Medical bacterial cellulose dressing with high water retention capacity and preparation method and application thereof

Abstract

The application provides a medical bacterial cellulose dressing with high water retention capacity and a preparation method and application thereof, and belongs to the technical field of medical supplies. The medical bacterial cellulose dressing with high water retention capacity is prepared by reacting a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of tetramethylphosphonium chloride, and the water retention time of the dressing is not less than 2000 min. The prepared medical bacterial cellulose dressing with high water retention capacity has great advantages in actual medical application. For burn and scald patients, traditional dressings are often difficult to keep the wound moist for a long time, and if they are removed after drying, secondary wound damage will be caused. However, the dressing can provide a persistent and stable moist environment for the wound and accelerate the repair and regeneration of damaged skin cells due to its excellent water retention capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical supplies, in particular to a medical bacterial cellulose dressing with high water retention capacity and a preparation method and application thereof. BACKGROUND

[0002] Bacterial cellulose (BC) is a natural polysaccharide synthesized by microorganisms such as acetic acid bacteria. Its unique physical and chemical properties make it an ideal material for modern wound dressings. Bacterial cellulose not only has good biocompatibility and excellent water absorption, but also has mechanical strength and antibacterial properties, playing multiple roles in the wound healing process. Unlike traditional plant-derived cellulose, bacterial cellulose is synthesized by bacteria, has a finer structure and higher purity. The fiber diameter of bacterial cellulose is generally between 20-100 nanometers, has a large specific surface area and extremely high water affinity.

[0003] The application of bacterial cellulose in wound dressings is mainly reflected in its physical and biological properties that promote the wound healing process. The mechanism of action can be summarized as follows: (1) Maintaining a moist environment: One of the important factors for wound healing is to maintain a proper moist environment. Bacterial cellulose has very high hydrophilicity, can absorb and hold a large amount of water, thus providing an ideal moist healing environment for the wound. A moist environment helps to accelerate cell migration and regeneration, preventing the wound from drying out and scabbing, thus promoting the healing process. Bacterial cellulose has a strong water-holding capacity, compared to traditional wound dressings, it can effectively avoid complications caused by dry wounds. Its water-holding and high water-absorbing properties also help to reduce the accumulation of exudates and prevent bacterial infection. (2) Promoting cell regeneration and supporting tissue repair: The three-dimensional network structure of bacterial cellulose provides support for cell growth, it can simulate the skin matrix and provide support for new cells. Studies have found that bacterial cellulose can promote the migration, proliferation and differentiation of skin epithelial cells and fibroblasts, accelerating wound healing. In addition, the microstructure of bacterial cellulose can reduce fibrosis and scar formation during wound healing. The high surface area and fiber structure of bacterial cellulose provide a broad adhesion surface for cells, promoting cell regeneration and migration. Its good mechanical strength allows it to withstand higher external forces, avoiding damage to the dressing when the wound is subjected to external pressure, thus effectively protecting the wound. (3) Antibacterial effect: The surface of bacterial cellulose has a wealth of hydrophilic groups, which help to adhere antibacterial components such as silver ions and antibiotics. The structure of bacterial cellulose also provides a good carrier for antibacterial components, allowing for sustained release of these antibacterial substances, thus reducing the risk of infection. In addition, bacterial cellulose can form a protective film layer when it comes into contact with the wound, isolating pathogenic bacteria from entering the wound. (4) Reducing scar formation: Bacterial cellulose helps to reduce the formation of scars by maintaining a moist environment, promoting cell regeneration and reducing excessive fibrosis. In some studies, wounds treated with bacterial cellulose dressing have fewer scars and smoother skin recovery. The mechanism of action is to promote the normal repair process of skin cells and avoid abnormal collagen deposition. Compared to traditional dry dressings, the moist environment provided by bacterial cellulose helps to reduce the proliferation of scars and facilitate smoother wound healing.

[0004] Bacterial cellulose has great potential in the field of wound dressings due to its excellent physical, chemical and biological properties. It can provide a moist environment for wounds, promote cell regeneration, reduce scar formation, and has antibacterial effects. With further research, the application of bacterial cellulose in wound care will further expand and become a new material for more extensive treatment of wounds.

[0005] The patent CN202310854206.X discloses a bacterial cellulose nanocrystal reinforced collagen-based material and its preparation method and application. EDC and NHS are used as cross-linking agent and catalyst to cross-link bacterial cellulose nanocrystal molecules and collagen molecules, and finally form a collagen-based material with improved mechanical properties. The patent introduces bacterial cellulose nanocrystal solution into the inside of the collagen-based material, which can improve the mechanical properties of the collagen-based material, but the effect of improving the water retention of the bacterial cellulose dressing is not obvious. SUMMARY

[0006] The present application is made in view of the short water retention time of the bacterial cellulose dressing in the prior art. The purpose is to provide a medical bacterial cellulose dressing with high water retention capacity and its preparation method and application, which can prolong the water retention time of the dressing to more than 2000 min, effectively reduce the loss of water, avoid excessive drying of the dressing, reduce the cost and operation of dressing replacement, and has significant clinical value.

[0007] Specifically, the first aspect of the present application provides a medical bacterial cellulose dressing with high water retention capacity, which is prepared by reacting a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of tetramethylphosphonium chloride. The water retention time of the dressing is not less than 2000 min.

[0008] Further, the dressing comprises:

[0009] a porous layer composed of a first bacterial cellulose skeleton, the internal pores and surface of the first bacterial cellulose skeleton being combined with sodium hyaluronate and cationic antibacterial polysaccharide through covalent bonds;

[0010] a dense layer composed of a second bacterial cellulose skeleton, the surface of the second bacterial cellulose skeleton being combined with sodium hyaluronate through covalent bonds. Further, the cationic antibacterial polysaccharide is quaternized chitosan.

[0011] Further, the density of sodium hyaluronate in the porous layer is higher than that in the dense layer.

[0012] Further, in the porous layer, the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is 1:1-10:1.

