Preparation method of fibroin hydrogel wound dressing compounded with trace elements and growth factors

Abstract

The invention relates to the technical field of biomedical materials, and discloses a preparation method of a fibroin hydrogel wound dressing compounded with trace elements and growth factors. On the basis of a silk fibroin solution, metal ions such as Cu < 2 + >, Zn < 2 + >, Mg < 2 + >, Mn < 2 + > and Sr < 2 + > and growth factors such as VEGF, bFGF and EGF are compounded to form the wound dressing with multiple biological activities, and the dressing can effectively promote proliferation and migration of wound cells and angiogenesis and remarkably shorten the wound healing time. In animal experiments, the fibroin hydrogel dressing compounded with the trace elements and the growth factors shows high tissue repair capacity, wound healing can be accelerated, and scar formation is reduced. The dressing not only has good biodegradability, but also can continuously release composite growth factors to further promote the wound healing process; the composite dressing provides a novel and effective material for clinical wound repair, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials, and in particular to a method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors. Background Art

[0002] Wound dressings are widely used in clinical trauma care to promote wound healing and prevent infection. Traditional wound dressings mostly rely on single functions, such as antibacterial and hemostatic, and lack comprehensive multi-functional materials that promote wound healing.

[0003] In recent years, gelatin, sodium alginate and other polymer hydrogel materials have shown certain effects in promoting wound healing, but they usually lack effective biostimulation functions and fail to fully mobilize the self-repair ability of cells in the wound area. Therefore, important biological processes such as cell proliferation, migration, collagen synthesis and angiogenesis in the wound healing process are still restricted, resulting in slow healing and even scar tissue formation.

[0004] Natural hydrogel biomaterials have good biocompatibility, but they usually have problems such as insufficient mechanical strength, too fast dissolution or immune response caused by degradation products. Existing hydrogel materials lack a composite material that can keep the wound site moist, provide physical support, and effectively and continuously release bioactive substances.

[0005] Growth factors such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) play a key role in the wound healing process. They can promote cell proliferation, migration and the formation of new blood vessels. However, these growth factors usually cannot maintain sufficient concentration and biological activity at the wound site, resulting in a delay in the healing process. At the same time, trace elements such as zinc (Zn2+), copper (Cu2+), magnesium (Mg2+), manganese (Mn2+) and strontium (Sr2+) are also insufficient. 2 +) play an important role in promoting cell function, enhancing collagen synthesis and regulating immune response, but are currently rarely used in hydrogels, and most of them lack long-term release and effective effects.

[0006] Therefore, there is an urgent need to design and invent a silk hydrogel wound dressing containing composite trace elements and growth factors based on silk gelatin solution, which can be used to accelerate the repair of skin wounds. Summary of the invention

[0007] In order to solve the above existing problems, the present invention discloses a method for preparing a silk hydrogel wound dressing with composite trace elements and growth factors, which can simultaneously promote wound healing, anti-inflammatory, antibacterial and repair functions, and has excellent mechanical properties, biocompatibility and biodegradability. It can solve the problems that the current treatment methods and materials for wound healing mostly rely on a single bioactive substance or matrix material, lack sufficient functional support, and the low mechanical strength of a single silk gel.

[0008] In one aspect, the present invention provides a method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors, comprising the steps of:

[0009] S1, preparing a silk fibroin solution: using a solvent to dissolve silk fibroin, dialyzing it with running water, and centrifuging to obtain a supernatant to obtain a silk fibroin solution;

[0010] S2, preparing a gelatin-silk fibroin solution: mixing the pig skin-derived gelatin with the silk fibroin solution to obtain a gelatin-silk fibroin solution;

[0011] S3, preparing a cross-linking solution: adding a photoinitiator to the gelatin silk solution, and mixing them evenly under light-proof conditions to obtain a cross-linking solution;

[0012] S4, loading growth factors and trace elements: adding growth factors and trace elements to the cross-linking solution, pouring it into a hydrogel mold, and irradiating it under blue light.

[0013] In some embodiments, in step S1, the solvent is a ternary solvent having a molar mass ratio of CaCl2, CH3CH2OH and H2O of 1:2:8.

