Long-lasting collagen fibers and methods of making the same

Abstract

The application belongs to the technical field of biomedical materials, and particularly relates to a long-acting collagen fiber and a preparation method thereof. The long-acting collagen fiber is prepared through an in-vitro 3D reconstruction process of the collagen fiber, and the collagen fiber has a precisely controlled morphology, is arranged in an order and is uniformly distributed, has a fiber diameter of 60-120 nm, and a fiber 3D network formed by the collagen fiber has a pore size of 200-220 mu m. The long-acting collagen fiber prepared by the method has not only a good growth environment for cells, but also excellent stability and significantly enhanced anti-enzymatic hydrolysis capacity. The long-acting collagen fiber prepared by the method can be applied to the fields of implant agents, artificial skin, artificial cartilage, hemostatic sponges, stent materials, medical devices and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a long-acting collagen fiber and a preparation method thereof. BACKGROUND

[0002] Collagen is the most abundant protein component in the human body, and is widely distributed in connective tissues such as tendons, bones, cartilages, skins, corneas, etc. At present, 29 different types of collagen have been found, all of which have the common characteristic of triple helix structure. Among them, types I, II and III are the most abundant collagen types, which can self-assemble to form characteristic fiber structures in the human body. The 3D scaffold formed by collagen fibers provides structural integrity and mechanical strength to the human body, and at the same time, they provide a suitable living environment for cells, promoting cell adhesion, proliferation and migration.

[0003] The biological function of collagen fibers is so important that its excessive degradation can lead to various pathological conditions of the skin, cartilage, etc. For the skin, collagen fibers are the most critical component of the dermis layer, and a large amount of degradation of collagen fibers in the skin can lead to skin aging phenomena such as decreased elasticity, relaxation, roughness, etc. Therefore, by injecting collagen fibers into orthopedic sites, the skin can be helped to return to a younger state, and collagen implants are widely used in medical cosmetology. However, the collagen fibers prepared by the prior art have the defects of poor stability and easy enzymatic degradation.

[0004] Chinese patent CN107812244A uses a method of directly freeze-drying collagen slurry to obtain collagen powder, and prepares a collagen filler. The morphology of the collagen fibers obtained by this method is not precisely controlled, and the size distribution of the collagen fibers is not uniform. Chinese patent CN104558675A adjusts the pH of the collagen solution to 5-10 with an alkaline solution to prepare a collagen microfiber hemostatic sponge. The size of the collagen fibers prepared by this method is also not well controlled. The collagen fibers prepared by the above methods are usually not highly stable and are easily degraded by enzymes. Therefore, Chinese patent CN101648989A and the like use glutaraldehyde and other chemical cross-linking agents to further cross-link the collagen fibers to prepare more long-acting collagen fibers. However, the potential toxicity risk of chemical cross-linking agents greatly affects the safety of cross-linked collagen.

[0005] In view of the above technical problems, the present application provides a long-acting collagen fiber and a preparation method thereof. The method adopts a series of in-vitro 3D reconstruction processes of collagen fiber, such as self-assembly, re-dissolution, incubation, homogenization, and sieving, to prepare the long-acting collagen fiber with precisely controlled morphology. The collagen fiber is uniformly distributed and closely arranged, and the fiber diameter is 60-120 nm. The long-acting collagen fiber prepared by the method has good stability and significantly enhanced anti-enzymatic ability, and can realize long-acting function, and has wide application potential in the fields of implant, artificial skin, artificial cartilage, hemostatic sponge, stent material, medical device, and the like. SUMMARY

[0006] The present application aims to provide a long-acting collagen fiber and a preparation method thereof, and specifically includes the following contents.

[0007] In a first aspect, the present application provides a preparation method of a long-acting collagen fiber, which comprises the following steps:

[0008] (1) adding sodium phosphate dibasic / sodium phosphate monobasic buffer solution, and / or sodium hydroxide solution, and / or potassium hydroxide solution to a collagen solution to react, and standing to obtain a collagen precipitate;

[0009] (2) re-dissolving the collagen precipitate obtained in step (1) into a collagen solution by using sodium phosphate dibasic / sodium phosphate monobasic buffer solution, and / or TRIS-HCl buffer solution, and / or HEPES buffer solution;

[0010] (3) incubating, homogenizing, and separating the re-dissolved collagen solution to obtain a long-acting collagen fiber.

[0011] Preferably, the configuration method of the collagen solution in step (1) is to completely dissolve the collagen in acetic acid solution, and / or hydrochloric acid solution, and / or phosphoric acid solution.

[0012] Preferably, the configuration method of the collagen solution is to dissolve the collagen in 0.5M acetic acid, and adjust the pH to 3.0-5.0 to obtain the collagen solution.

[0013] Preferably, the concentration of the collagen solution in step (1) is 1.0-8.0 mg / mL.

[0014] Preferably, the concentration of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution, and / or sodium hydroxide solution, and / or potassium hydroxide solution in step (1) is 10-100 mM, and the pH is 6.0-9.0.

