A collagen fiber for biomedical materials having a mesoscopic aggregate as a structural unit and a method for preparing the same

Abstract

The present application belongs to the field of biomedical materials, and particularly relates to collagen fibers with mesoscopic aggregates as structural units for biomedical materials and a preparation method thereof. The preparation method comprises the following steps in sequence: a step of splitting natural collagen aggregates from animal tissues, a step of regulating the mesoscopic size of the natural collagen aggregates, a step of assembling a fiber preparation device, a step of preparing collagen fibers with the natural collagen aggregates as structural units, and a step of twisting the collagen fibers. Compared with conventional solution method for preparing collagen fibers, the present application uses mesoscopic aggregates as structural units for preparing collagen fibers, and the obtained fibers have excellent mechanical properties, good biocompatibility and wound healing promoting performance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically referring to collagen fibers with mesoscale aggregates as structural units for use in biomedical materials and their preparation methods. Background Technology

[0002] Traditional surgical sutures are mostly made of synthetic materials such as polyester and nylon. While they possess good stability and reliability, they cannot be broken down or absorbed by the human body, requiring secondary surgery for removal and causing damage and inflammation to surrounding tissues. Therefore, finding new materials to replace traditional surgical sutures has become a hot topic in medical research. In recent years, research on collagen-based biodegradable surgical sutures has attracted considerable attention. Collagen is a natural structural protein widely found in human and mammalian tissues, possessing good biocompatibility and bioactivity. It can be broken down and absorbed by the body without causing adverse reactions to surrounding tissues. Therefore, the preparation of biodegradable surgical sutures using collagen has become a popular research direction. Furthermore, due to the bioactivity of collagen, the sutures can promote wound healing and tissue regeneration, which is highly beneficial for the recovery of surgical patients.

[0003] The commonly used processing method for preparing collagen-based surgical sutures is to obtain soluble collagen from animal tissues and then prepare the material. That is, the aggregated structure of natural collagen is broken down by using acid solutions, salt solutions or ionic liquids, so that the collagen is dispersed in a molecular state to form a solution, and then the collagen molecules are constructed into fiber materials through regeneration and self-assembly. This processing method of constructing collagen raw materials at the molecular scale has many technical bottlenecks: (1) The natural aggregated structure of collagen is destroyed during the dissolution process, losing its performance advantages at the nanoscale and microscale (referred to as the mesoscale in this patent). Therefore, compared with natural collagen raw materials, materials prepared using collagen solutions have disadvantages such as weak mechanical properties, low thermal stability and poor structural stability. For example, natural collagen fibers from animal tissues have a very high elastic modulus, while the modulus of collagen fibers obtained by electrospinning collagen solutions will be greatly reduced. (2) Although modification methods such as "chemical modification", "chemical crosslinking" and "blending" can enhance the interaction between collagen molecules and improve the structural stability and mechanical properties of collagen, these methods also have some problems that cannot be ignored, such as the biosafety issues caused by the use of chemical crosslinking agents, the loss of collagen properties caused by chemical modification, and the low compatibility of blended systems. (3) The assembly behavior of collagen molecules in vitro is affected by many factors such as telopeptide type, sequence length, temperature, pH value, ionic strength and medium type, and these factors usually influence and restrict each other, making it difficult to achieve the orderly and controllable assembly of collagen molecules in vitro. (4) The steps of collagen extraction, dissolution and assembly are extremely complicated, and the storage conditions of the prepared solution are harsh. This also limits the application and large-scale production of collagen fiber materials to a certain extent. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems and overcome the technical bottleneck of constructing collagen fibers from the collagen molecular scale. When preparing mesoscale aggregate collagen fibers for biomedical materials, the aggregate scale is controllable and the operation steps are relatively simple. This application provides a collagen fiber with mesoscale aggregates as structural units for biomedical materials and its preparation method, resulting in a collagen fiber with high mechanical properties and biocompatibility, without any modifier components, and with good market application prospects.

