CN110950333A - Method for preparing graphene oxide material - Google Patents
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Abstract
The invention provides a method for preparing a graphene oxide material, which comprises the following steps: 1) preparing graphene oxide: sequentially adding concentrated sulfuric acid, a solid mixture of graphite powder and ammonium nitrate and potassium permanganate into an ice water bath, stirring for reaction, adding hydrogen peroxide, filtering and washing; 2) dispersing the obtained graphene oxide in water to obtain a graphene oxide dispersion liquid; 3) adding a collagen solution into the obtained graphene oxide dispersion liquid, uniformly blending, and carrying out vacuum freeze drying to obtain a blend; 4) dissolving the obtained blend in acetic acid, stirring uniformly, centrifuging, freeze-drying and cross-linking. The preparation method of the graphene oxide material provided by the invention can prepare the graphene oxide material which is high in conductivity, good in stitching strength and resistant to degradation in one step, is simple to operate, stable in process, rich in raw material source and low in cost, and is easy to industrialize.
Description
Technical Field
The invention belongs to the field of medical biomaterials, medical instruments and nerve repair, and particularly relates to a method for preparing a graphene oxide material.
Background
The graphene is formed by passing sp through carbon atoms2Hybridization forms a hexagonal honeycomb of two-dimensional nanostructures. The unique structure endows graphene with unique physical sumElectrical property, excellent mechanical property, conductivity, larger specific surface area, good biocompatibility and the like. In addition, the graphene has good antibacterial activity and can remarkably promote the growth, differentiation and proliferation of stem cells.
The pure collagen material has poor mechanical property and electrical conductivity, high degradation rate and no antibacterial property, and is not beneficial to the application of the pure collagen material in tissue engineering. The graphene material is doped with collagen to prepare the conductive nerve repair material, however, the graphene and collagen doped composite material provided in the prior art cannot study the difference between the conductive performance and the mechanical performance, and cannot significantly improve the degradation resistance of the collagen.
The three-dimensional graphene-collagen composite scaffold is prepared by CN107432952A through a chemical vapor deposition method, an electrostatic spinning method is adopted for film forming, chemical crosslinking is adopted for crosslinking, the preparation process is complex, chemical residues are likely to exist, and the method is not suitable for industrial production. CN109833516A and CN109453430A provide a graphene catheter and a collagen-graphene oxide material containing a hydroxyapatite coating, respectively, and although both contain graphene, they fail to provide a material having better conductive effect, repair capability and good degradation resistance.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a preparation method of a graphene oxide material which is naturally absorbable, has good biocompatibility, certain three-dimensional pores, plasticity and mechanical strength and good degradation resistance, and can prolong the nerve repair time when being used for nerve repair, and the method comprises the following steps:
1) preparing graphene oxide: sequentially adding concentrated sulfuric acid, a solid mixture of graphite powder and ammonium nitrate and potassium permanganate into an ice water bath, stirring for reaction, adding hydrogen peroxide, filtering and washing;
2) dispersing the graphene oxide obtained in the step 1) in water to obtain a graphene oxide dispersion liquid;
3) adding a collagen solution into the graphene oxide dispersion liquid obtained in the step 2), uniformly blending, and performing vacuum freeze drying to obtain a blend;
4) dissolving the blend obtained in the step 3) in acetic acid, stirring uniformly, centrifuging, freeze-drying and crosslinking.
In one embodiment, the graphene oxide in step 1) may be prepared by the following method:
under the condition of ice-water bath, graphite and NaNO are mixed3Put into a three-neck flask, and concentrated sulfuric acid is slowly added into the three-neck flask. Starting stirring, and keeping the temperature in the three-neck flask to be lower than 5 ℃, adding KMnO4Adding into three-neck flask slowly at intervals of 12min for three times, and raising temperature until the temperature in three-neck flask reaches 35 deg.C, and maintaining at the temperature for 6 h. After the reaction is finished, adding deionized water into the three-neck flask, and continuously heating to 95-100 ℃. Finally, pouring the solution in the three-neck flask into a 2000mL beaker, adding deionized water, and gradually dropwise adding a large amount of H2O2Removing excess KMnO4And finally, the solution is bright yellow and no bubbles are generated. Standing until complete precipitation, pouring off the supernatant, and washing twice with hydrochloric acid solution and deionized water respectively. And finally, centrifuging the product to be neutral, freezing and drying to obtain the product, and sealing and storing.
Further, in the blend obtained in the step 2), the mass ratio of the graphene oxide to the collagen is 0.5-20%.