[0015] Preferably, the solution is sodium phosphate dibasic / sodium phosphate monobasic buffer solution.

[0016] Preferably, the concentration of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 20 mM.

[0017] Preferably, the pH of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 6.0-8.0.

[0018] Preferably, the pH of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 7.4-8.0.

[0019] Preferably, the concentration of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution, and / or the TRIS-HCl buffer solution, and / or the HEPES buffer solution in step (2) is 10-100 mM, and the pH is 6.0-9.0.

[0020] Preferably, the solution is a sodium phosphate dibasic / sodium phosphate monobasic buffer solution.

[0021] Preferably, the concentration of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 20 mM.

[0022] Preferably, the pH of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 6.0-8.0.

[0023] Preferably, the pH of the sodium phosphate dibasic / sodium phosphate monobasic buffer solution is 7.4-8.0.

[0024] Preferably, the constant temperature incubation condition in step (3) is incubation at 4-25°C for 0.1-72 hrs.

[0025] Preferably, the constant temperature incubation temperature is 8-25°C.

[0026] Preferably, the constant temperature incubation temperature is 17-25°C.

[0027] Preferably, the constant temperature incubation time is 2-19 hrs.

[0028] Preferably, the constant temperature incubation time is 8-19 hrs.

[0029] Preferably, the homogenization condition in step (3) is homogenization at 4-25°C for 0.1-10 hrs at a speed of 1000-10000 rpm, and the separation includes: passing the homogenized collagen through a 40-60 mesh sieve, and collecting the sieved collagen solution to obtain collagen fibers by centrifugation.

[0030] Preferably, the homogenization temperature is 4°C.

[0031] Preferably, the homogenization time is 20-30 min.

[0032] Preferably, the homogenization speed is 10000 rpm.

[0033] Preferably, the method is:

[0034] (1) dissolving collagen in water, adding acetic acid, adjusting pH to 3.0-5.0 to obtain a collagen solution of 0.1-5 mg / mL; adding a pH 6.0-9.0, 10-100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to the prepared collagen acidic solution, standing for 0.1-8.0 hrs, then centrifuging to collect collagen precipitate;

[0035] (2) redissolving the obtained collagen precipitate with a pH 6.0-9.0, 10-100 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to form a collagen solution;

[0036] (3) incubating the redissolved collagen solution at 4-25℃ for 1-24 hrs; homogenizing at 1000-10000 rpm for 0.1-2 hrs; sieving the homogenized collagen using a 40-60 mesh sieve; collecting the sieved collagen solution and centrifuging to obtain long-acting collagen fibers.

[0037] Preferably, the concentration of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution in steps (1) and (2) is 20 mM.

[0038] Preferably, the pH of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution is 6.0-8.0.

[0039] Preferably, the pH of the disodium hydrogen phosphate / sodium dihydrogen phosphate buffer solution is 7.4-8.0.

[0040] Preferably, the volume ratio of the collagen solution to the buffer solution in step (1) is 1:2.

[0041] In a second aspect, the present application provides long-acting collagen fibers prepared by the method of the first aspect.

[0042] In a third aspect, the present application provides use of the long-acting collagen fibers of the second aspect in the preparation of implants, artificial skin, artificial cartilage, hemostatic sponges, stent materials, and medical devices.

[0043] In a fourth aspect, the present application provides a collagen implant, which is prepared by dissolving the long-acting collagen fibers of the second aspect in a physiological saline phosphate buffer containing 0.3% lidocaine hydrochloride.

[0044] The beneficial effects of the present invention are: (1) The present invention prepares long-acting collagen fibers with precise morphological control through an in vitro 3D reconstruction process of collagen fibers. The collagen fibers are tightly and orderly arranged and evenly distributed; (2) Compared with the collagen fibers prepared by the control method, the long-acting collagen fibers prepared by the present invention have excellent stability and significantly enhanced anti-enzymatic ability. It is a long-acting collagen fiber that can be applied to implants, artificial skin and other fields; (3) The long-acting collagen fibers prepared by the method do not require the use of chemical cross-linking agents and have good biocompatibility; (4) The long-acting collagen fibers form a good network structure, providing a good growth environment for cells; (5) The long-acting collagen fibers are evenly dispersed, have good injectability, and can be prepared into collagen implants. Attached Figure Description

[0045] Figure 1 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 1;

[0046] Figure 2 Differential scanning calorimetry of the long-lasting collagen fibers prepared in Example 1;

[0047] Figure 3 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 2;

[0048] Figure 4 Differential scanning calorimetry of the long-lasting collagen fibers prepared in Example 2;

[0049] Figure 5 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 3;

[0050] Figure 6 Differential scanning calorimetry of the long-lasting collagen fibers prepared in Example 3;

[0051] Figure 7 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 4;

[0052] Figure 8 Differential scanning calorimetry of the long-lasting collagen fibers prepared in Example 4;

[0053] Figure 9 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 5;

[0054] Figure 10 Differential scanning calorimetry (DSC) of the long-lasting collagen fibers prepared in Example 5;