[0014] In some embodiments, in step S2, the gelatin strength of the pig skin-derived gelatin is 300 g Bloom.

[0015] In some embodiments, in step S3, the photoinitiator includes terpyridine ruthenium (II) chloride and potassium persulfate.

[0016] It is worth noting that although growth factors play a key role in the wound healing process, trace elements play an important role in promoting cell function, enhancing collagen synthesis and regulating immune response, the effects of the two cannot be achieved simultaneously by directly stacking them. Only by adopting the method of the present invention for preparation can the effects of growth factors and trace elements be exerted simultaneously.

[0017] In some embodiments, in step S4, the trace elements include Cu 2+ 、Zn 2+ Mg 2+ , Mn2+ and Sr 2 + one or more.

[0018] In some embodiments, in step S4, the concentration of the trace element is 0.5-2%.

[0019] In some embodiments, in step S4, the growth factor comprises one or more of vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor.

[0020] In some embodiments, in step S4, the concentration of the growth factor is 1 μg / mL.

[0021] Preferably, the combination of growth factors and trace elements includes: vascular endothelial growth factor (VEGF) and copper (Cu2+); basic fibroblast growth factor (bFGF) and zinc (Zn2+); epidermal growth factor (EGF) and magnesium (Mg2+). The silk hydrogel wound dressing prepared by the above three combinations has a significantly higher wound healing effect than the dressings prepared by other combinations, specifically, the wound healing speed is faster and the wound healing area is larger.

[0022] In some embodiments, in step S4, the wavelength of the blue light is 450 nm.

[0023] The present invention has carried out optimization experiments in multiple dimensions: on the one hand, in the cross-linking process, only by adding terpyridine ruthenium (II) chloride and potassium persulfate at the same time and cross-linking under 450nm wavelength of blue light can the silk molecules form a cross-linked structure that is conducive to the simultaneous loading of growth factors and trace elements. If other methods are adopted, although they are also possible, their cross-linked structure has difficulties in loading growth factors and trace elements, and is also not conducive to the release of growth factors and trace elements, and is therefore not conducive to wound healing; on the other hand, in view of the above cross-linked structure, theoretically not all growth factors and trace elements have good effects in the present invention. After experiments, Cu 2+ 、Zn 2+ Mg 2+ , Mn2+ and Sr 2 + and vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor have good effects, while Ag 2+ And insulin-like growth factor cannot play a good effect in the present invention, which is determined by the physical and chemical structure.

[0024] On the other hand, the present invention provides a silk hydrogel wound dressing containing composite trace elements and growth factors prepared according to the method.

[0025] In summary, the present invention has the following beneficial technical effects:

[0026] 1. The present invention discloses a method for preparing a silk hydrogel wound dressing containing composite trace elements and growth factors. The preparation process is simple and easy, the raw materials are easily available, and the method has good mass production capacity and broad application prospects. The growth factors and trace elements in the silk hydrogel wound dressing can significantly promote cell proliferation, migration and angiogenesis, and significantly improve the speed and quality of wound healing. The silk hydrogel wound dressing can effectively and slowly release growth factors and trace elements, ensuring that these active substances continue to play a role in the wound healing process, thereby improving the treatment effect.

[0027] 2. The present invention has discovered through experiments that Cu 2+ 、Zn 2+ Mg 2+ , Mn2+ and Sr 2 + and vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor. The silk hydrogel wound dressing prepared according to the method of the present invention has a good effect and significantly improves the speed and quality of wound healing. The above trace elements and growth factors can be slowly released and have a long-lasting effect on the wound. In addition, the following three combinations of growth factors and trace elements have been optimized through experiments, including: vascular endothelial growth factor (VEGF) and copper (Cu2+); basic fibroblast growth factor (bFGF) and zinc (Zn2+); epidermal growth factor (EGF) and magnesium (Mg2+). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Schematic diagram of the preparation method of the silk hydrogel wound dressing containing composite trace elements and growth factors of the present invention and the experimental diagram, wherein: Figure 1 A is a schematic diagram of the extraction method of silk fibroin. Figure 1 B is a schematic diagram of the preparation method of the silk hydrogel wound dressing containing composite trace elements and growth factors. Figure 1 C is a schematic diagram of the wound of mice treated with silk hydrogel wound dressing containing composite trace elements and growth factors. The three figures below are the results of the wound of mice treated with silk hydrogel wound dressing containing composite trace elements and growth factors, which are respectively the reduction of bacterial count (antibacterial property), the reduction of inflammatory factors (anti-inflammatory property) and wound healing (improving the speed and quality of wound healing);

