A bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing and its preparation method

Abstract

The present invention belongs to the technical field of biomedical hydrogels, and specifically discloses a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing and a preparation method thereof. The activated bacterial cellulose-producing strain is inoculated into a culture medium to obtain bacterial cellulose, which is repeatedly soaked and washed with pure water until the color is close to white and the pH is neutral. The bacterial cellulose (BC) is soaked in polyvinyl alcohol solutions with a mass concentration of 0.5-2% and dialdehyde carboxyl starch solutions with different oxidation degrees for 2-4 days to obtain different bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressings. Compared with the prior art, the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing of the present invention has stronger wound healing promoting ability, new granulation tissue growth promoting ability and collagen production promoting ability during long-term (2 weeks) use.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical hydrogels, in particular to a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing and a preparation method thereof. Background Art

[0002] A wound dressing is a protective layer used to cover a wound, which plays a role in preventing wound infection, promoting cell proliferation, and avoiding secondary trauma during the wound healing process. The hydrogel dressing has a unique three-dimensional network structure. Its soft elasticity and high hydrophilicity make the dressing easy to disassemble, prevent adhesion, prevent secondary trauma, help provide a moist environment for the wound, and promote the growth of epithelial granulation tissue. Bacterial cellulose (BC) is a natural polymer synthesized by microorganisms and has a nano-scale fiber network structure. As a hydrogel material, it has good biocompatibility, high plasticity, and relatively high porosity, and has application potential in the field of wound dressings.

[0003] Currently, the preparation method of bacterial cellulose-based hydrogel wound dressings is mainly to use bacterial cellulose as the matrix material and enhance its performance by adding reinforcing materials. Bacterial cellulose is often compounded with other materials with antibacterial, anti-inflammatory and other functions to prepare bacterial cellulose composite hydrogel wound dressings, so as to improve its mechanical properties, antibacterial properties or / and wound healing promotion ability.

[0004] The application of bacterial cellulose-based hydrogels in the medical field is becoming more and more extensive, but there are still certain limitations. Its mechanical strength and mechanical properties are still insufficient compared with dressings such as natural plants or synthetic fibers. At the same time, although components such as nano-silver and phenytoin in the composite material have strong antibacterial, anti-inflammatory and high wound healing promotion abilities, they have certain toxicity to human cells or tissues and organs. How to improve the mechanical properties of bacterial cellulose-based hydrogel wound dressings and reduce the cytotoxicity of the composite material on the premise of maintaining antibacterial properties and wound healing promotion properties is the development direction of bacterial cellulose-based hydrogel wound dressings.

[0005] A bacterial cellulose-xyloglucan-dextran composite hydrogel wound dressing with the application number 202310679359.5 applied in the early stage by us and its preparation method. The activated bacterial cellulose-producing bacteria are inoculated into the seed liquid culture medium to obtain the seed liquid; the obtained seed liquid is inoculated into a fermentation medium containing 0.2-1% (mass concentration) of xyloglucan and 0.2-1% (mass concentration) of dextran, and the bacterial cellulose-xyloglucan-dextran composite is harvested by culturing. It is repeatedly soaked and washed with pure water for many times until the color is close to white, and the pH of the composite is washed with pure water to neutrality to obtain the bacterial cellulose-xyloglucan-dextran composite hydrogel wound dressing. This bacterial cellulose-xyloglucan-dextran composite hydrogel wound dressing has good fitting degree, softness and ductility, and the ability to accelerate wound healing; however, the production process is relatively troublesome, and the abilities of promoting wound healing, promoting the growth of new granulation tissue and promoting collagen production still need to be improved.

[0006] Dialdehyde-carboxyl-amylose (DACA) is an oxidized starch with high water solubility, antibacterial property and good biocompatibility, and has the potential to form a new composite hydrogel wound dressing by combining with bacterial cellulose.

[0007] Therefore, it is urgent to try to prepare a bacterial cellulose-dialdehyde-carboxyl-amylose composite hydrogel wound dressing. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a bacterial cellulose-dialdehyde-carboxyl-amylose composite hydrogel wound dressing and its preparation method.

