Bletilla striata polysaccharide hydrogel for promoting wound healing as well as preparation method and application of bletilla striata polysaccharide hydrogel

Abstract

The invention discloses bletilla striata polysaccharide hydrogel for promoting wound healing as well as a preparation method and application of the bletilla striata polysaccharide hydrogel, and belongs to the technical field of biological medicines. The bletilla striata polysaccharide hydrogel comprises an adhesion functional layer and an anti-adhesion surface layer, the active ingredients of the adhesion functional layer comprise methacrylated bletilla striata polysaccharide, gallic acid and nano zinc oxide; the active component of the anti-adhesion surface layer comprises polyhedral oligomeric silsesquioxane modified by methacryloyl. The invention constructs the asymmetrically adhered bletilla striata polysaccharide hydrogel, and experiments prove that the hydrogel has ideal pore structure, mechanical strength and stable asymmetrically adhered double-layer characteristics. In-vivo and in-vitro researches prove that the hemostatic material is good in mechanical property, short in hemostasis time, high in antibacterial rate, capable of effectively preventing bacteria from invading, capable of promoting cell proliferation and migration and accelerating wound healing, and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a bletilla striata polysaccharide hydrogel for promoting wound healing, and a preparation method and application thereof. Background Art

[0002] Natural biological tissues, such as skin, often have asymmetric structures, and this asymmetry plays an important role in the biological world. Specifically, the surface layer of the skin is dense and has a certain hydrophobicity, which can effectively prevent the invasion of external pathogens; while the interior maintains good hydrophilicity, which is conducive to water exchange and tissue repair. Inspired by this, in recent years, researchers have tried to improve the comprehensive performance of wound dressings by constructing bionic asymmetric structures to meet the needs of complex wound care. Among these dressings, hydrogels have been widely studied and applied in biomedical fields such as wound healing due to their excellent properties such as high water content, softness and good biocompatibility. However, traditional hydrogels still have some problems in practical applications, such as low mechanical strength, insufficient antibacterial properties, limited adhesion and poor hemostasis.

[0003] Natural polysaccharides, as biomacromolecules, have been widely used in the field of hydrogels. Bletilla striata polysaccharide (BSP), a natural plant polysaccharide, has been widely used in tissue repair and drug delivery due to its excellent biocompatibility and moisturizing properties. However, its inherent low mechanical strength limits its application in hydrogels. Furthermore, there are currently no reports on the preparation of asymmetric adhesive hydrogels using Bletilla striata polysaccharide. Summary of the Invention

[0004] The present invention aims to provide a Bletilla striata polysaccharide hydrogel for promoting wound healing, as well as its preparation method and application, to address the aforementioned problems of the prior art. The present invention constructs an asymmetrically adhesive Bletilla striata polysaccharide hydrogel. Experimental studies have demonstrated that the hydrogel possesses ideal pore structure, mechanical strength, and stable asymmetrically adhesive bilayer properties. In vitro and in vivo studies have confirmed its excellent mechanical properties, rapid hemostasis, high antibacterial rate, and ability to effectively prevent bacterial invasion. It also promotes cell proliferation and migration, accelerating wound healing, demonstrating broad application prospects.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a bletilla striata polysaccharide hydrogel for promoting wound healing, wherein the bletilla striata polysaccharide hydrogel comprises an adhesion functional layer and an anti-adhesion surface layer;

[0007] The active ingredients of the adhesive functional layer include methacryloylated bletilla striata polysaccharide, gallic acid and nano zinc oxide;

[0008] Bletilla striata polysaccharide (BSP) is a natural plant polysaccharide with excellent biocompatibility and moisturizing properties. In the Bletilla striata polysaccharide hydrogel of the present invention, after methacrylation, Bletilla striata polysaccharide can be cured into a gel under the action of a photoinitiator, significantly improving the mechanical properties of the hydrogel.

[0009] Gallic acid (GA) is a natural phenolic compound. Due to its unique pyrogallol structure, it can form hydrogen and covalent bonds with various biological matrices, such as proteins and polysaccharides. In the Bletilla striata polysaccharide hydrogel of the present invention, the addition of GA can improve the hydrogel's stability, mitigate the hydrogel collapse caused by methacrylylated Bletilla striata polysaccharide, and enhance the hydrogel's adhesion and antioxidant activity.

[0010] Nano-zinc oxide (ZnO NPs) have broad-spectrum antibacterial properties and can inhibit bacterial growth through multiple mechanisms, including the release of Zn 2+ Ions damage bacterial cell membranes and induce the production of reactive oxygen species (ROS), causing oxidative damage to bacteria. The addition of ZnO NPs to the Bletilla striata polysaccharide hydrogel of the present invention can improve the hydrogel's stability and mitigate the hydrogel collapse caused by methacryloylation of Bletilla striata polysaccharide. It can also impart antibacterial properties to the hydrogel and promote the proliferation and migration of fibroblasts, thereby accelerating wound healing.

[0011] The active ingredient of the anti-adhesion surface layer includes methacryl-modified cage-type polysilsesquioxane.

[0012] Cage-type polysilsesquioxane (POSS) has a stable cage structure and good hydrophobicity. In the Bletilla striata polysaccharide hydrogel of the present invention, methacryloyl-modified POSS (MA-POSS) serves as the main component of the hydrogel's outer anti-adhesion surface layer. Its dense nanostructure can form a protective surface layer, effectively preventing bacterial invasion and unnecessary adhesion.

[0013] Furthermore, the adhesive functional layer is obtained by ultraviolet light-induced polymerization of acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, a photoinitiator and a crosslinking agent;

[0014] The anti-adhesion surface layer is obtained by polymerization of methacryl-modified cage-type polysilsesquioxane, a photoinitiator and a crosslinking agent through ultraviolet light initiation.

[0015] Furthermore, the preparation method of the methacryloylated Bletilla striata polysaccharide is as follows: under ice bath conditions, methacrylic anhydride is added dropwise to a Bletilla striata polysaccharide solution, reacted, and dialyzed to obtain the methacryloylated Bletilla striata polysaccharide;

[0016] The concentration of the bletilla striata polysaccharide solution is 8-12 g / L; the volume ratio of the methacrylic anhydride to the bletilla striata polysaccharide solution is 6:400-600;

[0017] The dropping speed is 0.5 mL / min;

[0018] The reaction time is 10-14 hours, the rotation speed is 400-600 rpm, and the pH is maintained at 7-8;

[0019] The molecular weight cut-off of the dialysis was 3500 Da.