[0055] Figure 11 Scanning electron microscope image of the long-lasting collagen fibers prepared in Example 6;

[0056] Figure 12Differential scanning thermogram of long-acting collagen fiber prepared in Example 6;

[0057] Figure 13 Scanning electron micrograph of collagen fiber prepared in Comparative Example 1;

[0058] Figure 14 Scanning electron micrograph of collagen fiber prepared in Comparative Example 2;

[0059] Figure 15 Scanning electron micrograph of collagen fiber prepared in Comparative Example 3;

[0060] Figure 16 Scanning electron micrograph of collagen fiber prepared in Comparative Example 4;

[0061] Figure 17 Cell proliferation result graph of long-acting collagen fiber prepared in the application. DETAILED DESCRIPTION

[0062] The application will be described in detail below through specific examples. Any technical solution that any person skilled in the art can think of on the basis of the application and in combination with common knowledge in the art belongs to the protection scope of the application.

[0063] The collagen protein described in the application includes type I, type II, and type III animal collagen protein, which can be commercially available or can be prepared according to the medical-grade yak collagen protein in Chinese patent CN112778412A as follows: The yak tendon is washed to remove foreign matter and is crushed into small pieces; the small pieces after crushing are defatted with 10% n-butanol, and the precipitate is taken and washed with water to neutral; the precipitate obtained in the previous step is decalcified with 0.5M hydrochloric acid, and after the decalcification is completed, the precipitate is taken and washed with water to neutral; the tissue precipitate obtained in the previous step is soaked in a 0.1M sodium hydroxide solution, stirred at 25°C for 4h, the precipitate is taken and washed with water to neutral; collagen protein is extracted with a 0.5M acetic acid solution containing 1g / L pepsin to obtain a crude collagen protein extract; the pH is adjusted to neutral for enzyme inactivation; dialysis is performed in an 8-14kDa dialysis bag, after the dialysis is completed, freeze-drying is performed, and finally low-endotoxin-content collagen protein is obtained.

[0064] The collagen protein used in the examples of the application is prepared according to the patent CN112778412A.

[0065] Example 1 Collagen fiber preparation 1

[0066] 1. Preparation of collagen fiber under different incubation times at pH 6

[0067] (1) Collagen powder was dissolved in water, 0.5M acetic acid was added, pH was adjusted to 3.0-5.0, to obtain a collagen solution, the concentration of the obtained collagen solution was 3.6mg / mL;

[0068] (2) pH 6.0, 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution was added to the collagen solution (the volume ratio of the collagen solution to the disodium hydrogen phosphate / sodium dihydrogen phosphate solution was 1:2), after standing for 1.5hrs, centrifugation was performed, and collagen precipitate was collected;

[0069] (3) The obtained collagen precipitate was uniformly dispersed with pH 6.0, 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution, to form a collagen solution with a concentration of 3mg / mL;

[0070] (4) The collagen solution after re-dissolution was incubated at 17°C for 2hrs, 8hrs and 19hrs, respectively;

[0071] (5) The incubated collagen was homogenized at 4°C at a speed of 10000rpm for 20min;

[0072] (6) The homogenized collagen was sieved using a 40-mesh sieve;

[0073] (7) The sieved collagen solution was centrifuged to obtain collagen fibers.

[0074] 2. Scanning electron microscope characterization of the prepared collagen fibers

[0075] Experimental procedure: The collagen fibers prepared in 1 above were freeze-dried, the freeze-dried sample was fixed on the sample stage of a scanning electron microscope, gold was sprayed for 25 seconds, and the sample morphology was detected at an operating voltage of 5.0kV.

[0076] Experimental results: The scanning electron microscope results of the collagen fibers prepared under different incubation times are shown in Figure 1 The pore size of the fiber network obtained by incubation for 2hrs was 200μm, the fiber diameter was 70±10nm; the pore size of the fiber network obtained by incubation for 8hrs was 205μm, the fiber diameter was 80±10nm; the pore size of the fiber network obtained by incubation for 19hrs was 209μm, the fiber diameter was 80±10nm. The results showed that the pore size of the fiber network prepared under incubation for 2-19hrs was similar, between 200-209μm; at the same time, with the incubation time increasing from 2hrs to 8-19hrs, thicker collagen fibers could be prepared.

[0077] 3. Thermal stability characterization of the prepared collagen fibers

[0078] Experimental procedure: The collagen fibers prepared in 1 above were freeze-dried, 5-10 mg of the freeze-dried sample was placed in an aluminum crucible, the heating rate was 5°C / min, and the thermal stability of the sample was determined using a differential scanning calorimeter (DSC) at 25-120°C.

[0079] Experimental results: The results of the DSC experiment are shown in Table 1 below. Figure 2 As shown in Table 1, the denaturation temperature of the collagen fibers obtained by incubation for 2 hrs was 101.4°C; the denaturation temperature of the collagen fibers obtained by incubation for 8 hrs was 102.1°C; and the denaturation temperature of the collagen fibers obtained by incubation for 19 hrs was 102.2°C. The results show that the stability of the prepared collagen fibers increased as the incubation time increased from 2 hrs to 8-19 hrs.