[0013] The second aspect of the present application provides a preparation method of a medical bacterial cellulose dressing, which is used to prepare the medical bacterial cellulose dressing with high water retention capacity. The preparation method comprises the following steps:

[0014] a. providing a first bacterial cellulose membrane and subjecting it to freeze-drying treatment to form a porous bacterial cellulose scaffold;

[0015] b. immersing the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and cationic antibacterial polysaccharide;

[0016] c. providing a second bacterial cellulose membrane, immersing it in a second solution containing sodium hyaluronate, wherein the concentration of sodium hyaluronate in the first solution is higher than that in the second solution;

[0017] d. superimposing the porous scaffold treated in step b and the second bacterial cellulose membrane treated in step c, placing them in a solution containing tetramethylammonium chloride, and allowing the bacterial cellulose membrane to crosslink with sodium hyaluronate, thereby obtaining a medical bacterial cellulose dressing.

[0018] Further, the freeze-drying treatment in step a comprises pre-freezing at a temperature lower than -30℃, and then freeze-drying under a temperature of -10℃ to -50℃ and a vacuum degree lower than 50 Pa.

[0019] Further, in step b, vacuum negative pressure assisted immersion is used to allow the first solution to fully penetrate into the internal pores of the porous scaffold.

[0020] Further, the crosslinking reaction in step d is carried out at 20-30℃ for 12-36h.

[0021] Further, the method further comprises the steps of cleaning, sterilizing and aseptically sealing the obtained dressing.

[0022] Further, the sterilization is Co60-γ ray irradiation sterilization.

[0023] The third aspect of the present application provides an application of the medical bacterial cellulose dressing with high water retention capacity, which is applied to medical devices for treating burns, scalds, chronic ulcers or wounds.

[0024] The present application has the following beneficial effects:

[0025] (1) The dressing of the present application is prepared by reacting bacterial cellulose membrane with sodium hyaluronate solution in the presence of crosslinking agent tetrahydroxymethyl phosphonium chloride. Sodium hyaluronate (HA) itself has strong water retention performance, and it can absorb 1000 times of its own weight of water. It is widely used in skin care and medical fields for moisturizing. When the bacterial cellulose membrane (BC) is immersed in the sodium hyaluronate solution, sodium hyaluronate molecules will adhere to the three-dimensional network structure of bacterial cellulose. Tetrahydroxymethyl phosphonium chloride (THPC) as a crosslinking agent can react with bacterial cellulose and sodium hyaluronate molecules to form chemical crosslinking bonds. These crosslinking bonds make sodium hyaluronate more firmly combined on the bacterial cellulose membrane, preventing sodium hyaluronate from falling off or losing from the membrane during use. At the same time, the crosslinking structure can also change the microstructure of the bacterial cellulose membrane, making it form a more dense network. This dense network can better lock in water and reduce the evaporation rate of water.

[0026] In a wound environment, the composite of bacterial cellulose membrane and sodium hyaluronate can provide a moist environment for the wound on the one hand, and the slow release of the large amount of water absorbed by sodium hyaluronate maintains the humidity of the wound. On the other hand, the presence of crosslinking structure enables the dressing to more effectively retain the liquid inside when absorbing wound exudate, rather than quickly losing it. Moreover, this crosslinking structure can also affect the interaction between water molecules and dressing molecules, such as increasing the hydrogen bonding between water molecules and dressing molecules, etc., further improving the binding ability of the dressing to water, thereby significantly prolonging the water retention time of the dressing, making its water retention time not less than 2000 min, even reaching 2000-3000 min, creating more favorable conditions for wound healing.

[0027] (2) The medical bacterial cellulose dressing with high water retention capacity prepared by the present application has shown great advantages in actual medical applications. For burn and scald patients, traditional dressings often have difficulty in maintaining the moisture of the wound for a long time, while the dressing with excellent water retention capacity can provide a persistent and stable moist environment for the wound, accelerating the repair and regeneration of damaged skin cells. For example, in the treatment of patients with large area burns, the dressing can effectively reduce the frequency of dressing change, and reduce the pain and infection risk of patients caused by dry and adhered dressing. For patients with chronic ulcers, a poor wound environment is an important reason for long-term non-healing of the wound. The high water retention property of the dressing can maintain the cleanliness and moisture of the wound, promote the growth of granulation tissue, and accelerate the healing of the ulcer surface. In the treatment of trauma, especially open trauma, it can prevent the wound from drying and scabbing, provide a good microenvironment for wound healing, and reduce scar formation. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is described and explained below in connection with embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and not to limit the present application. Based on the embodiments provided by the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0029] Obviously, the following description is only some examples or embodiments of the present application, and for those of ordinary skill in the art, the present application can also be applied to other similar situations without creative labor. In addition, it can be understood that although the efforts made in this development process can be complex and lengthy, for those of ordinary skill in the art related to the content disclosed in the present application, some design, manufacture or production changes based on the technology disclosed in the present application are only routine technical means and should not be understood as insufficient disclosure of the present application.

[0030] The first aspect of the present application provides a medical bacterial cellulose dressing with high water retention capacity, which is prepared by reacting a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of tetramethylammonium chloride phosphate, and the water retention time of the dressing is not less than 2000 min.

[0031] The dressing of the present application is prepared by reacting a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of tetramethylammonium chloride phosphate, wherein sodium hyaluronate (HA) itself has strong water retention performance, it can absorb 1000 times its own weight of water, and is widely used in skin care and medical fields for moisturizing. When the bacterial cellulose membrane (BC) is immersed in the sodium hyaluronate solution, sodium hyaluronate molecules will adhere to the three-dimensional network structure of bacterial cellulose. And tetramethylammonium chloride phosphate (THPC) as a crosslinking agent, it can react with bacterial cellulose and sodium hyaluronate molecules to form chemical crosslinking bonds. These crosslinking bonds make sodium hyaluronate more firmly combined on the bacterial cellulose membrane, preventing sodium hyaluronate from falling off or losing from the membrane during use. At the same time, the crosslinked structure can also change the microstructure of the bacterial cellulose membrane, making it form a more dense network. This dense network can better lock water and reduce the evaporation rate of water.

