Bletilla striata polysaccharide hydrogel dressing as well as preparation method and application thereof

Abstract

The invention discloses a bletilla striata polysaccharide hydrogel dressing and a preparation method and application thereof.The preparation method of the bletilla striata polysaccharide hydrogel dressing comprises the following steps that a carboxymethyl chitosan solution, a bletilla striata polysaccharide solution and cinnamyl aldehyde are mixed and stirred to be uniform at the temperature of 20-30 DEG C, a mixed solution system is prepared, and then standing is conducted to form gel; wherein the volume ratio of the carboxymethyl chitosan solution to the bletilla striata polysaccharide solution is (1-5): 1, and the concentration of the cinnamyl aldehyde in the mixed solution system is 0.05-5 wt%. The preparation process is simple, the raw materials are all green and natural sources, and the prepared hydrogel is good in biocompatibility, has good antibacterial, anti-inflammatory and angiogenesis promoting effects and can accelerate diabetic wound repair.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials. More specifically, the present invention relates to a bletilla striata polysaccharide hydrogel dressing and its preparation method and application. Background Art

[0002] Diabetic wounds are one of the common complications of diabetes. Due to the existence of the underlying diabetic disease, the wound is in a state of excessive inflammation, and angiogenesis is impaired, resulting in an extended wound healing cycle. Long-term unhealed wounds exposed to the environment are prone to bacterial colonization on the wound surface, causing wound infection and further hindering wound healing. Wound dressings are the standard method for wound treatment. Clinically, gauze dressings are often used to cover wounds, but this type of dressing only has the basic function of protecting the wound surface. As a new type of wound dressing, hydrogel has good protective and therapeutic effects. Using monomers with pharmacological value to construct hydrogels can achieve the purposes of hemostasis, antibacterial, anti-inflammatory, and promoting angiogenesis, and thus play a good therapeutic role at all stages of wound healing. The prior art, such as the invention patent with the authorization announcement number CN113413483B, discloses an adhesion-non-adhesion integrated hydrogel and its preparation method and application in wound repair. The prepared adhesion-non-adhesion integrated hydrogel has bactericidal properties and can promote wound repair, and can be used as a wound dressing. However, diabetic patients' wounds have a lot of exudate, and the above-mentioned adhesion-non-adhesion integrated hydrogel has poor air permeability and water absorption, which is not conducive to the absorption and discharge of wound exudate, and there are areas for improvement. Summary of the Invention

[0003] An object of the present invention is to provide a bletilla striata polysaccharide hydrogel dressing and its preparation method and application. The bletilla striata polysaccharide hydrogel dressing has good air permeability and water absorption, is conducive to the absorption and discharge of wound exudate, and can effectively promote the wound healing of diabetic patients.

[0004] In order to achieve these objects and other advantages of the present invention, according to one aspect of the present invention, a preparation method of a bletilla striata polysaccharide hydrogel dressing is provided, including the following steps: mixing a carboxymethyl chitosan solution, a bletilla striata polysaccharide solution and cinnamaldehyde at 20-30°C and stirring evenly to obtain a mixed solution system, and then standing to form a gel; wherein, the volume ratio of the carboxymethyl chitosan solution to the bletilla striata polysaccharide solution is (1-5):1, and the concentration of cinnamaldehyde in the mixed solution system is 0.05-5 wt%.

[0005] Preferably, the concentration of the carboxymethyl chitosan solution is 2-8 wt%.

[0006] Preferably, the concentration of the bletilla striata polysaccharide solution is 0.5-2 wt%.

[0007] Preferably, it further includes: Prepare a gelatin template, in which a plurality of cylinders with different diameters are arranged; Immerse the gelatin template in the mixed solution system, perform ultrasonic treatment at a frequency of 40 - 60 kHz for 5 - 10 min, then take out the gelatin template filled with the mixed solution system, and place it in a vacuum environment with a vacuum degree of -0.08~-0.1 MPa for degassing; Let it stand to form a gel, degrade the gelatin template, and finally obtain a Bletilla striata polysaccharide hydrogel dressing with a porous structure.

