CN110960723B

CN110960723B - Preparation method and application of ascidian conductive hydrogel

Info

Publication number: CN110960723B
Application number: CN201911067711.XA
Authority: CN
Inventors: 邱小忠; 何玉童; 侯鸿浩; 王乐禹
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2021-03-16
Anticipated expiration: 2039-11-04
Also published as: CN110960723A

Abstract

The invention discloses a preparation method and application of ascidian conductive hydrogel, which is a method for obtaining cellulose hydrogel by treating ascidian shells with simple acid and alkali. Putting the mixture into a pyrrole solution, and polymerizing the mixture by using an iron ion solution to obtain the conductive cellulose hydrogel. The ascidian hydrogel prepared by the invention has good biocompatibility and no toxicity to cells; the obtained sea squirt hydrogel has abundant pores and good elasticity. The obtained ascidian hydrogel is used for repairing skin wound, and the porous structure of the hydrogel can form a loose and breathable environment for the wound, avoid the reproduction of anaerobic pathogenic bacteria and promote the synthesis of collagen, thereby being beneficial to the recovery of the wound and accelerating the skin repair. The raw material adopts the sea squirts, so that the polluted sea squirts can be changed into valuable, the sea squirts can be reasonably utilized, and the loss caused by the sea squirts can be reduced.

Description

Preparation method and application of ascidian conductive hydrogel

Technical Field

The invention belongs to the technical field of hydrogel, and particularly relates to preparation of ascidian conductive hydrogel and repair and application of the ascidian conductive hydrogel in skin injury.

Background

Sea squirt belongs to tunicate, is a marine animal, and cellulose and protein are symbiotic in its tunicate. The sea squirts can be attached to the bottom of the ship, and when the attached quantity is too large, the speed of the ship is influenced, and the oil consumption of the ship is increased; when the sea squirt is attached to the underwater pipeline, the pipeline can be blocked, the smooth water flow is influenced, and the harm is caused. In a marine farm, the ascidians are often attached to the cultivation apparatus, which seriously affects the production of marine products, so that the ascidians are always used as byproducts of marine cultivation, resulting in great resource waste.

The preparation of the hydrogel is mainly achieved by three modes of chemical crosslinking, physical crosslinking and enzyme crosslinking.

(1) Chemical crosslinking is generally performed by means of a polyfunctional crosslinking agent, a metal ion coordinate bond, or the like. Chemical crosslinking of hydrogel means that under the action of a chemical crosslinking agent or an initiator, a high molecular polymer or various monomer small molecules are combined through covalent bonds or ionic bonds to form a three-dimensional network structure, but the forming process is generally irreversible. The hydrogel prepared by chemical crosslinking may be a chemically stable non-degradable hydrogel or a degradable hydrogel that can be hydrolyzed or enzymatically hydrolyzed. Since the chemical crosslinking method can improve the mechanical properties of the biopolymer hydrogel by adjusting the degree of crosslinking, researchers often adopt the method to prepare the hydrogel. However, most chemical cross-linking agents or initiators can generate toxic action on cells, and the prepared hydrogel has potential toxicity on human bodies, so that the application of the hydrogel in the aspect of biomedicine is greatly limited.

(2) Physical crosslinking of hydrogels typically employs hydrogen bonding, multiple hydrogen bonding, topological interlocking, crystallization, configuration transformation, entanglement, and the like. Polymeric hydrogels tend to exist in a variety of cross-linked forms. Physically crosslinked hydrogels are crosslinked by intermolecular forces, such as intermolecular chain crosslinking of molecules by hydrophobic interactions, electrostatic interactions, hydrogen bonding, or van der waals forces. The hydrogel formed by physical crosslinking has a reversible gel-sol transition process, and the structure of the hydrogel can be damaged by changing the physical environment (pH, temperature and ionic strength) of the gel. Therefore, the hydrogel prepared by physical crosslinking has no crosslinking agent, but it is difficult to obtain a uniform, stable hydrogel having a desired degree of crosslinking.