[0032] In the wound environment, the composite of bacterial cellulose membrane and sodium hyaluronate can provide a moist environment for the wound on one hand, and the slow release of the large amount of water absorbed by sodium hyaluronate can maintain the humidity of the wound; on the other hand, the presence of the cross-linked structure enables the dressing to more effectively retain the liquid inside when absorbing the wound exudate, instead of quickly losing. Moreover, the cross-linked structure can also affect the interaction between water molecules and dressing molecules, such as increasing the hydrogen bonding between water molecules and dressing molecules, etc., further improving the water retention capacity of the dressing, thereby significantly prolonging the water retention time of the dressing, and making the water retention time not less than 2000 min, even reaching 2000-3000 min, to create more favorable conditions for wound healing.

[0033] In the present embodiment, the dressing comprises:

[0034] a porous layer composed of a first bacterial cellulose skeleton, the internal pores and surface of the first bacterial cellulose skeleton being combined with sodium hyaluronate and cationic antibacterial polysaccharide through covalent bonds;

[0035] a dense layer composed of a second bacterial cellulose skeleton, the surface of the second bacterial cellulose skeleton being combined with sodium hyaluronate through covalent bonds.

[0036] The porous layer of the present application is on the side of the contact environment, contains sodium hyaluronate (HA) and cationic antibacterial polysaccharide (quaternized chitosan (HACC)), and has the functions of super liquid absorption, water retention (porous structure) and active antibacterial function, can quickly absorb wound exudate, and use its antibacterial property to inhibit the breeding of bacteria at the source, and prevent the dressing itself from becoming a source of infection.

[0037] The dense layer is on the side of the contact wound, and is used for keeping dense, moist and anti-adhesion, wherein the low concentration of sodium hyaluronate provides basic moisturizing, and the dense nanofiber structure effectively isolates the wound surface, avoids the growth of new granulation tissue into the dressing, and causes no pain and secondary damage when replaced.

[0038] In the present application, the internal pores and surface of the first bacterial cellulose skeleton of the porous layer are combined with sodium hyaluronate and cationic antibacterial polysaccharide through covalent bonds, so that the dressing has long-acting antibacterial capacity (quaternized chitosan is introduced) while maintaining excellent water retention, and due to the presence of different electric molecules, it can be more conducive to cell adhesion and growth, and its effect is far beyond the simple superposition of "water retention + antibacterial".

[0039] The mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide in the porous layer is 1-10:1. This specific mass ratio can fully exert the antibacterial effect of the cationic antibacterial polysaccharide while ensuring the water retention performance of the dressing. When the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is within this range, on the one hand, sodium hyaluronate can continue to exert its strong water retention capacity to provide a persistent moist environment for the wound; on the other hand, the cationic antibacterial polysaccharide can effectively inhibit the growth and reproduction of bacteria, reducing the risk of wound infection.

[0040] The cationic antibacterial polysaccharide is quaternized chitosan (HACC). Quaternized chitosan has good biocompatibility and broad-spectrum antibacterial properties. Quaternized chitosan molecules contain positively charged quaternary ammonium groups, which can interact with negatively charged components on the surface of bacteria (such as phospholipids on the bacterial cell membrane). This electrostatic attraction allows quaternized chitosan to bind tightly to the surface of bacteria, disrupting the integrity of the bacterial cell membrane and causing bacterial contents to leak, thereby achieving the purpose of inhibiting and killing bacteria. At the same time, quaternized chitosan has good biocompatibility, meaning it does not cause significant immune or toxic reactions when in contact with human tissues. During the wound healing process, it can avoid causing additional irritation and damage to normal tissues around the wound, facilitating normal repair and healing of the wound. In addition, quaternized chitosan can be covalently bonded to the first bacterial cellulose framework, thanks to its active groups in the molecular structure. Under the action of the crosslinking agent tetramethylammonium chloride phosphorus, the active groups of quaternized chitosan can react with bacterial cellulose and sodium hyaluronate molecules to form stable chemical cross-linking bonds. This covalent bonding method ensures the stability and durability of quaternized chitosan in the dressing, allowing it to continuously exert its antibacterial effect throughout the wound healing process.

[0041] In actual medical scenarios, the antibacterial properties of quaternized chitosan play an important role in different types of wounds, such as burns, scalds, chronic ulcers, or trauma. For example, in burn and scald wounds, where the skin barrier is damaged and bacteria can easily invade, quaternized chitosan can inhibit bacterial growth and reduce the risk of infection. In chronic ulcer and trauma wounds, it can help maintain a clean environment for the wound and promote wound healing. Moreover, compared with traditional antibacterial drugs, quaternized chitosan is less likely to develop drug resistance, making it have a broader application prospect in long-term wound treatment.

[0042] Embodiments of the second aspect of the application provide a preparation method of a medical bacterial cellulose dressing, for preparing the medical bacterial cellulose dressing with high water retention capacity, the preparation method comprising the following steps:

[0043] a. providing a first bacterial cellulose membrane and subjecting it to freeze-drying treatment to form a porous bacterial cellulose scaffold;

[0044] b. immersing the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and cationic antibacterial polysaccharide;

[0045] c. providing a second bacterial cellulose membrane and immersing it in a second solution containing sodium hyaluronate, wherein the concentration of sodium hyaluronate in the first solution is higher than that in the second solution;

[0046] d. superimposing the porous scaffold treated in step b and the second bacterial cellulose membrane treated in step c, and placing them in a solution containing tetramethylammonium chloride, so that the bacterial cellulose membranes and sodium hyaluronate are cross-linked to obtain a medical bacterial cellulose dressing.