[0020] The present invention also provides a method for preparing the above-mentioned Bletilla striata polysaccharide hydrogel, comprising the following steps:

[0021] (1) Preparation of hydrogel pre-solution ①: Using water as solvent, acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, photoinitiator and cross-linking agent were mixed uniformly;

[0022] (2) Preparation of hydrogel pre-solution ②: Using ethyl acetate as solvent, uniformly mix the methacryloyl-modified cage-type polysilsesquioxane, photoinitiator, and crosslinker;

[0023] (3) When the hydrogel preliquid ① is irradiated with ultraviolet light to an incompletely solidified state, the hydrogel preliquid ② is added and irradiated with ultraviolet light until it is completely solidified to obtain the Bletilla striata polysaccharide hydrogel.

[0024] Furthermore, in step (1), the mass ratio of acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, photoinitiator and cross-linking agent is 10:4:2:0.05:1:0.15;

[0025] The concentration of the methacryloylated Bletilla striata polysaccharide in the hydrogel pre-solution ① is 3%-5%.

[0026] Furthermore, in step (2), the mass ratio of the methacryloyl-modified cage-type polysilsesquioxane, the photoinitiator and the cross-linking agent is 3:1:0.15;

[0027] The concentration of the methacryloyl-modified cage-type polysilsesquioxane in the hydrogel pre-solution ② is 2%-4%.

[0028] Furthermore, in step (3), the volume ratio of the hydrogel pre-liquid ① to the hydrogel pre-liquid ② is 18-22:1.

[0029] Optionally, in step (3), the ultraviolet light irradiation time for the incomplete solidification state is 2-3 minutes; the ultraviolet light irradiation time for the complete solidification state is 2-3 minutes.

[0030] The present invention also provides use of the Bletilla striata polysaccharide hydrogel or the Bletilla striata polysaccharide hydrogel prepared by the above preparation method in preparing products for promoting wound healing.

[0031] The present invention also provides use of the Bletilla striata polysaccharide hydrogel or the Bletilla striata polysaccharide hydrogel prepared by the above preparation method in preparing hemostatic products.

[0032] The present invention also provides use of the Bletilla striata polysaccharide hydrogel or the Bletilla striata polysaccharide hydrogel prepared by the above preparation method in the preparation of antibacterial products.

[0033] The present invention discloses the following technical effects:

[0034] The present invention adopts a one-pot two-step method to prepare an asymmetric adhesive Bletilla striata polysaccharide hydrogel. The inner layer (adhesion functional layer) of the hydrogel is formed by photopolymerization of methacryloylated Bletilla striata polysaccharide and acrylamide to form a stable cross-linked network, ensuring excellent mechanical properties and water absorption; at the same time, gallic acid (GA) is added to enhance adhesion, and nano zinc oxide (ZnO NPs) provides antibacterial and cell migration promotion capabilities. The outer layer (anti-adhesion surface layer) is constructed by methacryloyl-modified cage-type polysilsesquioxane (MA-POSS) to construct a hydrophobic barrier, effectively preventing bacterial invasion and adhesion. Experiments have shown that the hydrogel has ideal pore structure, mechanical strength and stable double-layer characteristics. In vitro and in vivo studies have confirmed that it has good mechanical properties, short hemostasis time, high antibacterial rate, can effectively prevent bacterial invasion, and can promote cell proliferation and migration, accelerate wound healing, showing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 The morphology and physicochemical characterization results of each hydrogel; a is the SEM image of each hydrogel (scale bar is 200 μm); b is the EDS image of MP / AMBGZ (scale bar is 250 μm);

[0037] Figure 2 The image of the inverted hydrogel vial experiment; the left vial contains a mixture of hydrogel pre-liquids ① and ②; the right vial contains the hydrogel MP / AMBGZ formed after UV irradiation;

[0038] Figure 3 are Fourier transform infrared spectra of each hydrogel; wherein, a is the FTIR spectra of each hydrogel; b is the FTIR spectra of BSP and BSPMA;

[0039] Figure 4 is the swelling ratio of each hydrogel;

[0040] Figure 5 The tensile stress-strain curves of each hydrogel and the tensile photos of MP / AMBGZ on a universal testing machine;

[0041] Figure 6 Storage modulus (G') and loss modulus (G") of each hydrogel vary with frequency and time;

[0042] Figure 7 The following are the experimental results of the adhesion properties of each hydrogel; among them, a is the asymmetric adhesion of MP / AMBGZ to pigskin; b is the adhesion of AMBGZ to different mouse internal organs (heart, liver, spleen, lung, kidney) and skin; c is the pigskin adhesion test image; d is the stress-strain curve of the pigskin adhesion test; e is the force-displacement curve of the pigskin adhesion test;

[0043] Figure 8 The antioxidant capacity test results of each hydrogel; a is the DPPH clearance rate; b is the ABTS clearance rate;

[0044] Figure 9 The in vitro antibacterial activity test results of each hydrogel are shown in Figure 2; a is the antibacterial coating result of each hydrogel; b is the statistical result of the antibacterial rate;

[0045] Figure 10 SEM images of Staphylococcus aureus and Pseudomonas aeruginosa co-incubated with AMBGZ (scale bar, 1 μm); the upper image shows normal Staphylococcus aureus and Pseudomonas aeruginosa, and the lower image shows Staphylococcus aureus and Pseudomonas aeruginosa co-incubated with AMBGZ;

[0046] Figure 11 SEM images of the antibacterial adhesion experiments of each hydrogel (scale bar is 5 μm);

[0047] Figure 12 is the hemolysis rate of each hydrogel;

[0048] Figure 13 is the cell survival rate after treatment with different concentrations of hydrogel extract (n=4);

[0049] Figure 14 Live and dead staining images of cells after treatment with each hydrogel extract (scale bar is 100 μm);

[0050] Figure 15 Images of L929 cell migration experiments after treatment with various hydrogel extracts (scale bar: 200 μm);

[0051] Figure 16 Statistical results of cell migration rate after treatment with each hydrogel;

[0052] Figure 17 The experimental image of intracellular antioxidant activity of AMBGZ (scale bar is 100 μm);