[0009] To achieve the above object, the present invention is implemented according to the following technical scheme:

[0010] One of the objects of the present invention is to provide a preparation method of a bacterial cellulose-dialdehyde-carboxyl-amylose composite hydrogel wound dressing, including the following steps:

[0011] S1: Using carboxyl starch as the raw material, dialdehyde-carboxyl-amylose is prepared by periodate oxidation;

[0012] S2: Inoculating the bacterial cellulose-producing bacteria strain into the fermentation medium, standing and culturing at 25-36 °C for 3-5 days to obtain the concentrated bacterial strain solution; the fermentation medium is composed of the following components: glucose 20-25 g / L, disodium hydrogen phosphate anhydrous 2.0-3. g / L, citric acid monohydrate 1.0-2.0 g / L, yeast extract 5-10 g / L, peptone 5-10 g / L, and the balance is deionized water, and the pH is adjusted to 4.0-5.0 with 1 mol / L hydrochloric acid solution;

[0013] S3: Inoculate the obtained concentrated bacterial liquid into the fermentation medium at a ratio of 8 - 15 (v / v)%, and place it in an incubator for static culture at 25 - 36 °C for 5 - 9 days to obtain bacterial cellulose. After soaking and washing it with pure water for multiple times until the color is close to white and the pH reaches neutrality, bacterial cellulose hydrogel is obtained. The fermentation medium consists of the following components: 20 - 25 g / L of glucose, 2.0 - 3.0 g / L of disodium hydrogen phosphate anhydrous, 1.0 - 2.0 g / L of citric acid monohydrate, 5 - 10 g / L of yeast extract, 5 - 10 g / L of peptone, and the balance is deionized water. Adjust the pH to 4.0 - 5.0 with 1 mol / L hydrochloric acid solution;

[0014] S4: Immerse the pure bacterial cellulose in a solution containing 0.5 - 2% polyvinyl alcohol and dialdehyde carboxyl starch by mass concentration for 3 - 5 days to obtain a bacterial cellulose - dialdehyde carboxyl starch composite hydrogel wound dressing.

[0015] Further, the preparation method of the carboxyl starch in step S1 is as follows: Starch is oxidized successively by 2,2,6,6 - tetramethylpiperidine - 1 - oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyzed to obtain carboxyl starch.

[0016] Further, the molar concentration of sodium periodate in step S1 is 0.5 M - 1.1 M, preferably 0.9 M.

[0017] Further, the genus of the bacterial cellulose - producing bacteria used in step S1 is one of Acetobacter, Gluconacetobacter, and Komagataeibacter xylinus, preferably Gluconacetobacter.

[0018] Further, in step S2, the total number of colonies of bacterial cellulose - producing bacteria in each milliliter of the concentrated bacterial liquid is 10 2 -10 4 CFU.

[0019] The second object of the present invention is to provide a bacterial cellulose - dialdehyde carboxyl starch composite hydrogel wound dressing prepared by the above method.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] When comparing the commercially available sterile gauze, commercially available hydrocolloid dressing, and bacterial cellulose hydrogel wound dressing, the bacterial cellulose - dialdehyde carboxyl starch composite hydrogel wound dressing has stronger wound - healing promoting ability, new granulation tissue growth promoting ability, and collagen production promoting ability during long - term (2 - week) use.

[0022] Comparing medical sterile gauze, commercially available hydrocolloid dressings, bacterial cellulose hydrogel wound dressings, and bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressings, the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing has a microscopic structure with fibrous porous characteristics, and at the same time has mechanical characteristics such as high water content, good softness, and good ductility; in addition, it also has certain antibacterial properties and excellent biocompatibility. Description of the Drawings

[0023] Figure 1 Scanning electron microscope (SEM) images of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)).

[0024] Figure 2 Fourier transform infrared spectroscopy (FTIR) spectra of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)).

[0025] Figure 3 Results of tensile experiments and creep-recovery experiments of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)).

[0026] Figure 4 Results of rheological amplitude sweep and frequency sweep of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)).

[0027] Figure 5 Results of rheological amplitude sweep and frequency sweep of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)).

[0028] Figure 6 Results of antibacterial rate tests of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA).

[0029] Figure 7 Results of cell compatibility tests of bacterial cellulose hydrogel (BC) and different bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA(E)). Detailed Embodiments

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further details the present invention in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available or self-made; among them, Acetobacter xylinum was purchased from the China Center for Microbial Conservation; the fermentation medium consists of the following components: 20 g / L of glucose, 2.7 g / L of disodium hydrogen phosphate anhydrous, 1.26 g / L of citric acid monohydrate, 5 g / L of yeast extract, 5 g / L of peptone, and the balance is deionized water. The pH is adjusted to 5.0 with 1 mol / L hydrochloric acid solution.