[0080] 4. Enzymatic digestion experiment of the prepared collagen fibers

[0081] Experimental procedure: The collagen fibers obtained in 1 above were freeze-dried, 10 mg of the freeze-dried sample was weighed, and the initial dry weight was recorded. Collagenase was prepared in a buffer solution (TES, 1 mM CaCl2, pH 7.4) at a concentration of 5 U / mL, and the enzymatic digestion experiment was performed at 37°C. After the enzymatic digestion was completed, the sample was washed with water and centrifuged, and the sample obtained after centrifugation was freeze-dried and weighed to calculate the enzymatic digestion rate. The enzymatic digestion rate (AW%) was calculated as AW% = (Wo-W) / W x 100%, where Wo is the initial weight of the sample before enzymatic digestion, and W is the weight of the sample after enzymatic digestion.

[0082] The experimental results are shown in Table 1 below: The enzymatic digestion rate of the collagen fibers prepared in this example was 62%-70% after 48 h of enzymatic digestion, indicating that the reconstituted collagen fibers had certain resistance to enzymatic digestion, and the resistance of the collagen fibers to enzymatic digestion increased as the incubation time increased.

[0083] Table 1 Enzymatic digestion experiment results of the collagen fibers prepared in this example

[0084] Incubation time Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 2 hrs 5.0 1.5 70％ 8 hrs 5.0 1.6 68％ 19 hrs 5.0 1.9 62％

[0085] Example 2 Preparation of collagen fibers 2

[0086] 1. Preparation of collagen fibers at different incubation temperatures at pH 6

[0087] (1) The collagen powder was dissolved in water, 0.5 M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a collagen solution, and the concentration of the obtained collagen solution was 3.6 mg / mL;

[0088] (2) Add pH 6.0, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to the collagen solution (volume ratio of collagen solution to disodium hydrogen phosphate / sodium dihydrogen phosphate solution is 1:2), stand for 1.5 hrs, centrifuge, and collect the collagen precipitate;

[0089] (3) Disperse the obtained collagen precipitate with pH 6.0, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to form a collagen solution with a concentration of 3 mg / mL;

[0090] (4) Incubate the resuspended collagen solution at 10°C, 17°C, and 25°C for 19 hrs;

[0091] (5) Homogenize the incubated collagen at 4°C at a speed of 10,000 rpm for 20 min;

[0092] (6) Screen the homogenized collagen using a 40-mesh sieve;

[0093] (7) Collect the screened collagen solution, centrifuge, and obtain collagen fibers.

[0094] 2. Scanning electron microscope characterization of the prepared collagen fibers

[0095] Experimental procedure: same as Example 1.

[0096] Experimental results: the results of the scanning electron microscope of the collagen fibers prepared at different incubation temperatures are shown in Figure 3 The fiber diameter obtained at 10°C is 60 ± 10 nm, the fiber diameter obtained at 17°C is 80 ± 10 nm, and the fiber diameter obtained at 25°C is 80 ± 10 nm. The results show that the incubation temperature has a significant effect on the collagen fibers, and thicker collagen fibers can be prepared at 17-25°C than at 10°C.

[0097] 3. Thermal stability characterization of the prepared collagen fibers

[0098] Experimental procedure: same as Example 1.

[0099] Experimental results: the results of the DSC experiment are shown in Figure 4 The denaturation temperature of the collagen fibers obtained at 10°C is 100.9°C, the denaturation temperature of the collagen fibers obtained at 17°C is 102.2°C, and the denaturation temperature of the collagen fibers obtained at 25°C is 102.2°C. The results show that the stability of the prepared collagen fibers increases as the incubation temperature increases from 10°C to 17-25°C.

[0100] 4. Enzymatic hydrolysis experiment of the prepared collagen fibers

[0101] Experimental procedure: same as Example 1.

[0102] The experimental results are shown in Table 2: the enzymolysis rate of the collagen fibers prepared in this example was 60%-72% after 48h of enzymolysis, indicating that the reconstructed collagen fibers had certain anti-enzymolysis ability, and the anti-enzymolysis ability of the collagen proteins was enhanced with the increase of the incubation temperature.

[0103] Table 2 Experimental results of enzymolysis of the collagen protein fibers prepared in this example

[0104] Incubation temperature Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 10℃ 5.0 1.4 72％ 17℃ 5.0 1.9 62％ 25℃ 5.0 2.0 60％

[0105] Example 3 Preparation of collagen protein fibers 3

[0106] 1. Preparation of collagen protein fibers under different incubation times at pH 7.4

[0107] (1) The collagen protein powder was dissolved in water, 0.5M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a collagen protein solution, and the concentration of the obtained collagen protein solution was 3.6mg / mL;

[0108] (2) A pH 7.4, 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution was added to the collagen protein solution (the volume ratio of the collagen protein solution to the disodium hydrogen phosphate / sodium dihydrogen phosphate solution was 1:2), and after standing for 1.5hrs, the collagen protein precipitate was collected by centrifugation;

[0109] (3) The obtained collagen protein precipitate was uniformly dispersed with a pH 7.4, 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to form a collagen protein solution with a concentration of 3mg / mL;

[0110] (4) The resuspended collagen protein solution was incubated at 17°C for 2hrs, 8hrs and 19hrs, respectively;

[0111] (5) The incubated collagen protein was homogenized at 4°C at a speed of 10000rpm for 20min;

[0112] (6) The homogenized collagen protein was sieved using a 40-mesh sieve;

[0113] (7) The sieved collagen protein solution was collected and centrifuged to obtain collagen protein fibers.