[0053] Figure 18 The experimental results of in vitro hemostatic performance of each hydrogel; a is the BCI result; b is the statistical result of in vitro coagulation time; c is the in vitro coagulation photo at 5 minutes; d is the SEM image of platelet and red blood cell adhesion;

[0054] Figure 19 The following are the results of the mouse hemostasis experiment; a1 is the image of the mouse tail amputation hemostasis experiment; a2 is the image of the mouse femoral artery injury hemostasis experiment; a3 is the image of the mouse liver injury hemostasis experiment; b is the statistical result of the blood loss in the mouse tail amputation hemostasis experiment; c is the statistical result of the blood loss in the mouse femoral artery injury hemostasis experiment; d is the statistical result of the blood loss in the mouse liver injury hemostasis experiment;

[0055] Figure 20 This is an image of a mouse wound healing experiment;

[0056] Figure 21 This is the statistical result of wound area change in the mouse wound healing experiment;

[0057] Figure 22 This is a stained image of a mouse wound section from a mouse wound healing experiment (scale bar: 400 μm);

[0058] Figure 23 H&E staining images of mouse skin and major organs after hydrogel implantation (scale bar: 100 μm). DETAILED DESCRIPTION

[0059] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0060] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0061] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0062] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0063] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0064] Example 1

[0065] 1. Experimental materials are shown in Table 1

[0066] Table 1 Reagents and materials

[0067]

[0068]

[0069] 2. Experimental instruments are shown in Table 2

[0070] Table 2 Experimental instruments

[0071]

[0072] 3. Experimental Animals

[0073] The animal experiment procedures strictly adhered to the requirements of laboratory animal welfare and ethics. All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang Ocean University (acceptance number: 2024093). ICR mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (male, 6–8 weeks old) and were fed in an SPF animal room for 1 week before the experiments.

[0074] 4. Preparation of Bletilla striata polysaccharide hydrogel

[0075] (1) Synthesis of methacrylylated Bletilla striata polysaccharide

[0076] 5 g of Bletilla striata polysaccharide (BSP) was dissolved in phosphate buffered saline (PBS, pH 7.4, 500 mL) and stirred until completely dissolved. Then, 6 mL of methacrylic anhydride (MA) was added dropwise to the solution at a rate of 0.5 mL / min at 0°C and stirred at 500 rpm for 12 h. An ice bath was maintained during the reaction, and the pH was adjusted to maintain the reaction at 0°C and pH 7-8. Finally, the reaction solution was diluted with an equal volume of PBS to terminate the reaction. The reaction solution was dialyzed in deionized water using a dialysis bag (molecular weight cutoff 3500 Da) for 5 days, with the water being changed 2-3 times a day. Finally, the obtained solution was fully freeze-dried to obtain a white methacryloylated Bletilla striata polysaccharide (BSPMA) sample, which was stored at -20°C for further use.

[0077] (2) Preparation of hydrogel

[0078] A one-pot method was used to prepare the double-layer hydrogel. First, two hydrogel pre-solutions were prepared:

[0079] ① To a 10% (w / v) acrylamide (AM) aqueous solution, add 1% (w / v) of the solution volume of the photoinitiator Iragure2929 and 0.15% (w / v) of the crosslinker MBA, 4% (w / v) BSPMA, 2% (w / v) GA, and 0.05% (w / v, 0.5 mg / mL) ZnO NPs, and mix thoroughly by ultrasonication.

[0080] ② Using ethyl acetate as solvent, prepare a 3% (w / v) MA-POSS solution, add 1% (w / v) of the solution volume of photoinitiator Iragure 2929 and 0.15% (w / v) of the cross-linker MBA, and mix by ultrasonication.

[0081] To ensure full hydrogel solidification, the preparation process was performed in two steps. A certain amount of pre-liquid ① was placed in a mold and irradiated with UV light for 3 minutes, resulting in an incompletely solidified hydrogel. A certain amount of pre-liquid ② was then added, and the hydrogel was irradiated with UV light for another 2 minutes to obtain the MP / AMBGZ hydrogel. The volume ratio of pre-liquid ① to pre-liquid ② was 20:1.

[0082] Furthermore, different components were sequentially added to the pre-liquid system (①) and cured under UV irradiation for 5 minutes to obtain hydrogels AM (AM + photoinitiator + cross-linker), AMB (AM + photoinitiator + cross-linker + BSPMA), AMBG (AM + photoinitiator + cross-linker + BSPMA + GA), and AMBGZ (AM + photoinitiator + cross-linker + BSPMA + GA + ZnO NPs). The component contents in each hydrogel were the same as those in the MP / AMBGZ hydrogel.

[0083] 5. Hydrogel morphology and physicochemical characterization

[0084] (1) Scanning electron microscopy (SEM)

[0085] The micromorphology of the hydrogels was observed using a scanning electron microscope (SEM). After freeze-drying, the prepared hydrogels were cut into 0.3 cm x 0.3 cm squares and adhered to the conductive adhesive on the sample stage. Each sample was sprayed with gold under vacuum conditions, and the surface morphology of each hydrogel group was observed at an accelerating voltage of 3 kV. Energy dispersive spectroscopy (EDS) was also used to analyze the elemental species on the hydrogel surface.

[0086] Figure 1 Figure a is a SEM image. The addition of different substances altered the microscopic pore structure of each hydrogel group. After the addition of BSPMA, the hydrogel's pore structure significantly increased, but exhibited some degree of collapse. In contrast, the addition of GA and ZnO effectively improved the hydrogel's stability, making its internal pores more complete and evenly distributed, indicating that these components enhance the network structure within the hydrogel system. This larger and more complete pore structure enables the hydrogel to absorb more fluid, facilitating its applications in tissue fluid absorption and hemostasis. The SEM image of MP / AMBGZ shows a clear boundary between the two hydrogel layers. The upper layer, the MA-POSS layer, exhibits distinct square particles, creating a rough, dense surface. This unique morphology imparts a certain degree of hydrophobicity to the hydrogel's surface, thereby reducing its adhesion. Furthermore, the MA-POSS layer lacks a distinct pore structure, forming a biomimetic bilayer hydrogel with asymmetric adhesion, in conjunction with the inner hydrogel layer, which is filled with microscopic pores. This is a skin-like structure, and the low-adhesion surface provides the basis for the hydrogel to reduce bioadhesion, retain moisture and extend usage time.