[0047] In this embodiment, a multifunctional porous hydrophilic layer is prepared in step a: after a piece of pure bacterial cellulose (BC) membrane is washed with deionized water to neutral, it is subjected to pre-freeze-drying treatment (-40°C for 2h, then freeze-drying at -20°C under a vacuum degree of <10 Pa for 6h) to form a BC scaffold with a porous network structure. The porosity of the porous network structure of the BC scaffold is greater than 85%, and the average pore size is 1-500μm. This step can make the bacterial cellulose membrane form a porous structure suitable for absorbing and locking exudate. The specific temperature and time setting of the pre-freeze-drying treatment, as well as the requirement of the vacuum degree, helps to accurately control the formation of the porous structure, ensures that the porosity and pore size are within the appropriate range, and provides a good foundation for the subsequent combination of sodium hyaluronate and cationic antibacterial polysaccharide.

[0048] In step b, the sodium hyaluronate solution is prepared: take sodium hyaluronate powder and quaternized chitosan, add distilled water, maintain the temperature at 10°C, stand for 39min, make the HA powder fully dissolve and swell, continue to add appropriate amount of distilled water, stir at 10°C for 1h until completely dissolved, add distilled water to 1L, respectively prepare a mixed solution containing high concentration of sodium hyaluronate (5-15g / L) and quaternized chitosan (1-3g / L). Immerse the porous BC scaffold obtained in step a in the mixed solution, and immerse it under a vacuum negative pressure of -0.08MPa to -0.1MPa for 10min, so that the solution can fully penetrate into the internal pores of the porous scaffold. This way of vacuum negative pressure immersion can effectively remove the air in the pores of the scaffold, so that the solution can better fill in, and ensure that sodium hyaluronate and quaternized chitosan can be evenly distributed in various parts of the porous scaffold. During the immersion process, sodium hyaluronate and quaternized chitosan will be combined with the first bacterial cellulose skeleton of the porous scaffold through physical adsorption and preliminary chemical action, laying a foundation for the subsequent cross-linking reaction.

[0049] In step c, the moisturizing dense layer is prepared: take another piece of pure BC membrane (not lyophilized, keep the natural dense nanofiber structure), immerse it in a low concentration sodium hyaluronate (0.5-2g / L) solution.

[0050] In step d, the superimposed double-layer structure is placed in a reaction container, a solution containing the cross-linking agent tetramethyl phosphonium chloride is added, a buffer solution with pH = 5.5 is added, the composite membrane is completely immersed, and the reaction is carried out at 20-30℃ for 12-36h. In this process, tetramethyl phosphonium chloride forms chemical cross-linking bonds with bacterial cellulose, sodium hyaluronate and cationic antibacterial polysaccharide molecules. Among them, the carboxyl group of sodium hyaluronate reacts with the hydroxyl group of bacterial cellulose, the amino group of quaternary ammonium chitosan and the hydroxyl group of bacterial cellulose, and the carboxyl group of sodium hyaluronate and the amino group of quaternary ammonium chitosan, forming a strong "BC-HA-HACC" ternary cross-linking network, firmly locked in the pores. For the porous layer, tetramethyl phosphonium chloride promotes the formation of stable covalent cross-linking between the first bacterial cellulose skeleton and sodium hyaluronate and cationic antibacterial polysaccharide, further enhancing the stability of the porous layer structure, making sodium hyaluronate and cationic antibacterial polysaccharide more firmly combined on the first bacterial cellulose skeleton, not only ensuring the durability of water retention and antibacterial properties, but also making the pore structure of the porous layer more regular, which is conducive to the rapid absorption and locking of exudate.

[0051] For the dense layer, the carboxyl group of sodium hyaluronate HA reacts with the hydroxyl group of bacterial cellulose BC to form a "BC-HA" two-dimensional cross-linked layer. Tetramethyl phosphonium chloride forms covalent cross-linking between the second bacterial cellulose skeleton and sodium hyaluronate, enhancing the binding force between sodium hyaluronate and the second bacterial cellulose skeleton, so that the dense layer can better play the role of isolating the wound and preventing adhesion.

[0052] At the same time, when the wet, solution pretreated porous layer and dense layer are superimposed, at the interface between the two, the first bacterial cellulose membrane, the second bacterial cellulose membrane, and the sodium hyaluronate and quaternary ammonium chitosan molecules carried by them will be in close contact, forming molecular level entanglement and contact points. Tetramethyl phosphonium chloride crosslinking agent "activates" the carboxyl group (-COOH), making it more easily react with the hydroxyl group (-OH) or the amino group (-NH2), thereby covalently connecting the two layers to form a complete dressing structure. This cross-linking reaction is carried out at a mild temperature of 20-25℃ for 12-36h, which can ensure that the reaction proceeds sufficiently.

[0053] The concentration of tetramethyl phosphonium chloride is 0.3%-0.8%, preferably 0.5%-0.6%. If the concentration of tetramethyl phosphonium chloride is too low, the cross-linking reaction is not sufficient, and the combination of sodium hyaluronate and bacterial cellulose membrane is not firm enough; if the concentration is too high, it may lead to excessive cross-linking, affecting the performance of the dressing.

[0054] After the reaction, the composite film needs to be removed and cleaned to remove unreacted substances. Deionized water, distilled water or normal saline can be used for cleaning, and the cleaning time is usually 3-5 times, 15 minutes each time, until no unreacted substances are detected in the cleaning solution. The bacterial cellulose composite dressing obtained after cleaning also needs to be sterilized and aseptically sealed to ensure the safety and effectiveness of the dressing. Sterilization uses Co60-γ ray irradiation sterilization, which can effectively kill various microorganisms and has no significant effect on the performance of the dressing. Aseptic packaging can prevent the dressing from being contaminated during storage and transportation.

[0055] The third aspect of the application provides a medical bacterial cellulose dressing with high water retention capacity. The medical bacterial cellulose dressing with high water retention capacity is used in medical devices for treating burns, scalds, chronic ulcers or wounds. This dressing has significant advantages in actual medical applications. In the treatment of burns and scalds, patients with large area burns have severe skin damage and excessive exudate, so the dressing needs to be changed frequently. The dressing of the application has a long water retention time, which can reduce the frequency of dressing change and reduce the pain and infection risk of patients. At the same time, the continuous provision of a moist environment is beneficial to the repair and regeneration of damaged skin cells and promotes wound healing.