Further, in the blend obtained in the step 2), the mass ratio of the graphene oxide to the collagen is 1% to 16%, preferably 1% to 4%, and particularly preferably 1%.
Further, the size of the graphene oxide in the step 2) is 2-100 μm, preferably 2-50 μm, and more preferably 2 μm.
Further, the mass concentration of the graphene oxide dispersion liquid in the step 2) is 10-25mg/ml, and preferably 20 mg/ml.
Further, the collagen solution obtained by dissolving collagen in acetic acid in the step 3) has a mass fraction of 0.5% to 0.8%, preferably 0.6% to 0.7%, and more preferably 0.68%.
Further, the concentration of acetic acid in the step 4) is 0.2-0.8M, preferably 0.5M.
Further, the blend in the step 4) is dissolved in acetic acid, so that the mass volume concentration of the collagen is 3-10%, preferably 4-8%, and more preferably 5%.
Further, the centrifugation in the step 4) is carried out at 2-10 ℃ and at 3000-5000rpm for 40-120 min. Preferably, the centrifugation condition is 4 ℃ of temperature, 4000rpm of rotation speed and 60min of time.
Further, the crosslinking treatment in the step 4) comprises heavy dehydration crosslinking (DHT) treatment, and the treatment is carried out at 110 ℃ for 1-4 h. In other embodiments, the crosslinking treatment may also be selected from glutaraldehyde vapor crosslinking, ultraviolet crosslinking, or high temperature vacuum crosslinking.
Further, in step 3) of the method, freeze-drying is divided into two steps of pre-freezing and freeze-drying, wherein the pre-freezing step is as follows: after the mould is fixed, the sample is slowly injected along the axial direction, and is frozen and formed. The speed of the uniform speed elevator is 10 r/min, the angle is adjusted for 9, the time interval is 5s, the temperature stops decreasing after being reduced to minus 60 ℃, the elevator is taken out when the temperature is reduced to minus 80 +/-10 ℃, and the temperature is adjusted to minus 20 ℃ in a freezing chamber of a refrigerator for more than 1 hour.
The lyophilization procedure was as follows:
further, the sterilization is also included in step 4) of the above method, and the sterilization method is ethylene oxide gas sterilization or cobalt 60 irradiation sterilization, preferably ethylene oxide gas sterilization.
In one embodiment, the preparation method of graphene oxide includes the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) respectively taking a graphene oxide aqueous solution, water and a collagen solution with the mass fraction of 0.68 percent dissolved in a 0.5M acetic acid solution according to the mass ratio of the graphene oxide to the collagen, blending, mechanically stirring, homogenizing uniformly, vacuumizing, and freeze-drying to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
On the other hand, the invention also provides the graphene oxide material prepared by the method.
On the other hand, the invention also provides application of the graphene oxide material and/or the graphene oxide material prepared by the method in preparation of degradation-resistant cell culture materials and/or nerve repair materials, such as substrate materials for cell growth and proliferation, induced growth materials for damaged nerve cells and the like.
Preferably, the graphene oxide material is a porous material, the pore directions of pores are the same, and the pores are 50-220 microns.
Further, the nerve repair material comprises a nerve conduit, a nerve repair scaffold and the like.
Further, the conductivity of the graphene oxide material prepared by the method is measured by an RTS-8 four-probe tester, and is not lower than 3.5 multiplied by 10-3S/m, preferably not less than 3.9X 10-3S/m, more preferably not less than 4.8X 10-3S/m, more preferably not less than 10X 10-3S/m。
Further, the repair strength of the graphene oxide material prepared by the method is measured by an MED-01 medical packaging performance tester, and the set condition is 10 mm/min. Experiments show that the maximum repair strength of the material is not less than 1.5N, preferably not less than 1.8N, more preferably not less than 2.2N, more preferably not less than 2.3N, more preferably not less than 2.5N.
Further, the graphene oxide material prepared by the method has the days for beginning to generate fragmentation degradation in Phosphate Buffered Saline (PBS) with pH 7 of not less than 1 day, preferably not less than 4 days; preferably, the total degradation days of the graphene oxide material provided by the application in the PBS solution are not less than 10 days, and more preferably, not less than 14 days.