[0087] Figure 1 Figure b is an EDS image of MP / AMBGZ. It can be seen that the hydrogel contains C, N, O, Zn, and Si. The uniform distribution of Zn indicates that the ZnO NPs are uniformly distributed throughout the hydrogel, contributing to its long-lasting antibacterial effect. The presence of Si only in the superstructure indicates that the hydrogel's bilayer structure has been successfully constructed.

[0088] Figure 2 Images of an inverted hydrogel vial experiment. The left vial shows the pre-solution (pre-solution ① and pre-solution ②) directly mixed (without UV irradiation). The right vial shows the hydrogel cured by UV irradiation. Due to the difference in solvent polarity, the pre-solution on the left shows stratification. After UV irradiation and curing, the hydrogel interface in the right vial is clearly defined, forming a well-defined and tightly connected bilayer of hydrogel.

[0089] (2) Fourier transform infrared spectroscopy (FTIR)

[0090] After the prepared hydrogels were freeze-dried, the KBr tablet method was used to scan and record the hydrogels in each group at a wavenumber range of 4000 cm -1 -400cm -1 .

[0091] Figure 3 a is the infrared spectrum of each hydrogel. In the spectrum of AMB prepared by compounding acrylamide with methacrylylated Bletilla striata polysaccharide, at 3447 cm -1 A broader OH absorption band is formed near 1000-1150 cm -1 The stretching vibration peak of the polysaccharide C-O-C bond is enhanced. -1 After adding gallic acid, the double bond absorption peak at 1700cm -1 The carboxyl C=O stretching peak and 1600-1500 cm -1 The aromatic ring skeleton vibration peak further indicates that hydrogen bonding may occur between phenolic hydroxyl groups and polysaccharides / amides. The introduction of nano zinc oxide can be -1 The Zn-O characteristic absorption was observed in the range of 1000-1100 cm-1, and the OH and amide bands in the high wavenumber region were slightly shifted or the peak shape changed. -1 and 460cm -1 Si-O-Si stretching and bending vibration peaks are displayed nearby, and partially overlap with the polysaccharide skeleton absorption bands.

[0092] Figure 3 b is the infrared spectrum of BSP and BSPMA. It can be seen that compared with BSP, the spectrum of BSPMA is at 1718cm -1 There is an obvious absorption peak enhancement at the C=O stretching peak, indicating that the grafting is successful.

[0093] 6. Swelling rate of hydrogel

[0094] Weigh the lyophilized hydrogel and record it as W0. Use deionized water as the medium. Place the lyophilized hydrogel in an equal amount of deionized water at room temperature, ensuring that the liquid completely covers the hydrogel. Carefully remove the hydrogel at the designated time, blot the surface moisture with filter paper, and weigh it and record it as W1. Calculate the hydrogel swelling ratio according to the following formula.

[0095]

[0096] Figure 4The swelling rates of AM, AMBGZ, and MP / AMBGZ are shown. The swelling rates of the AM and AMBGZ hydrogels were faster within 0-6 hours, indicating that they have strong hydrophilicity and can quickly absorb wound fluid. The swelling rate of the AMBGZ group was significantly higher than that of the AM group, which indicates that more hydrophilic groups were introduced into AMBGZ, thereby significantly improving the water absorption capacity, which helps to quickly absorb fluid in wounds with acute bleeding or more secretions. In contrast, the MP / AMBGZ hydrogel showed a lower swelling rate in the initial stage. This is due to the hydrophobicity and compact structure of the hydrogel's anti-adhesion layer. Good hydrophobicity and compact structure are conducive to enhancing the mechanical stability and structural integrity of the hydrogel.

[0097] 7. Mechanical properties of hydrogels

[0098] (1) Tensile test

[0099] The tensile properties of the hydrogels were evaluated using a universal testing machine. Each group of hydrogels was molded into a standard dumbbell shape and subjected to a tensile test at a tensile rate of 20 mm / min.

[0100] Figure 5 Figures 2 and 3 show the tensile stress-strain curves of the hydrogel and a photograph of MP / AMBGZ stretched on a universal testing machine. Compared to AM, AMBGZ exhibits higher stress and strain at break, indicating greater strength, ductility, and toughness. This is likely due to the addition of the polymer BSPMA improving the hydrogel's toughness, while the addition of GA and ZnO imparts greater strength and stability, making the hydrogel less susceptible to fracture. MP / AMBGZ exhibits higher stress and strain at break, with an elongation exceeding 1000%. Its excellent ductility is also evident in the photographs of MP / AMBGZ stretched on a universal testing machine. These excellent mechanical properties may be due to the hydrogel's tightly bound bilayer structure, which effectively distributes stress during stretching and enhances its overall mechanical strength. It is noteworthy that the addition of nanomaterials generally increases a material's strength but sacrifices its toughness to some extent. However, the addition of MA-POSS not only enhances the hydrogel's strength but also maintains its toughness. This may be because it is chemically cross-linked with AMBGZ through ultraviolet light, forming a uniform and stable network structure at the molecular level, which enables the hydrogel to effectively resist fracture when subjected to external force and maintain good deformation ability under high strain.

[0101] (2) Rheological test

[0102] Rheological tests were performed on the hydrogels using a rheometer. The storage modulus (G') and loss modulus (G") of the hydrogels were recorded as a function of frequency and time. In the frequency sweep experiment, the temperature was set at 25°C, the strain was 1%, and the sweep frequency range was 0.1 to 100 rad / s. In the time sweep experiment, the frequency was fixed at 10 rad / s (1 Hz), the strain was 1%, and the test time range was 0 to 300 s.

[0103] Figure 6 The storage modulus (G') and loss modulus (G") of the hydrogel change with frequency and time. In the test results of the storage modulus (G') and loss modulus (G") of the hydrogel changing with frequency, G' is always greater than G", showing good viscoelasticity. In the results of the change with time, the G' and G" of the hydrogel remain almost unchanged, indicating its excellent stability.