[0114] 2. Scanning electron microscope characterization of the prepared collagen protein fibers

[0115] Experimental procedure: same as Example 1.

[0116] Experimental results: the scanning electron microscope results of the prepared collagen protein fibers are shown inFigure 5 As shown in Table 2, the pore size of the fiber network obtained by incubation for 2 hrs was 200 μm, and the fiber diameter was 90 ± 10 nm; the pore size of the fiber network obtained by incubation for 8 hrs was 203 μm, and the fiber diameter was 110 ± 10 nm; the pore size of the fiber network obtained by incubation for 19 hrs was 214 μm, and the fiber diameter was 110 ± 10 nm. The results show that the pore size of the fiber network prepared under the condition of incubation for 2-19 hrs is similar, being between 200-214 μm; and with the increase of the incubation time from 2 hrs to 8-19 hrs, thicker collagen fibers can be prepared.

[0117] 3. Thermal stability characterization of the prepared collagen fibers

[0118] Experimental procedure: same as Example 1.

[0119] Experimental results: the results of the DSC experiment are shown in Table 3. Figure 6 As shown in Table 3, the denaturation temperature of the collagen fibers obtained by incubation for 2 hrs was 104.5 °C; the denaturation temperature of the collagen fibers obtained by incubation for 8 hrs was 106.3 °C; and the denaturation temperature of the collagen fibers obtained by incubation for 19 hrs was 106.8 °C. The results show that with the increase of the incubation time from 2 hrs to 8-19 hrs, the stability of the prepared collagen fibers is increased.

[0120] 4. Enzymatic hydrolysis experiment of the prepared collagen fibers

[0121] Experimental procedure: same as Example 1.

[0122] Experimental results: as shown in Table 3, the enzymatic hydrolysis rate of the collagen fibers prepared in this example was 34%-50% at 48 h of enzymatic hydrolysis, indicating that the reconstituted collagen fibers have certain anti-enzymatic hydrolysis ability, and with the increase of the incubation time, the anti-enzymatic hydrolysis ability of the collagen fibers is enhanced.

[0123] Table 3 Enzymatic hydrolysis experiment results of the collagen fibers prepared in this example

[0124] Incubation time Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 2 hrs 5.0 2.5 50％ 8 hrs 5.0 3.1 38％ 19 hrs 5.0 3.3 34％

[0125] Example 4 Preparation of collagen fibers 4

[0126] 1. Preparation of collagen fibers at different incubation temperatures under the condition of pH 7.4

[0127] (1) Dissolve the collagen powder in water, add 0.5 M acetic acid, adjust the pH to 3.0-5.0, and obtain a collagen solution, the concentration of the obtained collagen solution being 3.6 mg / mL;

[0128] (2) Add pH 7.4, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to the collagen solution (volume ratio of collagen solution to disodium hydrogen phosphate / sodium dihydrogen phosphate solution is 1:2), stand for 1.5 hrs, centrifuge, and collect the collagen precipitate;

[0129] (3) Disperse the obtained collagen precipitate with pH 7.4, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution to form a collagen solution with a concentration of 3 mg / mL;

[0130] (4) Incubate the resuspended collagen solution at 10°C, 17°C, and 25°C for 19 hrs;

[0131] (5) Homogenize the incubated collagen at 4°C at a speed of 10,000 rpm for 20 min;

[0132] (6) Screen the homogenized collagen using a 40-mesh sieve;

[0133] (7) Collect the screened collagen solution, centrifuge, and obtain collagen fibers.

[0134] 2. Scanning electron microscope characterization of the prepared collagen fibers

[0135] Experimental procedure: same as Example 1.

[0136] Experimental results: the results of the scanning electron microscope of the collagen fibers prepared at different incubation temperatures are shown in Figure 7 , the fiber diameter obtained at 10°C is 90 ± 10 nm, the fiber diameter obtained at 17°C is 110 ± 10 nm, and the fiber diameter obtained at 25°C is 110 ± 10 nm. The results show that the incubation temperature has a significant effect on the collagen fibers, and thicker collagen fibers can be prepared at 17-25°C than at 10°C.

[0137] 3. Thermal stability characterization of the prepared collagen fibers

[0138] Experimental procedure: same as Example 1.

