1. The composite hydrogel is characterized by comprising fibroin, silver nanoparticles and aloe gel.

2. The composite hydrogel according to claim 1, wherein the molecular weight of the fibroin is < 3500 MW.

3. The method for preparing the composite hydrogel according to claim 1 or 2, comprising the steps of: (1) preparing the fibroin into a fibroin solution; (2) then mixing the Ag + and the aloe gel to obtain a mixed solution; (3) and carrying out photochemical reaction on the mixed solution.

4. The method according to claim 3, wherein the fibroin concentration in the mixed solution is 4-6 wt%, the Ag + solution is 0.4-0.6mg/ml, and the aloe vera gel concentration is 1-9 mg/ml.

5. The preparation method according to claim 3, wherein the mass ratio of the fibroin solution to the Ag + and aloe gel is 250: 2-2.5: 5-15.

6. The method according to claim 4 or 5, wherein Ag + is AgNO₃。

7. The method of claim 3, wherein the photochemical reaction is: continuously illuminating under 35-45W of incandescent light, wherein the illumination time is more than or equal to 24 h.

8. The method of claim 3, wherein the photochemical reaction is: or in a photochemical reaction instrument or a photochemical reaction kettle, a mercury lamp, a xenon lamp or a metal halide lamp is selected as a reaction light source, the illumination power is set to be 35-45W, the reaction temperature is 20-30 ℃, and the continuous illumination is more than or equal to 24 h.

9. Use of a composite hydrogel according to any one of claims 1 to 2 as or in the preparation of one or more of a wound healing dressing, an angiogenesis promoting agent, a TGF- β expression promoting agent, an EGF expression promoting agent, a VEGF expression promoting agent.

10. A method for improving the antibacterial/biological properties of fibroin, which comprises preparing fibroin into a fibroin composite hydrogel by using the preparation method of any one of claims 3-5.

Background

The skin is the largest organ of the human body, consisting of the epidermis and dermis, with a complex network of nerves and blood vessels. Thus, skin damage can cause physiological discomfort and functional problems for the patient. The skin plays a crucial role in protecting the human body from external mechanical damage. Skin is the first barrier against the external environment, and thus, damage to the skin can lead to serious physiological and functional disorders. Wound healing is a dynamic process that includes four links of hemostasis, immunity, proliferation and remodeling. Human skin has a certain self-healing capacity, however, the long repair process inevitably increases the risk of chronic inflammation. Chronic inflammatory reactions local to the wound can impede fibroblast migration and ECM formation, ultimately slowing the process of wound healing. Autologous skin grafting is considered to be the most effective method for treating traumatic skin defects. However, there are still certain difficulties in the clinical practice for the care and recovery of wounds in the donor area. At present, the gold standard for solving this problem is still the majority of treatments with wound dressings. Skin defects are a very delicate clinical problem, and incorrect treatment may lead to irreversible damage. An ideal multifunctional wound dressing must have the following conditions: good biocompatibility, capability of completely blocking the external environment, capability of providing a relatively humid environment for wound healing, air permeability, easiness in separation and no secondary damage. With reference to the above criteria, a variety of biological dressings have been developed. Natural materials, synthetic materials, and composites of the two are three categories that are currently in major use. In recent years, natural biomaterials have received increasing attention in scientific research. Therefore, it is crucial to construct a wound dressing with superior bioactivity.

Among a plurality of dressings constructed based on natural biomaterials, fibroin (SF) has attracted extensive attention because it can regulate the expression of certain signal pathways and further promote wound healing, and is a natural biomaterial with development prospects. However, the antibacterial property of the fibroin still needs to be improved, and the biological performance of the fibroin can be further enhanced, so that the ideal multifunctional wound dressing can be prepared.

Disclosure of Invention

In view of the above, the present invention provides a composite hydrogel, which is a fibroin-based gel that can be used in a wound dressing. Fibroin can regulate and control the expression of certain signal paths to promote wound healing, silver nanoparticles are a natural antibacterial agent with superior performance, aloe gel is a product extracted from natural herbal aloe and has remarkable biological activities of oxidation resistance, antibiosis, inflammation resistance, ulcer resistance and the like, no data report exists at present, and fibroin obtains a composite hydrogel based on fibroin under the action of the silver nanoparticles and the aloe gel through certain process conditions and can effectively cure wounds when used as a dressing. Compared with the prior wound dressing, the invention uses two antibacterial agents to improve the antibacterial performance of the wound, and evaluates the healing promotion effect of the wound from the aspects of histology and biochemistry.