[0005] The technical solution of the present invention is implemented according to the following steps:

[0006] A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials includes the following steps:

[0007] (1) Method for separating natural collagen aggregates from animal tissues: one or two of fresh pig skin or fish skin are used as raw materials for preparing collagen aggregates. The pretreated animal tissues are obtained by degreasing, decellularization, swelling, pulverization and freeze-drying.

[0008] (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent and stripping conditions (temperature, stripping agent ratio and stripping time).

[0009] (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion coagulation using fiber preparation equipment.

[0010] (4) Twist the collagen fibers from step (3) for use in biomedical materials.

[0011] The specific method for obtaining pretreated animal tissue in step (1) is as follows: Fat in pig or fish skin is removed by manual cutting or soaking in acetone, and the tissue is then granulated. The granules are soaked in an aqueous solution containing 0.5–2 wt% polyethylene glycol octylphenyl ether and 0.5–2 mmol / L thiobetaine at 4–6°C for 20–30 hours to remove animal cells. The decellularized animal tissue granules are then soaked in a 0.1–0.3 wt% sodium acetate solution at 4–10°C for 3–6 hours to promote tissue expansion. The expanded tissue is then processed in a high-speed mixer at 1000–1200 rpm for 5–10 minutes to obtain animal tissue slurry. After centrifugation at 1000–1200 rpm and washing with water 5–8 times to remove alkaline reagents, the slurry is then freeze-dried to obtain pretreated animal tissue.

[0012] There are four liquid phase exfoliation methods in step (2), and any one of them can obtain collagen aggregates with mesoscale, specifically:

[0013] Peeling Method 1: Place the pretreated animal tissue obtained in step (1) into a 4-6 wt% sodium acetate / 3-5 wt% thiourea / 0.1-0.5 wt% zinc chloride aqueous solution of peeling agent ①, freeze at -5℃ to -10℃ for 8-10 hours. After the system is thawed, the sample is thoroughly centrifuged and washed with water to remove the peeling agent components and obtain collagen aggregates with mesoscale.

[0014] Method 2: The pretreated animal tissue obtained in step (1) is stirred at room temperature for 5 to 15 minutes in lithium chloride / dimethyl sulfoxide with a mass ratio of 1:30 to 1:50 for the No. ② stripping agent at a stirring speed of 400 to 800 rpm. After centrifugation and washing with water, collagen aggregates with mesoscale are obtained.

[0015] Method 3: The pretreated animal tissue obtained in step (1) is stirred in the No. 3 stripping agent N-methylmorpholine-N oxide at 40-60°C for 0.5-3 hours at a stirring speed of 400-800 rpm, and then centrifuged and washed with water to obtain collagen aggregates with mesoscale.

[0016] Method 4: The pretreated animal tissue obtained in step (1) is treated with the No. 4 stripping agent N,N,N-triethyl-3,6-dioxaneheptylammonium salt at 40-60°C for 0.5-2 hours with stirring speed of 400-800 rpm. After centrifugation and washing, collagen aggregates with mesoscale are obtained.

[0017] The fiber preparation equipment in step (3) includes an injection pump, a syringe, a coagulation bath, a gradient dehydration bath series device, and a traction collection device. Collagen fibers are obtained by spinning and include:

[0018] a. Preparation of spinning solution: The collagen aggregates with mesoscopic scale obtained in step (2) are dispersed in a 0.3-0.5 v / v% acetic acid aqueous solution or a 0.05-0.1 wt% hydrochloric acid aqueous solution, wherein the mass fraction of collagen aggregates is 1.5-1.8 wt%, and the ambient temperature of the spinning solution is 4-10℃;

[0019] b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution;

[0020] c. Coagulation of spinning solution: After extrusion, the spinning solution is placed in a rotary coagulation bath. The coagulation bath liquid is a 0.5-2 wt% sodium bicarbonate aqueous solution, and the coagulation time is 1-3 hours.

[0021] d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

[0022] e. Traction collection: Traction rate 0.3-1 m / min, draw ratio 1-2.