(3) Most enzymes participate in biochemical reactions as catalysts, and most enzymes can also catalyze macromolecules to form a cross-linked network, such as glutamine transaminase (mTG), tyrosinase, horseradish peroxidase and the like. The principle is that proteins or polypeptides containing amino acid residues can form peptide bond cross-linked protein or polypeptide networks through transferase catalysis. And the polysaccharide macromolecules bonded and introduced with the phenolic hydroxyl groups can be a polysaccharide macromolecule network formed by catalyzing the oxidative crosslinking of the phenolic hydroxyl groups through oxidase and hydrogen peroxide.

The hydrogel can contain a large amount of water due to the characteristic of porous structure, so that nutrient substances and oxygen can freely pass through the hydrogel, and the hydrogel is similar to natural biological tissues, thereby being widely applied to tissue engineering. For example, skin tissue engineering for wound repair, myocardial tissue engineering for myocardial infarction repair, and the like.

From the viewpoint of utilization of biological resources, reuse of ascidians is advantageous for environmental development, and the loss due to ascidians can be reduced, and development of an ascidian hydrogel is a problem to be solved.

Disclosure of Invention

The invention aims to provide a conductive nanocellulose hydrogel which is simpler, faster, higher in strength and good in biocompatibility.

It is still another object of the present invention to provide a method for preparing the above electrically conductive nanocellulose hydrogel.

It is a further object of the present invention to provide the use of the above-described electrically conductive nanocellulose hydrogel.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, a method for preparing a conductive nanocellulose hydrogel is provided, comprising the following steps:

1) mixing the ascidian shell with an acidic solution;

2) mixing the mixed sea squirt shell with an alkaline solution for reaction, and washing the sea squirt shell to be neutral by water to obtain nano cellulose hydrogel;

3) polymerizing the nano-cellulose hydrogel obtained in the step 2) with pyrrole;

4) mixing the polymerized nano-cellulose hydrogel obtained in the step 3) with an iron ion solution to obtain the conductive nano-cellulose hydrogel.

According to an embodiment of the present invention, the acidic solution in step 1) is sulfuric acid or nitric acid.

According to the embodiment of the invention, the concentration of the acidic solution in the step 1) is 0.5-1.5 mol/L; in some embodiments, the concentration of the acidic solution is 0.3-1.5 mol/L; preferably, the concentration of sulfuric acid is 0.5 mol/L.

According to the embodiment of the invention, the mixing reaction in the step 1) is carried out for 48-72 h, and the mixing reaction temperature is 15-37 ℃; preferably, the mixing reaction temperature is 20-25 ℃.

According to an embodiment of the present invention, the alkaline solution in step 2) is sodium hydroxide or potassium hydroxide.

According to the embodiment of the invention, the concentration of the sodium hydroxide in the step 2) is 1-3 mol/L; in some embodiments, the concentration of sodium hydroxide is 1-2 mol/L.

According to the embodiment of the invention, the mixing reaction in the step 2) is carried out for 5-12 h, and the mixing reaction temperature is 15-37 ℃; preferably, the mixing reaction time is 5-12 h, and the temperature is 20-25 ℃.

According to the embodiment of the invention, the polymerization in the step 3) is carried out for 12-24 h at 0-4 ℃; the concentration of the pyrrole is 5-30 mmol/L; more preferably 10 to 15 mmol/L.

According to an embodiment of the present invention, the iron ion solution of step 4) is FeCl₃、APS。

According to the embodiment of the invention, the concentration of the iron ion solution in the step 4) is 3-14 mmol/L.

In a second aspect, the present invention provides the use of a conductive nanocellulose hydrogel prepared by any one of the foregoing methods as a skin lesion repair agent, said skin lesion being a blunt full-thickness lesion.

In a third aspect of the invention, the invention provides a biological auxiliary material prepared by any one of the preparation methods.

Compared with the traditional hydrogel preparation method, the method can remove the protein and lipid of the sea squirt shell through acid-base treatment; meanwhile, a porous structure only containing cellulose is obtained, and a large number of pores can endow the ascidian hydrogel with the capacity of containing a large amount of water. The invention can obtain stable hydrogel without adding toxic initiator. The sea squirt is taken as a raw material from natural biological tissues, and the hydrogel obtained by combining acid-base treatment and other reagents has similar performance to the natural biological tissues and has higher biocompatibility.

The invention has the beneficial effects that:

1. the experimental raw material sea squirt is simple, economic and renewable.

2. The sea squirts are treated and applied, so that the influence of the sea squirts on the culture of a fishing ground is reduced.

