Medical bacterial cellulose dressing with high water retention capacity as well as preparation method and application of medical bacterial cellulose dressing

Abstract

The invention 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 tetrakis hydroxymethyl phosphonium chloride, and the water retention time of the dressing is not less than 2000min. The medical bacterial cellulose dressing with high water retention capacity prepared by the invention shows great advantages in actual medical application. For patients with burns and scalds, traditional dressings are often difficult to keep moist of wounds for a long time, and if the dressings are uncovered after being dried to cause secondary wound injury, the dressings can provide a lasting and stable moist environment for the wounds and accelerate repair and regeneration of damaged skin cells by virtue of the excellent water retention capacity of the dressings.

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 with 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 extend 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: 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; 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.

[0009] Further, the density of sodium hyaluronate in the porous layer is higher than that in the dense layer.

[0010] Further, in the porous layer, the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is 1:1-10:1.

[0011] 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: a. providing a first bacterial cellulose membrane and subjecting it to freeze-drying treatment to form a porous bacterial cellulose scaffold; b. immersing the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and cationic antibacterial polysaccharide; 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; 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 membrane and sodium hyaluronate are cross-linked to obtain a medical bacterial cellulose dressing.

[0012] Further, the freeze-drying treatment in step a comprises pre-freezing under a condition of below -30℃, and then performing freeze-drying under a condition of -10℃ to -50℃ and a vacuum degree of below 50 Pa.

[0013] Further, the vacuum negative pressure assisted immersion in step b enables the first solution to fully penetrate into the internal pores of the porous scaffold.

[0014] Further, the cross-linking reaction in step d is performed at 20-30℃ for 12-36h.

[0015] Further, the application further comprises the steps of cleaning, sterilizing and aseptically sealing the obtained dressing.

[0016] Further, the sterilization is Co60-γ ray irradiation sterilization.

[0017] The third aspect of the application provides an application of the medical bacterial cellulose dressing with high water retention capacity, and the medical bacterial cellulose dressing with high water retention capacity is applied to medical devices for treating burns, scalds, chronic ulcers or wounds.

[0018] The application has the following beneficial effects: (1) The dressing is prepared by reacting a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of a cross-linking agent tetramethylammonium chloride, wherein sodium hyaluronate (HA) itself has strong water retention performance, and it can absorb 1000 times of water of its own weight, and is widely used in moisturizing in the fields of skin care and medicine. 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 the bacterial cellulose. And tetramethylammonium chloride (THPC) as a cross-linking agent, it can react with bacterial cellulose and sodium hyaluronate molecules to form chemical cross-linking bonds. These cross-linking 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 cross-linking 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.

[0019] In the wound environment, the compound of bacterial cellulose film and sodium hyaluronate can provide a moist environment for the wound on one hand, the large amount of water absorbed by sodium hyaluronate is slowly released, maintaining the humidity of the wound; on the other hand, the presence of cross-linked structure enables the dressing to retain the liquid inside more effectively when absorbing wound exudate, instead of quickly losing. Moreover, this 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 binding capacity 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. (2) The medical bacterial cellulose dressing with high water retention capacity prepared by the application has 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 can provide a persistent and stable moist environment for the wound due to its excellent water retention capacity, 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 the adhesion of dry dressings. For patients with chronic ulcers, a poor wound environment is an important reason for long-term non-healing of wounds, and 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

[0020] 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 combination with embodiments. It should be understood that the specific embodiments described herein 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.

[0021] 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 scenarios 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 technical content disclosed in the present application are only routine technical means and should not be understood as insufficient disclosure of the present application.

[0022] 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, and the water retention time of the dressing is not less than 2000 min.

[0023] 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, wherein sodium hyaluronate (HA) itself has strong water retention performance and can absorb 1000 times its own weight of water, and is widely used in skin care and medicine 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 the bacterial cellulose. And tetramethylammonium chloride (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 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.

[0024] In a 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 maintains the humidity of the wound; on the other hand, the presence of crosslinking structure makes the dressing more effectively retain the liquid inside when absorbing wound exudate, rather than quickly losing. 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 capacity 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.

[0025] In the present embodiment, the dressing comprises: 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; 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.