Specifically, fibroin is a natural biological material isolated from silkworm cocoons and comprises eighteen amino acids, wherein the ratio of alanine, glycine and serine is the highest. These amino acids are ordered by disulfide bond arrangement to form a 1: 1 ratio of heavy and light chains. The large number of amino acid arrays not only provides a large number of binding sites, but also ensures structural stability. Fibroin is a biological material with a great application prospect, and has been applied to the fields of tissue engineering (such as artificial ligaments, artificial blood vessels and the like) and other biomedicine fields due to the properties of good biocompatibility, weak immunogenicity, no biotoxicity, no carcinogenicity, mechanical stability, controllable degradation rate and high benefit. The polypeptide sequence Arg-Gly-Asp (RGD) contained in the fibroin can support the proliferation, migration and differentiation of various cells such as epidermal cells, endothelial cells, fibroblasts and the like. Fibroin has a variety of application forms: foams, hydrogels, electrospun micro-felts, membranes, scaffolds, and the like. Among the many application forms, hydrogels are the most suitable choice for wound dressings. The hydrogel formed by the fibroin can be tightly attached to the skin on the premise of no exogenous adhesive. Fibroin can promote wound healing by regulating proliferation, migration and differentiation of various cells at different stages of wound healing, however, its biological properties can still be significantly improved by the introduction of other bioactive substances.

The silver nano-particles are natural antibacterial agents with outstanding performance, have broad-spectrum antibacterial property and no drug resistance, can kill harmful bacteria in a short time, have strong permeability, can quickly penetrate into the subcutaneous skin through pores, and have strong killing effect on common bacteria, fungi and drug-resistant bacteria. Besides good antibacterial property, the silver nanoparticles can improve the microenvironment around the wound, effectively accelerate cell growth and reduce scar formation. The silver nanoparticle coated silk-based biomaterial is effective against Escherichia coli and Staphylococcus aureus. In addition, the close fit between the SF hydrogel and the human skin ensures that the fibroin-loaded nanoparticles can penetrate from the stratum corneum into the inner layer.

Aloe vera gel is a product extracted from the natural herb aloe vera, and its earliest record of use dates back to ancient roman times and even earlier. The aloe gel consists of tannin, saponin, flavonoid compounds, steroid and glucomannan, stimulates the proliferation and migration of fibroblasts by activating skin macrophages and influencing fibroblast growth factors, and has good wound healing potential. In addition, the aloe gel has obvious biological activities of oxidation resistance, antibiosis, anti-inflammation, ulcer resistance and the like. Glucose-6-phosphate and mannose-6-phosphate exhibit very strong wound healing and anti-inflammatory activities in aloe vera gel, and trace elements and anthraquinone compounds therein can decompose toxins and eliminate inflammation. Other SF-like amino acids like glycine, serine, aspartic acid confer potential for AV application as an external dressing during amino acid renewal in the skin.

The composite hydrogel comprises fibroin solution, silver nanoparticles and aloe gel.

Preferably, the molecular weight of the fibroin in the fibroin solution is < 3500 MW.

Furthermore, the invention also provides a preparation method of the composite hydrogel, the preparation method is to reduce Ag + into Ag NPs in situ in the presence of high-concentration SF through photochemical reaction, so that the Ag NPs are uniformly embedded on the surface of the SF, and the loose and porous structure of the hydrogel promotes cell adhesion and extracellular matrix (ECM) formation. And the preparation method is simple and feasible, and can realize large-scale production.

The preparation method comprises the following steps: (1) preparing the fibroin into a fibroin solution; (2) then mixing the Ag + and the aloe gel to obtain a mixed solution; (3) and carrying out photochemical reaction on the mixed solution.

Preferably, the molecular weight of the fibroin is < 3500 MW.

Preferably, in the mixed solution, the concentration of the fibroin is 4-6 wt%, the concentration of the Ag + is 0.4-0.6mg/ml, and the concentration of the aloe gel is 1-9 mg/ml. Preferably 1-3mg/ml, more preferably 3 mg/ml.

Preferably, in the mixed solution, the concentration of the fibroin is 5 wt%, the concentration of the Ag + is 0.5mg/ml, and the concentration of the aloe gel is 1-3 mg/ml.

Preferably, in the mixed solution, the concentration of the fibroin is 5 wt%, the concentration of the Ag + is 0.5mg/ml, and the concentration of the aloe gel is 3 mg/ml.

Preferably, the mass ratio or volume ratio of the fibroin solution, the silver nanoparticles and the aloe gel is 250: 2-2.5: 5-15.

In some embodiments, the composite gel is prepared by adding 20-30mg of AgNO3 and 50-150mg of aloe vera lyophilized powder to 50ml of a 4-6 wt% fibroin solution, mixing thoroughly, and exposing to light.