[0023] In step (4), the collagen fibers are obtained by spinning, including twisting the collagen fibers obtained in step (3) for use in biomedical materials. Two to four collagen monofibrils are fixed on a twisting machine and twisted at a speed of 400 to 600 r / min for 1 to 3 minutes to obtain twisted fibers.

[0024] The steps also include pulling and gathering collagen fibers and twisting them into strands of the required length and dimensions.

[0025] A collagen fiber with mesoscale aggregates as structural units for use in biomedical materials.

[0026] This invention offers the following advantages: It provides a method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials. No modifiers are added, and the aggregate scale can be stably controlled. The obtained collagen fibers exhibit excellent mechanical properties and biocompatibility, significantly shortening the wound healing cycle. The tensile stress and modulus of the obtained single fibers, as well as the mechanical properties of the twisted fibers, are far superior to those of fibers prepared using collagen solutions. Therefore, it overcomes the technical bottlenecks of weak mechanical properties, low thermal stability, low compatibility, and uncontrollable structure in constructing collagen fibers at the molecular scale. Furthermore, the raw materials used are widely available and inexpensive, and the preparation process is green, environmentally friendly, and relatively simple, thereby improving productivity and further reducing production costs, demonstrating promising market application prospects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the coagulation and extrusion molding of collagen fibers.

[0029] Figure 2 This is a top view of the rotary groove.

[0030] Figure 3 This is a photograph of the extruded spinning solution in a rotary coagulation bath.

[0031] Figure 4 SEM images of collagen aggregates obtained by pretreatment of porcine skin tissue with a 4wt% CH3COONa / 3wt% CH4N2S / 0.5wt% ZnCl2 aqueous solution as a pretreatment agent.

[0032] Figure 5 SEM images of collagen aggregates obtained by pretreatment of fish skin tissue with LiCl / DMSO at a mass ratio of 1:30 using No. 2 peeling agent.

[0033] Figure 6 SEM images of collagen aggregates obtained by pretreatment of pig and fish skin tissues with NMMO (exfoliating agent No. 3).

[0034] Figure 7 SEM images of collagen aggregates obtained by pretreatment of porcine skin tissue with peeling agent No. 4 M(OEt)2OAC at 50℃ for 0.5h.

[0035] Figure 8This is a SEM image of the surface of collagen fibers obtained using the extrusion coagulation method.

[0036] Figure 9 This is a SEM image of a cross-section of collagen fibers obtained using the extrusion coagulation method.

[0037] Figure 10 The graph shows the fold change in metabolic activity of L929 cells cultured in a tissue culture plate (Control) and in well plates containing collagen fibers, commercially available collagen suture fibers, commercially available silk suture fibers, and commercially available nylon suture fibers on days 3 and 5.

[0038] Figure 11 The collagen fibers obtained by the extrusion coagulation method are twisted to hold a 100g weight.

[0039] Figure 12 H&E staining and Masson staining results of different types of suture fibers implanted subcutaneously in mice one month later.

[0040] Figure 13 This is a comparative diagram showing the results of the rat epidermal suturing experiment. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1

[0043] A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials includes the following steps:

[0044] (1) Method for separating natural collagen aggregates from animal tissues: Fresh pig skin was used as the raw material for preparing collagen aggregates. The pretreated animal tissues were obtained by degreasing, decellularization, swelling, pulverization and freeze-drying.

[0045] (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent, stripping temperature, stripping agent ratio and stripping time.

[0046] (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion coagulation using fiber preparation equipment.

[0047] (4) Twist the collagen fibers from step (3) for use in biomedical materials.

[0048] The specific method for obtaining pretreated animal tissue in step (1) is as follows: the fat in the pig skin is removed by manual cutting and the tissue is granulated. Then, the tissue is soaked in an aqueous solution containing 0.5 wt% polyethylene glycol octylphenyl ether and 2 mmol / L thiobetaine at 4°C for 30 h to remove animal cells. The decellularized animal tissue granules are soaked in a 0.3 wt% sodium acetate solution at 10°C for 3 h to promote tissue expansion. The expanded tissue is then placed in a high-speed mixer and crushed at 1200 rpm for 5 min to obtain animal tissue slurry. The slurry is centrifuged at 1200 rpm and washed with water 8 times to remove alkaline reagents. Finally, the pretreated animal tissue is obtained by freeze drying.