3. The nanocellulose hydrogel obtained by acid-base treatment of ascidians has high yield and can not cause waste.

4. The prepared nano-cellulose hydrogel can keep stable for a long time.

5. The prepared nano-cellulose hydrogel has good biocompatibility and elasticity.

6. The ascidian hydrogel has good skin wound repairing effect.

Drawings

FIG. 1 is a schematic view showing different treatments, wherein A is the outer shell of the sea squirt of comparative example 1 without any treatment, B is the cellulose hydrogel treated with sulfuric acid and sodium hydroxide of comparative example 2, and C is the conductive nanocellulose hydrogel having conductivity of example 1.

FIG. 2 is an electron micrograph of different treatments, wherein A is the sea squirt shell of comparative example 1 without any treatment, B is the cellulose hydrogel of comparative example 2 after treatment with sulfuric acid and sodium hydroxide, and C is the conductive nanocellulose hydrogel having conductivity of example 1.

FIG. 3 is a scanning electron micrograph of the sea squirt shells after reaction with pepsin, wherein A is the sea squirt shells treated with 2% pepsin at 37 ℃; b is sea squirt shell treated with 2% pepsin at normal temperature of 25 deg.C.

FIG. 4 is the SEM images of the degradation of sea squirt shell, cellulose hydrogel and conductive nanocellulose hydrogel in PBS solution at 37 deg.C for 6 weeks.

FIG. 5 is a graph of dead-live staining for different treatments, wherein A is the sea squirt shell of comparative example 1 without any treatment, B is the cellulose hydrogel of comparative example 2 after treatment with sulfuric acid and sodium hydroxide, and C is the conductive nanocellulose hydrogel having conductivity of example 1, wherein the scale is 100 μm.

FIG. 6 is a graph showing the biocompatibility and elasticity of the hydrogel prepared in example 1 of the present invention, wherein A is a hydrogel, B is a compressed hydrogel, and C is a released hydrogel.

FIG. 7 shows the results of repairing deep skin wounds of rats, wherein A to C are established skin wound models, D is the sea squirt shell which is not treated in comparative example 1, E is the cellulose hydrogel treated with sulfuric acid and sodium hydroxide in comparative example 2, and F is the conductive nanocellulose hydrogel having conductivity in example 1.

FIGS. 8 a-f show the massson and HE staining of tissue sections after skin wound repair with differently treated hydrogels. Wherein a and c are Masson staining and HE staining, respectively, of skin wound repair without any treatment; b. e is the results of Masson staining and HE staining after wound repair with ascidian hydrogel; d. the f-chart shows the results of Masson staining and HE staining after wound repair of the electrically conductive ascidian hydrogel.

Detailed Description

The invention provides a preparation method of ascidian conductive hydrogel, which comprises the following steps:

1) mixing the ascidian shell with an acidic solution;

In some embodiments, the acidic solution of step 1) is sulfuric acid, nitric acid.

In some embodiments, the concentration of the acidic solution of step 1) is 0.5 mol/L.

In some embodiments, the mixing reaction in step 1) is carried out for 48-72 hours, and the mixing reaction temperature is 20-25 ℃.

In some embodiments, the alkaline solution of step 2) is sodium hydroxide, potassium hydroxide.

In some embodiments, the concentration of sodium hydroxide is 1-2 mol/L.

In some embodiments, the mixing reaction time in the step 2) is 5-12 hours, and the reaction temperature is 20-25 ℃.

In some embodiments, the polymerization in step 3) is carried out at 0-4 ℃ for 12-24 hours; the concentration of the pyrrole is 10-15 mmol/L.

In some embodiments, the iron ion solution of step 4) is FeCl₃APS (ammonium persulfate).

In a second aspect, the present invention provides the use of any one of the above electrically conductive ascidian hydrogels as a repair agent for skin lesions, wherein the skin lesions are blunt full-thickness lesions.

The technical solution of the present invention is clearly and completely illustrated below with reference to the following examples, but is not limited thereto.

Example 1

Early preparation: removing the inner capsule of sea squirt, removing impurities attached to the outer shell of sea squirt, washing with deionized water, and cutting into size of 1cm × 1cm × 0.3 cm.

Preparation of reagents: 0.5mol/L sulfuric acid and 2mol/L sodium hydroxide solution.

