Preparation of conductive hydrogel for delivering recombinant human amelogenin and application of conductive hydrogel in damage repair

Abstract

The invention discloses preparation of conductive hydrogel for delivering recombinant human amelogenin and application of the conductive hydrogel in damage repair. The method comprises the following steps: (1) stirring and dissolving hyaluronic acid into a buffer solution to obtain a hyaluronic acid solution; (2) adding the recombinant human amelogenin solution into a hyaluronic acid solution to obtain an amelogenin-loaded hyaluronic acid solution; and (3) under a dark condition, adding the phytic acid solution into the hyaluronic acid solution loaded with the amelogen protein, and standing to obtain the conductive hydrogel for delivering the recombinant human amelogen protein. The hydrogel prepared in the invention has good self-healing property, stretchability, conductivity and shape adaptability, and can promote healing of common acute wounds or chronic refractory wounds caused by diabetes.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and particularly relates to the preparation of a conductive hydrogel for delivering recombinant human amelogenin and its application in damage repair. Background Art

[0002] The skin is the largest organ in the human body and is divided into the epidermis, dermis, and subcutaneous tissue. Because it can directly contact the external environment, it is extremely vulnerable to damage. Generally, the healing of skin wounds is divided into four stages: hemostasis, inflammation, proliferation, and remodeling. Ordinary acute wounds will heal within a few weeks (such as cuts, accidental scratches, etc.). However, some chronic wounds such as burns, diabetes, and bacterial infections are in a malignant inflammatory response stage for a long time, and it is difficult to generate new blood vessels, and it takes several months to recover. At present, for chronic refractory wounds, debridement, negative pressure, skin transplantation, growth factor delivery, traditional dressings and other treatments are usually used. However, the above treatment methods have the disadvantages of complex process, high cost, complex operation and poor prognosis. Hydrogel dressings can not only improve the above disadvantages, but also have a three-dimensional porous structure containing water like extracellular matrix, which can provide a moist environment for diabetic wounds and absorb wound exudates. So far, a variety of hydrogel dressings have been developed for refractory wounds, but most dressings have complex synthesis processes, difficult to obtain raw materials, and single functions.

[0003] At present, some bioactive molecules that promote wound repair have also been reported, such as enamel matrix protein (EMP) synthesized during the development of tooth enamel, which has a repairing effect on periodontal tissue and can also promote wound healing. Among the effective components of EMP, amelogenin accounts for 90%. Therefore, artificially synthesizing amelogenin in large quantities through genetic engineering means while trying to maintain the biological activity of amelogenin and improve its bioavailability has important application value.

[0004] Hyaluronic acid (HA) is a natural straight-chain mucopolysaccharide that contains abundant carboxyl and hydroxyl groups. It is one of the key components of the extracellular matrix and has become one of the commonly used raw materials for hydrogel synthesis. However, since HA is difficult to form a hydrogel network alone, chemical modification is often required.

[0005] Phytic acid (PA) is a non-toxic natural organic macromolecule composed of six phosphate groups. It is widely found in plant seeds and germs and is mostly used in flame retardants, metal coatings and other fields. PA contains abundant hydroxyl groups, which are conducive to hydrogen bonding with water and can also ionize a large amount of H + , which can improve the performance of hydrogels to a certain extent, but the excessive cross-linking ability of phytic acid may cause excessive cross-linking of biopolymers and reduce the mechanical properties of hydrogels.

[0006] Therefore, the development of a multifunctional hydrogel with green and efficient raw materials and processes, good biocompatibility, and excellent anti-inflammatory, angiogenesis-promoting, and clinical transformation capabilities has great application prospects. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a conductive hydrogel for delivering recombinant human amelogenin.

[0008] Another object of the present invention is to provide a conductive hydrogel for delivering recombinant human amelogenin prepared by the method.

[0009] Another object of the present invention is to provide the application of the conductive hydrogel for delivering recombinant human amelogenin.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for preparing a conductive hydrogel for delivering recombinant human amelogenin comprises the following steps:

[0012] (1) stirring and dissolving hyaluronic acid in a buffer solution to obtain a hyaluronic acid solution;

[0013] (2) adding the recombinant human amelogenin solution to the hyaluronic acid solution, stirring and mixing evenly to obtain a hyaluronic acid solution loaded with amelogenin;

[0014] (3) Under light-proof conditions, the phytic acid solution is added to the hyaluronic acid solution loaded with amelogenin, allowed to stand (to form a hydrogel), and then excess phytic acid is removed to obtain a hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel, i.e., the conductive hydrogel for delivering recombinant human amelogenin.