[0008] Preferably, the gelatin template is prepared from gelatin with a concentration of 15%wt by 3D printing technology, the printing temperature is 60~70℃, and the plurality of cylinders include a first cylinder and a second cylinder evenly distributed in a grid pattern, where each second cylinder is located at the center of four first cylinders distributed in a rectangle, and a plurality of third cylinders are randomly connected between the side walls of every two adjacent first cylinders and second cylinders. The diameter of the first cylinder is 300~500μm, the diameter of the second cylinder is 50~200μm, and the diameter of the third cylinder is 1~20μm, so that large, medium, and small three - level pore structures are distributed in the Bletilla striata polysaccharide hydrogel dressing. The large pores are distributed in a grid pattern, each medium pore is located at the center of four large pores distributed in a rectangle, and the small pores randomly connect adjacent large pores and medium pores. The distance between every two adjacent large pores is 500~1000μm.

[0009] Preferably, the specific process of degrading the gelatin template is as follows: After standing to form a gel, freeze - dry the gelatin template filled with Bletilla striata polysaccharide hydrogel to obtain a gelatin template with solidified Bletilla striata polysaccharide hydrogel, and then immerse it in a gelatinase solution with a concentration of 0.05~0.15 mg / mL, and oscillate and react in a constant - temperature shaker at 30~40℃. Take a small amount of solution for detection every 1 h, and monitor the degradation degree of gelatin by protein quantitative analysis method. After the gelatin is completely degraded, repeatedly rinse the Bletilla striata polysaccharide hydrogel with deionized water to remove the residual enzyme and degradation products while re - hydrating the gel.

[0010] Preferably, the specific process of freeze - drying is as follows: Place the gelatin template filled with Bletilla striata polysaccharide hydrogel at -80℃ for pre - freezing for 1~2 h to completely freeze the solution, and then perform sublimation drying. Set the drying temperature to -60~40℃, the vacuum degree to 10 Pa, and the drying time to 12~24 h.

[0011] The present invention also provides a Bletilla striata polysaccharide hydrogel dressing, which is prepared by the preparation method of the above - mentioned Bletilla striata polysaccharide hydrogel dressing.

[0012] The present invention also provides an application of the Bletilla striata polysaccharide hydrogel dressing in the preparation of a drug for promoting the repair of diabetic wounds.

[0013] The present invention has at least the following beneficial effects: First, the present invention combines carboxymethyl chitosan and cinnamaldehyde to construct a hydrogel. Cinnamaldehyde has good antibacterial properties, enabling the hydrogel to exert a strong antibacterial effect. Incorporating polysaccharide from Bletilla striata into the hydrogel system, polysaccharide from Bletilla striata has good hemostatic and proliferation-promoting effects and can play a role in the hemostasis and proliferation stages of wound healing, promoting wound healing by promoting cell proliferation and collagen fiber deposition.

[0014] Second, the hydrogel dressing provided by the present invention is made from natural raw materials, is non-toxic, easily obtainable, and has good biocompatibility.

[0015] Third, the present invention can provide a preparation method for the hydrogel dressing, which can achieve precise control of the pore shape and spatial distribution, and the finally prepared dressing has a three-level pore structure of large, medium, and small pores. When the hydrogel is used as a wound dressing, substances such as salts and proteins in the wound exudate will make the osmotic pressure of the exudate higher than that inside the hydrogel, thereby promoting the hydrogel to absorb the exudate. The large pores are responsible for quickly capturing the exudate, the medium pores conduct preliminary liquid transmission and distribution, and the small pores achieve fine liquid diffusion and storage. This hierarchical structure can efficiently process according to the amount and nature of the exudate, improve the adaptability of absorption and drainage, and effectively promote the rapid healing of diabetic wounds.

[0016] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0017] Figure 1 is the transmission electron micrograph of the polysaccharide from Bletilla striata hydrogel dressing prepared in Example 1; Figure 2 is the infrared spectrogram of the polysaccharide from Bletilla striata hydrogel dressing prepared in Example 1, as well as polysaccharide from Bletilla striata, carboxymethyl chitosan, cinnamaldehyde, the mixture of polysaccharide from Bletilla striata and carboxymethyl chitosan, and the hydrogel dressing prepared in Comparative Example 1; Figure 3 is the rheogram of the polysaccharide from Bletilla striata hydrogel dressing prepared in Example 2; Figure 4 is the comparison chart of the water loss rate test results of the polysaccharide from Bletilla striata hydrogel dressing prepared in Example 2 and the hydrogel dressing prepared in Comparative Example 1; Figure 5 is the comparison chart of the hemolysis rate of the extraction solution of the polysaccharide from Bletilla striata hydrogel dressing prepared in Example 2 and the extraction solution of the hydrogel dressing prepared in Comparative Example 1; Figure 6It is a comparison chart of the survival of L929 fibroblasts in the extract of the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the extract of the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide; Figure 7 It is a comparison chart of the antibacterial experiments of the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide; Figure 8 It is a comparison chart of the healing conditions of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide for 3, 7, and 10 days; Figure 9 It is a comparison chart of the healing rates of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide for 3, 7, and 10 days; Figure 10 It is a comparison chart of H&E staining of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide for 3, 7, and 10 days; Figure 11 It is a comparison chart of Masson staining of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide for 3, 7, and 10 days; Figure 12 It is a comparison chart of immunohistochemistry of inflammatory factors of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide for 3 and 7 days; Figure 13 It is a comparison chart of immunohistochemistry of angiogenesis and collagen deposition of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide; Figure 14 It is a comparison chart of the immunohistochemistry results of angiogenesis and collagen deposition of diabetic rat skin wounds after treatment with the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2, the hydrogel dressing prepared in Comparative Example 1, and Bletilla striata polysaccharide. Detailed Embodiments