Preferably, the Ag + and the fibroin solution are mixed and stirred, and then the aloe gel is added for mixing and stirring.

Preferably, the Ag + is AgNO₃。

Preferably, the photochemical reaction is: continuously illuminating under 40W of incandescent light, wherein the illumination time is more than or equal to 24 h; or in a photochemical reaction instrument or a photochemical reaction kettle, a mercury lamp, a xenon lamp or a metal halide lamp is selected as a reaction light source, the illumination power is set to be 35-45W, the reaction temperature is 20-30 ℃, and the continuous illumination is more than or equal to 24 h.

Specifically, the preparation method of the fibroin is a preparation method which is conventional for a person skilled in the art, such as lithium bromide (LiBr) preparation, calcium chloride-absolute ethyl alcohol-deionized water solution (CaCl)₂-C₂H₅OH-ddH₂0) A preparation method.

In some embodiments, the fibroin is prepared by a method comprising: cutting silkworm cocoon into small pieces, and adding 0.02M Na₂CO₃Boiling in the solution for 60min to remove surface sericin component; washing with deionized water, and drying at 60 deg.C; dissolving silk in 9.3M LiBr in water bath for four hours until the silk is completely dissolved; dialyzing the dissolved solution in a dialysis bag (molecular weight cut-off is 3500MW) for 72h to obtain pure fibroin solution; after centrifugation for 15min, the precipitate was removed by filtration.

The invention also aims to provide application of the composite hydrogel. The composite hydrogel improves the antibacterial property and the biological property of the fibroin and can be well used as a wound dressing.

Further, the composite hydrogel may be used directly as a wound healing dressing; can also be used as active, and then added with some synergistic auxiliary materials to prepare the wound healing dressing; or adding some outer layers to prepare medical supplies such as wound dressings and the like.

Specifically, the wound referred to herein may be a lesion on the skin in different ways, such as a fall, burn, or knife. The composite hydrogel can induce weak immune response and enhance the formation of new blood vessels in histology; the biochemical results show that the expression levels of TGF-beta, EGF and VEGF can be up-regulated, so that the healing of wounds is accelerated.

Therefore, further, the composite hydrogel can also be directly used as an angiogenesis promoter or directly used as a cytokine expression promoter of one or more of TGF-beta, EGF and VEGF; or the composite hydrogel is used as an active agent, and some auxiliary materials are added to prepare an angiogenesis promoter or prepare one or more cell factor expression promoters of TGF-beta, EGF and VEGF.

Or preparing angiogenesis promoter or TGF-beta, EGF, VEGF expression promoter.

The invention also aims to provide a method for improving the antibacterial property/biological property of the fibroin, which is characterized in that silver nanoparticles with antibacterial property and aloe gel with biological activities of oxidation resistance, antibiosis, anti-inflammation, ulcer resistance and the like are added into a fibroin solution to creatively combine to form a composite hydrogel, so that the antibacterial property/biological property of the fibroin is improved. The silver nanoparticles and the aloe gel in the composite hydrogel exert a synergistic effect to improve the antibacterial performance of the fibroin.

Specifically, the biological properties are conventional terms of those skilled in the art, including but not limited to cytotoxicity, hemocompatibility and biocompatibility, and the present invention mainly improves the cytotoxicity and biocompatibility.

The method comprises the following steps: (1) preparing the fibroin into a fibroin solution; (2) then mixing the Ag + and the aloe gel to obtain a mixed solution; (3) and carrying out photochemical reaction on the mixed solution.

Preferably, the molecular weight of the fibroin is < 3500 MW.

Preferably, in the mixed solution, the concentration of the fibroin is 4-6 wt%, the concentration of the Ag + is 0.4-0.6mg/ml, and the concentration of the aloe gel is 1-9 mg/ml. Preferably 1-3mg/ml, more preferably 3 mg/ml.

Preferably, in the mixed solution, the concentration of the fibroin is 5 wt%, the concentration of the Ag + is 0.5mg/ml, and the concentration of the aloe gel is 1-3 mg/ml.

Preferably, in the mixed solution, the concentration of the fibroin is 5 wt%, the concentration of the Ag + is 0.5mg/ml, and the concentration of the aloe gel is 3 mg/ml.

Preferably, the mass ratio or volume ratio of the fibroin solution, the silver nanoparticles and the aloe gel is 250: 2-2.5: 5-15.

In some embodiments, the composite gel is prepared by mixing 20-30mg AgNO₃Adding 50-150mg of aloe gel lyophilized powder into 50ml of 4-6 wt% fibroin solution, and irradiating.

Preferably, the Ag + and the fibroin solution are mixed and stirred, and then the aloe gel is added for mixing and stirring.

