CN107118361A - A kind of fibroin albumen/carboxymethyl chitosan plural gel and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of fibroin albumen/carboxymethyl chitosan plural gel and preparation method thereof.The plural gel is mainly made up of the composition of fibroin albumen and carboxymethyl chitosan, and mass ratio is (0.05~2)：1；Method is first to be handled to obtain fibroin fiber with cocoon layer, then prepares silk fibroin solution, carboxymethyl chitosan solution is made, and silk fibroin solution and carboxymethyl chitosan solution are well mixed and obtain mixed solution；Add a certain amount of crosslinking agent, then solution is stirred to pour into immediately after the short time in mould on magnetic stirring apparatus stood；Immerse again in ethanol water, finally give fibroin/carboxymethyl chitosan plural gel.The present invention prepares the gel with stronger mechanical property and better stability with better simply technique, shorter time, and the preparation method for improvement fibroin albumen plural gel provides new thinking, has also further expanded the application approach of fibroin albumen.

Description

A kind of silk fibroin/carboxymethyl chitosan composite gel and preparation method thereof

technical field

The invention belongs to a processing technology of a regenerated silk fibroin solution, in particular to a silk fibroin/carboxymethyl chitosan composite gel and a preparation method thereof.

Background technique

Recently, gel materials have emerged in the field of biomedicine, and have been used in tissue engineering scaffolds, drug delivery carriers, and wound dressings, and have been favored by researchers. , natural polymer biomaterial with good biocompatibility and high safety performance. The regenerated silk fibroin solution is a thermodynamically unstable lysizing sol, which will transform from a random coil structure to a β-sheet structure and spontaneously transform into a gel state without any external action. Generally, this transformation takes 1 to 2 months, and the mechanical properties of the obtained gel are extremely poor, which greatly limits the application of silk fibroin gel as an excellent biomaterial. The modification of silk fibroin gel materials has become a research hotspot, mainly to improve the mechanical properties and stability of the gel. But at present, there is no silk fibroin gel prepared by a method that can exhibit higher mechanical properties with a simpler process and a shorter gel time. Carboxymethyl chitosan is a water-soluble chitosan derivative. Carboxymethyl chitosan composite materials have the advantages of guiding nerve regeneration, preventing tissue adhesion, promoting wound healing, and reducing scar formation. The resulting gel material is too strong in water absorption, brittle and brittle, making it difficult to apply on a large scale in the field of clinical medicine.

1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCL) has the characteristics of activating carboxyl groups, because of its mild reaction conditions, high yield and good selectivity, it is suitable for Environmentally friendly and other characteristics, it is widely used as a condensation agent and crosslinking agent in polymer modification ([J] Desalination, 2008, 234: 134-143).

Contents of the invention

In order to solve the problems in the background technology, the present invention aims to provide a silk fibroin/carboxymethyl chitosan composite gel and its preparation method, the process is simple, time-consuming, and the composite gel has excellent mechanical properties .

The technical scheme adopted in the present invention comprises the steps:

One, a kind of silk fibroin/carboxymethyl chitosan composite gel:

The composite gel is mainly composed of silk fibroin and carboxymethyl chitosan.

In the composite gel, the mass ratio of silk fibroin and carboxymethyl chitosan is (0.05-2):1, preferably (0.5-2):1.

Two, a preparation method of silk fibroin/carboxymethyl chitosan composite gel:

The silk fiber obtained by degumming was dissolved in LiBr solution, and the regenerated silk solution was obtained by dialysis, which was then mixed with carboxymethyl chitosan solution, and then the cross-linking agent 1-ethyl-(3 -Dimethylaminopropyl) carbodiimide hydrochloride, finally prepared into silk fibroin/carboxymethyl chitosan composite hydrogel under the effect of ethanol.

The specific process of the method is as follows:

(1) Treat with cocoon layer to obtain silk fibroin fiber, then prepare and obtain silk fibroin solution;

(2) carboxymethyl chitosan is dissolved in water to obtain carboxymethyl chitosan solution;

(3) the silk fibroin solution obtained in the step (1) is mixed with the carboxymethyl chitosan solution obtained in the step (2) to obtain a mixed solution;

(4) Add a certain amount of cross-linking agent to the mixed solution obtained in step (3), then pour the solution into the mold immediately after stirring on a magnetic stirrer for a short time;

(5) Immerse in the aqueous ethanol solution again to finally obtain the silk fibroin/carboxymethyl chitosan composite gel.