[0032] Example 1

[0033] S1: Starch was successively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyzed to obtain carboxylated starch; secondly, using carboxylated starch as the raw material, dialdehyde carboxylated starch (DACA1) was prepared by oxidation with 0.5 M periodate.

[0034] S2: The activated Acetobacter xylinum was inoculated into the fermentation medium and statically cultured at 30 °C for 3 days to obtain a concentrated bacterial solution.

[0035] S3: The obtained concentrated bacterial solution was inoculated into the fermentation medium-containing medium and statically cultured in an incubator at 30 °C for 5 days. Bacterial cellulose hydrogel was harvested and repeatedly soaked and washed with pure water for multiple times until the color was close to white and the pH was neutral to obtain bacterial cellulose hydrogel.

[0036] S4: The bacterial cellulose was soaked in a solution containing 1% polyvinyl alcohol and dialdehyde carboxylated starch (DACA1) by mass concentration for 3 days to obtain a bacterial cellulose-dialdehyde carboxylated starch composite hydrogel (BC-DACA1(E)) wound dressing.

[0037] Example 2

[0038] S1: Starch was successively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyzed to obtain carboxylated starch. Secondly, using carboxylated starch as the raw material, dialdehyde carboxylated starch (DACA2) was prepared by oxidation with 0.7 M periodate.

[0039] S2: The activated Acetobacter xylinum was inoculated into the fermentation medium and statically cultured at 30 °C for 3 days to obtain a concentrated bacterial solution.

[0040] S3: The obtained concentrated bacterial solution was inoculated into the fermentation medium-containing medium and statically cultured in an incubator at 30 °C for 5 days. Bacterial cellulose hydrogel was harvested and repeatedly soaked and washed with pure water for multiple times until the color was close to white and the pH was neutral to obtain bacterial cellulose hydrogel.

[0041] S4: Soak the bacterial cellulose in a solution containing 1% polyvinyl alcohol and dialdehyde carboxyl starch (DACA2) by mass concentration for 3 days to obtain a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA2(E)) wound dressing.

[0042] Example 3

[0043] S1: Oxidize starch successively with 2,2,6,6-tetramethylpiperidine-1-oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyze to obtain carboxyl starch; Next, use carboxyl starch as the raw material to prepare dialdehyde carboxyl starch (DACA3) by oxidation with 0.9 M periodate.

[0044] S2: Inoculate the activated Acetobacter xylinum into the fermentation medium and statically culture at 30 °C for 3 days to obtain a concentrated bacterial solution.

[0045] S3: Inoculate the obtained concentrated bacterial solution into the fermentation medium, place it in an incubator and statically culture at 30 °C for 5 days, harvest the bacterial cellulose hydrogel, soak and wash it repeatedly with pure water for many times until the color is close to white and the pH is neutral to obtain the bacterial cellulose hydrogel.

[0046] S4: Soak the bacterial cellulose in a solution containing 1% polyvinyl alcohol and dialdehyde carboxyl starch (DACA3) by mass concentration for 3 days to obtain a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA3(E)) wound dressing.

[0047] Example 4

[0048] S1: Oxidize starch successively with 2,2,6,6-tetramethylpiperidine-1-oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyze to obtain carboxyl starch; Next, use carboxyl starch as the raw material to prepare dialdehyde carboxyl starch (DACA4) by oxidation with 1.1 M periodate.

[0049] S2: Inoculate the activated Acetobacter xylinum into the fermentation medium and statically culture at 30 °C for 3 days to obtain a concentrated bacterial solution.

[0050] S3: Inoculate the obtained concentrated bacterial solution into the fermentation medium, place it in an incubator and statically culture at 30 °C for 5 days, harvest the bacterial cellulose hydrogel, soak and wash it repeatedly with pure water for many times until the color is close to white and the pH is neutral to obtain the bacterial cellulose hydrogel.

[0051] S4: Soak the bacterial cellulose in a solution containing 1% polyvinyl alcohol and dialdehyde carboxyl starch (DACA4) by mass concentration for 3 days to obtain a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA4(E)) wound dressing.

[0052] Comparative Example 1

[0053] S1: Inoculate the activated Acetobacter xylinum into a fermentation medium and statically culture it at 30 °C for 3 days to obtain a concentrated bacterial solution.