The hydrogel was prepared as follows:

1) mixing and reacting the sea squirt shell with 0.5mol/L sulfuric acid at 20 ℃ for 72 hours;

2) taking out the sea squirt shell reacted in the step 1), and mixing the sea squirt shell with 2mol/L sodium hydroxide for reaction for 8 hours; (removing protein and lipid of animals by acid and alkali), and washing with deionized water until the pH value is 7 to obtain the nano-cellulose hydrogel;

3) mixing the nano-cellulose hydrogel with 10mmol/L pyrrole and standing overnight at 4 ℃;

4) then mixed with 10mmol/L FeCl₃Mixing and reacting for 12h to obtain the conductive nano cellulose hydrogel.

Example 2

Preparation of reagents: 1.5mol/L sulfuric acid and 3mol/L sodium hydroxide solution.

The hydrogel was prepared as follows:

1) mixing and reacting the sea squirt shell with 1.5mol/L sulfuric acid at 37 ℃ for 48 hours;

2) mixing the sea squirt shell reacted in the step 1) with 3mol/L sodium hydroxide at 37 ℃ for reaction for 6h, and washing with deionized water until the pH value is 7, thus obtaining the nano cellulose hydrogel;

3) mixing the nano-cellulose hydrogel with 30mmol/L pyrrole and standing overnight at 4 ℃;

4) then mixed with FeCl of 30mmol/L₃Mixing and reacting for 12h to obtain the conductive nano cellulose hydrogel with good conductivity.

Example 3

Preparation of reagents: 1mol/L sulfuric acid and 2mol/L sodium hydroxide solution.

The hydrogel was prepared as follows:

1) mixing and reacting the sea squirt shell with 1mol/L sulfuric acid at 15 ℃ for 72 hours;

2) mixing the sea squirt shell reacted in the step 1) with 2mol/L sodium hydroxide at 25 ℃ for 8h, and washing with deionized water until the pH value is 7, thus obtaining the nano cellulose hydrogel;

3) mixing the nano-cellulose hydrogel with 5mmol/L pyrrole and standing overnight at 4 ℃;

4) then mixed with 5mmol/L FeCl₃Mixing and reacting for 12h to obtain the nano-cellulose hydrogel with good conductivity.

Example 4

Preparation of reagents: 1mol/L sulfuric acid and 1mol/L sodium hydroxide solution.

The hydrogel was prepared as follows:

2) mixing the sea squirt shell reacted in the step 1) with 1mol/L sodium hydroxide at 20 ℃ for reaction for 10h, and washing with deionized water until the pH value is 7, thus obtaining the nano cellulose hydrogel;

3) mixing the nano-cellulose hydrogel with 15mmol/L pyrrole and standing overnight at 4 ℃;

4) then mixed with 15mmol/L FeCl₃Mixing and reacting for 12h to obtain good electric conductionA conductive nanocellulose hydrogel.

Example 5

Preparation of reagents: 0.3mol/L nitric acid and 2mol/L potassium hydroxide solution.

The hydrogel was prepared as follows:

1) mixing and reacting the sea squirt shell with 0.3mol/L nitric acid at 15 ℃ for 72 hours;

2) mixing the sea squirt shell reacted in the step 1) with 2mol/L potassium hydroxide at 20 ℃ for reaction for 9h, and washing with deionized water until the pH value is 7, thus obtaining the nano cellulose hydrogel;

3) mixing the nano-cellulose hydrogel with 12mmol/L pyrrole and standing overnight at 4 ℃;

4) and then mixed with 12mmol/L APS (ammonium persulfate) for reaction for 12h to obtain the conductive nano cellulose hydrogel with good conductivity.

Comparative example 1

Comparative example 2

1) Mixing and reacting the sea squirt shell with sulfuric acid at 25 ℃ for 72 hours;

2) mixing the sea squirt shell reacted in the step 1) with sodium hydroxide for reaction for 8 hours;

3) washing the sea squirt shell reacted in the step 2) with deionized water until the pH value is 7, and obtaining the nano cellulose hydrogel.

Comparative example 3

1) Mixing sea squirtRemoving the inner capsule, removing impurities attached to the sea squirt shell, cleaning with deionized water, and cutting into pieces with cross-sectional area of 1cm²；

2) Respectively reacting the sea squirt shell with the prepared 2% pepsin solution at 25 ℃ and 37 ℃ for 72 hours;

3) washing with deionized water, and observing under an electron microscope.

