fibers in step (2), and immerse them in 10X PBS buffer solution containing 0.5% (W / V) glutaraldehyde at 37°C for 48 h.

[0097] (4) Use supercritical carbon dioxide to dry the collagen fibers in step (3) and remove the residual glutaraldehyde at the same time to obtain dry collagen fibers; in the supercritical carbon dioxide drying, control the extraction kettle pressure to be 35 MPa, the SCCO2 flow rate to be 5 L / min, and the extraction time to be maintained for 4 h.

[0098] Example 4

[0099] This example provides a method for preparing collagen fibers, which includes the following steps:

[0100] (1) Dissolve the collagen with a complete triple helix structure in a phosphoric acid aqueous solution with pH = 2.5 at 4°C to make the collagen concentration 1.5% (W / V) to obtain a collagen solution.

[0101] (2) Load the collagen solution prepared in step (1) into an injection pump and connect the injection needle, and then slowly inject the collagen solution at 4°C into the flowing 10X DPBS buffer solution at 40°C. When the injected collagen solution enters the buffer solution, it quickly self-assembles to form collagen fibers and flows with the buffer solution;

[0102] Among them, the inner diameter of the injection needle is 0.159 mm (30G gauge), the needle length is 1.5 cm, the pushing speed of the regulating injection pump is adjusted to make the flow rate of the collagen solution in the injection needle 150 cm / min; the flow rate of the 10X DPBS buffer solution relative to the injection needle is 150 cm / min, and the flow direction is the same as the extrusion and injection direction of the collagen solution.

[0103] (3) Collect the collagen fibers in step (2), place them in a vacuum box for drying at room temperature. After the collagen fibers are completely dried, heat-treat them at 115°C for 48 h under vacuum conditions to obtain collagen fibers.

[0104] Example 5

[0105] This example provides a method for preparing collagen fibers, which includes the following steps:

[0106] (1) Dissolve the collagen with a complete triple - helix structure in a phosphoric acid aqueous solution with pH = 2 at 4 °C to make the collagen concentration 1% (W / V), obtaining a collagen solution.

[0107] (2) Load the collagen solution prepared in step (1) into a syringe pump and connect an injection needle. Then slowly inject the 4 °C collagen solution into the flowing 10X DPBS buffer at 40 °C. When the injected collagen solution enters the buffer, it quickly self - assembles into collagen fibers and flows along with the buffer;

[0108] Among them, the inner diameter of the injection needle is 0.108 mm (32G specification), the needle length is 1.5 cm. Regulate the pushing speed of the syringe pump so that the flow rate of the collagen solution in the injection needle is 30 cm / min; the flow rate of the 10X DPBS buffer relative to the injection needle is 30 cm / min, and the flow direction is the same as the extrusion and injection direction of the collagen solution.

[0109] (3) Collect the collagen fibers in step (2), immerse them in an aqueous solution containing 0.5% (W / V) transglutaminase, and perform enzymatic cross - linking at 37 °C for 12 h.

[0110] (4) Wash the collagen fibers in step (3) with pure water, and then perform freeze - drying to obtain dry collagen fibers.

[0111] Example 6

[0112] This example refers to the preparation method of Example 3, with the only difference being that: in step (2), the size of the injection needle is different, and the injection speed of the collagen solution is different.

[0113] In this example, the inner diameter of the injection needle is 0.514 mm (21G specification), the needle length is 3 cm, and the injection speed of the collagen solution is 300 cm / min.

[0114] Example 7

[0115] This example refers to the preparation method of Example 3, with the only difference being that: in step (2), the size of the injection needle is different and the injection speed of the collagen solution is different.

[0116] In this example, the inner diameter of the injection needle is 0.686 mm (19G specification), the needle length is 3 cm, and the injection speed of collagen is 350 cm / min.

[0117] Example 8

[0118] This example refers to the preparation method of Example 5, with the only difference being that: in step (2), the injection speed of the collagen solution is different.

[0119] In this example, the flow rate of the collagen solution in the injection needle and the flow rate of the buffer solution are both 200 cm / min.

[0120] Example 9

[0121] This example refers to the preparation method of Example 3, with the only difference being that in step (2), the size of the injection needle is different.

[0122] In this example, the inner diameter of the injection needle is 0.260 mm (25G gauge), and the needle length is 5 cm.

[0123] Example 10

[0124] This example refers to the preparation method of Example 2, with the only difference being that in step (2), the size of the injection needle is different.

[0125] In this example, the inner diameter of the injection needle is 2.159 mm (12G gauge), and the needle length is 10 cm.

[0126] Comparative Example 1

[0127] Comparative Example 1 refers to the preparation method of Example 3, with the difference being that in step (2), the size of the injection needle is different.