Described step (1) is specifically:

(1) Degumming the cocoon layer in 0.5% Na ₂ CO ₃ boiling aqueous solution twice for 30 minutes each time, washing the obtained silk fibers with deionized water, wringing them dry, and drying in an oven;

(2) Place the silk fiber obtained in step (1) in a 9M LiBr solution, and dissolve it at 60°C for 4 hours. After filtering, the obtained liquid is put into a dialysis bag and dialyzed in deionized water for 4 days. water, the resulting silk fibroin solution.

The silk fibroin solution concentration of the step (1) is 0.5～10% w/v, the carboxymethyl chitosan solution concentration of the step (2) is 1～5% w/v, the step (3) The mixing volume ratio of silk fibroin solution and carboxymethyl chitosan solution is 1:1.

Finally, the mass ratio of solute silk fibroin and carboxymethyl chitosan after mixing should be in the range of (0.05-2):1. Preferably, the concentration ratio of the silk fibroin solution and the carboxymethyl chitosan solution is (0.1-2):1.

The degree of deacetylation of the carboxymethyl chitosan is 55%-99%, and the degree of substitution is 60%-99%.

Preferably, the concentration of the silk fibroin solution in the step (1) is 0.5-8% w/v, the concentration of the carboxymethyl chitosan solution in the step (2) is 1-4% w/v, and the carboxymethyl chitosan solution is 1-4% w/v. The degree of deacetylation of chitosan is 90%, and the degree of substitution is 90%.

More preferably, the concentration of the silk fibroin solution in the step (1) is 0.5-4% w/v and the concentration of the carboxymethyl chitosan solution in the step (2) is 4% w/v.

As most preferably, the concentration of the silk fibroin solution of the present invention is 4% w/v and the concentration of the carboxymethyl chitosan solution is 4% w/v, and the mixing volume ratio is 1:1, thus making the silk fibroin of the present invention The preparation of the plain and carboxymethyl chitosan solution is simpler and easier to control, the mixing is more uniform, the obtained composite gel has better molding, stronger mechanical properties, and excellent water absorption stability of the composite gel.

The molecular weight of the dialysis bag in the step (1) is 8000-14000.

In the step (4), the cross-linking agent adopts the solid powder of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCL), and the addition amount of the cross-linking agent It is 0.3-0.75% w/v of the system.

System refers to the mixed solution obtained in step (4).

Preferably, the addition of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCL) is 0.45% of the silk fibroin/carboxymethyl chitosan solution system ~0.6% w/v.

More preferably, the addition amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCL) is 0.6 of the silk fibroin/carboxymethyl chitosan solution system %w/v. Therefore, the cross-linking effect of the obtained composite gel is excellent, and the excess of the cross-linking agent will not be caused.

There are two ways to add the crosslinking agent: one is to directly add the solid powder to the system, and the other is to dissolve the solid powder in water and then add it to the system, as long as the crosslinking agent powder can be dissolved and the volume of water added must be as large as possible. as small as possible.

As a preference, the adding method of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCL) in the step (5) of the present invention is that the solid powder is dissolved in A small amount of water is added to the system, so that the distribution of the crosslinking agent in the mixed solution is more uniform, the reaction will be more rapid and efficient, and the obtained composite gel has a tighter structure and better performance.

In the described step (4), the stirring time is 20s, and the standing time is 6-12h; in the described step (5), the ethanol concentration in the ethanol aqueous solution is 60～90% v/v, and the time for immersing in the ethanol aqueous solution is 12h.

Preferably, the standing time for the solution system to be poured into the mold in the step (4) is 12 hours.

Preferably, the ethanol aqueous solution has an ethanol concentration of 75% v/v.