Preferably, the Ag + is AgNO₃。

Preferably, the photochemical reaction is: performing a photochemical reaction using incandescent light; more preferably, the illumination is continued under a 40W incandescent lamp, and the illumination time is more than or equal to 24 h; or in a photochemical reaction instrument or a photochemical reaction kettle, a mercury lamp, a xenon lamp or a metal halide lamp is selected as a reaction light source, the illumination power is set to be 35-45W, the reaction temperature is 20-30 ℃, and the continuous illumination is not less than 24h

Specifically, the preparation method of the fibroin is a preparation method which is conventional for a person skilled in the art, such as lithium bromide (LiBr) preparation, calcium chloride-absolute ethyl alcohol-deionized water solution (CaCl)₂-C₂H₅OH-ddH₂0) A preparation method.

In some embodiments, the fibroin is prepared by a method comprising: cutting silkworm cocoon into small pieces, and adding 0.02M Na₂CO₃Boiling in the solution for 60min to remove surface sericin component; washing with deionized water, and drying at 60 deg.C; dissolving silk in 9.3M LiBr in water bath for four hours until the silk is completely dissolved; dialyzing the dissolved solution in a dialysis bag (molecular weight cut-off is 3500MW) for 72h to obtain pure fibroin solution; after centrifugation for 15min, the precipitate was removed by filtration.

The invention has the beneficial effects that

The composite hydrogel provided by the invention has a good healing effect on a wound model animal as a wound dressing, and the wound has an obvious treatment effect in 14 days compared with a control group.

The histological observation results of different organs of the model animal show that the composite hydrogel provided by the invention has reliable biological safety, such as good cytotoxicity, good cell proliferation and migration capacity, and the like.

The composite hydrogel provided by the invention can obviously promote the generation of new blood vessels by observing wounds in histology, and in histological analysis, the composite hydrogel induces a weaker immune response and enhances the formation of new blood vessels; the biochemical result shows that the hydrogel can up-regulate the expression levels of TGF-beta, EGF and VEGF, and can be used as a wound dressing with development prospect to solve the future clinical problem.

Drawings

FIG. 1 is a schematic diagram of the construction process of Ag-AV-SF hydrogel.

Fig. 2 is a schematic diagram of the application of hydrogel in a wound animal model.

Fig. 3 is a representation of a fibroin-based hydrogel.

FIG. 4 is a graph of the ability of aloe vera gel to promote cell proliferation.

FIG. 5 shows FTIR results for SF hydrogels versus Ag-AV-SF hydrogels.

Fig. 6 shows the results of thermogravimetric analysis of four hydrogels.

FIG. 7 shows the release rate of silver element in Ag-AV-SF hydrogel.

FIG. 8 shows an adhesion performance evaluation model and a tissue adhesion state.

FIG. 9 shows frozen sections at 1h and 24h after adhesion of pigskin and fluorescence imaging thereof.

FIG. 10 is a fluorescence characterization of in vivo diffusion of Ag-AV-SF hydrogels.

FIG. 11 is a schematic representation of the antimicrobial ring diameters of different hydrogels.

FIG. 12 is a graph showing the statistical analysis of the size of the diameter of the antibacterial ring.

FIG. 13 is a diagram showing the experimental conditions of CCK-8.

FIG. 14 is a fluorescence characterization image of cell proliferation.

Fig. 15 is a graph showing the scratch test.

FIG. 16 is the mean decrease in cell spacing.

Fig. 17 is an image of an animal living body in a wound healing state.

FIG. 18 is a statistical plot of wound healing rates at 14 days and 28 days.

FIG. 19 is an immunohistochemical staining of HE staining, CD31 and CD 68.

FIG. 20 is a massson staining of tissues at day 14.

Fig. 21 is a blood vessel count.

Fig. 22 shows the number of positive results for CD 31.

Fig. 23 shows the number of positive results for CD 68.

FIG. 24 shows the relative expression level of TGF-. beta.at day seven.

FIG. 25 is the relative expression amounts of VEGF at day 28.

FIG. 26 is a graph showing the relative expression amounts of EGF at day 28.

FIG. 27 shows HE staining results of different organs (heart, brain, spleen, lung, testis, kidney).

In FIG. 3, a-d are the morphological changes of the fibroin solution after 24h of continuous illumination; e is a scanning electron microscope image of the reduced silver nanoparticles; f is an internal loose porous structure of the Ag-AV-SF composite hydrogel; g is the first letter of Chongqing medical university formed by mixing Ag-AV-SF composite hydrogel with haematochrome and injecting: chong Qi Medical University (CQMU); h-f is the EDX spectrum of the elements of the Ag-AV-SF hydrogel.