[0018] The following further elaborates on the present invention in conjunction with the accompanying drawings and specific embodiments, enabling those skilled in the art to implement it with reference to the description in the specification.

[0019] It should be understood that terms such as "having", "comprising", and "including" as used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0020] Example 1 A preparation method of a Bletilla striata polysaccharide hydrogel dressing (BCC), comprising the following steps: Step 1, prepare a carboxymethyl chitosan solution: dissolve carboxymethyl chitosan in deionized water to obtain a carboxymethyl chitosan solution; the concentration of the carboxymethyl chitosan solution is 5 wt%. Step 2, prepare a Bletilla striata polysaccharide solution: dissolve Bletilla striata polysaccharide in deionized water to obtain a Bletilla striata polysaccharide solution; the concentration of the Bletilla striata polysaccharide solution is 1.2 wt%. Step 3, prepare a polysaccharide-based hydrogel: mix the carboxymethyl chitosan solution with cinnamaldehyde and the Bletilla striata polysaccharide solution at 25 °C and stir evenly, then let it stand to form a gel. In the mixed solution system, the volume ratio of the Bletilla striata polysaccharide solution to the carboxymethyl chitosan solution is 3:1; the concentration of cinnamaldehyde in the system is 3 wt%.

[0021] Example 2 A preparation method of a Bletilla striata polysaccharide hydrogel dressing (BCC), comprising the following steps: Step 1, prepare a carboxymethyl chitosan solution: dissolve carboxymethyl chitosan in deionized water to obtain a carboxymethyl chitosan solution; the concentration of the carboxymethyl chitosan solution is 5 wt%. Step 2, prepare a Bletilla striata polysaccharide solution: dissolve Bletilla striata polysaccharide in deionized water to obtain a Bletilla striata polysaccharide solution; the concentration of the Bletilla striata polysaccharide solution is 1.2 wt%. Step 3, prepare a polysaccharide-based hydrogel: mix the carboxymethyl chitosan solution with cinnamaldehyde and the Bletilla striata polysaccharide solution at 25 °C and stir evenly to obtain a mixed solution system. In the mixed solution system, the volume ratio of the Bletilla striata polysaccharide solution to the carboxymethyl chitosan solution is 3:1; the concentration of cinnamaldehyde in the system is 3 wt%.

[0022] Step 4, at 65 °C, prepare a gelatin template by 3D printing technology with a 15% wt gelatin. There are multiple cylinders with different diameters in the gelatin template, including a first cylinder and a second cylinder evenly distributed in a grid pattern. Each second cylinder is located at the center of four first cylinders distributed in a rectangle, and multiple third cylinders are randomly connected between the side walls of every two adjacent first cylinders and second cylinders. The diameter of the first cylinder is 400 μm, the diameter of the second cylinder is 120 μm, and the diameter of the third cylinder is 12 μm, so that the Bletilla striata polysaccharide hydrogel dressing has large, medium, and small three-level pore structures. The large pores are distributed in a grid pattern, each medium pore is located at the center of four large pores distributed in a rectangle, and the small pores randomly connect adjacent large pores and medium pores. The distance between every two adjacent large pores is 800 μm; Step 5: Immerse the gelatin template in the mixed solution system, ultrasonically treat it at a frequency of 50 kHz for 8 min, then take out the gelatin template filled with the mixed solution system and place it in a vacuum environment with a vacuum degree of -0.09 MPa for degassing; Step 6: Let it stand to form a gel. Freeze-dry the gelatin template filled with the Bletilla striata polysaccharide hydrogel to obtain a gelatin template with solidified Bletilla striata polysaccharide hydrogel. Then immerse it in a gelatinase solution with a concentration of 0.1 mg / mL and oscillate and react it in a constant temperature shaker at 35°C. Take a small amount of solution for detection every 1 h, monitor the degradation degree of gelatin by protein quantitative analysis method. After the gelatin is completely degraded, repeatedly rinse the Bletilla striata polysaccharide hydrogel with deionized water to remove the residual enzyme and degradation products and rehydrate the gel at the same time, and finally obtain a Bletilla striata polysaccharide hydrogel dressing with a porous structure.