[0026] 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)), has super liquid absorption, water locking (porous structure) and active antibacterial functions, can quickly absorb wound exudate, and inhibit bacterial growth at the source by using its antibacterial property, preventing the dressing itself from becoming a source of infection.

[0027] The dense layer is on the side of the contact wound, is used for keeping dense, moist and anti-adhesion, wherein a low sodium hyaluronate concentration provides basic moisturizing, and a 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.

[0028] In the present application, the internal pores and the surface of the first bacterial cellulose skeleton of the porous layer are covalently bonded with sodium hyaluronate and cationic antibacterial polysaccharide, so that the dressing has excellent water retention while having long-acting antibacterial ability (due to the introduction of quaternized chitosan), and due to the presence of different electric molecules, it may be more conducive to cell adhesion and growth, and its effect is far beyond the simple superposition of "water retention + antibacterial".

[0029] In the porous layer, the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is 1-10:1. This specific mass ratio can ensure the water retention performance of the dressing while fully exerting the antibacterial effect of the cationic antibacterial polysaccharide. When the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is in this range, on the one hand, sodium hyaluronate can continue to exert its strong water retention ability to provide a persistent moist environment for the wound; on the other hand, cationic antibacterial polysaccharide can effectively inhibit the growth and reproduction of bacteria, reducing the risk of wound infection.

[0030] The cationic antibacterial polysaccharide is quaternary ammonium chitosan (HACC). Quaternary ammonium chitosan has good biocompatibility and broad-spectrum antibacterial properties. Quaternary ammonium 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, etc.). This electrostatic attraction allows quaternary ammonium 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, quaternary ammonium chitosan has good biocompatibility, which means that it will 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, which is conducive to the normal repair and healing of the wound. In addition, quaternary ammonium 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 quaternary ammonium 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 quaternary ammonium chitosan in the dressing, allowing it to continuously exert its antibacterial effect throughout the wound healing process.

[0031] In actual medical scenarios, the antibacterial properties of quaternary ammonium chitosan can play an important role in different types of wounds, such as burns, scalds, chronic ulcers, or trauma, etc. For example, in burn and scald wounds, due to the damage to the skin barrier, bacteria are easily invaded, and quaternary ammonium chitosan can timely inhibit bacterial growth and reduce the occurrence 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, quaternary ammonium chitosan is less likely to develop drug resistance, which makes it have a broader application prospect in long-term wound treatment. Embodiments of the second aspect of the present 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: a. providing a first bacterial cellulose membrane and subjecting it to freeze-drying treatment to form a porous bacterial cellulose scaffold; b. immersing the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and a cationic antibacterial polysaccharide; 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; d. Superimpose the porous scaffold treated in step b with the second bacterial cellulose film treated in step c, and place in a solution containing tetramethylammonium chloride, so that the bacterial cellulose film is cross-linked with sodium hyaluronate, thereby obtaining a medical bacterial cellulose dressing.

[0032] In this embodiment, the multifunctional porous hydrophilic layer is prepared in step a: after a piece of pure bacterial cellulose (BC) film is washed with deionized water to neutral, pre-freeze drying treatment is performed (-40℃ quick freezing for 2h, and then freeze drying under the condition of -20℃ and vacuum degree <10 Pa for 6h), forming 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 film 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 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 antimicrobial polysaccharide.

[0033] In step b, the preparation of sodium hyaluronate solution: take sodium hyaluronate powder and quaternized chitosan, add distilled water, maintain the temperature at 10℃, stand for 39min, so that the HA powder is fully dissolved and swelled, continue to add appropriate amount of distilled water, stir at 10℃ for 1h until completely dissolved, add distilled water to 1L, and prepare a mixed solution containing high concentration of sodium hyaluronate (5-15g / L) and quaternized chitosan (1-3 g / L) respectively. Immerse the porous BC scaffold obtained in step a in the mixed solution, and immerse for 10min under a vacuum negative pressure of -0.08MPa to -0.1MPa, so that the solution can fully penetrate into the internal pores of the porous scaffold. This vacuum negative pressure immersion method can effectively remove the air in the pores of the scaffold, so that the solution can better fill the pores, and ensure that sodium hyaluronate and quaternized chitosan can be uniformly 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.