[0049] The liquid phase exfoliation method in step (2) is as follows: the pretreated animal tissue obtained in step (1) is placed in an aqueous solution of 4wt% sodium acetate / 3wt% thiourea / 0.5wt% zinc chloride using exfoliant No. 10, and frozen at -8℃ for 9 hours. After thawing, the sample is thoroughly centrifuged and washed with water to remove the exfoliant components, obtaining collagen aggregates. The scanning electron microscope (SEM) image of the collagen aggregates is shown below. Figure 4 As shown, its outer wall is smooth, its structure is intact, and it has a mesoscopic scale.

[0050] The fiber preparation equipment in step (3) is as follows: Figure 1 As shown, it includes an injection pump, syringe, coagulation bath, gradient dehydration bath series device, and traction collection device; the coagulation bath is a rotary tank made entirely of acrylic material, with an overall width of 30cm, length of 45cm, and height of 10cm. A top view of the rotary tank is shown below. Figure 2 As shown, there are 8 rotating partitions in the middle, each partition is 20cm long and 8cm high, and the partitions are spaced about 5cm apart.

[0051] Collagen fibers are obtained by spinning, including:

[0052] a. Preparation of spinning solution: The collagen aggregates with mesoscopic scale obtained in step (2) are dispersed in a 0.5 v / v% acetic acid aqueous solution, wherein the mass fraction of collagen aggregates is 1.8 wt%, and the ambient temperature of the spinning solution is 10℃;

[0053] b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution;

[0054] c. Coagulation of spinning solution: such as Figure 3 As shown, after the spinning solution is extruded, it is placed in a rotary coagulation bath. The coagulation bath liquid is a 2wt% sodium bicarbonate aqueous solution, and the coagulation time is 1 hour.

[0055] d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

[0056] e. Traction collection: Traction rate 1 m / min, draw ratio 1.

[0057] The surface morphology of the obtained collagen fibers is from Figure 8 The SEM images show that its surface is continuous, smooth, and uniform in thickness; the cross-sectional morphology of the collagen fibers obtained from the site is... Figure 9 The SEM images show that its texture is dense; L929 cells were cultured in well plates containing the collagen fibers of this invention, commercial collagen suture fibers, commercial silk suture fibers, and commercial nylon suture fibers. The fold change in metabolic activity of the cells on days 3 and 5 is as follows. Figure 10 As shown in Table 1, the experimental results demonstrate that the obtained collagen fibers have good biocompatibility. The mechanical properties of the single collagen fibers prepared in this invention are compared with those of previously reported collagen fibers. The tensile stress of the single fibers obtained in this invention can reach 350±20MPa and the modulus can reach 4735±1500MPa, which shows good mechanical properties.

[0058] In step (4), the collagen fibers are obtained by spinning, including twisting the collagen fibers from step (3) for use in biomedical materials. Four collagen monofibrils are fixed on a twisting machine and twisted at a speed of 400 r / min for 3 minutes to obtain twisted fibers. Figure 11 As shown, the twisted collagen fibers can hold a 100g weight, and the mechanical properties can reach 0.284N / tex.

[0059] Using the obtained twisted fibers to suture wounds can significantly shorten the wound healing period.

[0060] Table 1 Comparison of mechanical properties of single collagen fibers prepared in this invention with those of previously reported collagen fibers.

[0061]

[0062] Example 2

[0063] A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials includes the following steps:

[0064] (1) Method for separating natural collagen aggregates from animal tissues: Fresh fish skin is used as the raw material for preparing collagen aggregates. The pretreated animal tissues are obtained by degreasing, decellularization, swelling, pulverization and freeze-drying.

[0065] (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent, stripping temperature, stripping agent ratio and stripping time.