[0023] Example 3 A preparation method of a Bletilla striata polysaccharide hydrogel dressing (BCC), comprising the following steps: Step 1: Prepare a carboxymethyl chitosan solution: Dissolve carboxymethyl chitosan in deionized water to obtain a carboxymethyl chitosan solution; the concentration of the carboxymethyl chitosan solution is 2 wt%; Step 2: Prepare a Bletilla striata polysaccharide solution: Dissolve Bletilla striata polysaccharide in deionized water to obtain a Bletilla striata polysaccharide solution; the concentration of the Bletilla striata polysaccharide solution is 0.5 wt%; Step 3: Prepare a polysaccharide-based hydrogel: Mix the carboxymethyl chitosan solution, cinnamaldehyde and the Bletilla striata polysaccharide solution at 20°C and stir evenly to obtain a mixed solution system. The volume ratio of the Bletilla striata polysaccharide solution to the carboxymethyl chitosan solution in the mixed solution system is 1:1; the concentration of cinnamaldehyde in the system is 0.05 wt%.

[0024] Step 4: At 60°C, prepare a gelatin template by 3D printing technology with 15%wt gelatin. The gelatin template is provided with a plurality of cylinders with different diameters, including a first cylinder and a second cylinder evenly distributed in a grid pattern. Each second cylinder is located at the center of four first cylinders distributed in a rectangle, and a plurality of third cylinders are randomly connected between the side walls of every two adjacent first cylinders and second cylinders. The diameter of the first cylinder is 300 μm, the diameter of the second cylinder is 50 μm, and the diameter of the third cylinder is 1 μm, so that large, medium and small three-level pore structures are distributed in the Bletilla striata polysaccharide hydrogel dressing, where the large pores are distributed in a grid pattern, each medium pore is located at the center of four large pores distributed in a rectangle, and the small pores are randomly connected to adjacent large pores and medium pores, and the distance between every two adjacent large pores is 500 μm; Step 5: Immerse the gelatin template in the mixed solution system, perform ultrasonic treatment at a frequency of 40 kHz for 5 min, then take out the gelatin template filled with the mixed solution system and place it in a vacuum environment with a vacuum degree of -0.08 MPa for degassing; Step 6: Let it stand to form a gel, freeze-dry the gelatin template filled with the Bletilla striata polysaccharide hydrogel to obtain a gelatin template with solidified Bletilla striata polysaccharide hydrogel, then immerse it in a gelatinase solution with a concentration of 0.05 mg / mL and oscillate and react in a constant temperature shaker at 30 °C. Take a small amount of solution for detection every 1 h, monitor the degradation degree of gelatin by protein quantitative analysis method. After the gelatin is completely degraded, repeatedly rinse the Bletilla striata polysaccharide hydrogel with deionized water to remove the residual enzyme and degradation products while rehydrating the gel, and finally obtain a Bletilla striata polysaccharide hydrogel dressing with a porous structure.

[0025] Example 4 A preparation method of a Bletilla striata polysaccharide hydrogel dressing (BCC), comprising the following steps: Step 1: Prepare a carboxymethyl chitosan solution: Dissolve carboxymethyl chitosan in deionized water to prepare a carboxymethyl chitosan solution; the concentration of the carboxymethyl chitosan solution is 8 wt%; Step 2: Prepare a Bletilla striata polysaccharide solution: Dissolve Bletilla striata polysaccharide in deionized water to prepare a Bletilla striata polysaccharide solution; the concentration of the Bletilla striata polysaccharide solution is 2 wt%; Step 3: Prepare a polysaccharide-based hydrogel: Mix the carboxymethyl chitosan solution with cinnamaldehyde and the Bletilla striata polysaccharide solution at 30 °C and stir evenly to obtain a mixed solution system. The volume ratio of the Bletilla striata polysaccharide solution to the carboxymethyl chitosan solution in the mixed solution system is 5:1; the concentration of cinnamaldehyde in the system is 5 wt%.