[0054] S2: Inoculate the obtained concentrated bacterial solution into a fermentation medium and statically culture it in an incubator at 30 °C for 3 days.

[0055] S3: Harvest bacterial cellulose, soak and wash it repeatedly with pure water for multiple times until the color is close to white, and wash the hydrogel with pure water until the color is close to white and the pH is neutral to obtain a bacterial cellulose (BC) wound dressing.

[0056] Take the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings prepared in Examples 1-4 and Comparative Example 1 and the bacterial cellulose (BC) wound dressing, and respectively make circular wound dressings with the same area. Their physical comparison diagrams are as Figure 1 shown, and the partial physical parameters of the wound dressings are shown in Table 1. The water content of all five is above 96%, with an extremely high moisture content. At the same time, the thickness is also significantly reduced, making the dressing more easily conformable to human skin.

[0057] Table 1

[0058]

[0059]

[0060] Note: Different letters in the same column indicate significant differences (p < 0.05).

[0061] Furthermore, take the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings prepared in Examples 1-4 and Comparative Example 1 and the bacterial cellulose (BC) wound dressing, cut them into small pieces of about 2×2 mm respectively, freeze-dry for 48 h, sputter gold coating, and then select appropriate fields of view and magnifications to observe the microscopic morphology under a scanning electron microscope and take pictures for record. The results are as Figure 1 shown. Different from BC fibers, more substances are attached to the fibers of BC-DACA(E). In particular, obvious sheet-like structures are presented at the fiber joints. Impregnation compounding enables the polymer to pass through the BC network, fills the free spaces between the fibers, reduces the distance between cellulose chains, promotes the combination of fiber bundles, reduces the porosity of the hydrogel, and makes the composite hydrogel have a denser and more filled structure than pure BC.

[0062] In this embodiment, the functional groups of bacterial cellulose (BC) wound dressings and bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA(E)) wound dressings were also determined as follows:

[0063] The transmittance of the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings and the dry film of bacterial cellulose (BC) wound dressings prepared in Examples 1-4 and Comparative Example 1 was tested using the attenuated total reflection (ATR) module of a Fourier transform infrared spectrometer (FTIR). The number of scans was 32, the resolution was 4, and the spectrum was recorded between 4000-400 cm -1 -1.

[0064] Figure 2 In the FTIR spectrum, the absorption peak of BC at 3345 cm -1 -1 is the vibration absorption peak of -OH. The absorption peak of BC-DACA(E) undergoes a blue shift, which is caused by the formation of hydrogen bonds. 1092 cm -1 -1 is the stretching vibration of -CO-. It merges with the 1040 cm -1 -1 of BC-DACA(E) to form a broad peak with an increased peak intensity, indicating an enhanced hydrogen bond interaction. A new absorption peak clearly appears at 1420 cm -1 -1. After combining with the peak at 1640 cm -1 -1, they respectively correspond to the symmetric vibration and antisymmetric vibration peaks of the carboxylic acid group, indicating that DACA is incorporated into the structural system.

[0065] To verify the softness and ductility of the wound dressings prepared by the present invention, the mechanical properties of the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings and the bacterial cellulose (BC) wound dressings were tested. The mechanical property test includes three parts: ① Tensile test; ② Creep-recovery test; ③ Amplitude sweep and frequency sweep test:

[0066] 1) Tensile test: According to the ISO37-4 standard, a gel was cut into dumbbell-shaped samples with a 6×35 mm end and a 2×10 mm middle part using a punching machine. Axial tension was performed using a texture analyzer or a universal testing machine at a speed of 10 mm / min and a maximum load of 5 N, and the tensile distance and tensile stress (σ, MPa) were recorded. The tensile distance was converted into tensile strain (ε, %), and the Young's modulus (E, MPa) was defined as the slope of the linear part of the strain-stress curve.

[0067] 2) Creep-recovery test: The test was carried out using a rotational rheometer. An initial shear stress (τ0) of 10 Pa was applied to the sample for 171 s, and then the stress was removed to allow the sample to recover for 171 s. The creep compliance (J, Pa -1 ) of the sample as a function of time was recorded, and the creep parameter α (α = 1 represents a perfectly viscous body, α = 0 represents a perfectly elastic body, and when 0 < α < 1, it is a viscoelastic body) was calculated using the following formula.