In FIG. 20, 4X (scale bar:500 μm) and 10X (scale bar:200 μm)

In fig. 1 to 27, P <0.05, P <0.01, and P <0.001 are shown.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

In the present example, the process of constructing the Ag-AV-SF hydrogel is shown in FIG. 1.

In the present example, the application process of the hydrogel in the animal model of trauma is shown in fig. 2.

In the embodiment of the invention, the composite biological hydrogel is prepared through photochemical reaction, and the silver nanoparticles and the aloe-modified SF hydrogel (Ag-AV-SF hydrogel) are adopted to accelerate the full-thickness healing of the wound.

In the examples of the present invention, the physical and chemical properties were characterized and studied by SEM, EDX, FTIR, TGA and ICP-MC.

In the examples of the present invention, the biological activity was verified using CCK-8, scratch test and fluorescent staining.

In the embodiment of the invention, a wound healing experiment is carried out in vivo, a wound model rat is treated by using the composite hydrogel, HE staining and immunohistochemical staining are carried out on CD31 and CD68, and the expression level of cytokines (TGF-beta, EGF and VEGF) in serum is analyzed, so that the treatment effect of the composite hydrogel on the wound model rat is analyzed.

In the examples of the present invention, in consideration of biosafety of hydrogel at the time of application, in vivo diffusion test and HE staining of various organs were performed.

Example 1 preparation of fibroin hydrogels

Cutting silkworm cocoon into small pieces, and adding 0.02M Na₂CO₃Boiling the solution for 60min to remove surface sericin component. Washed clean by deionized water and dried at 60 ℃. Silk was dissolved in 9.3M LiBr in a water bath for four hours until the silk was completely dissolved. Dialyzing the dissolved solution in a dialysis bag (molecular weight cut-off is 3500MW) for 72h to obtain pure fibroin solution. After centrifugation for 15min, the precipitate was removed by filtration. Finally, the fibroin solution concentration was adjusted to 5 wt%. The three gels of Ag-SF, AV-SF and Ag-AV-SF are respectively processed by passing AgNO₃AV and mixed systems of both were dissolved in the fibroin solution. Ensuring the safest biotoxicity and complete reduction of Ag (I) to Ag (0) and AgNO₃The concentration was set to 0.5 mg/ml.

Wherein the preparation of Ag-AV-SF is as follows: 25mg of AgNO₃Mixing with 150mg Aloe gel lyophilized powder, adding into 50ml 5 wt% fibroin solution, and mixingGel solution, pre-gel solution was transferred to petri dish and light was continued for 24h under 40W incandescent lamp to obtain final hydrogel.

The preparation of the Ag-SF, AV-SF, SF hydrogels was as described above.

The conditions during hydrogel formation are shown in fig. 3 as a, b, c and d, the pre-hydrogel solution (a-b) is successfully converted from a transparent clear solution into a viscous dark yellow hydrogel (c-d), and the hydrogel is attached to the bottom of the sample bottle after being illuminated.

The silver nanoparticles prepared by in situ reduction of fibroin were observed by scanning electron microscope (SU8010, Hitachi, ltd. tokyo, Japan), and the spatial structure of hydrogel was observed after freeze-drying, and as shown by e in fig. 3, the prepared Ag NPs were uniformly dispersed on the protein, demonstrating that the Ag NPs were obtained by SF in situ reduction in the composite system.

In order to verify that the SF solution successfully reduces the silver nanoparticles, the Ag-AV-SF mixed solution is subjected to SEM characterization, the condition is shown as f in figure 3, and the SEM result of the Ag-AV-SF hydrogel shows that the internal structure of the hydrogel is loose and porous.

Subsequently, the hydrogel was mixed with a red pigment and the initials of Chongqing university of medicine (CQMU) were drawn on clear glass to show that the hydrogel had good plasticity, as shown by g in FIG. 3.

The EDX spectrum characteristic peak of the Ag-AV-SF hydrogel shows that SF successfully exerts the reduction action to form Ag NPs as shown in the h part in figure 3.

Elemental mapping analysis as shown in section i of fig. 3, well-dispersed nitrogen (blue), silver (green), and carbon (pale yellow) confirmed that Ag NPs are embedded on the SF surface, indicating that the final Ag NPs are obtained by in-situ reduction in the presence of high concentration of SF.

Example 2 screening of optimum concentration of Aloe gel

L929 cells were seeded in 96-well plates. Taking aloe gel solutions with different concentrations as stimulating factors, adding a CCK-8 reagent after acting for 24h, incubating for 2h at 37 ℃, detecting the absorbance value, and selecting the optimal concentration of the aloe gel.