The invention has the beneficial effects that:
the preparation method of the graphene oxide material provided by the invention can prepare the graphene oxide material which is high in conductivity, good in stitching strength and resistant to degradation in one step, is simple to operate, stable in process, rich in raw material source and low in cost, and is easy to industrialize. Experiments show that the preparation method provided by the invention can show better conductive effect, suture ability and degradation resistance under the condition of proper mass ratio of collagen to graphene oxide, so that the preparation method is closer to an ideal biological cell culture, repair and growth induction material, and particularly when the preparation method contains 5% of collagen by mass and 1% (mass ratio of graphene oxide to collagen) of graphene oxide, the conductive effect, suture ability and degradation resistance of the preparation method are relatively better.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
fig. 1 is a Scanning Electron Microscope (SEM) image of a graphene oxide material having a collagen mass-volume concentration of 5% and a graphene oxide-to-collagen mass ratio of 1%;
fig. 2 is a Scanning Electron Microscope (SEM) image of pure collagen material.
Detailed Description
In order to more clearly explain the overall concept of the present application, the following detailed description is given by way of example. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. It will be apparent, however, to one skilled in the art, that the present application may be practiced without one or more of these specific details. In other instances, well-known features of the art have not been described in order to avoid obscuring the present application.
Unless otherwise specified, the starting materials and reagents in the following examples are all commercially available; in the performance test, RTS-8 four-probe tester is adopted as experimental equipment for measuring the conductivity, MED-01 medical package performance tester is adopted for measuring the suture strength, Phosphate Buffered Saline (PBS) for degradation experiment is provided by national pharmaceutical group chemical reagent limited, pH: 7.4.
in the following examples, the following steps were used for freeze-drying:
first-step pre-freezing: after the mould is fixed, the sample is slowly injected along the axial direction, and is frozen and formed. The speed of the uniform speed elevator is 10 r/min, the angle is adjusted for 9, the time interval is 5s, the temperature stops decreasing after being reduced to minus 60 ℃, the elevator is taken out when the temperature is reduced to minus 80 +/-10 ℃, and the temperature is adjusted to minus 20 ℃ in a freezing chamber of a refrigerator for more than 1 hour. And a second step of freeze-drying, wherein the freeze-drying process is as follows:
in the following examples DHT (heavy dehydration) crosslinking was used, wherein the crosslinking conditions were: the temperature is 110 ℃, and the fixing time is 2 h.
In the following examples, the method for oxidizing graphene in step 1) is as follows:
under the condition of ice-water bath, 5g of 300-mesh graphite and 5g of NaNO are mixed3The mixture was placed in a 500mL three-necked flask, and 150mL of concentrated sulfuric acid was slowly added to the three-necked flask. Starting stirring, and keeping the temperature in the three-neck flask below 5 ℃, adding 15g of KMnO4Adding into three-neck flask slowly at intervals of 12min for three times, and raising temperature until the temperature in three-neck flask reaches 35 deg.C, and maintaining at the temperature for 6 h. After the reaction is completed, 200mL of deionized water is added into the three-neck flask, and the temperature is continuously increased to 95-100 ℃. Finally, pouring the solution in the three-neck flask into a 2000mL beaker, adding 700mL deionized water, and gradually dropwise adding a large amount of H2O2Removing excess KMnO4And finally, the solution is bright yellow and no bubbles are generated. Standing until complete precipitation, pouring off the supernatant, and washing twice with 500mL of hydrochloric acid solution and deionized water. And finally, centrifuging the product to be neutral, freezing and drying to obtain the product, and sealing and storing.
Example 1
The method for preparing the graphene oxide material provided by the embodiment comprises the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) 0.3196ml of graphene oxide aqueous solution, 5.6804ml of water and 94ml of collagen solution with the mass fraction of 0.68 percent dissolved in 0.5M acetic acid solution are blended, mechanically stirred and homogenized evenly, and then vacuumized and freeze-dried to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent and the mass ratio of the graphene oxide to the collagen is 1 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
Example 2
The method for preparing the graphene oxide material provided by the embodiment comprises the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) 0.6292ml of graphene oxide aqueous solution, 5.3608ml of water and 95ml of collagen solution with the mass fraction of 0.68 percent dissolved in 0.5M acetic acid solution are blended, mechanically stirred and homogenized evenly, and subjected to vacuum pumping and freeze drying to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent and the mass ratio of the graphene oxide to the collagen is 2 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
Example 3
The method for preparing the graphene oxide material provided by the embodiment comprises the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) 1.2784ml of graphene oxide aqueous solution, 4.7216ml of water and 94ml of collagen solution with the mass fraction of 0.68 percent dissolved in 0.5M acetic acid solution are blended, mechanically stirred and homogenized evenly, and then vacuumized and freeze-dried to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent and the mass ratio of the graphene oxide to the collagen is 4 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
Example 4
The method for preparing the graphene oxide material provided by the embodiment comprises the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) 2.5568ml of graphene oxide aqueous solution, 3.4432ml of water and 94ml of collagen solution with the mass fraction of 0.68 percent dissolved in 0.5M acetic acid solution are blended, mechanically stirred and homogenized evenly, and then vacuumized and freeze-dried to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent and the mass ratio of the graphene oxide to the collagen is 8 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
Example 5
The method for preparing the graphene oxide material provided by the embodiment comprises the following steps:
(1) preparing graphene oxide;
(2) dispersing graphene oxide in pure water to prepare a 20mg/ml graphene oxide aqueous solution;
(3) 5.1136ml of graphene oxide aqueous solution, 0.8864ml of water and 94ml of collagen solution with the mass fraction of 0.68 percent dissolved in 0.5M acetic acid solution are blended, mechanically stirred and homogenized evenly, and then vacuumized and freeze-dried to obtain a blend;
(4) dissolving the obtained blend in 0.5M acetic acid solution to ensure that the final mass volume concentration of the collagen is 5 percent and the mass ratio of the graphene oxide to the collagen is 16 percent, and mechanically stirring and homogenizing uniformly;
(5) centrifuging, controlling the condition temperature at 4 ℃, rotating at 4000rpm, and keeping the time for 60 min;
(6) and (4) taking the precipitate, injecting the precipitate into a mold, and carrying out DHT crosslinking after freeze drying.