[0104] 8. Adhesion properties of hydrogel

[0105] The adhesion performance of the hydrogel was evaluated by pigskin adhesion test. The cut pigskin was washed and dried at room temperature. The hydrogel to be tested was used to bond two pieces of pigskin and applied about 0.5N / cm 2 Press and compact for 5 minutes. Use a universal testing machine to peel at a speed of 0.5 mm / s and record the data to evaluate the material adhesion performance.

[0106] Figure 7 The asymmetric adhesion test results of MP / AMBGZ on pigskin. Figure 7 As can be seen from a, different sides of MP / AMBGZ exhibit different adhesion to pig skin. This shows its advantage in wound application: the strong adhesion of the inner layer in contact with the wound enables the hydrogel to adhere well to the skin and seal the wound; while the outer anti-adhesion layer has no adhesion to the skin, showing an anti-adhesion effect. Figure 7 The results of these two experiments indicate that MP / AMBGZ has promising applications in wound treatment: it can effectively seal visceral wounds and prevent secondary damage caused by adhesion of the dressing in the complex internal environment.

[0107] Figure 7 Figures c, d, and e show the adhesion test results of the hydrogel using a universal testing machine, using pigskin adhesion tests. Both the stress-strain curve and the force-displacement curve show that the adhesion of AMBGZ is significantly improved compared to AM and AMB, indicating that the addition of GA significantly enhances the adhesion strength of the hydrogel.

[0108] 9. Antioxidant capacity of hydrogel

[0109] (1) DPPH clearance experiment

[0110] The antioxidant capacity of the hydrogel samples was evaluated using the DPPH free radical scavenging method. A 0.1 mM DPPH solution was prepared in ethanol. 3 mL of the DPPH solution and 200 mg of the hydrogel sample were placed in a centrifuge tube and thoroughly shaken to mix. The mixture was then allowed to react at room temperature in the dark for 30 minutes to ensure that the free radicals and antioxidant components fully interacted. After the reaction, the absorbance of the solution was measured at a wavelength of 517 nm using a UV spectrophotometer, and the DPPH free radical scavenging rate of the sample was calculated according to the following formula.

[0111]

[0112] Where A C represents the absorbance of the blank control group (untreated DPPH solution) at 517 nm, A S Represents the absorbance of DPPH solution at 517 nm after treatment with each group of hydrogel samples.

[0113] (2) ABTS clearance experiment

[0114] The antioxidant capacity of the hydrogel samples was evaluated by the ABTS free radical decolorization method. First, 7 mM ABTS was mixed with 2.45 mM potassium persulfate and reacted at room temperature for 24 h in the dark to generate a stable and sufficient amount of ABTS. + Free radicals. After the reaction is completed, the ABTS obtained + The solution was diluted to ensure that the absorbance of the solution at 734 nm was about 0.7 ± 0.02. Then, 3 mL of the diluted ABTS was taken. + The solution was mixed with the hydrogel sample to be tested and allowed to react in the dark for 30 minutes to allow the antioxidant components in the sample to fully react with the free radicals. Finally, the absorbance of the solution was measured at a wavelength of 734 nm using a UV spectrophotometer, and the ABTS free radical scavenging rate of the sample was calculated according to the following formula.

[0115]

[0116] Where A i represents the absorbance of the free radical solution after being treated with each group of hydrogel samples, A j represents the absorbance of the solution without free radicals, A C represents the absorbance of the untreated free radical solution.

[0117] Figure 8 The addition of GA significantly improved the antioxidant capacity of the hydrogel, with the scavenging rates of the two free radicals reaching over 99%.

[0118] 10. In vitro antibacterial activity and antibacterial mechanism of hydrogel

[0119] The antibacterial activity of the hydrogel was evaluated by the plate coating method. Each experiment was repeated three times. 200 μL of hydrogel pre-liquid was prepared into hydrogel samples of the same shape using a mold and sterilized by ultraviolet irradiation for 30 minutes. The hydrogels were grouped into AM, AMB, AMBG, and AMBGZ to investigate the effect of each component on the antibacterial activity of the hydrogel. Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) were inoculated into liquid culture medium and cultured at 37°C for 24 hours, and the bacterial solution was diluted to a concentration of 10 with PBS. 5 CFU / mL. The concentration of the bacterial solution was determined by McFadden turbidimetry. The hydrogel samples were then co-cultured with 3 mL of bacterial solution for 2 hours. After the culture was completed, 20 μL of the supernatant was evenly spread on the solid culture medium. The untreated bacterial solution was used as the blank control group. After culturing in a bacterial incubator at 37°C for 24 hours, the colonies were photographed and counted, and the inhibition rate was calculated. At the same time, the bacterial suspension obtained after hydrogel treatment was fixed with 2.5% glutaraldehyde solution for 1 hour, and then dehydrated with different concentrations of ethanol for 30 minutes each time. Finally, the sample was preserved with anhydrous ethanol, and the bacterial morphology was observed under SEM.

[0120] Figure 9 Figure 4 shows the results of plating and antibacterial rate analysis after incubation of different hydrogels with bacterial solutions. The addition of BSPMA imparts moderate antibacterial activity to the AMB hydrogel (60%-70% inhibition rate), while the addition of gallic acid (GA) and nano-zinc oxide (ZnONPs) further enhances the hydrogel's antibacterial properties, raising the inhibition rate to over 99%. The hydrogel exhibits broad-spectrum, highly effective antibacterial activity.

[0121] Studies have shown that the pyrogallol structure of GA can bind to the bacterial cell wall and destroy its integrity, while ZnONPs release Zn 2+ Ions cause damage to bacterial cell membranes and DNA, thereby effectively inhibiting bacterial growth. Figure 10 The SEM images of normal Staphylococcus aureus and Pseudomonas aeruginosa and those after co-incubation with AMBGZ are shown. Compared to normal, plump bacteria, the treated bacteria have wrinkled and cracked epidermis, and the bacterial cells are significantly damaged, demonstrating the hydrogel's killing and inhibitory effects on both bacteria.

[0122] 11. Antibacterial adhesion properties of hydrogels

[0123] The hydrogel was prepared into a size of 0.5 cm × 0.5 cm by using a mold and immersed in 1 mL of 10 8A 24-well plate containing a bacterial suspension of Staphylococcus aureus or Pseudomonas aeruginosa at a concentration of CFU / mL was placed in the wells of the plate and shaken at 37°C for 3 hours. The sample was removed, washed three times with PBS, and fixed with 2.5% glutaraldehyde for 2 hours. Dehydration was then performed using a gradient of ethanol with varying concentrations, each for 15 minutes. Finally, the resulting hydrogel samples were dried at 37°C and observed for bacterial adhesion using a scanning electron microscope (SEM).