[0026] Step 4: At 70 °C, prepare a gelatin template by 3D printing technology with 15%wt gelatin. The gelatin template is provided with a plurality of cylinders with different diameters, including a first cylinder and a second cylinder uniformly distributed in a grid pattern. Each second cylinder is located at the center of four first cylinders distributed in a rectangle, and a plurality of third cylinders are randomly connected between the side walls of every two adjacent first cylinders and second cylinders. The diameter of the first cylinder is 500 μm, the diameter of the second cylinder is 200 μm, and the diameter of the third cylinder is 20 μm, so that large, medium and small three-level pore structures are distributed in the Bletilla striata polysaccharide hydrogel dressing, where the large pores are distributed in a grid pattern, each medium pore is located at the center of four large pores distributed in a rectangle, and the small pores are randomly connected to adjacent large pores and medium pores, and the distance between every two adjacent large pores is 1000 μm; Step 5: Immerse the gelatin template in the mixed solution system, ultrasonically treat it at a frequency of 60 kHz for 10 min, then take out the gelatin template filled with the mixed solution system and place it in a vacuum environment with a vacuum degree of -0.1 MPa for degassing; Step 6: Let it stand to form a gel, freeze-dry the gelatin template filled with the Bletilla striata polysaccharide hydrogel to obtain a gelatin template with solidified Bletilla striata polysaccharide hydrogel, then immerse it in a gelatinase solution with a concentration of 0.15 mg / mL, and oscillate and react in a constant temperature shaker at 40 °C. Take a small amount of solution for detection every 1 h, monitor the degradation degree of gelatin by protein quantitative analysis method. After the gelatin is completely degraded, repeatedly rinse the Bletilla striata polysaccharide hydrogel with deionized water to remove the residual enzyme and degradation products while rehydrating the gel, and finally obtain a Bletilla striata polysaccharide hydrogel dressing with a porous structure.

[0027] Comparative Example 1 A preparation method of a hydrogel dressing (CC), which is different from Example 2 in that Bletilla striata polysaccharide is not added, and specifically includes the following steps: Step 1: Prepare a carboxymethyl chitosan solution: Dissolve carboxymethyl chitosan in deionized water to prepare a carboxymethyl chitosan solution; the concentration of the carboxymethyl chitosan solution is 5 wt%; Step 2: Mix the carboxymethyl chitosan solution and cinnamaldehyde at 25 °C and stir evenly to obtain a mixed solution system, and the concentration of cinnamaldehyde in the system is 3 wt%.

[0028] Structure characterization determination Scanning electron microscopy detection: Observe the microstructure of the Bletilla striata polysaccharide hydrogel dressing prepared in Example 1 through a transmission electron microscope.

[0029] Fourier transform infrared spectroscopy detection: Use Fourier transform infrared spectroscopy to analyze the chemical structures of other monomer components of the Bletilla striata polysaccharide hydrogel dressing prepared in Example 1, as well as the simple mixture of Bletilla striata polysaccharide and carboxymethyl chitosan, and scan in the wavelength range of 400 - 4000 cm -1 Wavelength range.

[0030] The results are as Figure 1-2 shown. It can be seen from Figure 1 that the Bletilla striata polysaccharide hydrogel dressing prepared in Example 1 presents irregular polygons under the transmission electron microscope. Figure 2 For the infrared spectrogram, it can be seen that the β-glycosidic bond and α-glycosidic bond with stretching vibrations at 884 cm -1 and 802 cm -1 of Bletilla striata polysaccharide (BSP), as well as the aldehyde group of cinnamaldehyde (CA) at 1680 cm -1 disappear after the gel formation reaction, which is due to the formation of Schiff base bonds.

[0031] Exudate Absorption and Transmission Performance Test Take the hydrogel dressings prepared in Examples 1-4 with the same mass and place them in petri dishes respectively. Slowly drip simulated diabetic wound exudate onto the dressings with a dropper, 0.1 mL each time, until the dressings reach the saturated state, and record the total volume of exudate (V) dripped. Every 10 minutes, weigh the mass of the dressings (m t ), and calculate the absorption amount of exudate by the dressings at different time points (Δm = m t - m 0 ). Observe the transmission of exudate in the dressings and record the roles of macropores, mesopores and micropores in the liquid transmission process. The results are shown in Table 1.