[0034] In step c, the preparation of the moisturizing dense layer: take another piece of pure BC film (not freeze-dried, maintaining the natural dense nanofiber structure), and immerse it in a low concentration sodium hyaluronate (0.5-2g / L) solution.

[0035] 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 to completely immerse the composite membrane, and the reaction is carried out at 20-30°C for 12-36h. In this process, tetramethyl phosphonium chloride forms chemical cross-linking bonds with the 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 quaternized chitosan and the hydroxyl group of bacterial cellulose, and the carboxyl group of sodium hyaluronate and the amino group of quaternized 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 bound to 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.

[0036] 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-linking 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.

[0037] At the same time, when the wet, solution-pre-treated 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 quaternized chitosan molecules carried by them will be in close contact, forming molecular-level entanglement and contact points. Tetramethyl phosphonium chloride cross-linking 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 and forming a complete dressing structure. This cross-linking reaction is carried out at a mild temperature of 20-25°C for 12-36h, which can ensure that the reaction proceeds sufficiently.

[0038] 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 will not be sufficient, and the combination of sodium hyaluronate and bacterial cellulose membrane will not be firm enough; if the concentration is too high, it may lead to excessive cross-linking, affecting the performance of the dressing.

[0039] In step d, after the reaction of the composite film, it is necessary to clean and remove the unreacted substances. The cleaning can use deionized water, distilled water or normal saline, and the cleaning times are generally 3-5 times, 15 min each time, until no unreacted substances are detected in the cleaning liquid. 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 obvious effect on the performance of the dressing. Aseptic packaging can prevent the dressing from being contaminated during storage and transportation.

[0040] The application of the third aspect of the embodiment provides a medical bacterial cellulose dressing with high water retention capacity. The medical bacterial cellulose dressing with high water retention capacity is applied to 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, and 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.

[0041] 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.

[0042] 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. Embodiment The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight. Unless otherwise stated, all reagents used in the examples are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, all instruments used in the examples are available commercially.

[0043] Example 1 A method for preparing a medical bacterial cellulose dressing includes the following steps: a: After rinsing a pure bacterial cellulose membrane with deionized water until neutral, pre-freeze-dry it, then freeze it at -40℃ for 2 hours, and then freeze-dry it at -20℃ and vacuum degree <10 Pa for 6 hours to form a porous bacterial cellulose scaffold. b: Immerse the porous scaffold obtained in step a in a first solution containing sodium hyaluronate and quaternized chitosan at -0.09 MPa for 10 min to ensure that the mixed solution fully penetrates into the depth of the pores; wherein, the concentration of sodium hyaluronate is 5 g / L and the concentration of quaternized chitosan is 1 g / L. c: Take another piece of pure BC membrane (not freeze-dried, retaining its natural dense nanofiber structure) and immerse it in a 0.5 g / L sodium hyaluronate solution; d: The porous scaffold treated in step b is superimposed with the second bacterial cellulose membrane treated in step c. The superimposed bilayer structure is placed in a solution containing tetrahydroxymethyl phosphorus chloride (THC) at a concentration of 0.5%, and crosslinked at 25°C for 24 hours to obtain a composite membrane. The composite membrane sample is taken out and rinsed three times with distilled water for 15 minutes each time. The obtained bacterial cellulose composite membrane sample is sterilized by Co60-γ irradiation and then aseptically sealed and stored at 5°C.

[0044] Example 2 This embodiment is basically the same as Embodiment 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.

[0045] Example 3 This embodiment is basically the same as Embodiment 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.

[0046] Comparative Example 1 A double-layered bacterial cellulose membrane was used, but no cross-linking treatment was performed.

[0047] Comparative Example 2 This comparative example is substantially the same as Example 1, except that in step d, the tetrahydroxymethyl phosphonium chloride is replaced by EDC / NHS (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) with a molar ratio of 4:1.

[0048] Comparative Example 3 This comparative example is substantially the same as Example 2, except that in step d, the tetrahydroxymethyl phosphonium chloride is replaced by EDC / NHS with a molar ratio of 4:1.