Comparative example 1
Comparative example 1 provides a pure collagen material having a collagen concentration of 5% by mass in 0.5M acetic acid, without graphene oxide, and the rest of the preparation method is the same as in example 1.
Example 6: performance testing
The conductivity of the graphene oxide material obtained in each of the above examples in a wet state was measured by a four-probe method, and the results obtained by comparing pure collagen without graphene oxide and pure graphene oxide obtained in step 1) are shown in table 1:
table 1 conductivity of each of the exemplary materials
| Examples of the invention | Collagen concentration | Mass ratio of | Conductivity (. times.10)-3S/m) | p value |
| Pure collagen | 5％ | - | 2.04 | - |
| Example 1 | 5％ | And (3) graphene oxide: 1% of collagen | 10.68 | 0.021 |
| Example 2 | 5％ | And (3) graphene oxide: 2% of collagen | 4.82 | 0.00002 |
| Example 3 | 5％ | And (3) graphene oxide: collagen protein 4% | 4.88 | 0.012 |
| Example 4 | 5％ | And (3) graphene oxide: collagen protein 8% | 3.91 | 0.026 |
| Example 5 | 5％ | And (3) graphene oxide: 16% of collagen | 3.58 | 0.022 |
| Pure graphene oxide | - | - | 8.34 | - |
Note: p < 0.05 shows significant differences compared to pure collagen, and p < 0.01 shows very significant differences compared to pure collagen
As can be seen from table 1, the graphene oxide materials provided in examples 1 to 5 all show higher conductivity than pure collagen, and all show significant differences compared to pure collagen, wherein example 2 has very significant differences compared to pure collagen. Meanwhile, the conductivity of example 1 is better than that of pure graphene oxide compared to other examples, and thus it can be shown that the addition of a proper amount of collagen to graphene oxide can have a synergistic effect on its conductivity.
The graphene oxide materials obtained in the examples were tested for their suture ability using the following method: the material obtained in the above embodiments is made into a tubular shape with the same size, one end of the tubular shape is fixed, a 5-0 nylon monofilament suture is used for puncturing the other end of the tubular shape which is 2mm away from the edge, the suture is connected with a tensile machine and is stretched at the speed of 10mm/min, the maximum bearing force when the tubular shape is instantly pulled off is recorded, namely the suture strength of the material, and the obtained result is shown in table 2:
TABLE 2 seam Strength of the materials of the examples
| Examples of the invention | Collagen concentration | Composition of matter | Stitching Strength (N) | p1 value | p2 value |
| Pure collagen | 5％ | - | 1.78±0.39 | - | - |
| Example 1 | 5％ | And (3) graphene oxide: 1% of collagen | 2.37±0.44 | 0.036 | - |
| Example 2 | 5％ | And (3) graphene oxide: collagen2% of protein | 2.26±0.35 | 0.022 | 0.640 |
| Example 3 | 5％ | And (3) graphene oxide: collagen protein 4% | 2.52±0.47 | 0.003 | 0.059 |
| Example 4 | 5％ | And (3) graphene oxide: collagen protein 8% | 1.87±0.35 | 0.630 | 0.055 |
| Example 5 | 5％ | And (3) graphene oxide: 16% of collagen | 1.58±0.36 | 0.321 | 0.006 |
Note: p1 < 0.05 indicated significant differences compared to pure collagen, p1 < 0.01 indicated very significant differences compared to pure collagen; p2 < 0.05 indicated significant differences compared to example 1, p2 < 0.01 indicated very significant differences compared to example 1
As can be seen from table 2, the graphene oxide materials obtained in examples 1 to 3 have better suture strength than pure collagen, and have significant difference compared with pure collagen, showing better advantage in suture performance. Among them, example 3 showed the highest seam strength, but at the same time, example 1 showed a seam strength that is less different from example 3, and other examples except example 5 were not significantly different from example 1.