[0139] Experimental results: the results of the DSC experiment are shown in Figure 8 , the denaturation temperature of the collagen fibers obtained at 10°C is 105.5°C, the denaturation temperature of the collagen fibers obtained at 17°C is 106.8°C, and the denaturation temperature of the collagen fibers obtained at 25°C is 107.0°C. The results show that the stability of the prepared collagen fibers slightly increases as the incubation temperature increases from 10°C to 17-25°C.

[0140] 4. Enzymatic digestion experiment of the prepared collagen fibers

[0141] Experimental procedure: same as Example 1.

[0142] The experimental results are shown in Table 4: the enzymolysis rate of the collagen fibers prepared in this example was 26%-48% at 48h of enzymolysis, indicating that the reconstructed collagen fibers had certain anti-enzymolysis ability, and the anti-enzymolysis ability of the collagen proteins was enhanced with the increase of the incubation temperature.

[0143] Table 4 Experimental results of enzymolysis of the collagen protein fibers prepared in this example

[0144] Incubation temperature Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 10℃ 5.0 2.6 48％ 17℃ 5.0 3.3 34％ 25℃ 5.0 3.7 26％

[0145] Example 5 Preparation of collagen protein fibers 5

[0146] 1. Preparation of collagen protein fibers under different incubation times at pH 8

[0147] (1) The collagen protein powder was dissolved in water, 0.5M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a collagen protein solution, and the concentration of the obtained collagen protein solution was 3.6mg / mL;

[0148] (2) 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution at pH 8.0 was added to the collagen protein solution (the volume ratio of the collagen protein solution to the disodium hydrogen phosphate / sodium dihydrogen phosphate solution was 1:2), and after standing for 1.5hrs, the collagen protein precipitate was collected by centrifugation;

[0149] (3) The obtained collagen protein precipitate was uniformly dispersed with 20mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution at pH 8.0, and the collagen protein solution was reconstituted to a concentration of 3mg / mL;

[0150] (4) The reconstituted collagen protein solution was incubated at 17°C for 2hrs, 8hrs and 19hrs, respectively;

[0151] (5) The incubated collagen protein was homogenized at 4°C at a speed of 10000rpm for 20min;

[0152] (6) The homogenized collagen protein was sieved using a 40-mesh sieve;

[0153] (7) The sieved collagen protein solution was collected and centrifuged to obtain collagen protein fibers.

[0154] 2. Scanning electron microscope characterization of the prepared collagen protein fibers

[0155] Experimental procedure: same as Example 1.

[0156] The experimental results: the scanning electron microscope results of the collagen protein fibers prepared under different incubation temperatures are shown inFigure 9 As shown in Table 4, the pore size of the fiber network obtained by incubation for 2 hrs was 214 μm, and the fiber diameter was 90 ± 10 nm; the pore size of the fiber network obtained by incubation for 8 hrs was 215 μm, and the fiber diameter was 110 ± 10 nm; the pore size of the fiber network obtained by incubation for 19 hrs was 218 μm, and the fiber diameter was 120 ± 10 nm. The results show that the pore size of the fiber network prepared under the condition of incubation for 2-19 hrs is similar, being between 214-218 μm; and with the increase of the incubation time from 2 hrs to 8-19 hrs, thicker collagen fibers can be prepared.

[0157] 3. Thermal stability characterization of the prepared collagen fibers

[0158] Experimental procedure: same as Example 1.

[0159] Experimental results: The results of the DSC experiment are shown in Table 5. Figure 10 As shown in Table 5, the denaturation temperature of the collagen fibers obtained by incubation for 2 hrs was 104.6 °C; the denaturation temperature of the collagen fibers obtained by incubation for 8 hrs was 105.4 °C; the denaturation temperature of the collagen fibers obtained by incubation for 19 hrs was 106.8 °C. The results show that with the increase of the incubation time from 2 hrs to 8-19 hrs, the stability of the prepared collagen fibers slightly increases.

[0160] 4. Enzymatic hydrolysis experiment of the prepared collagen fibers

[0161] Experimental procedure: same as Example 1.

[0162] The experimental results are shown in Table 5: the enzymatic hydrolysis rate of the collagen fibers prepared in this example at 48 h of enzymatic hydrolysis was 34%-50%, indicating that the reconstituted collagen fibers have certain anti-enzymatic hydrolysis ability, and with the increase of the incubation temperature, the anti-enzymatic hydrolysis ability of the collagen fibers is enhanced.