[0049] Comparative Example 4 This comparative example is substantially the same as Example 3, except that in step d, the tetrahydroxymethyl phosphonium chloride is replaced by EDC / NHS with a molar ratio of 4:1.

[0050] Experimental Case Preparation of simulated body fluid: NaCl 8.298g, CaCl20.275g, and a suitable amount of distilled water were dissolved and diluted to 1L.

[0051] 1. Dehydration time comparison test The composite film samples in Examples 1-3 and Comparative Examples 1-4 were carefully cut into 10x10 cm pieces with scissors, and three parallel tests were performed for each sample. The surface free water was removed with a glass rod, and the pre-prepared simulated body fluid was taken out. The simulated body fluid was preheated to 37°C using a constant temperature water bath, and 40 times the weight of the sample was weighed and poured into a clean dish. The sample was placed in the dish and moved into a drying oven, which was set to a temperature of (37±1) °C. The sample was soaked for 30 min, and then a pair of tweezers was used to carefully pick up one corner of the film piece and hold it at a distance from the table to allow the excess water to drip naturally. The film piece was hung for 30 s, and the weight was recorded as M1. The sample film was placed in a dry dish and placed in a hot air drying oven, which was set to a temperature of (37±1) °C and a relative humidity RH of less than 20%. The sample was taken out every 1 h and weighed until the weight was constant. The corresponding time and weight Mt were recorded. The dehydration rate was calculated as follows: dehydration rate = [(M1-Mt) / M1]x100%. According to the dehydration time and the corresponding weight, the percentage of water loss was calculated, and the results are shown in Table 1.

[0052] Table 1 Dehydration time comparison test results

[0053] From Table 1, when the concentration of sodium hyaluronate solution in step b is 8 g / L and the concentration of sodium hyaluronate solution in step c is 1 g / L, the BC composite film (BC-THPC cross-linking-HA) formed by using tetramethylammonium phosphate chloride (THPC) as a cross-linking 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 sodium hyaluronate solution in step b to 12 g / L and the concentration of sodium hyaluronate solution in step c to 1.5 g / L does not prolong the dehydration time. In addition, THPC as a cross-linking agent greatly outperforms EDC / NHS cross-linking agent in prolonging the dehydration time of the BC film.

[0054] The reason may be that the cross-linking 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 cross-linking 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 cross-linking structure formed by EDC / NHS may not be stable enough, and the distribution of cross-linking points is not uniform enough, resulting in defects in the network structure, which cannot effectively lock water molecules like the structure formed by THPC.

[0055] In addition, from the perspective of chemical properties, tetramethylammonium phosphate chloride has unique chemical activity. Its reaction process with sodium hyaluronate and bacterial cellulose film is more gentle and controllable, and can achieve good cross-linking effect without damaging the original structure and performance of the molecules. In contrast, EDC / NHS may cause some side reactions during the reaction process, causing damage to the structure of sodium hyaluronate and bacterial cellulose film to some extent, thereby affecting the water retention performance and dehydration time of the dressing.

[0056] Furthermore, the structure formed by THPC cross-linking has stronger affinity for water molecules. Its surface may have some special chemical groups that can form more hydrogen bonds or other interactions with water molecules, increasing the binding force between water molecules and 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.

[0057] 2.30 min water loss rate test 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 prepared for each sample. The surface of the film was pressed with a glass rod to remove the free water on the surface. The simulated body fluid was pre-prepared and preheated to 37°C in a constant temperature water bath. 40 times the weight of the sample was weighed and poured into a clean dish. The sample was placed in the dish and moved into a drying oven, set to (37±1) °C, and soaked for 30 min. The sample was removed, and one corner of the film was carefully clamped with tweezers at a distance from the table to allow the excess water to drip naturally. The film was hung for 30 s and weighed as M1. The samples were placed in 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 removed and the film weight was recorded as M2. The 30 min water loss rate was calculated as follows: 30 min water loss rate = [(M1-M2) / M1]x100%. The results are shown in Table 2.

[0058]

[0059] As can be seen from Table 2, 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 makes 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 may be that when the HA concentration reaches a certain value, the three-dimensional network structure of the bacterial cellulose film has been basically saturated with sodium hyaluronate molecules. Further increasing the HA concentration, the excess sodium hyaluronate molecules cannot effectively adhere to the bacterial cellulose film, 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 may aggregate or entangle, forming some structures that are not conducive to water retention. This makes water molecules more likely to lose in this high concentration system, rather than being stably bound inside the dressing.