[0015] The molecular weight of the hyaluronic acid described in step (1) is 800,000 to 1,000,000.

[0016] The stirring conditions in step (1) are: stirring at 500-1000 rpm for 20-40 min; preferably: stirring at 900 rpm for 30 min.

[0017] The buffer solution described in step (1) is preferably a PBS buffer solution; more preferably a PBS buffer solution with a pH of 7.4.

[0018] The concentration of the hyaluronic acid solution in step (1) is 40-60 mg / mL, preferably 50 mg / mL.

[0019] The concentration of recombinant human amelogenin in the amelogenin-loaded hyaluronic acid solution described in step (2) is 50 to 250 μg / mL, preferably 200 μg / mL.

[0020] The concentration of hyaluronic acid in the hyaluronic acid solution loaded with amelogenin in step (2) is 30 to 50 μg / mL, preferably 40 μg / mL.

[0021] The phytic acid solution described in step (3) is a phytic acid aqueous solution with a concentration of 40-60 mg / mL, preferably 50 mg / mL.

[0022] In step (3), the phytic acid solution added is excessive, that is, the volume of the phytic acid solution added is greater than the volume of the hyaluronic acid solution loaded with amelogenin.

[0023] The standing condition described in step (3) is: standing at 0-4°C for 8-24 hours; preferably: standing at 4°C for 12 hours.

[0024] A conductive hydrogel for delivering recombinant human amelogenin is prepared by any of the methods described above.

[0025] The conductive hydrogel for delivering recombinant human amelogenin is used in the preparation of a product for promoting the repair of skin wound damage (promoting skin wound repair and treating skin wounds).

[0026] The skin wounds include common acute wounds or chronic wounds that are difficult to heal.

[0027] The chronic and difficult-to-heal wounds include ulcer wounds caused by diabetes.

[0028] The diabetes mellitus is type Ⅰ diabetes mellitus.

[0029] The conductive hydrogel for delivering recombinant human amelogenin has conductive properties, good self-healing properties and stable mechanical properties, can promote the migration of endothelial cells, can promote endothelial cell angiogenesis, can promote the transformation of macrophages from type M1 to type M2, reduce inflammatory response, and promote wound healing.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] 1. The hyaluronic acid-phytic acid hydrogel loaded with recombinant human amelogenin in the present invention is obtained by loading the recombinant human amelogenin active molecule into the hydrogel network HA-PA composed of hyaluronic acid and phytic acid. The raw materials used are all green and natural substances with high biological safety. Among them, hyaluronic acid has excellent water retention capacity. The moist environment it creates promotes the migration of cells in the wound site and is also one of the main synthetic raw materials for hydrogels; phytic acid is an organic macromolecule separated from plants and has good antioxidant properties.

[0032] 2. The present invention uses an osmotic method to place a phytic acid solution on hyaluronic acid wrapped with recombinant human amelogenin, and after standing for a period of time, the target hydrogel can be obtained. This process does not have any chemical reaction, is green and efficient, and has a high degree of clinical conversion. However, simply blending hyaluronic acid and phytic acid cannot obtain a hydrogel with uniform texture.

[0033] 3. The recombinant human amelogenin hydrogel constructed by the present invention has high self-healing, stretchability, conductivity, and shape adaptability, and can adapt to wounds of different shapes. In vitro experiments have shown that it has good ability to promote human umbilical vein endothelial cell proliferation, migration, and angiogenesis. In addition, the hydrogel also has excellent ability to promote diabetic wound healing, anti-inflammatory, and induce macrophage polarization from M1 phenotype to M2 phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a synthesis route map of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention.

[0035] Figure 2 It is a characterization diagram of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention; wherein A is a scanning electron microscope image; and B is a FT-IR image of the hydrogel and its components.