[0068]

[0069] where J(t) is the creep compliance, γ(t) is the strain during the test, τ0 is the instantaneous stress applied, λ1 and λ2 represent the reciprocals of the gel elastic modulus of the sample during the creep and recovery stages, respectively, Γ(x) is the gamma function, t is the test time, and t m is the start time of the recovery stage, and H(x) is the Heaviside step function.

[0070] 3) Amplitude sweep and frequency sweep tests: The tests were carried out using a rotational rheometer. For the amplitude sweep, the shear strain amplitude was increased logarithmically from 0.01% to 100% at a constant frequency of 10 rad / s to perform an amplitude sweep on the sample, and the storage modulus (G') and loss modulus (G”) were recorded. For the frequency sweep, with a fixed amplitude of 0.01%, the frequency was increased from 0.4 rad / s to 400 rad / s, and the storage modulus (G') and loss modulus (G”) at different frequencies were recorded.

[0071] The tensile test, creep-recovery test results, and rheological amplitude sweep and frequency sweep results of the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings and bacterial cellulose (BC) wound dressings are shown respectively in Figure 3 、 Figure 4 as follows.

[0072] Figure 3Shows the mechanical properties of BC and BC-DACA(E) hydrogel wound dressings. Compared with BC hydrogel, the Young's modulus of BC-DACA(E) decreases and the fracture strain increases significantly, indicating that the BC-DACA(E) composite hydrogel becomes softer than the BC hydrogel; the tensile stress of BC-DACA4(E) increases significantly, indicating that the addition of DACA4 improves the ductility of the composite hydrogel while maintaining a certain stiffness. The α value of the BC-DACA4(E) composite hydrogel is the largest (0.82), indicating that its viscous property is higher than that of other composite hydrogels. Compared with BC (44.71%), the recovery rates of BC-DACA3 (39.77%) and BC-DACA4 (28.84%) composite hydrogels decrease, indicating that the addition of DACA makes the composite hydrogels more easily deformed under the action of external stress.

[0073] Figure 4 Shows the rheological properties of BC and BC-DACA(E) hydrogel wound dressings. The critical shear strain of BC is 1.38%, and the critical shear strains of other BC-DACA(E) generally show a decreasing trend (0.83 - 1.55%), indicating that the BC-DACA(E) composite hydrogels are softer and more easily deformed. The initial storage modulus and initial loss modulus of BC are 9.66 kPa and 1.34 kPa respectively. Among them, the initial storage modulus and initial loss modulus of BC-DACA4(E) both decrease, and the loss factor increases, indicating that the addition of DACA makes the composite hydrogel more inclined to viscous properties, that is, BC-DACA4(E) is the softest among all composite hydrogels, which is consistent with the results of tensile experiments and creep experiments.

[0074] In order to determine the antibacterial rate of the wound dressing prepared by the present invention, antibacterial rate tests were carried out on bacterial cellulose-aldehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings and bacterial cellulose (BC) wound dressings. The specific experimental process is as follows:

[0075] The three activated bacterial suspensions: Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) were diluted with sterile water to about 10 5 CFU / mL. 1 mL of the bacterial suspension was added to each well of a 24-well plate. BC and BC-DACA(E) composite hydrogels were cut into circles with a diameter of 1 cm and immersed in the bacterial suspension. Using the bacterial suspension as a control, the optical density (OD = 600 nm) value of the bacteria after 24 h of culture was measured using an ultraviolet spectrophotometer, and the antibacterial rate was calculated.

[0076] The antibacterial rate test results of bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings are as follows Figure 5 shown.

[0077] As Figure 5 can be seen, the BC-DACA3(E) composite hydrogel has the best antibacterial effect against Staphylococcus aureus, and BC-DACA4(E) has the best inhibitory effect on Escherichia coli and Pseudomonas aeruginosa. Other composite hydrogels have certain antibacterial properties and have a relatively prominent inhibitory effect on Pseudomonas aeruginosa. The BC-DACA(E) prepared by non-in-situ synthesis has certain antibacterial properties, and DACA with a high degree of oxidation has a better inhibitory effect on bacteria, which is caused by the carboxyl and aldehyde groups of DACA.