The product of the present invention has obtained higher mechanical properties and stable water retention performance through the tests of the examples, the elastic modulus reaches (363.94 ± 45.57) kPa, the compression recovery rate reaches 100%, and the volume in the phosphate buffer solution of pH=7.4 Relatively stable, the swelling rate is (9.32±0.09)×100%.

The invention promotes the silk fibroin/carboxymethyl chitosan to form a composite gel through the cross-linking effect of EDC·HCL and the characteristic that ethanol promotes the beta transformation of the silk fibroin. A gel with strong mechanical properties and good stability was prepared with a simpler process and a shorter time, which provided a new idea for improving the preparation method of silk fibroin composite gel, and further expanded the field of silk fibroin. Approach.

Compared with the prior art, the present invention has the following advantages and effects:

1. The process is simple: the preparation of silk fibroin composite gel in the prior art often requires methods such as organic solvents, ultrasonic treatment, and optical cross-linking, while the present invention can be completed at room temperature;

2. Short gelation time: the preparation of silk fibroin composite gel generally takes a long time in the prior art, but the present invention only needs about 1 to 2 days, which greatly improves the efficiency;

3. The cross-linking agent required by the present invention is used as an active agent, which can be easily removed by water washing, and has high biological safety;

4. The composite gel prepared by the present invention has strong mechanical properties, good water absorption stability and moisture retention, and is superior to silk fibroin composite gel prepared by other methods. The composite gel prepared by the invention can be used in the fields of medicine and sanitation, tissue engineering, environmental protection, etc., expands the application path of silk fibroin, and provides a new idea and a new method for preparing high-strength composite gel.

detailed description

The present invention will be described in further detail below through examples, and the following examples are explanations of the present invention and the present invention is not limited to the following examples.

Example 1

The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in this embodiment includes the following steps in sequence.

(1) Take by weighing 10g of shredded cocoon layer and degumming twice in _0.5 % _Na2CO3 boiling aqueous solution, each time for 30min, the obtained silk fibroin fiber is washed with deionized water and wrung dry, and dried in an oven medium drying;

(2) Put the silk fiber obtained in step (1) in 9M LiBr solution, dissolve it at 60°C for 4 hours, and put the obtained liquid into a dialysis bag (molecular weight 8000-14000) in deionized water after filtration (3) An appropriate amount of carboxymethyl chitosan (90% degree of deacetylation, 90% degree of substitution) was dissolved in water to prepare a carboxymethyl chitosan solution with a concentration of 4% w/v.

(4) the concentration obtained in the step (2) is the carboxymethyl chitosan solution of 4% w/v in the silk fibroin solution of 0.5% w/v and the concentration obtained in the step (3) by volume ratio 1: 1 Mix well to obtain a mixed solution.

(5) Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCL) in the mixed solution obtained in step (4), There are two adding methods: one is to add the solid powder directly to the system, and the other is to dissolve the solid powder in a small amount of water before adding to the system. The amount of cross-linking agent added is 0.3-0.75% w/v of the system, preferably 0.45-0.6% w/v, most preferably 0.6% w/v. The best way to add cross-linking agent is the second one.

(6) Add EDC·HCL to the silk fibroin/carboxymethyl chitosan composite solution under constant stirring by a magnetic stirrer. After 20 seconds, pour the solution into the mold and let it stand for 6-12 hours, preferably 12 hours.

(7) Immerse it in 60-90% v/v ethanol aqueous solution for 12 hours, the optimal concentration of ethanol is 75% v/v, finally obtain silk fibroin/carboxymethyl chitosan composite gel.

(8) In this example, a mechanical universal testing machine (Shimadzu, AGS-J series) was used to perform uniaxial compression (compression rate: 1 mm/min) to measure the mechanical properties of the gel. The swelling performance of the gel was tested by mass weighing method: first, the composite gel material was soaked in deionized water or phosphate buffer for ₂₄ hours to make it reach swelling equilibrium, and then the surface moisture was absorbed by filter paper, and its mass was called M1 (g), and then place the composite gel in an oven at 60°C to dry, and measure its mass as M ₂ (g). The calculation formula is as follows: swelling rate=(M ₁ -M ₂ )/M ₂ ×100%.