[0036] Figure 3 It is a graph showing the experimental results of the ductility and self-healing properties of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention; wherein A is a photograph of the hydrogel in its original state, in its bent state at the finger joints, and in a stretched state of different strengths; B is a photograph of the self-healing property of the hydrogel; and C is a photograph of a hydrogel that has not formed a uniform hydrogel.

[0037] Figure 4 It is a test result diagram of the conductivity of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention; wherein A is the CV curve of the hydrogel; and B is the conductivity of the hydrogel.

[0038] Figure 5 It is a measurement graph of the cell migration and angiogenesis promoting abilities of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention; wherein, A is a graph showing the scratch test results at 0h, 12h, and 24h; B is a graph showing the quantitative analysis results of the scratch test; C is a graph showing the Transwell test results; D is a graph showing the quantitative analysis results of the Transwell test; E is a graph showing the angiogenesis situation; F is a graph showing the quantitative analysis results of the number of blood vessels in the angiogenesis test; and G is a graph showing the quantitative analysis results of the blood vessel length in the angiogenesis test.

[0039] Figure 6 This is the wound healing status and wound superposition diagram of the experimental rats within 14 days.

[0040] Figure 7 This is a quantitative analysis result of the wound area of ​​experimental rats.

[0041] Figure 8 This is a diagram showing the effect of the hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel in the present invention on macrophage polarization; wherein A is a diagram showing the double-labeled immunofluorescence staining results of CD68 and CD206; B is a diagram showing the quantitative analysis results of CD68; C is a diagram showing the quantitative analysis results of CD206; D is a diagram showing the TNF-α immunofluorescence staining results; and E is a diagram showing the quantitative analysis results of TNF-α. DETAILED DESCRIPTION

[0042] The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0043] The recombinant human amelogenin (rhAM) involved in the embodiments of the present invention is purified by the acetic acid method (reference: T.Qiao, Y.Yi, Z.Kang, Z.Huang, J.Wan, Y.WangC.Qian, Recombinant human amelogenin promotes wound healing by enhancing angiogenesis, Biochem.Biophys.Res.Commun.734(2024)150462.).

[0044] The hyaluronic acid involved in the embodiments of the present invention is selected to be cosmetic grade, and has a molecular weight of 800,000 to 1,000,000.

[0045] Example 1 Preparation of hyaluronic acid-phytic acid hydrogel network delivery of recombinant human amelogenin and its application in repairing diabetic wounds

[0046] 1. Preparation of HA-PA and HA-PA-rhAM hydrogels

[0047] 1.1 Hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel was prepared by the following steps ( Figure 1 ):

[0048] Slowly add 40 mg of hyaluronic acid (HA) to 0.8 mL of PBS buffer (pH = 7.4) and stir at room temperature until completely dissolved (900 rpm, 30 min). Then add 200 μL of recombinant human amelogenin (rhAM) mother solution (1 mg / mL) and quickly mix it with the hyaluronic acid solution. Add a phytic acid (PA) solution (50 mg / mL) larger than the volume of the hyaluronic acid solution loaded with amelogenin in a dark place and place it at 4°C overnight. The next day, remove the excess phytic acid to obtain a hyaluronic acid-phytic acid-recombinant human amelogenin (HA-PA-rhAM) hydrogel.

[0049] 1.2 Preparation of hyaluronic acid-phytic acid (HA-PA) hydrogel: refer to the above step 1.1, except that recombinant human amelogenin is not added, and the remaining steps are the same.

[0050] 2. Performance testing of HA-PA and HA-PA-rhAM hydrogels

[0051] 2.1 The newly synthesized HA-PA and HA-PA-rhAM hydrogels were freeze-dried and the internal structure of the hydrogels was observed using a scanning electron microscope (SEM, Zeiss, Germany). It was found that the interior of the HA-PA and HA-PA-rhAM hydrogels were three-dimensional porous structures ( Figure 2 A). The pore size inside the HA-PA-rhAM hydrogel is smaller and the arrangement is more dense, indicating that the addition of amelogenin increases the number of hydrogen bonds inside the hydrogel. In addition, the Fourier transform infrared spectra (FT-IR) of the hydrogel and its components are shown in Figure 2. Figure 2 As shown in B, in the spectra of HA-PA and HA-PA-rhAM, the -1 The characteristic absorption peak of P=O bond was observed at 3700-3300cm -1 The peak at 40° is the hydrogen bond absorption peak. In the spectrum of HA-PA, the characteristic peak of this band becomes wider because the phosphate groups in PA form a large number of hydrogen bonds with the OH groups in HA. After adding amelogenin, more hydrogen bonds are generated under the joint action of the amino groups of amelogenin, the phosphate groups of PA, and the hydroxyl groups of HA, causing the absorption peak of this band to become wider and slightly move to the right.