[0032] Table 1 As can be seen from Table 1, through the above table data, it can be clearly seen that Examples 2, 3, and 4 have significant advantages in exudate absorption performance compared with Example 1. In terms of the absorption amount per unit time, the absorption amounts of Examples 2, 3, and 4 in each 10-minute time interval are higher than those of Example 1, indicating that the dressings of Examples 2, 3, and 4 can absorb exudate faster. In terms of the total absorption amount in the saturated state, Examples 2, 3, and 4 can absorb more exudate, indicating that they are superior to Example 1 in absorption capacity. Generally speaking, the Bletilla striata polysaccharide hydrogel dressings prepared in Examples 2, 3, and 4 are significantly superior to Example 1 in exudate absorption performance, and can absorb exudate faster and in larger amounts. This is because the Bletilla striata polysaccharide hydrogel dressings prepared in Examples 2-4 have a three-level pore structure of large, medium and small pores, which provides a more reasonable channel for the absorption and transmission of exudate. Macropores can quickly capture exudate and rapidly introduce a large amount of liquid into the interior of the dressing; mesopores are responsible for the preliminary transmission and distribution of liquid, spreading the liquid to a wider area; micropores realize fine liquid diffusion and storage, making the liquid evenly distributed in the dressing and avoiding local fluid accumulation. However, Example 1 does not have this fine hierarchical pore structure, and the absorption and transmission of liquid are relatively limited. This has positive significance for dealing with wound exudate, keeping the wound dry and promoting wound healing in practical applications.

[0033] Physicochemical Property Determination Rheological property test: Use a rheometer to test the rheological properties of the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2. The frequency sweep fixes the strain at 1%, and the sweep range is 0.1-10 Hz.

[0034] Water loss rate test: Weigh the hydrogel dressings prepared in Example 2 and Comparative Example 1 and place them at 37°C. Weigh the samples every two hours. The water loss rate calculation formula is as follows: ; Figure 3It is a frequency scanning spectrum. The results show that G’ > G’’, and it does not change with frequency, indicating that the Bletilla striata polysaccharide hydrogel dressing prepared in Example 2 has good elasticity and stability, can fill irregular wounds, and provide physical support. Figure 4 It is a comparison chart of the water loss rates of the Bletilla striata polysaccharide hydrogel dressing (BCC) in Example 2 and the hydrogel dressing (CC) in Comparative Example 1. The water loss rate of BCC after 24 hours is 26.08 ± 1.05%, indicating that the good water retention performance of the Bletilla striata polysaccharide hydrogel dressing enables it to maintain a moist wound environment and can be easily removed from the wound without causing secondary damage.

[0035] The Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2, the hydrogel dressing (CC) prepared in Comparative Example 1, as well as the blank control group (Control) and the Bletilla striata polysaccharide (BSP) group under the same conditions were subjected to the following tests.

[0036] Biocompatibility determination Red blood cells were obtained by centrifugation after blood collection and diluted to 5% with physiological saline. The extraction solution of the Bletilla striata polysaccharide hydrogel dressing (BCC) was extracted at a ratio of 1 g of gel to 10 ml of physiological saline. 500 μl of the extraction solution and 500 μl of the 5% red blood cell suspension were gently mixed and placed at 37 °C for 4 hours, and the samples were inverted 2 times per hour. After 4 hours, the samples were centrifuged to obtain the supernatant, and the absorbance at 540 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader. The positive control group was 0.1% Triton X-100, and the negative control was physiological saline (NS). The hemolysis rate calculation formula is as follows: ; The hydrogel dressings prepared in Example 2 and Comparative Example 1 were subjected to cytotoxicity determination, and the blank control group (Control) and the Bletilla striata polysaccharide (BSP) group under the same conditions were set: the extraction solution of the hydrogel dressing was sterilized by ultraviolet light and filtered through a filter membrane to remove bacteria for standby. L929 fibroblasts were passaged into 96-well plates at a density of 5,000 cells / well, and live-dead staining and photography were performed after culturing for 24 h, 48 h, and 72 h.

[0037] Figure 5 It is a hemolysis rate chart of the extraction solution of the Bletilla striata polysaccharide hydrogel dressing (BCC), and its hemolysis rate is 0.84 ± 0.44%. The results show that the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 of the present invention has good biocompatibility. Figure 6 It is a live-dead cell staining chart. The green fluorescence represents live cells, and the red fluorescence represents dead cells. The survival rate of cells in the extraction solution of the Bletilla striata polysaccharide hydrogel dressing (BCC) in Example 2 is higher than that in Comparative Example 1 after culturing for 72 h.