[0060] In addition, the THPC cross-linking 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 film 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 may interfere with the formation of this cross-linked network, resulting in defects in the network structure and reducing the binding capacity of water.

[0061] In contrast, the structure formed by EDC / NHS crosslinker is inherently less stable and desirable. Even at different HA concentrations, its water retention ability is less than that of THPC crosslinker. Side reactions that can occur during EDC / NHS crosslinking can destroy the original structure of the bacterial cellulose film and sodium hyaluronate, making it easier for water to escape from these damaged structures.

[0062] In practical applications, the selection of appropriate HA concentration and crosslinker is crucial for the preparation of medical bacterial cellulose dressings with high water retention capacity. Based on the test results of dehydration time and 30 min water loss rate, the concentration of sodium hyaluronate solution in step b is 8 g / L, the concentration of sodium hyaluronate solution in step c is 1 g / L and THPC is used as the crosslinker is a more desirable combination.

[0063] 3. Saturated water content determination According to the 30 min absorption equilibrium condition proposed in most contact dressings in YYT 0471-2004 Test Methods for Contact Wound Dressings, the saturated water content of the film was determined using the constant weight method, and the results were presented in the form of saturated water content. The sample films of Example 2, Comparative Example 1 and Comparative Example 3 were carefully cut into squares of 5 cm * 5 cm using scissors, and three samples were taken from each group. The solution used for soaking the samples was pre-prepared simulated body fluid (SBF), which was preheated to 37°C in a constant temperature water bath. The cut films were placed in 200 ml beakers, 150 ml of 37°C SBF solution was added, and each film was suspended in the SBF solution to avoid adhesion. The beakers were placed in a forced air drying oven set at 37°C, and the solution was allowed to absorb for 30 min. The beakers were removed, and the cut films were weighed using an electronic balance. Before weighing, the film was lifted by the corner using a toothless forceps, and the water was allowed to drip naturally for 30 s. The film was then laid flat on a dry glass plate and placed in a vacuum drying oven. The weight was recorded as M2. The saturated water content of the wet film was calculated according to the following formula: [(M1-M2) / M2] x 100%, and the results are shown in Table 3.

[0064]

[0065] From Table 3, it can be seen that the use of crosslinking agent has no significant effect on the saturated water content of BC membrane. The reason could be that the bacterial cellulose membrane 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 in this network structure, so that the membrane reaches a 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 membrane to form a crosslinked structure, this crosslinked structure mainly affects the release speed of water, rather than the absorption amount of water. That is, the crosslinked structure mainly acts to limit the escape of water molecules from the membrane, and has little effect on the process of water molecules entering the inside of the membrane. In practical applications, although the crosslinking agent has little effect on the saturated water content, it can be seen from the test results of dehydration time and 30 min water loss rate that the crosslinking agent has an important influence on the water retention performance of the dressing.

[0066] 4. Water vapor transmission rate determination Water vapor transmission rate standard test cup, add enough water at room temperature to form a water seal with the sample after placement, prepare 5 test samples 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 in a drying oven, and keep the temperature at (37±1)℃. After 24h, take out each container from the drying oven, and record the experimental time (T) to 5min. Immediately reweigh the container, sample and liquid, and 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: grams per square meter per 24h (g·m -2 ·24h -1 ); W1—mass of container, sample and liquid, unit: grams (g); W2—container after the experiment; T—experimental period, unit: h (h), the results are shown in Table 4.

[0067] Table 4 Water vapor transmission rate determination results

[0068] As shown in Table 4, the use of crosslinking agent has no significant effect on the water vapor transmission rate of BC membrane. The reason could be that the water vapor transmission of bacterial cellulose membrane is mainly realized through the pores in its three-dimensional network structure. Water molecules can diffuse freely in these pores, thereby completing the water vapor transmission process. When crosslinking structure is formed using crosslinking agent, although the crosslinking structure will have a certain effect on the microstructure of the membrane, it does not significantly change the porosity and pore size distribution of the membrane. Therefore, the diffusion path and diffusion rate of water molecules in the membrane do not change significantly, so that the water vapor transmission rate remains basically stable.