The material obtained in each of the above examples was cut into squares of the same size while immersed in 200ml of PBS buffer at pH 7, and the time at which the material began to crack was observed and counted, wherein the results obtained for pure collagen are shown in table 3:
TABLE 3 PBS onset fragmentation times for each of the exemplary materials
| Examples of the invention | Collagen concentration | Composition of matter | Time to begin disintegration (day) |
| Pure collagen | 5％ | - | 4.5 (complete degradation) |
| Example 1 | 5％ | And (3) graphene oxide: 1% of collagen | 5 |
| Example 2 | 5％ | And (3) graphene oxide: 2% of collagen | 4 |
| Example 3 | 5％ | And (3) graphene oxide: collagen protein 4% | 2.5 |
| Example 4 | 5％ | And (3) graphene oxide: collagen protein 8% | 1.5 |
| Example 5 | 5％ | And (3) graphene oxide: 16% of collagen | 0.5 |
The experimental result shows that the pure collagen begins to generate fragmentation in the PBS solution in less than 1 day and is completely degraded in about 4.5 days. While examples 1-4 began to show chipping after 1.5 days, especially example 1, started to show cracking on day 5 and was completely degraded on day 14, showing excellent resistance to degradation. In addition, although the suture strength of the graphene oxide material with the content of 4% in example 3 is slightly increased, there is no significant difference compared with the content of 1%, and the in vitro degradation starting rupture time is shorter, and in combination with the consideration that the content of graphene oxide is too high, which may cause unexpected toxicity, example 1 is preferred.
By combining the above, a material with better conductive effect, suture ability and degradation resistance can be obtained by adding a certain amount of collagen into graphene oxide, so that the material is closer to an ideal biological cell culture, repair and induced growth material, and particularly, when the material contains 5% by mass of collagen and 1% by mass of graphene oxide (mass ratio of graphene oxide to collagen), the conductive effect, suture ability and degradation resistance of the material are relatively better.
The above description is only an example of the present application and is not intended to limit the present application. Various modifications and changes may occur to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims (10)
1. A method for preparing a graphene oxide material, comprising the steps of:
1) preparing graphene oxide: sequentially adding concentrated sulfuric acid, a solid mixture of graphite powder and ammonium nitrate and potassium permanganate into an ice water bath, stirring for reaction, adding hydrogen peroxide, filtering and washing;
2) dispersing the graphene oxide obtained in the step 1) in water to obtain a graphene oxide dispersion liquid;
3) adding a collagen solution into the graphene oxide dispersion liquid obtained in the step 2), uniformly blending,
and vacuum freeze-drying to obtain a blend;
4) dissolving the blend obtained in the step 3) in acetic acid, stirring uniformly, centrifuging, freeze-drying and crosslinking.
2. The method according to claim 1, wherein the mass ratio of the graphene oxide to the collagen in the blend obtained in the step 2) is 0.5-20%.
3. The method according to claim 2, wherein the mass ratio of the graphene oxide to the collagen in the blend obtained in the step 2) is 1-16%.
4. The method according to claim 1, wherein the graphene oxide in the step 2) has a size of 2-100 μm.
5. The method according to claim 1, wherein the mass concentration of the graphene oxide dispersion liquid in the step 2) is 10-25 mg/ml.
6. The method as claimed in claim 1, wherein the collagen solution in step 3) is a solution obtained by dissolving collagen in acetic acid, and the mass fraction of the collagen solution is 0.5% -0.8%.
7. The method as claimed in claim 1, wherein the concentration of acetic acid in the step 4) is 0.2-0.8M.
8. The method as claimed in claim 1, wherein the blend in step 4) is dissolved in acetic acid so that the collagen concentration is 3-10% by mass/volume.
9. The method as claimed in claim 1, wherein the centrifugation in step 4) is performed at 2-10 deg.C and 3000-5000rpm for 40-120 min.
10. The method as claimed in claim 1, wherein the crosslinking treatment in the step 4) comprises a heavy dehydration crosslinking treatment under the condition of 110 ℃ for 1-4 h.
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