[0124] Figure 11 SEM images of bacterial adhesion experiments on AM and MP / AMBGZ hydrogels. A large number of bacteria adhere to the surface of the AM hydrogel, which lacks the MA-POSS anti-adhesion layer, indicating that the uncoated hydrogel is susceptible to bacterial invasion. However, no bacteria are evident on the surface of the MP / AMBGZ hydrogel, demonstrating excellent antibacterial adhesion properties and preventing bacterial invasion of the hydrogel dressing and wound.

[0125] 12. In vitro biocompatibility of hydrogels

[0126] (1) Hemolysis test

[0127] Add 20 mg of hydrogel sample to a 2% red blood cell (RBC) suspension and place in a centrifuge tube. The negative control group is PBS, and the positive control group is deionized water. The tubes are incubated in a 37°C shaker for 3 hours, removed, and photographed. Centrifuge, collect the supernatants, and read the OD value of each supernatant at 540 nm using a microplate reader. The hemolysis rate is calculated according to the following formula.

[0128]

[0129] Where, OD S Represents the OD value of the sample, OD N and OD P represent the OD values ​​of the negative control group and the positive control group, respectively.

[0130] Figure 12 The following are photos and statistical results of the hemolysis rates of each group of hydrogels. The hemolysis rates of each group of hydrogels were all below the standard of 5%, demonstrating good in vitro blood compatibility.

[0131] (2) Cytotoxicity assay

[0132] The cytotoxicity of the hydrogels was evaluated by the MTT assay. 15 mg of each hydrogel was added to 30 mL of culture medium to obtain a 2 mg / mL hydrogel extract. The extract was sterilized by filtration and diluted with culture medium to 50 μg / mL to 400 μg / mL for later use. L929 cells were plated at 8 × 10 cells per well in a 96-well plate. 3Cells were seeded at a density of 100 μl and cultured for 24 hours until the cells adhered. 20 μl of different extracts were then added to each well. 20 μl of culture medium was added to the blank control group. After 24 hours of culture, 20 μl of MTT solution (5 mg / mL) was added to each well in the dark and cultured for another 4 hours. The culture medium was then discarded, 150 μl of DMSO was added to each well, the mixture was shaken for 10 minutes, and the absorbance was measured at 490 nm. Cell viability was calculated as follows.

[0133]

[0134] Where, OD S and OD C represent the absorbance of sample and control, respectively.

[0135] Figure 13 Figure 2 shows the cell survival rates after treatment with hydrogel extracts. With the exception of the AM group, the cell survival rates of all hydrogel groups exceeded 95%, demonstrating good cell biocompatibility. The relatively low cell survival rate in the AM group may be due to incomplete polymerization of acrylamide, leaving residual monomers with some cytotoxicity. The hydrogels prepared by chemical polymerization of BSPMA and AM achieve complete monomer reaction, reducing cytotoxicity. Furthermore, the cell survival rates of the high-concentration extracts in the AMB, AMBG, and AMBGZ groups exceeded 100%, indicating that the hydrogel extracts promoted cell proliferation. This promotes tissue growth and accelerates wound healing. The MP / AMBGZ group also exhibited good cytocompatibility, but the cell survival rate decreased after treatment with the high-concentration extract. This may be related to the toxicity of residual solvents and additives in the MP / AMBGZ anti-adhesion layer. However, as the surface layer of the hydrogel, the anti-adhesion layer does not come into direct contact with the wound, and the near-100% cell survival rate suggests that the residual substances are less toxic to cells. In summary, the hydrogel has good cell biocompatibility and can promote cell proliferation to a certain extent.

[0136] (3) Live and dead cell fluorescence staining

[0137] Cell morphology was assessed by AO / EB live-dead fluorescence staining. 1 × 10 cells were plated per well in a 12-well plate. 5 L929 cells were seeded at a density of 1:1 and cultured for 24 hours. Then, 200 μL of sponge extract was added to each well. An equal amount of culture medium was added to the control group. Culture was continued for another 24 hours. The culture medium was discarded, and the cells were washed three times with PBS in the dark. AO / EB stain was added, and cell morphology was observed using a fluorescence microscope.

[0138] Figure 14Live-dead staining images of AO / EB cells after treatment with hydrogel extracts. All cells in each group exhibited healthy green fluorescence and spindle-shaped morphology, with virtually no dead cells, demonstrating the hydrogel's good cell biocompatibility. Furthermore, compared to the blank control group, the number of cells in each hydrogel group increased significantly, consistent with the cell viability results from the MTT assay.

[0139] 13. Effect of hydrogel on cell migration ability

[0140] The cell scratch test was used to evaluate the ability of the hydrogel to promote cell migration. 5 L929 cells were seeded at a density of 100 μl and incubated in a 37°C incubator for 24 hours to allow the cells to fully adhere. A 200 μL pipette tip was used to vertically scratch the adherent cell layer. The cells were then washed twice with PBS to remove any free cells. The hydrogel extract was added and the cells were incubated in a 37°C incubator. Cell migration was observed under a microscope at 0, 12, and 24 hours.

[0141] Figure 15 The following are photos of L929 cell migration after treatment with different hydrogel extracts (scale bar is 200 μm). The addition of BSPMA, GA, and ZnO all accelerated cell migration to a certain extent, among which the effect of ZnO was the most significant. Figure 16 Judging from the cell migration results, the migration rate of AMBGZ is significantly higher than that of AMB and AMBG, which also shows that the addition of ZnO provides the hydrogel with a strong ability to promote cell migration, which is beneficial to the application of hydrogel in promoting wound healing.

[0142] 14. Intracellular antioxidant activity of hydrogels

[0143] L929 cells were seeded in a six-well plate and cultured in a 37°C constant temperature incubator for 24 hours to allow the cells to fully adhere to the wall and grow. Subsequently, hydrogel was added to the wells to allow it to fully contact the cells. Culture medium containing 200 μM H2O2 was added to each well and incubated for another 3 hours to induce oxidative stress in the cells. After the incubation, the culture medium in each well was removed and gently rinsed with PBS to remove residual H2O2. The positive control group consisted of cells with oxidative stress, and the negative control group consisted of untreated cells. Culture medium containing 20 μM DCFH probe was added and observed under a fluorescence microscope.