The elastic modulus of the composite gel reaches (29.46±16.70) kPa, and the compression recovery rate reaches 100%. The swelling rate of the gel in deionized water reaches (103.63±4.91)×100%, and the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (20.79±2.64)×100%.

Example 2

The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in this embodiment includes the following steps in sequence.

(1) Take by weighing 10g of shredded cocoon layer and degumming twice in _0.5 % _Na2CO3 boiling aqueous solution, each time for 30min, the obtained silk fibroin fiber is washed with deionized water and wrung dry, and dried in an oven medium drying;

(2) Put the silk fiber obtained in step (1) in 9M LiBr solution, dissolve it at 60°C for 4 hours, and put the obtained liquid into a dialysis bag (molecular weight 8000-14000) in deionized water after filtering After dialysis for 4 days, the water was changed continuously during the period, and the concentration of the finally obtained silk fibroin solution was 1.0% w/v.

(3) An appropriate amount of carboxymethyl chitosan (90% degree of deacetylation, 90% degree of substitution) was dissolved in water to prepare a carboxymethyl chitosan solution with a concentration of 4% w/v.

(4) the concentration obtained in the step (2) is the carboxymethyl chitosan solution of 4% w/v in the silk fibroin solution of 1.0% w/v and the concentration obtained in the step (3) by volume ratio 1: 1 Mix well to obtain a mixed solution.

(5) Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCL) in the mixed solution obtained in step (4), The addition method is to dissolve the solid powder in a small amount of water and then add it to the system. The amount of crosslinking agent added is 0.6% w/v of the system.

(6) Add EDC·HCL to the silk fibroin/carboxymethyl chitosan composite solution under constant stirring by a magnetic stirrer. After 20 seconds, pour the solution into the mold and let it stand for 12 hours.

(7) Immerse it in 75% v/v ethanol aqueous solution for 12 hours to finally obtain silk fibroin/carboxymethyl chitosan composite gel.

(8) By testing in the same manner as in Example 1, the elastic modulus of the composite gel obtained reaches (78.89 ± 5.67) kPa, the compression recovery rate reaches 100%, and the gel's water absorption swelling rate in deionized water reaches (56.95 ± 5.67) kPa. 6.34)×100%, the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (19.17±4.30)×100%.

Example 3

The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in this embodiment includes the following steps in sequence.

(1) Take by weighing 10g of shredded cocoon layer and degumming twice in _0.5 % _Na2CO3 boiling aqueous solution, each time for 30min, the obtained silk fibroin fiber is washed with deionized water and wrung dry, and dried in an oven medium drying;

(2) Put the silk fiber obtained in step (1) in 9M LiBr solution, dissolve it at 60°C for 4 hours, and put the obtained liquid into a dialysis bag (molecular weight 8000-14000) in deionized water after filtering After dialysis for 4 days, the water was changed continuously during the period, and the concentration of the finally obtained silk fibroin solution was 1.5% w/v.

(3) An appropriate amount of carboxymethyl chitosan (90% degree of deacetylation, 90% degree of substitution) was dissolved in water to prepare a carboxymethyl chitosan solution with a concentration of 4% w/v.

(4) the concentration obtained in the step (2) is the carboxymethyl chitosan solution of 4% w/v in the silk fibroin solution of 1.5%w/v and the concentration obtained in the step (3) by volume ratio 1: 1 Mix well to obtain a mixed solution.

(5) Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCL) in the mixed solution obtained in step (4), The addition method is to dissolve the solid powder in a small amount of water and then add it to the system. The amount of crosslinking agent added is 0.6% w/v of the system.

(6) Add EDC·HCL to the silk fibroin/carboxymethyl chitosan composite solution under constant stirring by a magnetic stirrer. After 20 seconds, pour the solution into the mold and let it stand for 12 hours.

(7) Immerse it in 75% v/v ethanol aqueous solution for 12 hours to finally obtain silk fibroin/carboxymethyl chitosan composite gel.

(8) By testing in the same manner as in Example 1, the elastic modulus of the composite gel obtained reaches (99.03 ± 9.72) KPa, the compression recovery rate reaches 100%, and the gel's water absorption swelling rate in deionized water reaches (51.74 ± 9.72) KPa. 10.03)×100%, the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (16.60±2.11)×100%.