[0056] For patients with chronic ulcers, long-term non-healing of the wound is often due to poor wound environment, lack of sufficient moisture and nutrients. The dressing of the application can maintain the cleanliness and moisture of the wound, promote the growth of granulation tissue and accelerate the healing of the ulcer surface. In the treatment of wounds, especially open wounds, the wound is prone to dry and scab, affecting healing. The dressing can prevent the wound from drying and scabbing, providing a good microenvironment for wound healing and reducing scar formation.

[0057] In summary, the medical bacterial cellulose dressing with high water retention capacity and its preparation method and application have significant advantages and clinical value. By reasonably controlling the concentration of sodium hyaluronate solution, the concentration of tetramethylphosphonium chloride, reaction temperature and time and other factors, a dressing with long water retention time and excellent performance can be prepared, providing an effective solution for the treatment of patients with burns, scalds, chronic ulcers and wounds.

[0058] Embodiment

[0059] The present disclosure is more specifically described by the following examples, which are intended merely to illustrate the disclosure, since various modifications and changes thereto will become apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported herein are based on weight. Unless otherwise stated, all reagents used in the examples are available commercially or are synthesized according to conventional methods and used as received without further purification. Unless otherwise stated, all instruments used in the examples are available commercially.

[0060] Example 1

[0061] A method for preparing a medical bacterial cellulose dressing, comprising the following steps:

[0062] a: a piece of pure bacterial cellulose film is washed to neutral with deionized water, then pre-frozen and dried, frozen at -40℃ for 2h, and freeze-dried at -20℃ under a vacuum degree of <10 Pa for 6h to form a porous bacterial cellulose scaffold;

[0063] b: the porous scaffold obtained in step a is immersed in a first solution containing sodium hyaluronate and quaternized chitosan at -0.09 MPa for 10 min to ensure that the mixed solution fully enters the deep pores; wherein the concentration of sodium hyaluronate is 5 g / L, and the concentration of quaternized chitosan is 1 g / L;

[0064] c: another piece of pure BC film (not freeze-dried, maintaining the natural dense nanofiber structure) is immersed in a 0.5 g / L sodium hyaluronate solution;

[0065] d: the porous scaffold treated in step b is stacked with the second bacterial cellulose film treated in step c, and the stacked double-layer structure is placed in a solution containing tetramethylammonium chloride at a concentration of 0.5% for crosslinking reaction at 25℃ for 24h to obtain a composite film; the composite film sample is washed with distilled water for 3 times, 15 min each time, and the obtained bacterial cellulose composite film sample is sterilized by Co60-γ irradiation and then aseptically sealed and stored at 5℃.

[0066] Example 2

[0067] This example is basically the same as Example 1, except that the concentration of the sodium hyaluronate solution in step b is 8 g / L, and the concentration of the sodium hyaluronate solution in step c is 1 g / L.

[0068] Example 3

[0069] This example is substantially the same as Example 1, except that the concentration of the sodium hyaluronate solution in step b is 12 g / L and the concentration of the sodium hyaluronate solution in step c is 1.5 g / L.

[0070] Comparative Example 1

[0071] A double-layered bacterial cellulose film was used without any cross-linking reaction treatment.

[0072] Comparative Example 2

[0073] This comparative example is substantially the same as Example 1, except that the tetramethylammonium chloride in step d is replaced with EDC / NHS (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide) at a molar ratio of 4:1.

[0074] Comparative Example 3

[0075] This comparative example is substantially the same as Example 2, except that the tetramethylammonium chloride in step d is replaced with EDC / NHS at a molar ratio of 4:1.

[0076] Comparative Example 4

[0077] This comparative example is substantially the same as Example 3, except that the tetramethylammonium chloride in step d is replaced with EDC / NHS at a molar ratio of 4:1.

[0078] Experimental Example

[0079] Preparation of simulated body fluid: NaCl 8.298 g, CaCl20.275 g, and distilled water to 1 L.

[0080] 1. Dehydration time comparison test

[0081] The composite film samples in Examples 1-3 and Comparative Examples 1-4 were carefully cut into 10x10 cm pieces with scissors, three parallel tests were performed for each sample, the surface part of free water was removed with a glass rod, the pre-prepared simulated body fluid was taken out, a constant temperature water bath was used to preheat to 37℃, 40 times the weight of the sample of simulated body fluid was weighed, poured into a clean dish, the sample was placed in the dish and placed in a dry box, the temperature was set to (37±1)℃, and soaked for 30 min; the sample was taken out, the corner of the film piece was carefully clamped with tweezers, a certain distance from the table, and the excess water was naturally dropped, suspended for 30 s, and weighed as M1; each sample film was placed on a dry dish and placed in a hot air drying oven, the temperature was set to (37±1)℃, and the relative humidity RH was less than 20%, the sample was taken out and weighed every 1 h until the weight was constant, and the corresponding time and weight Mt were recorded. The dehydration rate of the sample was calculated as: dehydration rate = [(M1-Mt) / M1]x100%. According to the dehydration time and the corresponding weight, the water loss percentage was calculated, and the results are shown in Table 1.

[0082] Table 1 Dehydration time comparison test results

[0083]

[0084] From Table 1, when the concentration of the sodium hyaluronate solution in step b is 8 g / L and the concentration of the sodium hyaluronate solution in step c is 1 g / L, the BC composite film (BC-THPC crosslinking-HA) formed by crosslinking with tetramethylammonium phosphate chloride (THPC) as the crosslinking agent has the longest dehydration time, reaching 3000 min, which is 6.25 times that of the untreated BC film. Further increasing the concentration of the sodium hyaluronate solution in step b to 12 g / L and the concentration of the sodium hyaluronate solution in step c to 1.5 g / L does not prolong the dehydration time. In addition, THPC as a crosslinking agent greatly outperforms EDC / NHS crosslinking agent in prolonging the dehydration time of the BC film.