[0052] 2.2HA-PA-rhAM hydrogel is transparent and soft in normal state, and can fit well in joints bent at different angles. Using tweezers to stretch the hydrogel at both ends, it was found that there was no breakage during the stretching process ( Figure 3 A), indicating that the hydrogel has excellent deformation ability and can cover wounds of various irregular shapes.

[0053] 2.3 HA-PA-rhAM hydrogel was stained with doxorubicin hydrochloride and placed close to unstained HA-PA-rhAM hydrogel for 1 min. The two immediately healed ( Figure 3 B), indicating that the hydrogel has excellent self-healing ability.

[0054] 2.4 In order to further study whether ordinary blending can form HA-PA-rhAM hydrogel, 40 mg hyaluronic acid (HA) was slowly added to 0.8 mL PBS buffer (pH = 7.4), stirred at room temperature until completely dissolved (900 rpm, 30 min), and then 200 μL recombinant human amelogenin (rhAM) mother solution (1 mg / mL) was added and quickly mixed with the hyaluronic acid solution to obtain a hyaluronic acid solution containing rhAM; then 50 mg / mL of phytic acid solution was added (the amount added was greater than the volume of the hyaluronic acid solution) and stirred at room temperature (800 rpm, 20 min). The results showed that the hydrogel formed by ordinary blending (i.e., stirring and mixing the hyaluronic acid solution containing rhAM and the phytic acid solution) could not be formed and did not have the properties of a wound dressing ( Figure 3 C).

[0055] 3. Testing the conductivity of hydrogels delivering recombinant human amelogenin

[0056] The conductivity of HA-PA and HA-PA-rhAM hydrogels was evaluated using cyclic voltammetry (CV) curves using an electrochemical workstation (cortest Instruments, China). Figure 4 As shown in the figure, hyaluronic acid and phytic acid molecules can effectively build a good ion conductive network. The introduction of amelogenin did not affect the conductivity of the hydrogel constructed by hyaluronic acid and phytic acid. The two hydrogels performed similarly, with a conductivity of about 1.75×10 -3 S / cm, which is within the conductivity range of conductive hydrogels.

[0057] IV. Promoting cell migration and angiogenesis at the cellular level by delivering recombinant human amelogenin hydrogel

[0058] 4.1 Testing the ability of hydrogels to promote cell migration

[0059] The effects of HA-PA and HA-PA-rhAM hydrogels on the migration ability of human umbilical vein endothelial cells (HUVECs) (purchased from ATCC cell bank) were studied.

[0060] (1) 1 g of HA-PA and HA-PA-rhAM hydrogels were incubated in 10 mL of DMEM for 24 hours. The samples were centrifuged (3000 rpm, 10 min), and the supernatant was filtered with a sterile filter to obtain a hydrogel extract.

[0061] (2) HUVECs were plated at 3×10 per well. 5 Cells were inoculated into 6-well plates. When the cell fusion rate reached 100%, a 200μL pipette was used to scratch the well plate in a certain direction perpendicular to the plane of the well plate. The original culture medium was removed, and the cells were gently rinsed twice with PBS. The hydrogel extract (2mL / well) was added to the groups (HA-PA, HA-PA-rhAM). The group without material was used as the control. The migration of cells in each group was observed with an inverted microscope at 0h, 12h, and 24h, and the migration area was analyzed using Image J. The experiment was repeated three times.

[0062] (3) To further verify the ability of the hydrogel to promote endothelial cell migration, HUVECs were plated at 2×10 4 Cells were inoculated into the upper chamber of Transwell, and 500 μl of hydrogel extract (HA-PA, HA-PA-rhAM) was added to the lower chamber. The group without material was used as the control. Incubated in a 5% CO2, 37°C incubator for 24 hours. The Transwell chamber was washed twice with PBS and fixed with paraformaldehyde for 20 minutes. Subsequently, 0.1% crystal violet staining was performed for 30 minutes, and the non-migrated cells were scraped off with a cotton swab. The cell migration was observed and recorded using an inverted microscope. The experiment was repeated three times.