As shown in FIG. 4, the aloe vera gel concentration is 1mg/ml to 9mg/ml, preferably 3 mg/ml.

EXAMPLE 3 hydrogel physical Properties study

(1) Fourier transform infrared spectroscopy

The binding of functional groups in the gel composite system was studied by Fourier transform Infrared Spectroscopy (FTIR) (Thermo Fisher Scientific-CN, USA) at 4000 cm-1-400 cm-1, and the changes in the binding of functional groups within different hydrogels are shown in FIG. 5: C-N stretching (1451.88cm-1) into newly formed C-N bonds (1017.68 cm-1); the action strength of C-O bond (3339.11cm-1), C-O bond (1634.70cm-1, 1249.80cm-1) and N-H tensile (1543.73cm-1) in the Ag-AV-SF system is obviously higher than that of SF hydrogel. Under an alkaline environment, alpha-amino nitrogen and alpha-carboxyl oxygen generated by tyrosine form a complex with silver ions through coordination bonds, and a stretching vibration phenomenon is formed.

(2) Thermogravimetric analysis

The thermal stability of the four hydrogels, SF, AV-SF, Ag-SF and Ag-AV-SF, was tested by measuring the relative weight change of the four composite gels during the temperature rise from room temperature to 500 ℃ with a thermal gravimetric analyzer (SDT650, TA, USA), and as shown in FIG. 6, the relative mass of the Ag-AV-SF hydrogel was still higher than that of the other hydrogels after the temperature rise from room temperature to 500 ℃, and this trend was maintained throughout the heating process. The TGA results indicate that the Ag-AV-SF hydrogel has good thermal stability, indicating that it can maintain structural integrity under storage and practical application conditions.

(3) Rate of silver ion release

Samples were collected at different time points, and the content of silver element in the samples was measured by an inductively coupled plasma source mass spectrometer (Agilent7900, USA) and calculated by a formula.

(M1: Total content of silver element; D: detection time point)

The release rate of Ag + is shown in FIG. 7, after PBS dialyzed for 7d (p H ═ 7.4), the release mass of Ag NPs reaches 60.256%; the release rate was about 10%/day. Furthermore, the Ag content in 7 consecutive detection spots is linear (R2 ═ 0.9628), indicating that the silver nanoparticles are continuous and uniform throughout the release process. In vivo experiments, the hydrogel dressing is injected repeatedly every 3 days, so that the silver nanoparticles released on the wound surface can effectively inhibit the growth of bacteria and improve the bioactivity.

Example 4 tissue adhesion test

The adhesion of the Ag-AV-SF hydrogel was evaluated using the model (a) shown in FIG. 8, where the Ag-AV-SF hydrogel was mixed with FITC and applied to the surface of a clean pig skin, and another pig skin of the same size was covered. Cryosections were taken at 1h and 24h, respectively, and observed under a fluorescence microscope. Coating Ag-AV-SF hydrogel on the surface of a clean pigskin, covering another pigskin with the same size on the surface of the pigskin, enabling the pigskin and the pigskin to be tightly combined, hanging one end of the pigskin on a rubber band, connecting the other end of the pigskin with a weight, and taking the maximum weight of the weight as an index for evaluating the adhesion degree. After hanging a weight of 15g, the two pigskins can still be tightly attached together (as shown in b in fig. 8), which shows that the hydrogel has good tissue adhesion and can be used for subsequent in vivo animal experiments.

After injecting FITC-labeled hydrogel between two pieces of pigskin for 1 hour and 24 hours, fluorescence images of frozen sections of the pigskin were taken, and as shown in FIG. 9, it can be clearly seen that the hydrogel was still firmly attached to the surface of each pigskin after 1 hour (c and d in FIG. 9) and after 24 hours (e and f in FIG. 9), indicating that the hydrogel prepared by the above method had good adhesion to the skin wound surface. In addition, the swelling properties of the hydrogel allow it to effectively expand and cover the diseased area. These results indicate that the composite hydrogel can be applied to a wound and tightly adhered to the surrounding tissue while protecting the wound tissue from the external environment.

Example 5 in vivo fluorescence diffusion experiment

After anesthetizing the rats, the hair on the back surface of the rats was shaved off, and a 0.5X 0.5cm2 piece of skin was taken. FITC-labeled Ag-AV-SF hydrogel was applied to the wound for diffuse fluorescence characterization. 0. After 4, 12, 24h, diffusion was recorded using a multifunctional laser scanning system, as shown in fig. 10, and the hydrogel was not diffused throughout the body, but rather was relatively fixed at the wound site.