[0038] Antibacterial performance evaluation The method of colony counting was used to evaluate the anti - Staphylococcus aureus effect of the hydrogel dressings prepared in Example 2 and Comparative Example 1, and a blank control group (Control) and a Bletilla striata polysaccharide (BSP) group under the same conditions were set up. 1 g of the gel was added to 5 ml of the culture medium, and then 200 μl of the bacterial solution with OD600 = 0.4 was added for co - culture for 24 h. The co - culture solution was diluted 10 6 times, and then 100 μl was taken for spreading on the plate. After the agar plate was cultured for 24 h, colony counting was carried out. The antibacterial rate calculation formula is as follows: ; Figure 7 Figure is the photo of the plate spreading and the antibacterial rate graph after co - culture of the Bletilla striata polysaccharide hydrogel dressing (BCC) and Staphylococcus aureus. The results show that the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 has excellent anti - Staphylococcus aureus performance, and its antibacterial rate can reach 97.88 ± 1.05%.

[0039] Promote wound healing assessment (1) Establishment and treatment of the diabetic rat skin wound model: Male SD rats weighing 190 - 220 g were selected. The feeding environment of the rats was at a constant temperature of 23 ± 2 °C, with a normal day - night cycle every 12 h. All animal experiments were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine. Streptozotocin was intraperitoneally injected for type I diabetes mellitus modeling. Blood glucose ≥ 16.8 was considered successful diabetes mellitus modeling. Two wounds with a diameter of 8 mm were made on the back skin of the rats. Dressing changes were carried out daily, including the hydrogel dressings prepared in Example 2 and Comparative Example 1, as well as the blank control group (Control) and the Bletilla striata polysaccharide (BSP) group under the same conditions. Photographs were taken and samples were collected at 0, 3, 7, and 10 days after modeling.

[0040] (2) Histopathological analysis: H&E and Masson staining were used to evaluate the tissue morphology of wound healing. After sampling, the samples were fixed, embedded, sectioned and stained.

[0041] (3) Immunohistochemical analysis: Molecular markers, cytokines and growth factors in the wound tissue were analyzed by immunohistochemical experiments. The principle of immunohistochemical experiments is the binding of antigens and antibodies. The following markers were used to evaluate wound healing: TNF - α, IL - 10, VEGF - A, CD31, Col I.

[0042] As Figure 8-9 shown, the wound healing speed of the Bletilla striata polysaccharide hydrogel dressing (BCC) group prepared in Example 2 was the fastest. At the end of the treatment, the wound healing areas of the control group and the Bletilla striata polysaccharide hydrogel dressing (BCC) group were 85.86 ± 3.72% and 94 ± 1.63% respectively. The Bletilla striata polysaccharide hydrogel dressing (BCC) group was better than the control group at each time point, indicating that it has good ability to promote rapid wound healing.

[0043] As shown by H&E staining Figure 10 as shown in Fig. 1, on the 3rd day, it was the early inflammatory stage, and signs of inflammatory cell infiltration were presented in each group. However, in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2, granulation tissue had already appeared, which could fill the wound defect and provide physical support for angiogenesis. On the 7th day, the epidermis of the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 had been completely covered, while no epidermis was formed in the control group. On the 10th day, complete epidermal regeneration occurred in each group, but regeneration of skin appendages (hair follicles and sebaceous glands) was observed in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2.

[0044] As shown by Masson staining Figure 11 as shown in Fig. 2, there were significant differences in the collagen deposition density between the control group and the other three groups. Although the epidermis was completely regenerated in the control group, there was little collagen fiber deposition. However, the collagen fibers in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 were cross-linked into bundles and arranged regularly, providing better tension for the skin tissue.

[0045] As shown by immunohistochemical staining Figure 12 as shown in Fig. 3, TNF-α is an important pro-inflammatory cytokine. At 3 days after skin injury, similar levels of TNF-α were expressed in all four groups. On the 7th day, the level of TNF-α in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 (5.74 ± 1.28%) was lower than that in the control group (11.01 ± 1.6%). IL-10 is an anti-inflammatory cytokine. At 3 days after skin injury, there was a statistically significant difference in the expression of IL-10 between the control group (1.66 ± 0.56%) and the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 (3.05 ± 0.59%). On the 7th day, the level of IL-10 in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 (4.56 ± 0.58%) was continuously higher than that in the control group (3.18 ± 0.55%). Through the quantitative analysis of the pro-inflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 in the wound tissue, it was verified that the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 had a good inflammatory regulation effect.