[0063] The scratch test results are as follows Figure 5 As shown in A, after HA-PA and HA-PA-rhAM hydrogel extracts were co-incubated with cells for 12 hours, the cells on both sides migrated faster than the control group, and the hydrogel with added amelogenin migrated faster. After 24 hours, the cells in the HA-PA-rhAM group were almost completely fused, with almost no blank areas in the field of view, and the healing rate could reach 96.72±0.80% ( Figure 5 B), indicating that amelogenin significantly promoted the migration of HUVEC. Figure 5 C, the results of the Transwell experiment also confirmed this. Quantitative analysis showed that the number of HUVECs that migrated after HA-PA and HA-PA-rhAM hydrogel treatment was 2.2 times and 3.2 times that of the control group, respectively ( Figure 5 D). In summary, HA-PA-rhAM hydrogel can significantly enhance the migration ability of HUVEC.

[0064] 4.2 Hydrogel promotes endothelial cell angiogenesis

[0065] To investigate the effects of HA-PA and HA-PA-rhAM hydrogels on angiogenesis, 50 μl of Matrigel was slowly added to each well of a pre-cooled 96-well plate. This process was performed on ice to avoid bubbles. The plate was then placed in a refrigerator overnight. The next day, the plate was placed in a 37°C incubator for 1 hour to allow the matrix gel to solidify. Then, 50 μl of HUVEC (2×10 4 The hydrogel extracts (HA-PA, HA-PA-rhAM) of 10 cells / well were added to a 96-well plate, and the group without material was used as a control. The plate was placed in an incubator and incubated for 4 hours. Subsequently, the tube formation was observed under an inverted microscope. The total length and number of blood vessels generated were quantitatively analyzed by Image J software. The experiment was repeated three times.

[0066] Angiogenesis Figure 5 As shown in E, the HA-PA-rhAM group has formed clearly visible tubules. Quantitative analysis showed that the number of tubules did not show significant difference in the HA-PA group compared with the control group, but the number of tubules in the HA-PA-rhAM group increased significantly ( Figure 5 F). At the same time, the total length of the tubules generated in the HA-PA-rhAM group was 1.52 times and 1.33 times that of the control group and the HA-PA group ( Figure 5 G). This indicates that HA-PA-rhAM hydrogel has a stronger ability to promote angiogenesis, which may be due to the fact that amelogenin can increase the production of VEGF and stimulate endothelial cells to promote angiogenesis.

[0067] 5. Repair ability of recombinant human amelogenin hydrogel on diabetic wounds

[0068] 5.1 Construction of type Ⅰ diabetes wound model

[0069] Female SD rats (purchased from Beijing Huafukang, 8-9 weeks old, weighing 200-250g) were used. After fasting for at least 14 hours, the blood glucose levels of the rats were measured and recorded. Subsequently, streptozotocin (STZ) solution (55mg / kg) was injected intraperitoneally to induce type 1 diabetes. Next, the blood glucose levels and body weights of SD rats were tested every two days. Rats with blood glucose levels ≥16.8mmol / L for two consecutive times were judged to have successfully established a type 1 diabetes model.

[0070] 5.2 Healing of diabetic wounds

[0071] The full-thickness skin wound model of diabetic rats was used to explore the repair ability of recombinant human amelogenin hydrogel for chronic wounds. The hair on the back of the rats was shaved, and chloral hydrate was injected intraperitoneally into the SD rats to anesthetize them. Four circular full-thickness skin wounds with a diameter of 1 cm were made on the back of the rats with a biopsy punch. The 12 rats were randomly divided into 3 groups, namely the material-free group (control group), the HA-PA hydrogel treatment group, and the HA-PA-rhAM hydrogel treatment group. The dosage of the treatment groups was 1g. Wound photos were taken on days 0, 3, 7, 10, and 14, and the wound area was calculated by Image J.