[0128] In Comparative Example 1, the inner diameter of the injection needle is 3.429 mm (8G gauge), and the needle length is 5 cm; the flow rate of the collagen solution in the injection needle and the flow rate of the buffer solution are both 300 cm / min. When prepared with these parameters, the collagen cannot quickly self-assemble to form oriented gel fibers, but instead aggregates together to gradually form gel blocks.

[0129] Experimental Example

[0130] Figure 1 This is a photograph of the highly oriented collagen fibers prepared in Example 4 of the present invention. As can be seen from the figure, the prepared collagen fibers exist in a helical form in the deposition solution and can be straightened during subsequent collection or use.

[0131] Figure 2 This is the test result of the mechanical properties of the collagen fibers prepared in different examples of the present invention (each sample was randomly sampled and tested twice in parallel). Among them, the curves marked 1 and 2 in the figure are the mechanical tensile curves of the collagen fibers prepared in Example 1, the curves marked 3 and 4 are the mechanical tensile curves of the collagen fibers prepared in Example 4, the curves marked 5 and 6 are the mechanical tensile curves of the collagen fibers prepared in Example 5, the curves marked 7 and 10 are the mechanical tensile curves of the collagen fibers prepared in Example 8, and the curves marked 8 and 9 are the mechanical tensile curves of the collagen fibers prepared in Example 9.

[0132] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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 various embodiments of the present invention.

Claims

1. A method for preparing highly oriented collagen fibers, characterized in that: The method comprises the following steps: injecting a collagen solution into a deposition liquid through an injection needle, and self-assembling and depositing in the deposition liquid to form highly oriented collagen fibers; The inner diameter D of the injection needle is 0.083 to 3.810 mm, the length L of the injection needle is 1.5 to 15 cm; the extrusion speed V1 of the injection needle is 5 to 500 cm / min; The inner diameter D, the length L and the extrusion speed V1 satisfy ; Among them, the unit of D is mm, the unit of L is cm, and the unit of V1 is cm / min.

2. The preparation method according to claim 1, characterized in that: The deposition liquid is fluid relative to the injection needle; Preferably, the flow velocity V2 of the deposition liquid is 5 to 500 cm / min.

3. The preparation method according to claim 2, characterized in that: The flow direction of the deposition liquid is the same as the extrusion direction of the injection needle; Preferably, the ratio V1 / V2 of the extrusion velocity V1 to the flow velocity V2 is 0.8 to 1.

2.

4. The preparation method according to claim 1, characterized in that: The collagen solution is mainly prepared from collagen and acid solution; Preferably, in the collagen solution, the concentration of collagen W / V is 0.1% to 20%; Preferably, the pH of the acid solution is 0.5-5.

5. The preparation method according to claim 1, characterized in that: The collagen solution also includes a photocrosslinking agent; Preferably, the concentration W / V of the photocrosslinking agent is 0.005% to 5%.

6. The preparation method according to claim 5, characterized in that: The photocrosslinking agent includes at least one of riboflavin, riboflavin sodium phosphate, camphorquinone, curcumin and eosin Y; Preferably, when the collagen solution includes a photocrosslinking agent, the preparation method further comprises: subjecting the collagen fibers to light treatment.

7. The preparation method according to claim 1, characterized in that: The pH of the sedimentation liquid is 6 to 9; Preferably, the concentration of the deposition liquid is 1X to 10X; Preferably, the temperature of the deposition liquid is 4-40°C.

8. The preparation method according to claim 1, characterized in that: The preparation method further comprises: collecting the collagen fibers and performing a modification process; the modification process comprises at least one of the following: (1) cross-linking the collagen fibers in a solution containing a chemical cross-linking agent; (2) subjecting the collagen fibers to an enzyme cross-linking treatment in an enzyme-containing solution; (3) subjecting the collagen fibers to thermal cross-linking treatment; Preferably, the chemical cross-linking agent comprises at least one of glutaraldehyde, formaldehyde, EDC-NHS, genipin, catechol and maleimide cross-linking agents; Preferably, the enzyme comprises at least one of transglutaminase, lysine oxidase and horseradish peroxidase; Preferably, in the thermal cross-linking treatment, the temperature of the collagen fibers in a vacuum state is 100 to 140° C., and the treatment time is 12 to 96 hours.

9. A highly oriented collagen fiber, characterized in that: The preparation method is described in any one of claims 1 to 8.

10. Use of the highly oriented collagen fiber according to claim 9 in the preparation of tendons, ligaments, surgical sutures, cosmetic lifting threads, cornea and / or blood vessel repair products.

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