[0085] The reason may be that the crosslinking structure formed by tetramethylammonium phosphate chloride (THPC) is more stable and reasonable. When THPC reacts with sodium hyaluronate and bacterial cellulose film, it can accurately form crosslinking bonds at appropriate positions and construct a uniform and dense network structure. This structure not only effectively wraps a large number of water molecules, but also ensures the slow release of water molecules under appropriate conditions, thereby prolonging the dehydration time. The crosslinking structure formed by EDC / NHS may not be stable enough, and the crosslinking points are not uniformly distributed, resulting in defects in the network structure, which cannot effectively lock water molecules like the structure formed by THPC.

[0086] In addition, from the perspective of chemical properties, THPC has unique chemical activity. Its reaction process with sodium hyaluronate and bacterial cellulose membrane is more gentle and controllable, and can achieve good cross-linking effect without destroying the original structure and performance of the molecules. In contrast, EDC / NHS may cause some side reactions in the reaction process, causing some damage to the structure of sodium hyaluronate and bacterial cellulose membrane, thereby affecting the water retention performance and dehydration time of the dressing.

[0087] Furthermore, the structure formed by THPC cross-linking has stronger affinity for water molecules. There may be some special chemical groups on its surface that can form more hydrogen bonds or other interactions with water molecules, increasing the binding force of water molecules with the dressing, making it more difficult for water to evaporate and lose. The structure formed by EDC / NHS cross-linking may lack this strong affinity for water molecules, resulting in easier loss of water from the dressing.

[0088] 2.30min water loss rate test

[0089] First, the composite film samples in Examples 1-3 and Comparative Examples 1-4 were cut into 10*10 cm size with scissors, and three parallel samples were made for each sample. The surface of the film was lightly pressed with a glass rod to remove the free water on the surface. The pre-prepared simulated body fluid was taken out and preheated to 37°C using a constant temperature water bath. 40 times the weight of the sample was weighed SBF solution, poured into a clean dish, and the sample was placed in the dish. It was moved into a drying oven, and the temperature was set to (37±1) °C. It was fully soaked for 30 min. The sample was taken out, and the corner of the film was carefully clamped with tweezers at a distance from the table, allowing the excess water to naturally drip. It was suspended for 30 s, and the weight was M1. The sample film was placed on a dry dish and placed in a hot air drying oven, set to (37±1) °C, and the relative humidity RH was less than 20%. After 30 min, the dish was taken out and weighed, and the weight was recorded as M2. The 30 min water loss rate was calculated according to the following method: 30 min water loss rate = [(M1-M2) / M1]x100%. The results are shown in Table 2.

[0090]

[0091] From Table 2, it can be seen that when the concentration of the sodium hyaluronate solution in step b is 8 g / L and the concentration of the sodium hyaluronate solution in step c is 1 g / L, the 30 min water loss rate of the BC-THPC cross-linked-HA composite material is the lowest. Compared with EDC / NHS, the cross-linking agent THPC can make the composite material less likely to lose water. When the concentration of the sodium hyaluronate solution in step b is 12 g / L and the concentration of the sodium hyaluronate solution in step c is 1.5 g / L, the 30 min water loss rate cannot be further reduced. The reason can be that when the HA concentration reaches a certain value, the three-dimensional network structure of the bacterial cellulose membrane has been basically saturated with sodium hyaluronate molecules. Continuing to increase the HA concentration, the excess sodium hyaluronate molecules cannot effectively adhere to the bacterial cellulose membrane, and thus cannot further enhance the water retention capacity of the dressing. From the perspective of molecular interaction, there is a certain interaction between sodium hyaluronate molecules. When the concentration is too high, these molecules can aggregate or entangle, forming some structures that are not conducive to water retention. This makes water molecules more likely to be lost in such a high-concentration system rather than being stably bound inside the dressing.

[0092] In addition, the THPC cross-linked structure plays a key role in water retention within a certain range. When the concentration of the sodium hyaluronate solution in step b is 8 g / L and the concentration of the sodium hyaluronate solution in step c is 1 g / L, the cross-linked network formed by THPC, sodium hyaluronate and bacterial cellulose membrane reaches a relatively ideal state. This network can effectively wrap water and also ensure the slow release of water molecules. When the HA concentration is too high, it can interfere with the formation of this cross-linked network, resulting in defects in the network structure and thus reducing the binding capacity of water.

[0093] In contrast, the structure formed by the EDC / NHS cross-linking agent is not stable and ideal itself. Even at different HA concentrations, its water retention capacity is not as good as that of the THPC cross-linking agent. The side reactions that can occur during the EDC / NHS cross-linking process can damage the original structure of the bacterial cellulose membrane and sodium hyaluronate, making water more likely to escape from these damaged structures.

[0094] In practical applications, selecting appropriate HA concentration and cross-linking agent is crucial for preparing medical bacterial cellulose dressings with high water retention capacity. Based on the test results of dehydration time and 30 min water loss rate, it is a relatively ideal combination to use 8 g / L of sodium hyaluronate solution in step b and 1 g / L of sodium hyaluronate solution in step c and use THPC as the cross-linking agent.

[0095] 3. Saturated water content determination

[0096] According to the most contact dressing can use 30 min to achieve the absorption equilibrium conditions proposed in YYT 0471-2004 contact wound dressing test method, using constant weight method to determine the saturated wet film moisture content, the results in the form of saturated moisture content. The sample film of example 2, comparative example 1 and comparative example 3 are carefully cut into square of 5cm*5cm by scissors, three samples in each group. The solution used for sample saturation is to use the prepared simulated body fluid (SBF), which needs to be heated to 37℃ in advance in constant temperature water bath. The cut film is put into 200ml beaker, 150ml, 37℃ SBF solution is added, each film is suspended in SBF solution, avoid sticking, the beaker is put into the hot air drying oven, the temperature is set to 37℃, the solution is fully absorbed for 30min. Take out each group of beaker, use electronic balance to weigh the cut piece, before weighing, use toothless forceps to lift one corner of the film, vertically away from the table a certain distance, let the water drop naturally, the suspension time is 30s, the weighed amount is recorded as M1, the film is laid on the dry glass flat dish, put into vacuum drying oven, take out and weigh every certain time, until the weight of the film does not change, record its weight as M2, calculate the saturated moisture content of the wet film according to the following formula: [(M1-M2) / M2]x100%, the results are shown in table 3.