[0072] The results are as follows Figure 6 As shown in the figure, the wound area of ​​all groups decreased with time, and the wounds of rats treated with HA-PA-rhAM hydrogel healed the fastest. In addition, the wound healing overlay diagram was used to more clearly show the excellent effect of HA-PA-rhAM hydrogel in treating wounds. It can be clearly seen from the wound area analysis diagram ( Figure 7 ), on the 7th day after treatment, the wound area of ​​the control group was more than twice that of the group treated with the hydrogel containing amelogenin. On the 14th day of treatment, the wounds treated with the HA-PA-rhAM hydrogel group had almost completely healed, which was significantly better than the other groups. This shows that amelogenin has an excellent ability to promote wound repair.

[0073] VI. Analysis of Macrophage Polarization and Inflammation in Diabetic Wounds

[0074] The mechanism of diabetic wound healing was investigated. To this end, wounds of the above experimental rats were collected on the 3rd and 7th days and immunofluorescence analysis of CD68, CD206 and TNF-α was performed. The results are as follows Figure 8 As shown in A, on the 3rd day, the expression of M1 macrophages in the HA-PA-rhAM group was the lowest. Since the wound was in the inflammatory stage, the number of M2 macrophages in each group was relatively small. On the 7th day, M1 macrophages were still clearly visible in the control group, but decreased in the HA-PA and HA-PA-rhAM groups, indicating that the diabetic wounds in the control group were still in the abnormal inflammatory stage. In addition, the M2 macrophages in the HA-PA-rhAM group were the most widely distributed. The results of immunofluorescence quantitative analysis of CD68 and CD206 were consistent with the above ( Figure 8 B and Figure 8 C). Further immunofluorescence staining of TNF-α on the third day of wound treatment revealed that the positive expression of TNF-α in both the HA-PA and HA-PA-rhAM groups was low, with the lowest in the HA-PA-rhAM group ( Figure 8 D and Figure 8E). These results indicate that HA-PA-rhAM hydrogels can promote the transformation of macrophages from M1 to M2, reduce inflammatory responses, and promote diabetic wound healing.

[0075] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a conductive hydrogel for delivering recombinant human amelogenin, characterized in that: The steps include: (1) stirring and dissolving hyaluronic acid in a buffer solution to obtain a hyaluronic acid solution; (2) adding the recombinant human amelogenin solution to the hyaluronic acid solution, stirring and mixing evenly to obtain a hyaluronic acid solution loaded with amelogenin; (3) Under light-proof conditions, the phytic acid solution is added to the hyaluronic acid solution loaded with amelogenin, the solution is allowed to stand, and then excess phytic acid is removed to obtain a hyaluronic acid-phytic acid-recombinant human amelogenin hydrogel, i.e., the conductive hydrogel for delivering recombinant human amelogenin.

2. The method according to claim 1, characterized in that: The concentration of recombinant human amelogenin in the hyaluronic acid solution loaded with amelogenin in step (2) is 50 to 250 μg / mL, and the concentration of hyaluronic acid is 30 to 50 μg / mL; The concentration of the phytic acid solution in step (3) is 40-60 mg / mL; The standing condition described in step (3) is: standing at 0-4°C for 8-24 hours.

3. The method according to claim 2, characterized in that: The concentration of recombinant human amelogenin in the hyaluronic acid solution loaded with amelogenin described in step (2) is 200 μg / mL, and the concentration of hyaluronic acid is 40 μg / mL; The concentration of the phytic acid solution described in step (3) is 50 mg / mL.

4. The method according to claim 1, characterized in that: The concentration of the hyaluronic acid solution in step (1) is 40-60 mg / mL; The molecular weight of the hyaluronic acid described in step (1) is 800,000 to 1,000,000.

5. The method according to claim 1, characterized in that: The stirring conditions described in step (1) are: stirring at 500-1000 rpm for 20-40 min; The buffer solution described in step (1) is PBS buffer.

6. A conductive hydrogel for delivering recombinant human amelogenin, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. Use of the conductive hydrogel for delivering recombinant human amelogenin as claimed in claim 6 in the preparation of products for repairing skin wound damage.

8. The use according to claim 7, characterized in that: The skin wounds include common acute wounds or chronic wounds that are difficult to heal.

9. The use according to claim 8, characterized in that: The chronic difficult-to-heal wounds include ulcer wounds caused by diabetes.

10. The use according to claim 9, characterized in that: The diabetes mellitus is type Ⅰ diabetes mellitus.

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