[0029] Figure 2 : Antibacterial test of silk hydrogel wound dressings with composite trace elements and growth factors, Figure 2 a is four groups of different Cu 2+ Actual image of silk hydrogel wound dressing with high concentration. Figure 2 b is the image of the above four groups of flat plate coating experiment results, Figure 2 c is the cell morphology images of four groups of bacteria under electron microscope. Figure 2d is the statistics of live bacteria of E. coli. Figure 2 e is the live bacteria statistics of Staphylococcus aureus S.aureus;

[0030] Figure 3 :Biocompatibility test of silk hydrogel wound dressings containing composite trace elements and growth factors, Figure 3 a is a cell growth image treated with silk hydrogel wound dressings containing four groups of composite trace elements and growth factors. Figure 3 b is the scratch test images of the above four groups, Figure 3 c is the cell viability test results of the above four groups. Figure 3 d is the detection result diagram of cell migration rate of the above four groups;

[0031] Figure 4 : A mouse wound test of silk hydrogel wound dressings containing complex trace elements and growth factors, Figure 4 a is a healing image of the mouse wound surface treated with four groups of silk hydrogel wound dressings containing complex trace elements and growth factors, and a superimposed image of the mouse wound surface. Figure 4 b is a cross-sectional diagram of tissue growth on the wound surface of mice treated by the above four groups, and a statistical diagram of wound healing rate;

[0032] In the accompanying drawings of the specification, the "*" mark represents that the difference between the test group and the control group was analyzed by SPSS with p<0.05, and the difference is statistically significant; the "**" mark represents that the difference between the test group and the control group was analyzed by SPSS with p<0.01, and the difference is extremely significant; the "***" mark represents that the difference between the test group and the control group was analyzed by SPSS with p<0.001, and the difference is more significant. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The materials, reagents, etc. used in the following examples are reagents and materials that can be obtained from commercial channels unless otherwise specified; the experiments performed in the following examples are all performed according to the experimental methods recorded in textbooks unless otherwise specified.

[0034] Figure 1 The figure is a schematic diagram of the preparation method of the silk hydrogel wound dressing containing composite trace elements and growth factors of the present invention and an experimental diagram, Figure 1 A is a schematic diagram of a method for extracting silk fibroin, specifically, firstly using sodium hydroxide to extract silk fibroin from silk, and then using a ternary solvent to dissolve the silk fibroin to obtain a silk fibroin solution; Figure 1B is a schematic diagram of the preparation method of the silk hydrogel wound dressing containing composite trace elements and growth factors, wherein a mixture containing silk, trace elements, gelatin and growth factors is irradiated and cross-linked under blue light under the catalysis of a photoinitiator, and the specific process will be described in Example 1; Figure 1 C is a schematic diagram of a mouse wound treated with a silk hydrogel wound dressing containing complex trace elements and growth factors. The three figures below are the results of a mouse wound treated with a silk hydrogel wound dressing containing complex trace elements and growth factors, which are respectively a reduction in the number of bacteria (antibacterial properties), a reduction in inflammatory factors (anti-inflammatory properties), and wound healing (improvement of the speed and quality of wound healing), which will be described in detail in Examples 2-4.