[0066] (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion coagulation using fiber preparation equipment.

[0067] (4) Twist the collagen fibers from step (3) for use in biomedical materials.

[0068] The specific method for obtaining pretreated animal tissue in step (1) is as follows: acetone is used to remove fat from fish skin and the tissue is granulated. Then, the tissue is soaked in an aqueous solution containing 2 wt% polyethylene glycol octylphenyl ether and 0.5 mmol / L thiobetaine at 6°C for 20 h to remove animal cells. The decellularized animal tissue particles are soaked in a 0.1 wt% sodium acetate solution at 4°C for 6 h to promote tissue expansion. The expanded tissue is then placed in a high-speed mixer and crushed at 1000 rpm for 10 min to obtain animal tissue slurry. The slurry is centrifuged at 1000 rpm and washed with water 5 times to remove alkaline reagents. Finally, the pretreated animal tissue is obtained by freeze-drying.

[0069] The liquid phase stripping method in step (2) involves stirring the pretreated animal tissue obtained in step (1) in a lithium chloride / dimethyl sulfoxide solution with a stripping agent mass ratio of 1:30 (②) at room temperature for 15 minutes at a stirring speed of 600 rpm. After centrifugation and washing with water, collagen aggregates are obtained. The SEM image of these collagen aggregates is shown below. Figure 5 As shown, its outer wall is smooth, its structure is intact, and it has a mesoscopic scale.

[0070] The fiber preparation equipment in step (3) is as follows: Figure 1 As shown, it includes an injection pump, syringe, coagulation bath, gradient dehydration bath series device, and traction collection device; the coagulation bath is a rotary tank made entirely of acrylic material, with an overall width of 30cm, length of 45cm, and height of 10cm. A top view of the rotary tank is shown below. Figure 2 As shown, there are 8 rotating partitions in the middle, each partition is 20cm long and 8cm high, and the partitions are spaced about 5cm apart.

[0071] Collagen fibers are obtained by spinning, including:

[0072] a. Preparation of spinning solution: The collagen aggregates with mesoscale obtained in step (2) are dispersed in a 0.1 wt% hydrochloric acid aqueous solution, wherein the mass fraction of collagen aggregates is 1.5 wt%, and the ambient temperature of the spinning solution is 4℃.

[0073] b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution;

[0074] c. Coagulation of spinning solution: such as Figure 3 As shown, after the spinning solution is extruded, it is placed in a rotary coagulation bath. The coagulation bath liquid is a 0.5wt% sodium bicarbonate aqueous solution, and the coagulation time is 3h.

[0075] d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

[0076] e. Traction collection: Traction rate 0.3 m / min, draw ratio 2.

[0077] In step (4), the collagen fibers are obtained by spinning, including twisting the collagen fibers obtained in step (3) for use in biomedical materials. Two collagen monofibrils are fixed on a twisting machine and twisted at a speed of 600 r / min for 1 min. The twisted fibers are then removed to obtain twisted fibers.

[0078] The resulting twisted fibers have good mechanical properties and biocompatibility, and when used to suture wounds, they can significantly shorten the wound healing cycle.

[0079] Example 3

[0080] A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials includes the following steps:

[0081] (1) Method for separating natural collagen aggregates from animal tissues: Fresh pig skin and fish skin are used as raw materials for preparing collagen aggregates. The pretreated animal tissues are obtained by degreasing, decellularization, swelling, pulverization and freeze-drying.

[0082] (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent, stripping temperature, stripping agent ratio and stripping time.

[0083] (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion coagulation using fiber preparation equipment.

[0084] (4) Twist the collagen fibers from step (3) for use in biomedical materials.