[0097]

[0098] From table 3, the use of crosslinking agent has no significant effect on the saturated moisture content of BC film. The reason may be that the bacterial cellulose film itself has a rich three-dimensional network structure, which provides a large amount of space for water storage. During the soaking process, water molecules can quickly fill the voids of these network structures, so that the film can reach the saturated water absorption state in a short time. When treated with crosslinking agent, although the crosslinking agent will react with sodium hyaluronate and bacterial cellulose film to form crosslinking structure, but this crosslinking structure mainly affects the release speed of water, not the water absorption amount. That is to say, the crosslinking structure mainly acts on the restriction of water molecules escaping from the film, and has little effect on the process of water molecules entering the film. In practical application, although the crosslinking agent has little effect on the saturated moisture content, but from the test results of dehydration time and 30min water loss rate, it can be seen that the crosslinking agent has an important influence on the water retention performance of the dressing.

[0099] 4. Water vapor transmission rate determination

[0100] Water vapor transmission rate standard test cup, add enough water at room temperature to form a water seal with the sample after placement, 5 test samples are prepared for the sample films of Example 2, Comparative Example 1 and Comparative Example 3. Weigh and record the mass of the container, sample and liquid W1 to 0.0001 g. Place the container into a drying box, keep the temperature at (37±1)℃. After 24h, take out each container from the drying box, and record the experimental time (T) to 5min. Immediately reweigh the container, sample and liquid, record the mass (W2) to 0.0001 g. Calculate the water vapor transmission rate of the sample according to the following formula: X=(W1-W2)×1000×(24 / T). In the formula: X—water vapor transmission rate (MVTR), unit is grams per square meter per 24h (g·m -2 ·24h -1 ); W1—mass of the container, sample and liquid, unit is grams (g); W2—container after the experiment; T—experimental period, unit is h (h), see Table 4.

[0101] Table 4 Water vapor transmission rate determination results

[0102]

[0103] As shown in Table 4, the use of crosslinking agent has no significant effect on the water vapor transmission rate of BC film. The reason may be that the water vapor transmission of bacterial cellulose film is mainly realized through the pores in its three-dimensional network structure. Water molecules can diffuse freely in these pores, thus completing the water vapor transmission process. When using crosslinking agent to form crosslinked structure, although the crosslinked structure will have some effect on the microstructure of the film, it does not significantly change the porosity and pore size distribution of the film. Therefore, the diffusion path and diffusion rate of water molecules in the film do not change significantly, so that the water vapor transmission rate remains basically stable.

[0104] 5. Tensile strength determination

[0105] Test according to GB / T1040.3-2006 standard. Cut the test film sample into dumbbell-shaped test pieces with narrow middle and wide ends, the middle part is 4mm wide. Hydrate the test pieces with purified water for 30min, measure the thickness of the middle part with three-point sampling, fix the material on the tensile testing machine, set the tensile testing machine to move at a speed of 100±10mm. The tensile strength is the maximum tensile force per unit area, take the average of three measurements, calculate the results as follows: Ts=F / S. In the formula, Ts: tensile strength, unit is MPa; F: the maximum tensile force borne by the test piece at break, unit is N; S: the area of the test piece fracture surface, unit is mm 2 . Results are shown in Table 5.

[0106]

[0107] As shown in Table 5, the use of crosslinking agent did not have a significant effect on the tensile strength of the BC film. The reason could be that the bacterial cellulose film itself has a high degree of crystallinity and orientation, so that there is a strong interaction force between the molecular chains, and this structure gives the bacterial cellulose film good mechanical properties. When the bacterial cellulose film is treated with a crosslinking agent, the crosslinking agent will react with the molecules in the film to form a crosslinking structure, but this crosslinking structure does not significantly change the original molecular chain arrangement and intermolecular force of the bacterial cellulose film. That is, the crosslinking structure does not have a substantial effect on the intrinsic mechanical properties of the bacterial cellulose film.

[0108] 6. Cytotoxicity test

[0109] The cytotoxicity test uses the agar overlay method. According to ISO 10993-5:2009 Biological evaluation of medical devices - Part 5: In vitro cytotoxicity tests (agar overlay method), the cytotoxicity test is performed on the composite film and BC film samples in each group to determine the potential toxic effects of the test substance on L929 cells.

[0110] The test method is as follows: (1) Sample, agar medium preparation and cell culture: 100 mm 2 of each group of samples were taken and sterilized for standby. At the same time, the negative control and positive control were placed in a 37°C incubator for 24 h. Sterilized 3% agar and 2% DMEM medium (20% fetal bovine serum) were mixed in equal volumes. L-929 cells were cultured in MEM medium containing 10% fetal bovine serum and placed in a 37°C, 5% CO2 incubator for culture. The cells were digested with 0.25% trypsin to prepare a single cell suspension, and then the cell suspension with a concentration of 1.3 x 10 5 / ml was inoculated into a flat dish, and 2 ml of the above cell suspension was added to each dish. After the cells grew into a monolayer, the original culture medium was aspirated, 2 ml of agar medium was added, and then the test samples, positive control and negative control were placed on the agar, and the culture was continued. (2) Cell morphology observation and cytotoxicity evaluation: after 24 h of culture in a 37°C, 5% CO2 incubator, the flat dish was taken out and the sample position was marked at the bottom with a marker pen, then the sample was discarded, 2 ml of neutral red was added to each dish, incubated for 1 h, the excess neutral red was aspirated, and observed under a microscope. Positive control: MEM medium containing 0.5% phenol and 10% fetal bovine serum (37°C for 24 h). Negative control: high-density polyethylene sheet. The size of cytotoxicity is indicated by the reaction grade, as shown in Table 6.

[0111]

[0112] According to ISO 10993-5:2009, samples ranked as 0, 1 or 2 are judged to be non-toxic, and samples ranked as 3 or 4 are judged to be toxic. The results are shown in Table 7.