[0078] In order to determine the biocompatibility of the wound dressing prepared by the present invention, biocompatibility tests of bacterial cellulose-dialdehyde carboxyl starch composite hydrogel (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings, bacterial cellulose (BC) wound dressings and commercially available hydrocolloid dressings were carried out. The specific experimental process is as follows:

[0079] Before the experiment, the hydrogel samples need to be extracted. Under aseptic operation, BC and BC-DACA(E) composite hydrogels were respectively mixed into DMEM medium containing 10% serum by volume at a ratio of 0.2 g / mL and extracted at 37°C for 24 h. The blank control was DMEM medium containing 10% serum by volume without hydrogel extraction, and the extraction solution was diluted 0, 2, 4, 6, and 8 times. HaCaT cells were inoculated into a 96-well plate at a concentration of 10,000 cells / well, and DMEM medium containing 10% serum by volume was added and cultured at 37°C and 5% CO2 by volume to make the cells completely adhere. A circle of PBS was added to the edge of the 96-well plate to prevent edge effects. After the cells grew to nearly cover the bottom of the 96-well plate, the blank medium was aspirated, and the blank control, positive control, and bacterial cellulose dressing extraction solution were added. The medium was changed to 200 μL of medium containing different concentrations of extraction solution (prepared freshly), and all concentrations were made in 3 parallels. The treated plate was incubated at 5% CO2 and 37°C for 72 h. After the reaction, the original medium was replaced, 100 μL of CCK-8 containing 10% was added to each well, and it was placed in a cell culture incubator for dark incubation for 2 h, and the culture temperature was 37°C. Finally, the OD value (OD = 450 nm) was measured with a multifunctional microplate reader, and the cells in each group were observed under a microscope for the culture results.

[0080] Biocompatibility test results of bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings, bacterial cellulose (BC) wound dressings and commercially available hydrocolloid dressings are as Figure 6 shown.

[0081] As Figure 6 can be seen, only the extract of the commercially available hydrocolloid has a certain inhibitory effect on the proliferation of human immortalized epidermal cells (HaCaT) (81.38 - 89.60%), and the extracts of BC and BC-DACA(E) diluted at any multiple can promote the proliferation of HaCaT cells. The results show that BC-DACA(E) composite hydrogels all have excellent biocompatibility.

[0082] On the basis of the above embodiments, in order to further verify the wound healing ability of the wound dressings prepared by the present invention, bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings, bacterial cellulose (BC) wound dressings and commercially available hydrocolloid dressings were used for animal experiments. The specific experimental process is as follows:

[0083] 8-week-old Sprague-Dawley (SD) rats were used for the wound healing experiment. The rats were anesthetized with sodium pentobarbital (80 mg / kg) and their hair was shaved. Three full-thickness wound incisions with a diameter of about 1 cm were cut along the outer edge of the circle with surgical scissors. The wounds were respectively covered with bacterial cellulose and bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressings, and medical sterile gauze and commercially available hydrocolloid dressings were used as controls. Tegaderm material and a layer of bandage were used to fix the hydrogel wound dressings on the wounds. Samples were changed and bandaged every 2 days from 0 to 6 days and every 4 days from 6 to 14 days at the beginning of the experiment. The wound healing situation was observed with a microscope, and the proportion of the wound healing area was calculated. At 7 days and 14 days, the skin tissues of the rats' wounds and the surrounding areas were fixed with 4% paraformaldehyde, and the fixed skin tissue samples were embedded in paraffin to prepare pathological sections. The tissue sections were stained with hematoxylin-eosin (H&E) and Masson to evaluate the wound healing degree at different time points.

[0084] The test results of the animal wound healing ability of bacterial cellulose-dialdehyde carboxyl starch composite hydrogels (BC-DACA1(E), BC-DACA2(E), BC-DACA3(E), BC-DACA4(E)) wound dressings, bacterial cellulose (BC) wound dressings and commercially available hydrocolloid dressings are as Figure 7 shown.