[0085] The specific method for obtaining pretreated animal tissue in step (1) is as follows: the fat in pig and fish skin is removed by soaking in acetone and then granulated. The tissue is then soaked in an aqueous solution containing 1 wt% polyethylene glycol octylphenyl ether and 1 mmol / L thiobetaine at 5°C for 25 h to remove animal cells. The decellularized animal tissue particles are then soaked in a 0.2 wt% sodium acetate solution at 7°C for 5 h to promote tissue expansion. The expanded tissue is then placed in a high-speed mixer and processed at 1100 rpm for 8 min to obtain animal tissue slurry. The slurry is centrifuged at 1100 rpm and washed with water 8 times to remove alkaline reagents, and then freeze-dried to obtain pretreated animal tissue.

[0086] The liquid phase exfoliation method in step (2) involves treating the pretreated animal tissue obtained in step (1) with exfoliating agent ③, N-methylmorpholine-N-oxide (NMMO), at 50°C for 2 hours with stirring at a stirring speed of 600 rpm. After centrifugation and washing, collagen aggregates are obtained. The SEM image of these collagen aggregates is shown below. Figure 6 As shown, its outer wall is smooth, its structure is intact, and it has a mesoscopic scale.

[0087] The fiber preparation equipment in step (3) is as follows: Figure 1 As shown, it includes an injection pump, syringe, coagulation bath, gradient dehydration bath series device, and traction collection device; the coagulation bath is a rotary tank made entirely of acrylic material, with an overall width of 30cm, length of 45cm, and height of 10cm. A top view of the rotary tank is shown below. Figure 2 As shown, there are 8 rotating partitions in the middle, each partition is 20cm long and 8cm high, and the partitions are spaced about 5cm apart.

[0088] Collagen fibers are obtained by spinning, including:

[0089] a. Preparation of spinning solution: The collagen aggregates with mesoscale obtained in step (2) were dispersed in a 0.5 v / v% acetic acid aqueous solution, wherein the mass fraction of the collagen aggregates was 1.6 wt%, and the ambient temperature of the spinning solution was 7℃.

[0090] b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution;

[0091] c. Coagulation of spinning solution: such as Figure 3 As shown, after the spinning solution is extruded, it is placed in a rotary coagulation bath. The coagulation bath liquid is a 1wt% sodium bicarbonate aqueous solution, and the coagulation time is 2h.

[0092] d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

[0093] e. Traction collection: Traction rate 0.6 m / min, draw ratio 2.

[0094] In step (4), the collagen fibers are obtained by spinning, including twisting the collagen fibers obtained in step (3) for use in biomedical materials. Three collagen monofibrils are fixed on a twisting machine and twisted at a speed of 500 r / min for 2 minutes to obtain twisted fibers.

[0095] The obtained collagen fibers have good mechanical properties and biocompatibility, and when used to suture wounds, they can significantly shorten the wound healing cycle.

[0096] Example 4

[0097] A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials includes the following steps:

[0098] (1) Method for separating natural collagen aggregates from animal tissues: Fresh pig skin was used as the raw material for preparing collagen aggregates. The pretreated animal tissues were obtained by degreasing, decellularizing, swelling, pulverizing and freeze-drying.

[0099] (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent, stripping temperature, stripping agent ratio and stripping time.

[0100] (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion coagulation using fiber preparation equipment.

[0101] (4) Twist the collagen fibers from step (3) for use in biomedical materials.

[0102] The specific method for obtaining pretreated animal tissue in step (1) is as follows: acetone is used to remove fat from pig skin and the tissue is granulated. Then, an aqueous solution containing 2 wt% polyethylene glycol octylphenyl ether and 2 mmol / L thiobetaine is used to soak the tissue at 4°C for 20 h to remove animal cells. The decellularized animal tissue particles are soaked in a 0.3 wt% sodium acetate solution at 4°C for 6 h to promote tissue expansion. The expanded tissue is placed in a high-speed mixer and crushed at 1200 rpm for 10 min to obtain animal tissue slurry. The slurry is centrifuged at 1200 rpm and washed with water 8 times to remove alkaline reagents. Then, the pretreated animal tissue is obtained by freeze drying.