[0113] Table 7 Results of cytotoxicity test of BC membrane after crosslinking

[0114]

[0115] As shown in Table 7, the results of toxicology test of the composite membrane after crosslinking treatment are non-toxic. This indicates that the crosslinking structure formed by the crosslinking agent, sodium hyaluronate and bacterial cellulose membrane does not have obvious toxic effect on cells. This may be because the chemical reaction between the crosslinking agent and the membrane material during the crosslinking reaction is relatively mild, and no toxic by-products are generated. At the same time, the bacterial cellulose membrane itself has good biocompatibility and can provide a relatively stable and safe microenvironment for cells.

[0116] From the process of cell culture and observation, after culturing cells on agar medium and contacting with samples, no obvious abnormal changes in cell morphology were observed. Even under microscopic observation, only a slight reaction zone appeared under the sample, and no massive cell death or severe degeneration occurred. This further proves that the composite membrane after crosslinking treatment meets the medical requirements in terms of cytotoxicity.

[0117] In actual medical scenarios, cytotoxicity is one of the important indicators for evaluating the safety of dressings. The test results show that this crosslinking treated medical bacterial cellulose dressing with high water retention capacity not only ensures its water retention performance, dehydration performance, saturated water content ratio, water vapor transmission rate and tensile strength, but also has good biological safety, which can provide a safe and reliable environment for wound healing. This has important significance for the development and application of new medical dressings, and is expected to be widely promoted and applied in clinical practice.

[0118] 8. Bacteriostatic test of dressing

[0119] The agar plate diffusion method according to GB / T 20944.1-2007 was used to evaluate the antibacterial properties of the prepared dressing. Two layers of agar medium were injected into the agar plate, with the lower layer being sterile medium and the upper layer being inoculated medium. The sample was placed on the two layers of medium, and after a certain period of incubation, the antibacterial properties of the sample were qualitatively evaluated according to the degree of bacterial reproduction at the contact between the medium and the sample. Representative samples were selected from the sample, with 4 pieces of circular sample (2 pieces on the front and 2 pieces on the back) for each bacterial test, with a diameter of 25 mm. The sample was placed in the center of the plate using sterile tweezers, and was pressed evenly on the agar medium until the sample and the agar medium were in good contact. After incubation in an incubator, the width of the antibacterial band was observed and measured, and then the sample was removed from the agar medium using tweezers, and the bacterial reproduction in the contact area under the sample was examined using a microscope.

[0120] Table 8 Antibacterial properties of the test sample

[0121]

[0122] The results show that the test sample of Example 2 has good inhibitory effect on both Escherichia coli and Staphylococcus aureus bacteria, while the control sample of Comparative Example 1 does not show antibacterial effect, indicating that the introduction of the antibacterial material enables the test sample to effectively exert antibacterial effect.

[0123] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and embodiments having substantially the same configuration and exerting the same effect as the technical idea within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the main idea of the present application, various modifications that can be thought of by those skilled in the art, and other ways constructed by combining part of the elements in the embodiments are also included in the scope of the present application.

Claims

1. A medical bacterial cellulose dressing with high water retention capacity, characterized in that, The dressing is prepared by reacting bacterial cellulose membrane with sodium hyaluronate solution in the presence of tetrahydroxymethylphosphoric acid, and the water retention time of the dressing is not less than 2000 min. The dressing includes: The porous layer is composed of a first bacterial cellulose skeleton, and the internal pores and surface of the first bacterial cellulose skeleton are covalently bonded with sodium hyaluronate and cationic antibacterial polysaccharides. The dense layer is composed of a second bacterial cellulose skeleton, the surface of which is covalently bonded with sodium hyaluronate. The density of sodium hyaluronate in the porous layer is higher than that in the dense layer.

2. The medical bacterial cellulose dressing with high water retention capacity according to claim 1, characterized in that, The cationic antibacterial polysaccharide is quaternized chitosan.

3. The medical bacterial cellulose dressing with high water retention capacity according to claim 1, characterized in that, In the porous layer, the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is 1-10:

1.

4. A method for preparing a medical bacterial cellulose dressing, characterized in that, The method for preparing the medical bacterial cellulose dressing with high water retention capacity according to any one of claims 1-3 includes the following steps: a. Provide a first bacterial cellulose membrane and freeze-dry it to form a porous bacterial cellulose scaffold; b. Immerse the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and cationic antibacterial polysaccharides; c. Provide a second bacterial cellulose membrane, impregnate it in a second solution containing sodium hyaluronate, wherein the concentration of sodium hyaluronate in the first solution is higher than that in the second solution; d. The porous scaffold treated in step b is stacked with the second bacterial cellulose membrane treated in step c, and placed in a solution containing tetrahydroxymethylphosphoric acid to allow the bacterial cellulose membrane to undergo a cross-linking reaction with sodium hyaluronate, thereby obtaining a medical bacterial cellulose dressing.

5. The method for preparing the medical bacterial cellulose dressing according to claim 4, characterized in that, The freeze-drying process described in step a includes: pre-freezing at a temperature below -30°C, and then freeze-drying at a temperature between -10°C and -50°C and a vacuum degree below 50 Pa.

6. The method for preparing the medical bacterial cellulose dressing according to claim 4, characterized in that, In step b, vacuum negative pressure is used to assist impregnation, so that the first solution can fully penetrate into the internal pores of the porous support.

7. The method for preparing the medical bacterial cellulose dressing according to claim 4, characterized in that, The crosslinking reaction described in step d is carried out at 20-30°C for 12-36 hours.

8. The method for preparing the medical bacterial cellulose dressing according to claim 4, characterized in that, It also includes the steps of cleaning, sterilizing and aseptically sealing the resulting dressing.

9. The application of a medical bacterial cellulose dressing with high water retention capacity, characterized in that, The medical bacterial cellulose dressing with high water retention capacity as described in any one of claims 1-3 is used in medical devices for treating burns, scalds, and chronic ulcers.

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