[0035] Example 1: Preparation of silk hydrogel wound dressing containing composite trace elements and growth factors

[0036] The present invention can prepare a silk hydrogel wound dressing loaded with a combination of various trace elements and growth factors. However, the antibacterial property, biocompatibility and wound healing performance of the prepared silk hydrogel wound dressing vary with the combination of different trace elements and growth factors. The following three silk hydrogel wound dressings are preferred:

[0037] 1. Silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+)

[0038] (1) Preparation of silk fibroin solution: Dissolve silk fibroin in a ternary solvent (CaCl2:CH3CH2OH:H2O) with a molar mass ratio of 1:2:8 in a water bath at 70±5°C, with a bath ratio of 1:10, and then dialyze in running water for 3 days. Centrifuge the obtained silk fibroin solution at 4°C and 10,000 r / min for 20 min. The supernatant is a silk fibroin solution with a concentration of about 2%;

[0039] (2) Preparation of gelatin-silk solution: Weigh 0.5 g of pig skin gelatin with a gelatin strength of 300 g Bloom, mix it with 20 mL of silk solution at 37°C, and gently invert to mix;

[0040] (3) adding 0.001 g of terpyridine ruthenium (II) chloride and 0.001 g of potassium persulfate (KPS) to 20 mL of gelatin silk solution, and mixing them evenly in a light-proof condition to promote the formation of a stable cross-linked structure between silk molecules;

[0041] (4) Pipette 1 mL of the cross-linking solution and add 1 μg of vascular endothelial growth factor (VEGF) and 0.001 g of copper sulfate pentahydrate (CuSO4·5H2O);

[0042] (5) A solution of vascular endothelial growth factor (VEGF) and copper sulfate pentahydrate (CuSO4·5H2O) was poured into a hydrogel mold and irradiated under a 450nm blue light LED for 5 minutes to form a hydrogel with the function of promoting wound repair.

[0043] 2. Silk hydrogel wound dressing containing basic fibroblast growth factor (bFGF) and zinc (Zn2+)

[0044] (1) Preparation of silk fibroin solution: Dissolve silk fibroin in a ternary solvent (CaCl2:CH3CH2OH:H2O) with a molar mass ratio of 1:2:8 in a water bath at 70±5°C, with a bath ratio of 1:10, and then dialyze in running water for 3 days. Centrifuge the obtained silk fibroin solution at 4°C and 10,000 r / min for 20 min. The supernatant is a silk fibroin solution with a concentration of about 2%;

[0045] (2) Preparation of gelatin-silk solution: Weigh 0.5 g of pig skin gelatin with a gelatin strength of 300 g Bloom, mix it with 20 mL of silk solution at 37°C, and gently invert to mix;

[0046] (3) adding 0.001 g of terpyridine ruthenium (II) chloride and 0.001 g of potassium persulfate (KPS) to 20 mL of gelatin silk solution, and mixing them evenly in a light-proof condition to promote the formation of a stable cross-linked structure between silk molecules;

[0047] (4) Pipette 1 mL of the cross-linking solution and add 1 μg of basic fibroblast growth factor (bFGF) and 0.001 g of zinc sulfate (ZnSO4);

[0048] (5) A solution of basic fibroblast growth factor (bFGF) and zinc sulfate (ZnSO4) is poured into a hydrogel mold and irradiated under a 450nm blue light LED for 5 minutes to form a hydrogel with the function of promoting wound repair.

[0049] 3. Silk hydrogel wound dressing loaded with epidermal growth factor (EGF) and magnesium (Mg2+)

[0050] (1) Preparation of silk fibroin solution: Dissolve silk fibroin in a ternary solvent (CaCl2:CH3CH2OH:H2O) with a molar mass ratio of 1:2:8 in a water bath at 70±5°C, with a bath ratio of 1:10, and then dialyze in running water for 3 days. Centrifuge the obtained silk fibroin solution at 4°C and 10,000 r / min for 20 min. The supernatant is a silk fibroin solution with a concentration of about 2%;

[0051] (2) Preparation of gelatin-silk solution: Weigh 0.5 g of pig skin gelatin with a gelatin strength of 300 g Bloom, mix it with 20 mL of silk solution at 37°C, and gently invert to mix;

[0052] (3) adding 0.001 g of terpyridine ruthenium (II) chloride and 0.001 g of potassium persulfate (KPS) to 20 mL of gelatin silk solution, and mixing them evenly in a light-proof condition to promote the formation of a stable cross-linked structure between silk molecules;

[0053] (4) Pipette 1 mL of the cross-linking solution and add 1 μg of epidermal growth factor (EGF) and 0.001 g of magnesium sulfate (MgSO4);

[0054] (5) A solution of epidermal growth factor (EGF) and magnesium sulfate (MgSO4) is poured into a hydrogel mold and irradiated under a 450nm blue light LED for 5 minutes to form a hydrogel with the function of promoting wound repair.