[0069] 5. Tensile strength determination The test was performed according to GB / T 1040.3-2006 standard. The test film sample was cut into dumbbell-shaped specimen with narrow middle and wide both ends, the middle part width was 4 mm. The specimen was hydrated with purified water for 30 min, the thickness of the middle part was measured by three-point sampling, the material was fixed on the tensile testing machine, and the tensile testing machine was set to move at a speed of 100 ± 10 mm. The tensile strength was the maximum tensile force per unit area, the average value of three measurements was calculated as follows: Ts = F / S. In the formula, Ts: tensile strength, unit MPa; F: the maximum tensile force borne by the specimen at the time of fracture, unit N; S: the area of the specimen fracture surface, unit mm 2 . The results are shown in Table 5.

[0070]

[0071] As shown in Table 5, the use of crosslinking agent had no significant effect on the tensile strength of the BC film. The reason may be that the bacterial cellulose film itself has high crystallinity and orientation, so that there is 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.

[0072] 6. Cytotoxicity test The cytotoxicity test used 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 was 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.

[0073] 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. 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 concentration was adjusted to 1.3 × 10 5 / ml of cell suspension 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, respectively, and then the test sample, 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 at 37℃ in a 5% CO2 incubator, the dish was taken out and the sample location 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 microscopic observation was performed. Positive control: MEM culture medium containing 0.5% phenol and 10% fetal bovine serum (37℃ for 24 h). Negative control: high-density polyethylene sheet. The size of cytotoxicity was expressed by reaction grading, see Table 6.

[0074]

[0075] According to ISO 10993-5:2009, samples graded as 0, 1 or 2 were judged to be non-toxic, and those graded as 3 or 4 were judged to be toxic, and the results are shown in Table 7.

[0076] Table 7 Cytotoxicity test results of BC membrane after crosslinking

[0077] As shown in Table 7, the toxicology test results of the composite membrane after crosslinking treatment were non-toxic. It is indicated that the crosslinking structure formed by the crosslinking agent and 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, bacterial cellulose membrane itself has good biocompatibility and can provide a relatively stable and safe microenvironment for cells.

[0078] From the process of cell culture and observation, after the cells were cultured on agar medium and contacted with the sample, 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.

[0079] In actual medical scenarios, cytotoxicity is one of the important indicators to measure 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 popularized and applied in clinical practice.

[0080] 8. Bacterial inhibition test of dressing 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 a sterile medium and the upper layer being an 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 side and 2 pieces on the back side) 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 evenly pressed onto the agar medium until good contact between the sample and the agar medium. After incubation in an incubator, the width of the antibacterial zone 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.

[0081] Table 8 Antibacterial properties of test samples

[0082] 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.

[0083] It should be noted that the present application is not limited to the above-described embodiments. The above-described embodiments are merely 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 of 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 a bacterial cellulose membrane with a sodium hyaluronate solution in the presence of tetrahydroxymethylphosphoric acid, and the water retention time of the dressing is not less than 2000 min.

2. The medical bacterial cellulose dressing with high water retention capacity according to claim 1, characterized in that, 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.

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

4. The medical bacterial cellulose dressing with high water retention capacity according to claim 2, characterized in that, The density of sodium hyaluronate in the porous layer is higher than that in the dense layer; and / or In the porous layer, the mass ratio of sodium hyaluronate to cationic antibacterial polysaccharide is 1-10:

1.

5. 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 1-4 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.

6. The method for preparing the medical bacterial cellulose dressing according to claim 5, 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.

7. The method for preparing the medical bacterial cellulose dressing according to claim 5, 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.

8. The method for preparing the medical bacterial cellulose dressing according to claim 5, characterized in that, The crosslinking reaction described in step d is carried out at 20-30°C for 12-36 hours.

9. The method for preparing the medical bacterial cellulose dressing according to claim 5, characterized in that, It also includes the steps of cleaning, sterilizing and aseptically sealing the resulting dressing.

10. 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 1-4 is used in medical devices for treating burns, scalds, chronic ulcers or trauma.

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