Example 6 antibacterial property test

Gram-positive staphylococcus aureus and gram-negative escherichia coli are used as model bacteria. The antibacterial activity of different hydrogels was evaluated using the modified K-B method. 100 mul of the bacterial suspension was inoculated on an agar plate and divided into 4 parallel control zones, SF, Ag-SF, AV-SF and Ag-AV-SF. Four hydrogels were applied to the corresponding areas, and the diameter of the hydrogels was controlled at 6 mm. After incubation for 24h at 37 ℃, the diameter of the antibacterial ring is determined, and the antibacterial ability is evaluated.

The results of the detection of the antibacterial loops of Escherichia coli and Staphylococcus aureus are shown in FIG. 11, and the results of the statistical analysis are shown in FIG. 12 (where b is Escherichia coli and c is Staphylococcus aureus). The Ag-AV-SF hydrogel has the largest diameter of a bacteriostatic ring for escherichia coli (13.92 +/-0.94 mm) and staphylococcus aureus (10.623 +/-0.61 mm), and the released active ingredients are the most, so that the bacteriostatic performance of the Ag-AV-SF hydrogel is superior to that of other groups, and the antibacterial performance of SF is improved by AV and Ag NPs.

Example 7 cytotoxicity assay of L929 cells

In the experiment, the cells were cultured in DMEM (37 ℃, 5% CO2) containing 1% P/S solution and 10% fetal bovine serum. The CCK-8 method is adopted to detect the cytotoxicity of different hydrogels. After inoculation of L929 cells, the cells were cultured in 96-well plates (5000/well). L929 cells were stimulated with four types of pre-gel solutions (10. mu.l/well) for 24h, and then incubated at 37 ℃ for 2h with the addition of CCK-8 solution (10. mu.l/well). The absorbance value at 450nm is measured by a spectrophotometer to evaluate the cytotoxicity of the wound, the result is shown in figure 13, the cell proliferation level of the Ag-AV-SF hydrogel treatment group is obviously higher than that of other parallel experiment groups, and the biological characteristic is favorable for the proliferation of wound cells in the recovery process and effectively reduces the wound.

EXAMPLE 8 in vitro cell proliferation of hydrogels

After culturing L929 cells in 6-well plates for 24 hours, the original medium was discarded, replaced with fresh medium, and incubated with medium containing SF, Ag-SF, AV-SF, or Ag-AV-SF pre-hydrogel solutions for an additional 24 hours. FITC-labeled phalloidin was used to show the morphology of the cytoskeleton and DAPI was used to show the location of the nuclei.

As a result, as shown in FIG. 14, L929 cells were subjected to fluorescent staining to evaluate the accelerating effect of the hydrogel on cell proliferation. After the hydrogel is added for 24 hours, the fluorescence intensity of cell nucleuses (blue) and cytoskeletons (green) of the Ag-AV-SF hydrogel treatment group shows the highest level, and the Ag-AV-SF hydrogel has a good cell proliferation promoting effect.

Example 9 cell migration assay

L929 cells were cultured in the same environment as described above. L929 cells were seeded onto 6-well plates and cultured until the cells were completely covered. Marking a gap of about 150 μm in the culture dish by using a ruler; the cells were washed 2 times with PBS, serum-free DMEM medium was added, 50. mu.l of a different pre-gel solution was added, and the cells were cultured in an incubator. After 24h, the primary and late scratch cell migration was observed under a microscope. The ability of the cell to promote cell migration was studied by scratch test.

As shown in fig. 15 and 16, the scratch width was most decreased (97.01 ± 18.96 μm) in the Ag-AV-SF hydrogel-treated group compared to the other experimental groups, and the decrease in relative distance between L929 cells indicates that the combination of silver nanoparticles and aloe has synergy to promote cell migration.

Example 10 wound healing experiments

45 rats (adult male SD rats, provided by the university of chongqing laboratory animals center) were randomly assigned to 5 groups (n ═ 9). After anaesthesia with isoflurane using an anaesthesia machine (RWD Life Science, Shenzhen, China), the surface of the back was shaved and 1.0 × 1.0cm2 of skin was taken from the back; then cleaned with iodophor and alcohol to avoid infection, and then the wound was covered with different hydrogels. Control groups were given iodoform and alcohol treatment only. Rats were individually housed under identical conditions to prevent bites and cross-contamination. Each hydrogel dressing was replaced every 3 days up to 28 days. On days 7, 14, 21 and 28, photographs of the wound were taken.