[0055] The above are the three preferred methods for preparing silk hydrogel wound dressings in this embodiment. Different trace elements and growth factors can be replaced according to the above methods to prepare corresponding silk hydrogel wound dressings.

[0056] Example 2: Antibacterial effect of silk hydrogel wound dressing

[0057] Taking the silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+) in Example 1 as an example, the amount of copper (Cu2+) added during the preparation process was adjusted to obtain silk hydrogel wound dressings containing no copper (Cu2+), 0.5%, 1.0% and 2.0% copper (Cu2+), respectively. Two groups were set up, and equal amounts of the above silk hydrogel wound dressings were evenly coated on the plates of the two groups, and then Escherichia coli E.coli and Staphylococcus aureus S.aureus were coated. The antibacterial effect was tested at 72 hours, and the results were as follows: Figure 2 shown.

[0058] Figure 2 a is the corresponding real image of silk hydrogel wound dressing. Figure 2 b is the image of the plate coating at 72 hours. It can be seen that with the increase of copper (Cu2+) content, the number of colonies on the plate decreases visibly, indicating that the silk hydrogel wound dressing with copper (Cu2+) added has antibacterial properties. Figure 2 c is the bacterial morphology under electron microscope. It can be seen that with the increase of copper (Cu2+) content, the bacterial cell structure is destroyed. Figure 2 d and Figure 2e are the live bacteria statistics of Escherichia coli E.coli and Staphylococcus aureus S.aureus, respectively, indicating that with the increase of copper (Cu2+) content, the live bacteria count of Escherichia coli E.coli and Staphylococcus aureus S.aureus decreased significantly, and there were significant differences among the groups containing 0.5%, 1.0% and 2.0% copper (Cu2+).

[0059] Example 3: Biocompatibility of silk hydrogel wound dressing

[0060] Taking the silk hydrogel wound dressing loaded with vascular endothelial growth factor (VEGF) and copper (Cu2+) in Example 1 as an example, the biocompatibility of the silk hydrogel wound dressing was tested, and the following four groups were set up respectively: a control group (Control), i.e., a blank control, without any treatment; a silk hydrogel wound dressing loaded with copper (CuSF), i.e., prepared by the method of Example 1, but without adding growth factors; Example 1 (CuSF@VEGF); a silk hydrogel wound dressing loaded with vascular endothelial growth factor (VEGF), i.e., prepared by the method of Example 1, but without adding copper. Mouse fibroblast (3T3) cells were used for cell compatibility test, and then the silk hydrogel wound dressings of each group were incubated with DMEM high glucose medium supplemented with 10% fetal bovine serum to culture cells, and the cell viability of 3T3 cells was tested by referring to the CCK-8 method and the scratch test method; human umbilical vein endothelial cells (HUVEC) were used for cell scratch migration test, and the cells with density greater than 90% were cultured with the silk hydrogel wound dressings of each group and DMEM high glucose medium supplemented with less than 2% fetal bovine serum to detect the migration effect of HUVEC. The results are as follows: Figure 3 shown.

[0061] Figure 3 a is the cell image after 24h, 48h and 72h of treatment with the extract of silk hydrogel wound dressing co-incubated with cell culture medium. The black part is the wound background and the gray part is the cell. Figure 3 b is the image of HUVEC treated with the extract of silk hydrogel wound dressing co-incubated with cell culture medium at 0h and 24h, and the dotted line is the initial state of cell scratch; Figure 3 c is the test result of cell viability, which shows that after 72 hours of treatment with the extract of silk hydrogel wound dressing co-incubated with cell culture medium, the cell viability of CuSF@VEGF group and VEGF group was significantly improved compared with other groups. Figure 3 d is the test result of cell migration rate, which shows that the cell migration rate of CuSF@VEGF group and VEGF group after the wound surface was treated with silk hydrogel wound dressing was significantly improved compared with other groups.