Example 4

The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in this embodiment includes the following steps in sequence.

(1) Take by weighing 10g of shredded cocoon layer and degumming twice in _0.5 % _Na2CO3 boiling aqueous solution, each time for 30min, the obtained silk fibroin fiber is washed with deionized water and wrung dry, and dried in an oven medium drying;

(2) Put the silk fiber obtained in step (1) in 9M LiBr solution, dissolve it at 60°C for 4 hours, and put the obtained liquid into a dialysis bag (molecular weight 8000-14000) in deionized water after filtering After dialysis for 4 days, the water was changed continuously during the period, and the concentration of the finally obtained silk fibroin solution was 2.0% w/v.

(3) An appropriate amount of carboxymethyl chitosan (90% degree of deacetylation, 90% degree of substitution) was dissolved in water to prepare a carboxymethyl chitosan solution with a concentration of 4% w/v.

(4) the concentration obtained in the step (2) is the carboxymethyl chitosan solution of 4% w/v in the silk fibroin solution of 2.0%w/v and the concentration obtained in the step (3) by volume ratio 1: 1 Mix well to obtain a mixed solution.

(5) Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCL) in the mixed solution obtained in step (4), Adding method First dissolve the solid powder in a small amount of water and then add to the system. The amount of crosslinking agent added is 0.6% w/v of the system.

(6) Add EDC·HCL to the silk fibroin/carboxymethyl chitosan composite solution under constant stirring by a magnetic stirrer. After 20 seconds, pour the solution into the mold and let it stand for 12 hours.

(7) Immerse it in 75% v/v ethanol aqueous solution for 12 hours to finally obtain silk fibroin/carboxymethyl chitosan composite gel.

(8) By testing in the same manner as in Example 1, the elastic modulus of the composite gel obtained reaches (248.60 ± 34.47) kPa, the compression recovery rate reaches 100%, and the water swelling rate of the gel in deionized water reaches (35.78 ± 34.47) kPa. 10.75)×100%, the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (13.18±0.53)×100%.

Example 5

The preparation method of the silk fibroin/carboxymethyl chitosan composite gel in this embodiment includes the following steps in sequence.

( ₁ ) Take by weighing 10g of shredded cocoon layer and degumming twice in 4.0% _Na2CO3 boiling aqueous solution, each time for 30min, the silk fibroin fiber obtained is wrung out after washing with deionized water, and dried in an oven medium drying;

(2) Put the silk fiber obtained in step (1) in 9M LiBr solution, dissolve it at 60°C for 4 hours, and put the obtained liquid into a dialysis bag (molecular weight 8000-14000) in deionized water after filtering After dialysis for 4 days, the water was changed continuously during the period, and the concentration of the finally obtained silk fibroin solution was 0.5% w/v.

(3) An appropriate amount of carboxymethyl chitosan (90% degree of deacetylation, 90% degree of substitution) was dissolved in water to prepare a carboxymethyl chitosan solution with a concentration of 4% w/v.

(4) the concentration obtained in the step (2) is the silk fibroin solution of 4.0%w/v and the concentration obtained in the step (3) is the carboxymethyl chitosan solution of 4%w/v by volume ratio 1: 1 Mix well to obtain a mixed solution.

(5) Add a certain amount of crosslinking agent 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCL) in the mixed solution obtained in step (4), The addition method is to dissolve the solid powder in a small amount of water and then add it to the system. The amount of crosslinking agent added is 0.6% w/v of the system.

(6) Add EDC·HCL to the silk fibroin/carboxymethyl chitosan composite solution under constant stirring by a magnetic stirrer. After 20 seconds, pour the solution into the mold and let it stand for 12 hours.

(7) Immerse it in 75% v/v ethanol aqueous solution for 12 hours to finally obtain silk fibroin/carboxymethyl chitosan composite gel.

(8) By testing in the same manner as in Example 1, the elastic modulus of the composite gel obtained reaches (363.99 ± 45.57) kPa, the compression recovery rate reaches 100%, and the water swelling rate of the gel in deionized water reaches (20.79 ± 45.57) kPa. 3.14)×100%, the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (9.32±0.09)×100%.