[0103] The liquid phase stripping method in step (2) involves treating the pretreated animal tissue obtained in step (1) with stripping agent No. ④, N,N,N-triethyl-3,6-dioxohepylammonium salt (M(OEt)2OAC), at 50°C for 0.5 h with stirring at a stirring speed of 800 rpm. After centrifugation and washing, collagen aggregates are obtained. A super-depth-of-field photograph of these collagen aggregates is shown below. Figure 7 As shown, the collagen aggregates prepared by this invention have intact and uniform structures, are well dispersed, and have mesoscopic scale.

[0104] The fiber preparation equipment in step (3) is as follows: Figure 1 As shown, it includes an injection pump, syringe, coagulation bath, gradient dehydration bath series device, and traction collection device; the coagulation bath is a rotary tank made entirely of acrylic material, with an overall width of 30cm, length of 45cm, and height of 10cm. A top view of the rotary tank is shown below. Figure 2 As shown, there are 8 rotating partitions in the middle, each partition is 20cm long and 8cm high, and the partitions are spaced about 5cm apart.

[0105] Collagen fibers are obtained by spinning, including:

[0106] a. Preparation of spinning solution: The collagen aggregates with mesoscale obtained in step (2) are dispersed in a 0.5 v / v% acetic acid aqueous solution, wherein the mass fraction of the collagen aggregates is 1.5 wt%, and the ambient temperature of the spinning solution is 4℃.

[0107] b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution;

[0108] c. Coagulation of spinning solution: such as Figure 3 As shown, after the spinning solution is extruded, it is placed in a rotary coagulation bath. The coagulation bath liquid is a 2wt% sodium bicarbonate aqueous solution, and the coagulation time is 1 hour.

[0109] d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

[0110] e. Traction collection: Traction rate 1m / min, draw ratio 2.

[0111] In step (4), the collagen fiber is obtained by spinning, including twisting the collagen fiber from step (3) for use in biomedical materials. Three collagen monofibrils are fixed on a twisting machine and twisted at a speed of 600 r / min for 1 min to obtain twisted fibers with good mechanical properties.

[0112] Figure 12In the figures, a1, b1, c1, and d1 show the H&E staining results of collagen fibers, commercial collagen suture fibers, commercial silk suture fibers, and commercial nylon suture fibers implanted subcutaneously in mice one month after the procedure. a2, b2, c2, and d2 show the Masson staining results of collagen fibers, commercial collagen suture fibers, commercial silk suture fibers, and commercial nylon suture fibers implanted subcutaneously in mice one month after the procedure. The comparison shows that the collagen fibers in this embodiment have good biocompatibility. Figure 13 The numbers 1, 2, 3, and 4 on the back of the mice represent the incision locations. Incision 1 was left untreated, incision 2 was sutured with twisted collagen fibers according to this embodiment, incision 3 was sutured with commercial collagen sutures, and incision 4 was sutured with commercial silk sutures. Experimental results show that, compared with silk sutures and existing commercial collagen sutures, the collagen fibers prepared by this invention can significantly shorten the wound healing cycle. Moreover, the raw materials used are widely available and inexpensive, and the preparation process is green, environmentally friendly, and relatively simple, thereby improving productivity and further reducing production costs.

[0113] The embodiments of the invention described above and shown in the accompanying drawings should not be construed as limiting the technical concept of the invention. The scope of protection of the invention is limited only by the contents set forth in the claims, and those skilled in the art can modify and alter the technical concept of the invention in various forms. Therefore, it will be apparent to those skilled in the art that such improvements and modifications will fall within the scope of protection of the invention.