[0062] Example 4: Mouse wound test of silk hydrogel wound dressing

[0063] Taking the silk hydrogel wound dressing loaded with vascular endothelial growth factor (VEGF) and copper (Cu2+) in Example 1 as an example, a mouse experiment was conducted to test its healing effect on mouse wounds; the following four groups were set up respectively: a control group (Control), i.e. a blank control, without any treatment; a silk hydrogel wound dressing loaded with copper (CuSF), i.e. prepared by the method of Example 1, but without adding growth factors; Example 1 (CuSF@VEGF); a silk hydrogel wound dressing loaded with vascular endothelial growth factor (VEGF), i.e. prepared by the method of Example 1, but without adding copper. After one week of adaptive feeding of ICR mice (8 weeks old), the mice were anesthetized with isoflurane for respiratory anesthesia. After anesthesia, the backs of the mice were depilated with a razor, and the mice were modeled with a circumcision knife. A circular wound with a diameter of 1 cm was made on the back of each mouse; the wound healing conditions were photographed and measured on the 0th, 3rd, 6th, 9th, and 12th days from the beginning of the experiment, and the results are shown as follows: Figure 4 shown.

[0064] Figure 4 a is the healing images and overlap images of the mouse wounds after being treated with four groups of silk hydrogel wound dressings. It can be seen that Example 1 (CuSF@VEGF) has the best healing effect on the mouse wounds, the fastest healing speed, and is almost completely healed on the 12th day; Figure 4 b is a cross-sectional view of the mouse wound and a wound treatment rate result diagram. The framed range is the range of tissue growth. It can be seen that Example 1 (CuSF@VEGF) has the fastest healing speed for the mouse wound. The wound treatment rate result diagram shows that there are significant differences in the healing effects among different groups, and Example 1 (CuSF@VEGF) has the best effect.

[0065] Example 5: Experiment on the effect of preparation method on the healing effect of wound dressing

[0066] Examples 2-4 show that the silk hydrogel wound dressing containing composite trace elements and growth factors of the present invention has good antibacterial properties, biocompatibility and healing effect on the wound surface. Under the preparation method of the present invention, the trace elements and growth factors have a synergistic effect. This example attempts to use other methods to prepare the silk hydrogel wound dressing containing composite trace elements and growth factors, and compares its healing effect on the wound surface.

[0067] Comparative Preparation Example 1: The preparation method of the silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+) is referred to Example 1, except that terpyridine ruthenium (II) chloride is not added;

[0068] Comparative Preparation Example 2: The preparation method of the silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+) is referred to Example 1, except that the wavelength of the blue light is 220 nm;

[0069] Comparative Preparation Example 3: Referring to the preparation method of the silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+) in Example 1, the difference is that the vascular endothelial growth factor (VEGF) is replaced by insulin-like growth factor (IGF);

[0070] Comparative Preparation Example 4: Referring to the preparation method of the silk hydrogel wound dressing carrying vascular endothelial growth factor (VEGF) and copper (Cu2+) of Example 1, the difference is that the copper (Cu2+) is replaced by silver (Ag2+), specifically silver nitrate;

[0071] The silk hydrogel wound dressings prepared by the above four methods and the silk hydrogel wound dressings carrying vascular endothelial growth factor (VEGF) and copper (Cu2+) in Example 1 were tested for wound healing in mice according to the method of Example 4. The healing conditions on the 3rd and 12th days were mainly tested, reflecting the healing rate and the comprehensive healing effect, respectively, measured in terms of wound rate, wound rate % = wound area / initial wound area. The results are shown in Table 1.

[0072] Table 1: Wound healing effects of Example 1 and comparative preparation examples on mice

[0073]

[0074]

[0075] The results show that the healing rate of the silk hydrogel wound dressing containing vascular endothelial growth factor (VEGF) and copper (Cu2+) prepared in Example 1 is the fastest and the comprehensive healing effect is the best; if terpyridine ruthenium (II) chloride is not added and only the photoinitiator potassium persulfate is used, the healing rate and the comprehensive healing effect are significantly reduced; the wavelength of blue light has a similar effect to the composition of the photoinitiator, both of which affect the cross-linking process and thus affect the healing effect of the final product on the wound. The present invention has also conducted tests on other blue light wavelengths and photoinitiators. Only under a specific 450nm blue light wavelength and photoinitiators of terpyridine ruthenium (II) chloride and potassium persulfate can the best effect be obtained; during the cross-linking process, the types of growth factors and trace elements will also have an impact. The present invention has also conducted tests on other growth factors and trace elements, and preferred trace elements are copper (Cu2+), zinc (Zn2+), magnesium (Mg2+), manganese (Mn2+), strontium (Sr2+), and zinc (Zn2+). 2 +), more preferably the combination of the three trace elements and growth factors in Example 1.