[0144] Figure 17These images show the antioxidant activity of the control and hydrogel groups under a fluorescence microscope. After H2O2-induced oxidative stress in the positive control group, the cells appeared abnormally round under a fluorescence microscope, and the probe emitted significant green fluorescence, indicating an increase in H2O2 content within the cells and a degree of cell damage due to oxidative stress. In the negative control group, there was almost no fluorescence. The fluorescence of the hydrogel-treated cells was weaker, and some cells returned to a normal morphology, close to that of the negative control group without oxidative stress. This indicates that the hydrogel effectively inhibited the damage to cells caused by reactive oxygen species and demonstrated strong antioxidant capacity at the cellular level.

[0145] 15. Evaluation of the in vitro hemostatic properties of hydrogels

[0146] (1) Determination of whole blood coagulation index (BCI)

[0147] A sponge of a defined volume (0.5 cm × 0.5 cm × 0.5 cm) was placed in a centrifuge tube containing 200 μL of anticoagulated whole blood. 100 μL of 0.2 M CaCl₂ solution was added. A control group consisted of untreated anticoagulated whole blood and CaCl₂ solution. The mixture was incubated at 37°C for 10 minutes, followed by the addition of 3 mL of deionized water. The supernatant was collected and absorbance was measured at 540 nm. The BCI was calculated according to the following formula.

[0148]

[0149] Where, OD S and OD N represent the absorbance of the sample and negative control group, respectively.

[0150] (2) Adhesion of platelets and blood cells

[0151] Take fresh anticoagulated whole blood, centrifuge at 1500rpm / min for 5 minutes, aspirate the supernatant, and retain the precipitate. Repeat several times until a red-free supernatant is obtained, which is the platelet. Take 2mL of anticoagulated mouse blood, centrifuge at 3000rpm to collect red blood cells (RBCs) and wash three times with PBS. Add the obtained platelets and RBCs dropwise onto the hydrogel AMBGZ and incubate at 37°C for 30 minutes. After washing with PBS, fix with 2.5% glutaraldehyde for 30 minutes. Dehydrate with ethanol gradient for 10 minutes each time. Dry the obtained hydrogel and observe the adhesion under SEM.

[0152] (3) Coagulation time

[0153] To analyze the effect of the hydrogel on blood clotting rates, an experiment was designed to measure clotting time. First, 50 mg of hydrogel sample was added to a centrifuge tube, followed by 200 μL of anticoagulated whole blood and 10 μL of CaCl₂ solution. The tube was inverted every 20 seconds until blood flow within the hydrogel aggregate ceased, and the corresponding time was recorded.

[0154] Figure 18 These are the experimental results for evaluating the in vitro hemostatic properties of the hydrogel. Figure 18 The a is the BCI result. A lower BCI indicates a higher coagulation ability. The BCI of AMB and AMBGZ is significantly lower than that of AM, indicating that the addition of BSPMA significantly reduces the BCI of the hydrogel and improves its coagulation ability. Figure 18 Figures b and c also illustrate this point: the coagulation time of the AMB and AMBGZ groups was within 100 s, indicating that the hydrogel can quickly aggregate and coagulate blood. Figure 18 Figure d shows an SEM image of platelet adhesion and red blood cell adsorption on AMBGZ. Platelets are activated, with pseudopodia growing; a large number of red blood cells are adsorbed by the sponge, and fibrin aggregates. These are prerequisites for rapid hemostasis. These experimental results demonstrate that the hydrogel possesses excellent coagulation capacity and can rapidly activate the hemostatic mechanism, making it suitable for hemostatic applications.

[0155] 16. Mouse hemostasis experiment

[0156] The hydrogel's hemostatic properties were evaluated using mouse tail amputation, femoral artery injury, and liver injury models. Mice were anesthetized and the different injury models were constructed. The tail amputation model involved cutting the tail 3 cm from the base of the tail. The femoral artery injury model involved removing hair from the groin, then incising the skin to expose the femoral artery, which was then severed with scissors. The liver injury model involved creating a 3-4 cm incision in the mouse's upper abdomen with a scalpel to expose the mouse's internal organs. The liver was located, carefully removed without damaging it, and placed on weighed filter paper. A wound was created in the liver using a needle to simulate liver injury and bleeding. After modeling, hemostasis was immediately performed using pre-weighed hydrogels from each group, and the mice were closely observed until bleeding stopped. The control group received no hemostatic treatment and used pre-weighed filter paper to absorb blood. After the wound stopped bleeding, the hemostatic hydrogel and filter paper were weighed and recorded to calculate blood loss.

[0157] Figure 19 Photos and blood loss results from hemostasis experiments in mice. Blood loss in the AMBGZ group in all three models was less than 300 mg, significantly lower than in the control group. This demonstrates that the hydrogel can rapidly seal wounds, achieve rapid hemostasis, and significantly reduce bleeding. This demonstrates the promising application of the AMBGZ hydrogel in treating acute bleeding.

[0158] 17. Mouse wound healing experiment

[0159] After anesthetizing the mice, depilatory cream was used to remove hair on the back. A 0.8 cm diameter circular punch was used to punch a hole in the center of the mouse's back and the skin was cut off according to the shape to establish a full-thickness wound model. An equal amount of hydrogel pre-liquid was taken, and the hydrogel was prepared into a circular patch of the same size as the wound using a mold, which was applied to the wound of the mouse after modeling. The control group was not treated. Each group was sprayed with equal concentrations of Staphylococcus aureus liquid to simulate bacterial invasion. After 1 day, samples were taken from the wound surface for plating. The wounds were photographed on days 0, 3, 7, 10, and 14, and the wound area was measured using Image J software. The wound area change rate was calculated according to the following formula. At the same time, skin sections from the wound were taken on days 7 and 14, and H&E and Masson staining were performed. The staining of the sections was observed under a microscope.

[0160]

[0161] Where A0 represents the wound area on day 0, and A1 represents the wound area on days 3, 7, 10, and 14.