Wound healing as shown in fig. 17 and 18, wound condition was recorded on days 0, 7, 14, and 28, respectively. On day 7, the wound area was not significantly reduced, as the early stages of healing were mainly associated with inflammatory responses. On the 14 th day, the healing area of the Ag-AV-SF mixed hydrogel treatment group is reduced more than that of other groups, which indicates that the Ag-AV-SF hydrogel has good antibacterial performance in the middle stage of wound repair. On day 28, the wound area was nearly completely closed (98.08% ± 1.89%) over the blank control (88.94% ± 7.60%), SF hydrogel treatment (92.20% ± 5.80%), AV-SF hydrogel treatment (92.15% ± 4.65%), and Ag-SF hydrogel treatment (93.36% ± 0.93%) (fig. S2). The result shows that the Ag-AV-SF hydrogel has good promotion effect in the early inflammatory reaction stage and the subsequent tissue repair, regeneration and remodeling stages. It can also be seen that the Ag-AV-SF hydrogel treatment group showed the fastest hair growth at day 28, suggesting that the hydrogel has the potential to promote hair regrowth.

Example 11 histological analysis

Tissue samples and surrounding 0.5cm of healthy skin were collected on days 7, 14, and 28. Wound surface tissues are taken at different time points after operation for histological evaluation so as to evaluate the in-vivo healing promotion effect of the wound surface tissues. Thereafter, HE staining, CD31 and CD68 immunohistochemical staining and Masson staining were used to analyze the effects of neovascularization and hydrogel on local immune responses during repair and regeneration, as well as collagen deposition, respectively.

To further analyze the tissue healing status, animal wound tissues were HE stained and immunohistochemically treated with CD31 and CD68 to demonstrate their wound healing promoting effects.

The HE staining of wound tissue is shown in fig. 19, and the masson staining at 14 days is shown in fig. 20, and the number of blood vessels and the skin tissue structure at the wound site can be observed. As shown in FIG. 21, the number of blood vessels in the Ag-AV-SF hydrogel treated group (28.7. + -. 2.5) was significantly higher than those in the control group (5.6. + -. 1.1), SF hydrogel- (11.3. + -. 3.5), Ag-SF hydrogel (12.6. + -. 2.8) and AV-SF hydrogel treated group (20.7. + -. 0.6).

The CD31 immunohistochemistry of tissue samples at day 7 reflected the reconstitution of the blood supply network at the wound site (fig. 19). Neovascular density (31.3 ± 3) was higher with Ag-AV-SF hydrogel treatment (as shown in figure 22) than in the other groups, probably due to reactive revascularization from mild immune response.

The CD68 immunohistochemistry of tissue samples at day 7 reflected the location and number of macrophages (fig. 19) for evaluation of the immune response during tissue regeneration. The lowest density of immunocytes (4.7 +/-0.6) in the Ag-AV-SF hydrogel treated group (as shown in FIG. 23) indicates that the hydrogel can reduce the immunoreaction intensity to some extent.

In addition, in the early stage of healing, moderate immune response to local wounds can induce reactive neovascularization, promote the restoration of vascular networks and the removal of damaged metabolites, and accelerate wound healing.

Example 12 cytokine assay

Venous blood was collected and serum was isolated for cytokine detection (TGF- β, VEGF, EGF) by ELISA kit to further study the mechanism of hydrogel in improving wound healing process.

To investigate the effect of different hydrogels on cell proliferation and differentiation, the relative expression of TGF-. beta.was determined on day 7 in this example. The results are shown in FIG. 24. As can be seen, the expression level of the Ag-AV-SF hydrogel-treated group was higher than that of the other groups (P < 0.05).

The relative expression levels of EGF and VEGF were measured in serum samples at day 28 and further analyzed for epidermal regeneration and vascular remodeling during the remodeling stage of the wound healing process. As shown in FIG. 25, the angiogenesis effect was the strongest in the Ag-AV-SF-treated group (P < 0.05).

However, the AV-SF-treated group also showed relatively high revascularization capacity, indicating that the most effective ingredient in the composite hydrogel was aloe. In addition, this high expression also favors transport of nutrients by epidermal tissues. The VEGF results also match the previous HE staining results, suggesting that the Ag-AV-SF hydrogel is beneficial to the reconstruction of the vascular network from a biochemical perspective.

The EGF detection result is consistent with the VEGF detection result, the result is shown in figure 26, the relative expression level of the Ag-AV-SF hydrogel treatment group is obviously higher than that of other test groups (P <0.001), and the Ag-AV-SF hydrogel has the strongest epidermal regeneration potential.

Example 13 in vivo biosafety assessment

On day 28, different organs (heart, liver, spleen, lung, kidney, testis, and brain tissue) of the animals were collected and examined for pathological changes by HE staining. The results are shown in fig. 27, and show that the organ structure was intact in the experimental group and the control group, and no significant pathological changes were observed, especially due to the deposition of silver nanoparticles.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.