The hydrogel prepared in the example was further tested for effects.

Hydrogel characterization experiment

The shape of the nanocellulose hydrogel of example 1 is shown in fig. 1, wherein a is the sea squirt shell which is not treated in comparative example 1, B is the cellulose hydrogel treated with sulfuric acid and sodium hydroxide in comparative example 2, and C is the conductive nanocellulose hydrogel having conductivity in example 1.

Hydrogel electron microscopy characterization experiment

The electron micrograph of the nanocellulose hydrogel obtained in example 1 is shown in FIG. 2, wherein A is the sea squirt shell which is not treated in comparative example 1, B is the cellulose hydrogel which is treated with sulfuric acid and sodium hydroxide in comparative example 2, and C is the conductive sea squirt hydrogel of example 1. The untreated sea squirt shell has a compact structure and a smooth surface; the shells after the acid and alkali treatment form porous hydrogel formed by nano fibers due to the removal of components such as protein, lipid and the like; after pyrrole monomer polymerization, the conductive nanoparticles are uniformly attached to the periphery of the nanofibers, so that the cellulose hydrogel has good conductivity. The conductive nano cellulose hydrogel after acid and alkali treatment and pyrrole polymerization can better play the role of a bracket.

The product of comparative example 3 was observed by scanning electron microscopy as shown in FIG. 3, wherein A was treated with 2% pepsin at 37 ℃ and B was treated with 2% pepsin at room temperature. The structure of the sea squirt shell treated by the protease A and the protease B is still compact, and ideal pores cannot be obtained. It is known that a suitable ascidian hydrogel cannot be obtained by treating only with a protease.

Fig. 4 shows that the simple sea squirt shell is unstable (a), changes obviously after 6 weeks of degradation, and the dense structure changes into a porous structure due to the degradation of proteins and lipids in the sea squirt shell. In comparison, the structures of the cellulose hydrogel and the conductive nanocellulose hydrogel prepared by the method are not changed too much, which shows that the material properties of the cellulose hydrogel and the conductive nanocellulose hydrogel are stable.

Hydrogel characterization experiment

The dead-live staining pattern of the nanocellulose hydrogel obtained in this example 1 is shown in FIG. 5. Wherein A is the sea squirt shell which is not treated in the comparative example 1, because the sea squirt shell is not treated, the surface of the sea squirt shell is smooth, cells are relatively difficult to attach to the surface, the number of the cells is small, and because the structure is compact, nutrient substances and oxygen are difficult to permeate, the cells at the bottom are easy to die due to oxygen deficiency; and B is cellulose hydrogel treated by sulfuric acid and sodium hydroxide in comparative example 2, and the good porous structure provides more attachment sites and sufficient nutrients for cells, so that the cells can grow well. C is the nanocellulose hydrogel with conductivity in example 1, and the good porous structure provides more attachment sites and sufficient nutrients for cells, so that the cells can grow well. (Scale: 100. mu.m).

The dead-live staining of fig. 5 shows that the hydrogel with conductive ascidians prepared in this example 1 also has good biocompatibility, is not cytotoxic, and can provide a good growth microenvironment for cells.

Hydrogel stability test

The scanning electron micrograph of the nanocellulose hydrogel obtained in example 1 degraded under simulated physiological conditions for 2 months is shown in fig. 5. The hydrogel obtained in the example is placed in PBS solution at 37 ℃ for 2 months, and the degradation condition of each group is observed through a scanning electron microscope. After 2 months, the scanning electron microscope shows that the morphological structure of the pure quilt cover shell has larger change, a plurality of holes appear, and the cellulose hydrogel and the conductive cellulose hydrogel have little change. The results indicate that the treated cellulose hydrogel and the conductive cellulose hydrogel have good stability.

As a result: the prepared conductive ascidian hydrogel can keep stable for a long time.

The hydrogel has good biocompatibility and elasticity

Fig. 6A to C are elasticity test results, which show that the hydrogel has good biocompatibility and elasticity through the experiment operation of continuous squeezing and relaxing for tens of times.