[0162] Figure 20 The following are photos of plates of samples taken from the wounds of mice treated with different hydrogel samples and photos of wound healing of mice treated with different hydrogel samples on days 0, 3, 7, and 14. As can be seen from the photos of plates of samples taken from the wound surface after 1 day, the bacterial shielding effect of MP / AMBGZ is better than that of other groups. Figure 21 As shown in the wound area statistics, on day 3, the wound area of ​​MP / AMBGZ-treated mice was 30.3%, significantly smaller than that of the other groups. This is due to the excellent anti-bacterial ability of MP / AMBGZ, which protects the wound from bacterial infection and reduces the initial inflammatory response. Both AMBGZ and MP / AMBGZ showed superior wound healing effects at day 14 compared to the AM and control groups, demonstrating the hydrogel's ability to promote wound healing.

[0163] Figure 22 The results of staining mouse wound sections. From the H&E staining results, it can be seen that on the 7th day, except for the MP / AMBGZ group, the other groups were still in an inflammatory state, and many inflammatory factors accumulated in the skin structure. In the MP / AMBGZ group, the skin inflammatory factors basically disappeared, the epidermis grew, and the skin structure was relatively complete. On the 14th day, the new skin epidermis of each group had grown, and the AMBGZ and MP / AMBGZ groups already had complete appendages in the skin structure. A similar trend was also seen in the Masson staining results. On the 7th day, the blue collagen deposition rate in the skin of the AMBGZ and MP / AMBGZ groups was faster, while the control group and AM group were mostly red areas where collagen was not deposited. The above experimental results show that the designed hydrogel can effectively prevent bacterial invasion, reduce wound infection, promote tissue regeneration, and accelerate wound healing.

[0164] 18. Biocompatibility of hydrogel in mice

[0165] After adaptive feeding, ICR mice were randomly divided into three groups. The hydrogel samples were implanted subcutaneously on the back of the mice and sutured with surgical suture. A blank control group received no implantation. On day 15, skin samples from the dorsal embedding area, as well as the liver, kidney, and spleen, were fixed, embedded, sectioned, and stained with H&E. Histological observations of the sections were performed using an optical microscope.

[0166] Figure 23 H&E staining of mouse skin and major organs after hydrogel implantation. Fifteen days after hydrogel implantation, the wound on the mouse's back was completely healed, and the epidermis and skin appendages were intact. Sections of major internal organs (liver, spleen, and kidney) showed intact structures and vascularity, with no obvious pathology. These results demonstrate the hydrogel's excellent in vivo biocompatibility.

[0167] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Bletilla striata polysaccharide hydrogel for promoting wound healing, characterized in that: The bletilla striata polysaccharide hydrogel comprises an adhesion functional layer and an anti-adhesion surface layer; The active ingredients of the adhesive functional layer include methacryloylated bletilla striata polysaccharide, gallic acid and nano zinc oxide; The active ingredient of the anti-adhesion surface layer includes methacryl-modified cage-type polysilsesquioxane.

2. The Bletilla striata polysaccharide hydrogel according to claim 1, characterized in that The adhesive functional layer is obtained by ultraviolet light-induced polymerization of acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, a photoinitiator and a cross-linking agent; The anti-adhesion surface layer is obtained by polymerization of methacryl-modified cage-type polysilsesquioxane, a photoinitiator and a cross-linking agent initiated by ultraviolet light.

3. The Bletilla striata polysaccharide hydrogel according to claim 1, characterized in that The preparation method of the methacryloylated Bletilla striata polysaccharide comprises the following steps: adding methacrylic anhydride dropwise to a Bletilla striata polysaccharide solution under ice bath conditions, reacting, and dialyzing to obtain the methacryloylated Bletilla striata polysaccharide; The concentration of the bletilla striata polysaccharide solution is 8-12 g / L; the volume ratio of the methacrylic anhydride to the bletilla striata polysaccharide solution is 6:400-600; The dropping speed is 0.5 mL / min; The reaction time is 10-14 hours, the rotation speed is 400-600 rpm, and the pH is maintained at 7-8; The molecular weight cut-off of the dialysis was 3500 Da.

4. The method for preparing the Bletilla striata polysaccharide hydrogel according to any one of claims 1 to 3, characterized in that: The steps include: (1) Preparation of hydrogel pre-solution ①: Using water as solvent, acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, photoinitiator and cross-linking agent were mixed uniformly; (2) Preparation of hydrogel pre-solution ②: Using ethyl acetate as solvent, uniformly mix the methacryloyl-modified cage-type polysilsesquioxane, photoinitiator, and crosslinker; (3) When the hydrogel preliquid ① is irradiated with ultraviolet light to an incompletely solidified state, the hydrogel preliquid ② is added and irradiated with ultraviolet light until it is completely solidified to obtain the Bletilla striata polysaccharide hydrogel.

5. The preparation method according to claim 4, characterized in that In step (1), the mass ratio of acrylamide, methacrylylated Bletilla striata polysaccharide, gallic acid, nano zinc oxide, photoinitiator and cross-linking agent is 10:4:2:0.05:1:0.15; The concentration of the methacryloylated Bletilla striata polysaccharide in the hydrogel pre-solution ① is 3%-5%.

6. The preparation method according to claim 4, characterized in that In step (2), the mass ratio of the methacryloyl-modified cage-type polysilsesquioxane, the photoinitiator and the cross-linking agent is 3:1:0.15; The concentration of the methacryloyl-modified cage-type polysilsesquioxane in the hydrogel pre-solution ② is 2%-4%.

7. The preparation method according to claim 4, characterized in that In step (3), the volume ratio of the hydrogel pre-liquid ① to the hydrogel pre-liquid ② is 18-22:

1.

8. Use of the Bletilla striata polysaccharide hydrogel according to any one of claims 1 to 3 or the Bletilla striata polysaccharide hydrogel prepared by the preparation method according to any one of claims 4 to 7 in the preparation of a product for promoting wound healing.

9. Use of the Bletilla striata polysaccharide hydrogel according to any one of claims 1 to 3 or the Bletilla striata polysaccharide hydrogel prepared by the preparation method according to any one of claims 4 to 7 in the preparation of hemostatic products.

10. Use of the Bletilla striata polysaccharide hydrogel according to any one of claims 1 to 3 or the Bletilla striata polysaccharide hydrogel prepared by the preparation method according to any one of claims 4 to 7 in the preparation of antibacterial products.

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