[0163] Table 5 Enzymatic hydrolysis experiment results of the collagen fibers prepared in this example

[0164] Incubation time Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 2 hrs 5.0 2.5 50％ 8 hrs 5.0 2.7 46％ 19 hrs 5.0 3.3 34％

[0165] Example 6 Preparation of collagen fibers 6

[0166] 1. Preparation of collagen fibers at different incubation temperatures under the condition of pH 8.0

[0167] (1) The collagen powder was dissolved in water, 0.5 M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a collagen solution, and the concentration of the obtained collagen solution was 3.6 mg / mL;

[0168] (2) Add pH 8.0, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution (volume ratio of collagen solution to disodium hydrogen phosphate / sodium dihydrogen phosphate solution is 1:2) to the collagen solution, stand for 1.5 hrs, then centrifuge, collect the collagen precipitate;

[0169] (3) Disperse the obtained collagen precipitate with pH 8.0, 20 mM disodium hydrogen phosphate / sodium dihydrogen phosphate solution, reconstitute to form a collagen solution with a concentration of 3 mg / mL;

[0170] (4) Incubate the reconstituted collagen solution at 10°C, 17°C, and 25°C for 19 hrs;

[0171] (5) Homogenize the incubated collagen at 4°C at a speed of 10,000 rpm for 20 min;

[0172] (6) Screen the homogenized collagen using a 40-mesh screen;

[0173] (7) Collect the screened collagen solution, centrifuge, and obtain collagen fibers.

[0174] 2. Scanning electron microscope characterization of the prepared collagen fibers

[0175] Experimental procedure: same as Example 1.

[0176] Experimental results: the results of scanning electron microscopy of the collagen fibers prepared at different incubation temperatures are shown in Figure 11 , the fiber diameter obtained at 10°C is 90 ± 10 nm, the fiber diameter obtained at 17°C is 120 ± 10 nm, and the fiber diameter obtained at 25°C is 120 ± 10 nm. The results show that the incubation temperature has a significant effect on the collagen fibers, and thicker collagen fibers can be prepared at 17-25°C than at 10°C.

[0177] 3. Thermal stability characterization of the prepared collagen fibers

[0178] Experimental procedure: same as Example 1.

[0179] Experimental results: the results of DSC experiments are shown in Figure 12 , the denaturation temperature of the collagen fibers obtained at 10°C is 104.5°C, the denaturation temperature of the collagen fibers obtained at 17°C is 106.8°C, and the denaturation temperature of the collagen fibers obtained at 25°C is 106.8°C. The results show that the stability of the prepared collagen fibers increases as the incubation temperature increases from 10°C to 17-25°C.

[0180] 4. Enzymatic digestion experiment of the prepared collagen fibers

[0181] Experimental procedure: same as Example 1.

[0182] The experimental results are shown in Table 6: the enzymolysis rate of the collagen fibers prepared in this example was 32%-50% after 48h of enzymolysis, indicating that the reconstructed collagen fibers had certain anti-enzymolysis ability, and the anti-enzymolysis ability of the collagen proteins was enhanced with the increase of the incubation temperature.

[0183] Table 6 Experimental results of the enzymolysis of the collagen protein fibers prepared in this example

[0184] Incubation temperature Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate 10℃ 5.0 2.5 50％ 17℃ 5.0 3.3 34％ 25℃ 5.0 3.4 32％

[0185] Example 7 Cell proliferation experiment of collagen protein fibers

[0186] HFF-1 human fibroblasts were cultured in high glucose DMEM medium containing 10% v / v fetal bovine serum and 1% v / v penicillin at 37°C, 5% CO2. The collagen protein fiber samples prepared by incubation at 17°C under different pH (6, 7.4, 8) conditions for 19 hrs were placed in DMEM high glucose culture solution, soaked at 37°C for 72 hrs, and extracts were prepared according to the standard cytotoxicity test ISO 10993-5. The cells were digested with 0.25% w / w trypsin, seeded in a 96-well plate at a density of 7000 per well, incubated at 37°C and 5% CO2 for 24 hrs, and then 100 μL / well of the extract of different samples was added to replace the culture medium, and incubated for 24 hrs, 48 hrs, and 72 hrs, respectively, followed by the addition of 10 μL of Cell Counting Kit-8, and incubation at 37°C for 1-3 hrs. The base medium was used as a blank control (n=6), and the OD value at 450 nm was read.

[0187] The cell survival rate was calculated as follows: cell survival rate (%) = (A-C) / (B-C) x 100%.

[0188] The experimental results are shown in Table 6: the enzymolysis rate of the collagen fibers prepared in this example was 32%-50% after 48h of enzymolysis, indicating that the reconstructed collagen fibers had certain anti-enzymolysis ability, and the anti-enzymolysis ability of the collagen proteins was enhanced with the increase of the incubation temperature. Figure 17 As shown in Table 6, the 3D reconstructed collagen protein fibers had no cytotoxicity and significantly promoted the proliferation of HFF-1 cells.

[0189] Comparative Example

[0190] 1. Comparative Example 1

[0191] The collagen protein powder was dissolved in water, 0.5M acetic acid was added, the pH was adjusted to 3.0-5.0, and a clear collagen protein solution was obtained, with a collagen protein concentration of 3.6 mg / mL.

[0192] 2. Comparative Example 2

[0193] The collagen powder was dissolved in water, 0.5M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a clear collagen solution with a collagen concentration of 3.6 mg / mL; 20 mM sodium phosphate dibasic / sodium phosphate monobasic solution (pH 6.0) was added to the prepared collagen solution, and the volume ratio of the collagen solution to the sodium phosphate dibasic / sodium phosphate monobasic solution was 1:2. After standing for 1.5 hrs, the collagen precipitate was collected by centrifugation.