[0076] In addition, the silk hydrogel wound dressings containing vascular endothelial growth factor (VEGF) and copper (Cu2+) prepared in the above-mentioned comparative preparation examples 1, 2 and Example 1 were tested for growth factor and trace element release. The test method was carried out according to the method of Example 4. Epithelial tissue was sampled at the wound surface of mice on the 3rd and 12th days of the experiment, and the growth factor and trace element content therein was detected by high performance liquid chromatography. The results are shown in Table 2.

[0077] Table 2: Growth factor and trace element release test

[0078] Grouping Example 1 Comparative Preparation Example 1 Comparative Preparation Example 2 Growth factors (day 3) ++++ - - Trace elements (day 3) ++++ + + Growth factors (day 12) ++++ - - Trace elements (day12) +++ - -

[0079] Note: "+" means it contains, each additional "+" means the content is one order of magnitude higher, "-" means it does not contain

[0080] The results show that only the silk hydrogel wound dressing loaded with vascular endothelial growth factor (VEGF) and copper (Cu2+) prepared in Example 1 of the present invention can stably release growth factors and trace elements, ensuring that the contents of vascular endothelial growth factor (VEGF) and copper (Cu2+) on the wound surface are always at a high value, which is beneficial to the healing of the wound, as well as the improvement of the speed and quality of wound healing; the dressings prepared by other methods no longer release growth factors on day 3, because the growth factor load is small and the growth factor loss rate cannot be reduced. No growth factors and trace elements are released on day 12, indicating that the trace elements in the content are exhausted, which is not conducive to the healing of the wound.

[0081] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto, and any equivalent modifications or substitutions that can be easily thought of by any technician familiar with the technical field within the technical scope disclosed by the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors, characterized in that: Includes steps: S1, preparing a silk fibroin solution: using a solvent to dissolve silk fibroin, dialyzing it with running water, and centrifuging to obtain a supernatant to obtain a silk fibroin solution; S2, preparing a gelatin-silk fibroin solution: mixing the pig skin-derived gelatin with the silk fibroin solution to obtain a gelatin-silk fibroin solution; S3, preparing a cross-linking solution: adding a photoinitiator to the gelatin silk solution, and mixing them uniformly under light-proof conditions to obtain a cross-linking solution; S4, loading growth factors and trace elements: adding growth factors and trace elements to the cross-linking solution, pouring it into a hydrogel mold, and irradiating it under blue light.

2. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors according to claim 1, characterized in that: In the step S1, the solvent is a ternary solvent with a molar mass ratio of CaCl2, CH3CH2OH and H2O of 1:2:

8.

3. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors according to claim 1, characterized in that: In step S2, the gelatin strength of the pigskin-derived gelatin is 300 g Bloom.

4. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors according to claim 1, characterized in that: In the step S3, the photoinitiator includes terpyridine ruthenium (II) chloride and potassium persulfate.

5. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors as claimed in claim 1, characterized in that: In step S4, the trace elements include Cu 2+ 、Zn 2+ Mg 2+ , Mn2+ and Sr 2 + one or more.

6. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors as claimed in claim 5, characterized in that: In step S4, the concentration of the trace elements is 0.5-2%.

7. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors according to claim 1, characterized in that: In step S4, the growth factor includes one or more of vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor.

8. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors as claimed in claim 7, characterized in that: In step S4, the concentration of the growth factor is 1 μg / mL.

9. The method for preparing a silk fibroin hydrogel wound dressing containing composite trace elements and growth factors according to claim 1, characterized in that: In step S4, the wavelength of the blue light is 450 nm.

10. A silk hydrogel wound dressing containing composite trace elements and growth factors prepared by the method according to any one of claims 1 to 9.

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