It can be seen that a new method for preparing silk fibroin composite gel provided by the present invention has simple preparation process, mild conditions, short gel time, and the composite gel obtained has better mechanical properties and water absorption stability: The elastic modulus reaches (363.94±45.57) kPa, and the compression recovery rate reaches 100%, that is, when a certain pressure is applied on it, the composite gel has a strong bearing capacity and is not crushed. The gel returns to its original state; the volume is relatively stable in the phosphate buffer solution of pH=7.4, and the swelling rate is (9.32±0.09)×100%, and the dried sample can quickly absorb a large amount of water and swell when placed in water, but it still maintains Good shape, not loose and broken.

What enumerate above is only some specific embodiments of the present invention, obviously, the present invention is not limited to above embodiment, also can have many deformations, all deformations that those of ordinary skill in the art can directly derive or think of from the disclosed content of the present invention , should be considered as the protection scope of the present invention.

Claims (10)

1. a kind of fibroin albumen/carboxymethyl chitosan plural gel, it is characterised in that：

The plural gel is mainly made up of the composition of fibroin albumen and carboxymethyl chitosan.

2. a kind of fibroin albumen/carboxymethyl chitosan plural gel according to claim 1, it is characterised in that：It is described multiple Close in gel, the mass ratio of fibroin albumen and carboxymethyl chitosan is (0.05~2)：1.

3. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel, it is characterised in that：

The fibroin fiber that degumming is obtained carries out dissolution process in LiBr solution, and regenerated silk solution is made by dialysis, It is mixed with carboxymethyl chitosan solution again, crosslinking agent 1- ethyls-(3- dimethylaminopropyls) carbodiimide is then added Hydrochloride, is finally prepared into fibroin/carboxymethyl chitosan composite aquogel in the presence of ethanol.

4. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：Methods described detailed process is as follows：

(1) handled with cocoon layer and obtain fibroin fiber, then prepare silk fibroin solution；

(2) it is carboxymethyl chitosan is soluble in water, carboxymethyl chitosan solution is made；

(3) carboxymethyl chitosan solution obtained in the silk fibroin solution obtained in step (1) and step (2) is well mixed and obtained Mixed solution；

(4) a certain amount of crosslinking agent is added in the mixed solution obtained in step (3), then solution is stirred on magnetic stirring apparatus Mix to pour into immediately after the short time in mould and stand；

(5) immerse again in ethanol water, finally give fibroin/carboxymethyl chitosan plural gel.

5. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：The step (1) is specially：

(1) cocoon layer is placed in the Na that mass fraction is 0.5%₂CO₃In boiling water degumming twice, each 30min, obtained silk Cellulose fiber is dried with being wrung out after deionized water rinsing in an oven；

(2) fibroin fiber obtained in step (1) is placed in 9M LiBr solution, dissolution process 4h, is obtained under the conditions of 60 DEG C Liquid be fitted into bag filter after filtering and dialysed in deionized water 4d, during which constantly change water, the silk fibroin solution finally obtained.

6. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：The silk fibroin solution concentration of the step (1) is 0.5~10%w/v, the carboxymethyl chitosan solution concentration of the step (2) For 1~5%w/v, silk fibroin solution and the mixed volume of carboxymethyl chitosan solution ratio are 1 in the step (3):1.

7. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：The deacetylation of the carboxymethyl chitosan is 55%~99%, and substitution value is 60%~99%.

8. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：In the step (4), crosslinking agent uses 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC HCL solid powder), the addition of crosslinking agent is 0.3~0.75%w/v of system.

9. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature exists In：The feed postition of the crosslinking agent is divided to two kinds：One kind is directly added into system for solid powder, and one kind is to be dissolved in solid powder System is added in water.

10. a kind of preparation method of fibroin albumen/carboxymethyl chitosan plural gel according to claim 3, its feature It is：In the step (4), mixing time is 20s, and time of repose is 6-12h；In the step (5), in ethanol water Concentration of alcohol is 60~90%v/v, and the time of immersion ethanol water is 12h.