[0194] 3. Comparative Example 3

[0195] The collagen powder was dissolved in water, 0.5M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a clear collagen solution with a collagen concentration of 3.6 mg / mL; 20 mM sodium phosphate dibasic / sodium phosphate monobasic solution (pH 7.4) was added to the prepared collagen solution, and the volume ratio of the collagen solution to the sodium phosphate dibasic / sodium phosphate monobasic solution was 1:2. After standing for 1.5 hrs, the collagen precipitate was collected by centrifugation.

[0196] 4. Comparative Example 4

[0197] The collagen powder was dissolved in water, 0.5M acetic acid was added, and the pH was adjusted to 3.0-5.0 to obtain a clear collagen solution with a collagen concentration of 3.6 mg / mL; 20 mM sodium phosphate dibasic / sodium phosphate monobasic solution (pH 8.0) was added to the prepared collagen solution, and the volume ratio of the collagen solution to the sodium phosphate dibasic / sodium phosphate monobasic solution was 1:2. After standing for 1.5 hrs, the collagen precipitate was collected by centrifugation.

[0198] 5. Scanning electron microscopy characterization of the collagen fibers prepared in Comparative Examples 1-4

[0199] Experimental procedure: same as in Example 1.

[0200] Experimental results: the results of the scanning electron microscopy of the collagen fibers prepared at different incubation temperatures are shown in Table 6. Figures 13-16 The pore size and diameter of the fibers prepared in Comparative Example 1 were not uniform; the diameter of the fibers prepared in Comparative Example 2 was 40 ± 10 nm; the diameter of the fibers prepared in Comparative Example 3 was 70 ± 10 nm; and the diameter of the fibers prepared in Comparative Example 4 was 70 ± 10 nm.

[0201] 6. Enzymatic hydrolysis experiment of the prepared collagen fibers

[0202] Experimental procedure: same as in Example 1.

[0203] The experimental results are shown in Table 7: the enzymatic hydrolysis rate of the collagen fibers prepared in Comparative Examples 1-4 was as high as 72%-98% at 48 h of enzymatic hydrolysis, indicating that the anti-enzymatic hydrolysis ability of the collagen or fibers prepared in the comparative examples was poor.

[0204] Table 7 Enzymolysis experimental results of collagen fibers prepared by the comparative examples

[0205] Initial mass (mg) Mass after enzymolysis (mg) Enzymolysis rate Comparative Example 1 5.0 0.1 98％ Comparative Example 2 5.0 0.7 86％ Comparative Example 3 5.0 1.3 74％ Comparative Example 4 5.0 1.4 72％

[0206] The results of the above comparative examples show that the collagen fibers prepared by the comparative examples are unevenly distributed and have a fast enzymolysis speed. In comparison with the comparative examples, the present application adopts a series of in vitro 3D reconstruction processes of collagen fibers, such as self-assembly, resolubilization, incubation, homogenization, and sieving, to prepare collagen fibers with precisely controlled morphology; the collagen fibers are uniformly distributed and closely and orderly arranged, and have a fiber diameter of 60-120 nm; the collagen fibers prepared by the present application exhibit good stability and significantly enhanced anti-enzymolysis capacity, and have wide application potential in the fields of implant agents, artificial skin, artificial cartilage, hemostatic sponges, scaffold materials, medical devices, and the like.

[0207] The above description is only the details of the individual exemplary embodiments of the present application, and the present application can have various modifications and changes according to specific preparation conditions in practical application for those skilled in the art, and is not used to limit the present application. Any modification within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing long-lasting collagen fibers, characterized in that, The method is as follows: (1) Dissolve collagen in acetic acid and adjust the pH to 3.0-5.0 to obtain a collagen solution of 0.1-5 mg / mL; add 2 times the volume of 10-100 mM disodium hydrogen phosphate and sodium dihydrogen phosphate solution with pH 6.0-9.0 to the prepared collagen solution, let stand for 0.1-8.0 hrs, centrifuge, and collect the collagen precipitate; (2) The collagen precipitate was reconstituted with a 10-100mM disodium hydrogen phosphate and sodium dihydrogen phosphate solution with a pH of 6.0-9.0 to form a collagen solution; (3) Incubate the reconstituted collagen solution at 4-25℃ for 1-24 hours; homogenize at 1000-10000 rpm for 0.1-2 hours. Use a 40-60 mesh sieve to sieve the homogenized collagen; collect the sieved collagen solution, centrifuge, and obtain long-lasting collagen fibers.

2. The long-lasting collagen fibers prepared by the method described in claim 1.

3. The application of the long-acting collagen fiber as described in claim 2 in the preparation of implants, artificial skin, artificial cartilage, hemostatic sponges, and scaffold materials.

4. The application of the long-lasting collagen fiber as described in claim 2 in the preparation of medical devices.

5. A collagen implant, characterized in that, The implant is prepared by dissolving the long-acting collagen fibers described in claim 2 in physiological saline phosphate buffer containing 0.3% lidocaine hydrochloride.

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