Hydrogel repair application experiments

The hydrogel repair application experiment was performed as follows:

a blank control group was set up without any treatment with B1, with B2 being the pure ascidian hydrogel group and B3 being the conducting ascidian hydrogel group (the well-conducting nanocellulose hydrogel prepared in example 1). B3 was previously rinsed with water to remove Fe from the hydrogel surface³⁺。

180g of 9 SD rats were divided into 3 groups; each SD rat was wounded with blunt full-thickness skin lesions on both sides of the back, each wound having a diameter of about 0.8 cm.

1) The rinsed B3 conductive nanocellulose hydrogel, B1 blank control, B2 were placed at the wounds, and then a pressure dressing (petrolatum gauze) was attached to each wound. Rats were placed in the same cage for observation and the dressing was checked daily to ensure integrity of the dressing.

2) Observing the wound condition every day, taking a picture of the mouse wound after 14 days, analyzing the picture by using Image J software, and comparing unhealed areas of different groups;

3) the rats were sacrificed and the wound tissue was removed and sectioned for massson and HE staining tests.

As a result: FIG. 7 shows the results of repairing deep skin wounds of rats, where A to C are established skin wound models, D is the sea squirt shell of comparative example 1 without any treatment, and it can be seen that a small part of inflammatory reaction still exists in the wounds; e is the cellulose hydrogel treated by sulfuric acid and sodium hydroxide in comparative example 2, the wound has no inflammatory reaction and new granulation tissues appear; f is the electroconductive nanocellulose hydrogel of example 1, and there was no inflammatory reaction in the wound, and there was also new granulation tissue. In conclusion, the nanocellulose hydrogel with conductivity has a remarkable effect on skin wound repair, does not generate inflammatory reaction, can promote the formation of new granulation tissues and promote the healing of wounds.

FIGS. 8 a-f show the massson and HE staining of tissue sections after skin wound repair of ascidian hydrogels prepared in example 1. a. c are Masson and HE staining results, respectively, for skin wound repair without any treatment; b. e-plot of Masson and HE staining after wound repair with ascidian hydrogel, showing better skin healing than the a, c untreated groups and the appearance of dermal and epidermal layers; d. the f picture shows the results of Masson and HE staining after wound repair of the conductive ascidian hydrogel, wherein the skin wound is well repaired and a plurality of new hair follicles appear. In conclusion, the ascidian hydrogel of example 1 can promote wound healing and has significant capability of promoting skin repair.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A preparation method of ascidian conductive hydrogel is characterized by comprising the following steps:

1) mixing the ascidian shell with an acidic solution;

4) mixing the nanocellulose hydrogel polymerized in the step 3) with an iron ion solution to obtain a conductive ascidian hydrogel;

the acid solution in the step 1) is sulfuric acid and nitric acid; the concentration of the acid solution is 0.25-1.0 mol/L;

step 2) the alkaline solution is sodium hydroxide and potassium hydroxide;

the polymerization in the step 3) is carried out for 12-24 hours at the temperature of 0-4 ℃; the concentration of the pyrrole is 5-30 mmol/L;

step 4) the iron ion solution is FeCl₃APS ammonium persulfate; the concentration of the iron ion solution is 3-14 mmol/L.

2. The method according to claim 1, wherein the concentration of the sulfuric acid is 0.5 mol/L.

3. The preparation method of claim 1, wherein the mixing reaction in the step 1) is carried out for 48-72 hours at a temperature of 15-37 ℃.

4. The method according to claim 3, wherein the mixing reaction temperature in the step 1) is 20 to 25 ℃.

5. The method according to claim 1, wherein the concentration of sodium hydroxide is 1 to 3 mol/L.

6. The method according to claim 5, wherein the concentration of sodium hydroxide is 1 to 2 mol/L.

7. The preparation method of claim 1, wherein the mixing reaction in the step 2) is carried out for 8-10 h, and the mixing reaction temperature is 15-37 ℃.

8. The method according to claim 7, wherein the mixing reaction temperature in the step 2) is 20 to 25 ℃.

9. Use of an ascidian conductive hydrogel prepared by the method of any one of claims 1 to 8 in the preparation of a skin injury repair agent, wherein the skin injury is blunt full-thickness injury.

10. A biological adjuvant, which is characterized by being prepared by the preparation method of any one of claims 1 to 8.