[0046] As Figure 13-14 shown in Fig. 4, VEGF-A (vascular endothelial growth factor A) and CD31 (platelet endothelial cell adhesion molecule) were used to evaluate angiogenesis, and Col I (collagen fiber I) was used to evaluate collagen deposition. The VEGF-A area in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 was higher than that in other groups. At 10 days, new blood vessels were formed in each group, but the blood vessels in the group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 were more mature with larger lumens. The group of the Bletilla striata polysaccharide hydrogel dressing (BCC) prepared in Example 2 could significantly enhance the deposition of collagen fiber I (Col I).

[0047] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and the examples shown and described herein.

Claims

1. A method for preparing a bletilla striata polysaccharide hydrogel dressing, characterized in that: The method comprises the following steps: mixing a carboxymethyl chitosan solution, a bletilla striata polysaccharide solution and cinnamaldehyde at 20-30° C. and stirring evenly to obtain a mixed solution system, and then standing to form a gel; wherein the volume ratio of the carboxymethyl chitosan solution to the bletilla striata polysaccharide solution is (1-5):1, and the concentration of the cinnamaldehyde in the mixed solution system is 0.05-5wt%; The bletilla polysaccharide hydrogel dressing has a three-level pore structure of large, medium and small, wherein the large pores are distributed in a grid shape, each medium pore is located at the center of four large pores distributed in a rectangular shape, and the small pores randomly connect adjacent large pores and medium pores, and the distance between every two adjacent large pores is 500-1000 μm.

2. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 1, characterized in that: The concentration of the carboxymethyl chitosan solution is 2-8 wt %.

3. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 1, characterized in that: The concentration of the bletilla striata polysaccharide solution is 0.5-2wt%.

4. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 1, characterized in that: Also includes: preparing a gelatin template, wherein a plurality of cylinders with different diameters are arranged in the gelatin template; The gelatin template is immersed in the mixed solution system, and ultrasonically treated at a frequency of 40-60 kHz for 5-10 minutes, and then the gelatin template filled with the mixed solution system is taken out and placed in a vacuum environment with a vacuum degree of -0.08 to -0.1 MPa for degassing; The gelatin template was degraded by standing to form gel, and finally a Bletilla striata polysaccharide hydrogel dressing with a porous structure was obtained.

5. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 4, characterized in that: The gelatin template is made of gelatin with a concentration of 15%wt by 3D printing technology, the printing temperature is 60~70°C, and the multiple cylinders include first cylinders and second cylinders uniformly distributed in a grid shape, wherein each second cylinder is located at the center of four first cylinders distributed in a rectangular shape, and multiple third cylinders are randomly connected between the side walls of each two adjacent first cylinders and second cylinders, the diameter of the first cylinder is 300~500μm, the diameter of the second cylinder is 50~200μm, and the diameter of the third cylinder is 1~20μm.

6. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 4, characterized in that: The specific process of degrading the gelatin template is as follows: after standing to gel, the gelatin template filled with the bletilla polysaccharide hydrogel is freeze-dried to obtain the gelatin template with solidified bletilla polysaccharide hydrogel, which is then immersed in a gelatinase solution with a concentration of 0.05-0.15 mg / mL, and oscillated in a constant temperature shaker at 30-40°C for reaction, a small amount of solution is taken for detection every 1 hour, and the degradation degree of gelatin is monitored by protein quantitative analysis. After the gelatin is completely degraded, the bletilla polysaccharide hydrogel is repeatedly rinsed with deionized water to remove residual enzymes and degradation products while rehydrating the gel.

7. The method for preparing the Bletilla striata polysaccharide hydrogel dressing according to claim 6, characterized in that: The specific process of freeze drying is: pre-freeze the gelatin template filled with Bletilla striata polysaccharide hydrogel at -80°C for 1-2 hours to completely freeze the solution, and then perform sublimation drying, setting the drying temperature to -60-40°C, the vacuum degree to 10Pa, and the drying time to 12-24 hours.

8. Bletilla striata polysaccharide hydrogel dressing, characterized in that: The dressing is prepared by the preparation method of the bletilla striata polysaccharide hydrogel dressing according to any one of claims 1 to 7.

9. Use of the Bletilla striata polysaccharide hydrogel dressing as claimed in claim 8 in the preparation of a drug for promoting diabetic wound repair.

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