[0085] It can be seen from Figure 7 that on the 4th day, except for the gauze group, new granulation tissue grew significantly in all wounds. In particular, for the BC-DACA(E) composite hydrogel, the wound healing area was significantly higher than that of other groups. On the 14th day, all wounds were basically healed, and there was no obvious large-area scab wound exposed. It can be found from the wound healing area diagram that the wound healing degree and rate of the gauze group were the slowest, and those of the BC-DACA group were significantly higher than those of other groups. After the 14th day, all wounds were basically healed, and the wound healing area of the BC-DACA4(E) group was significantly higher than that of other groups. There was no significant difference in the healing area between the commercially available hydrocolloid and other sample groups. Generally speaking, the sample groups showed good performance in terms of healing degree and rate. In particular, BC-DACA3(E) showed the fastest healing rate, and BC-DACA4(E) showed the highest healing degree, indicating that the BC-DACA(E) composite hydrogel has the performance of promoting wound healing. By observing the results of H&E tissue staining, it can be found that 7 days after the operation, the skin tissue structure of the wound surface in the gauze group and the hydrocolloid group was still severely abnormal; the epidermal layer was missing in the field of view, the dermis was exposed (as shown by the red arrow), a large area of collagen fibers in the dermis was missing (as shown by the blue arrow), a large number of inflammatory cells infiltrated the tissue (as shown by the yellow arrow), a large number of cells were necrotic, and the cell nuclei were fragmented and deeply stained (as shown by the black arrow). There was local tissue bleeding in the BC group (as shown by the brown arrow); there was also a lack of the epidermal layer in the BC-DACA3(E) and BC-DACA4(E) groups, accompanied by varying degrees of tissue inflammation. 14 days after the operation, the epidermal layer structure in the field of view of the BC-DACA4(E) group was complete, the spinous layer was thin and uniform, no obvious inflammatory reaction was seen, it was closest to the normal skin around the incision, and the thickness of the newly formed granulation tissue was significantly higher than that of other groups. By observing the results of Masson tissue staining, it can be known that the BC-DACA3(E) group and the BC-DACA4(E) group had the highest collagen content and were arranged relatively regularly.

[0086] In summary, compared with medical sterile gauze, commercially available hydrocolloid dressings, and bacterial cellulose hydrogel wound dressings, the bacterial cellulose-bisaldehyde carboxyl starch composite hydrogel wound dressing has faster and higher wound healing-promoting ability, ability to promote the growth of new granulation tissue, and ability to promote collagen production during long-term (2 weeks) use.

[0087] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A preparation method of a bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing, characterized in that, Comprising the following steps: S1: Starch is successively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl radical, sodium bromide, and sodium hypochlorite, and then dialyzed to obtain carboxymethyl starch; using carboxymethyl starch as a raw material, dialdehyde carboxymethyl starch is prepared by oxidation with sodium periodate with a molar concentration of 0.5M - 1.1M; S2: Inoculate the bacterial cellulose-producing bacterial strain into the fermentation medium, and statically culture it at 25 - 36 °C for 3 - 5 days to obtain a concentrated bacterial strain solution; the fermentation medium consists of the following components: glucose 20 - 25 g / L, disodium hydrogen phosphate anhydrous 2.0 - 3.0 g / L, citric acid monohydrate 1.0 - 2.0 g / L, yeast extract 5 - 10 g / L, peptone 5 - 10 g / L, and the balance is deionized water, and the pH is adjusted to 4.0 - 5.0 with 1 mol / L hydrochloric acid solution; S3: Inoculate the obtained concentrated bacterial strain solution into the fermentation medium at a ratio of 8 - 15 (v / v)%, and statically culture it in an incubator at 25 - 36 °C for 5 - 9 days to obtain bacterial cellulose. After soaking and washing it with pure water for multiple times until the color is close to white and the pH is neutral, a bacterial cellulose hydrogel is obtained; the fermentation medium consists of the following components: glucose 20 - 25 g / L, disodium hydrogen phosphate anhydrous 2.0 - 3.0 g / L, citric acid monohydrate 1.0 - 2.0 g / L, yeast extract 5 - 10 g / L, peptone 5 - 10 g / L, and the balance is deionized water, and the pH is adjusted to 4.0 - 5.0 with 1 mol / L hydrochloric acid solution; S4: Soak the pure bacterial cellulose in a solution containing polyvinyl alcohol and dialdehyde carboxymethyl starch with a mass concentration of 0.5 - 2% for 3 - 5 days to obtain a bacterial cellulose-dialdehyde carboxymethyl starch composite hydrogel wound dressing.

2. The preparation method of the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing according to claim 1, wherein, The bacterial cellulose-producing bacterial strain used in the step S1 is one of the genera Acetobacter, Gluconacetobacter, and Komagataeibacter xylinus.

3. The preparation method of the bacterial cellulose-dialdehyde carboxyl starch composite hydrogel wound dressing according to claim 1, characterized in that, In the step S2, the total number of colonies of the bacterial cellulose-producing bacteria in each milliliter of the concentrated bacterial solution is 10 2 -10 4 CFU.

4. A bacterial cellulose-dialdehyde carboxymethyl starch composite hydrogel wound dressing prepared by the method according to any one of claims 1 - 3.

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