Claims

1. A method for preparing collagen fibers with mesoscale aggregates as structural units for use in biomedical materials, characterized in that, Includes the following steps: (1) Method for separating natural collagen aggregates from animal tissues: one or two of fresh pig skin or fish skin are used as raw materials for preparing collagen aggregates. The pretreated animal tissues are obtained by degreasing, decellularization, swelling, pulverization and freeze-drying. (2) Using the pretreated animal tissue from step (1), collagen aggregates with mesoscale are obtained by liquid phase stripping. The size of the aggregates can be controlled by adjusting the type of stripping agent, stripping temperature, stripping agent ratio and stripping time. (3) Using the collagen aggregates with mesoscale in step (2) as structural units, collagen fibers are formed by extrusion, coagulation and dehydration using fiber preparation equipment; (4) Twist the collagen fibers from step (3) for use in biomedical materials; There are two liquid phase exfoliation methods in step (2), and either method can obtain collagen aggregates with mesoscale, specifically: Method 2: The pretreated animal tissue obtained in step (1) is stirred at room temperature for 5 to 15 minutes in lithium chloride / dimethyl sulfoxide with a mass ratio of 1:30 to 1:50 for the No. ② stripping agent at a stirring speed of 400 to 800 rpm. After centrifugation and washing with water, collagen aggregates with mesoscale are obtained. Method 4: The pretreated animal tissue obtained in step (1) is stirred in the No. 4 stripping agent N,N,N-triethyl-3,6-dioxane-heptylammonium salt at 40~60℃ for 0.5~2h at a stirring speed of 400~800 rpm, and then centrifuged and washed with water to obtain collagen aggregates with mesoscale.

2. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to claim 1, characterized in that: The specific method for obtaining pretreated animal tissue in step (1) is as follows: the fat in pig skin or fish skin is removed by manual cutting or soaking in acetone and then granulated. The granules are then soaked in an aqueous solution containing 0.5-2 wt% polyethylene glycol octylphenyl ether and 0.5-2 mmol / L thiobetaine at 4-6°C for 20-30 h to remove animal cells from the tissue. The decellularized animal tissue granules are then soaked in a 0.1-0.3 wt% sodium acetate solution at 4-10°C for 3-6 h to promote tissue expansion. The expanded tissue is then placed in a high-speed mixer and crushed at 1000-1200 rpm for 5-10 min to obtain animal tissue slurry. The slurry is then centrifuged and washed with water to remove alkaline reagents, and then freeze-dried to obtain pretreated animal tissue.

3. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to claim 1, characterized in that: The fiber preparation equipment in step (3) includes an injection pump, a syringe, a coagulation bath, a gradient dehydration bath series device, and a stretching and collecting device; In step (3), the collagen fibers are obtained by spinning, including: a. Preparation of spinning solution: The collagen aggregates with mesoscopic scale obtained in step (2) are dispersed in a 0.3~0.5 v / v% acetic acid aqueous solution or a 0.05~0.1wt% hydrochloric acid aqueous solution, wherein the mass fraction of collagen aggregates is 1.5~1.8wt%, and the ambient temperature of the spinning solution is 4~10℃; b. Extrusion of spinning solution: Place the spinning solution in a syringe, fix the syringe in the injection pump, and extrude the spinning solution; c. Coagulation of spinning solution: The extruded spinning solution is placed in a coagulation bath. The coagulation bath liquid is a 0.5~2wt% sodium bicarbonate aqueous solution, and the coagulation time is 1~3h. d. Dehydration treatment: The extruded fibers are placed in isopropanol and anhydrous ethanol for gradual dehydration. After the solvents evaporate, the collagen fibers are collected.

4. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to claim 2, characterized in that: The centrifugal washing involves multiple centrifugal washings, with 5 to 8 washes performed at a centrifuge speed of 1000 to 1200 rpm. Specifically, the supernatant from each centrifugation is poured out and replaced with a suitable volume of deionized water, i.e., the sample and water volume does not exceed 2 / 3 of the volume of the centrifuge tube or flask, and the operation is repeated.

5. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to claim 3, characterized in that: The extrusion of the spinning solution uses a syringe needle of 19-22G and an injection pump with a linear speed of 3-5 ml / min.

6. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to claim 3, characterized in that: The coagulation bath is a rotary tank with a rotary partition plate in the middle.

7. The method for preparing collagen fibers with mesoscale aggregates as structural units for biomedical materials according to any one of claims 1 to 6, characterized in that: The collagen fibers also include a traction and collection process, and a twisting process to form strands of the required length and dimensions.

Images