Microbial mineralization self-power-generation antibacterial wound dressing as well as preparation method and application thereof

Abstract

The invention discloses a microbial mineralization self-power-generation antibacterial wound dressing and a preparation method and application thereof. The microbial mineralization self-power-generation antibacterial wound dressing comprises an outer breathable protective layer, an outer carbon adhesive conducting layer, a positive and negative separation type paper-based microbial fuel cell assembly and an inner carbon adhesive conducting layer which are sequentially arranged from outside to inside. The three-in-one synergistic function of mineralization repairing, natural antibiosis and self-electricity-generating stimulation is achieved in a breakthrough mode, the key problems that a traditional dressing is single in function, an electric stimulation dressing depends on an external power source, and the mechanical strength of hydrogel is insufficient are solved, the 3D printed micro-grid structure enables spores to be distributed more evenly, mineralization is more even, and the application range is wide. The hydrogel has the advantages of excellent biocompatibility, high adhesion, low cost, biodegradability and the like, and is particularly suitable for intelligent nursing of chronic refractory wounds such as diabetic foot ulcer, pressure ulcer, burn and the like.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials technology, specifically relating to a microbial mineralization self-generating antibacterial wound dressing, its preparation method, and its application. Background Technology

[0002] Wound healing is a complex process involving inflammatory response, cell proliferation, and tissue remodeling. Chronic wounds, in particular (such as diabetic foot ulcers and pressure ulcers), pose a challenge to clinical nursing due to issues such as local microcirculation disorders, high risk of infection, and weak repair capacity.

[0003] Currently, commonly used wound dressings in clinical practice mainly include traditional gauze, hydrogel dressings, and silver ion antibacterial dressings, but all of them have obvious drawbacks: traditional gauze can only passively absorb exudate and has no active repair function, easily adheres to the wound and has a high risk of infection; hydrogel dressings have good moisturizing properties but insufficient mechanical strength and limited antibacterial effect; although silver ion dressings can be antibacterial, there is a risk of heavy metal residue, which can easily lead to bacterial resistance, and the cost is high, making it difficult to meet the needs of primary healthcare and long-term care.

[0004] In addition, bioelectric stimulation dressings, due to their ability to accelerate wound healing through microcurrents, have gradually become a research hotspot in chronic wound care. However, existing bioelectric stimulation dressings face significant technical bottlenecks:

[0005] ① External power dependence and poor clinical applicability: Most electrical stimulation dressings require external batteries or power adapters to provide power, which not only increases the size and weight of the dressing, leading to decreased wearing comfort and susceptibility to movement interference, but also poses a risk of leakage. They are difficult to adapt to chronic wounds on active areas such as joints and feet (such as diabetic foot ulcers). A few passive electrical stimulation dressings rely on piezoelectric / triboelectric effects, which require external pressure to trigger the generation of electricity. They cannot achieve continuous and stable micro-electric stimulation and cannot meet the long-term repair needs of chronic wounds.

[0006] ② Limited functionality and weak synergistic repair capabilities: Existing products focus only on the single function of "electric stimulation" and do not integrate key nursing mechanisms such as antibacterial and wound closure. They need to be used with separate antibacterial dressings or gels, which increases the complexity and cost of nursing operations. Although some products have added antibacterial functions, they mostly rely on heavy metal components such as silver ions, which pose risks of drug resistance and tissue residue.

[0007] ③ High cost and difficulty in industrialization: The manufacturing process of core components (such as micro batteries and precision electrodes) is complex and the cost of raw materials is high, far exceeding the cost affordability of primary healthcare and home care, making it difficult to promote on a large scale.

[0008] Studies have shown that CaCO3 particles generated by MICP have good biocompatibility, and calcium ion release can promote cell proliferation. However, its application in wound care is still in the exploratory stage. Existing research only focuses on its mineralization and repair function and has not combined it with other care mechanisms. Microbial fuel cell (MFC) technology converts chemical energy into electrical energy through microbial metabolism. Paper-based MFCs show potential in the field of medical sensors due to their low cost and flexibility. However, current technologies lack a wound dressing that integrates multiple functions such as "repair, antibacterial, and irritation," is cost-effective, and meets clinical needs. Summary of the Invention

[0009] Purpose of the Invention: Addressing the problems of existing technologies, this invention discloses a microbial mineralization self-generating antibacterial wound dressing. This invention is the first to integrate microbial induced mineralization (MICP) technology with paper-based biofuel cell (MFC) technology into a wound dressing. It is the first to fuse MIP and paper-based MFC technologies, using *S. pasteurii* to induce the formation of a dynamic mineralization membrane from CaCO3. This membrane can adaptively close the wound in response to pH changes in wound exudate and continuously release calcium ions to aid tissue regeneration, overcoming the limitations of traditional dressings that only offer dual functions of 'antibacterial + electrical stimulation'. This invention achieves a breakthrough in realizing a three-in-one synergistic function of "mineralization repair—natural antibacterial—self-generating stimulation," solving key problems such as the single function of traditional dressings, the dependence of electrical stimulation dressings on external power sources, and insufficient mechanical strength of hydrogels.

[0010] The present invention also provides a method for preparing and applying the microbial mineralization self-generating antibacterial wound dressing.

[0011] Technical Solution: To achieve the above objectives, the present invention provides a microbial mineralization self-generating antibacterial wound dressing, comprising, from the outside to the inside, an outer breathable protective layer, an outer carbon conductive layer, a forward and reverse separated paper-based microbial fuel cell assembly, and an inner carbon conductive layer; the forward and reverse separated paper-based microbial fuel cell assembly comprises: cellulose chromatography paper, a wax isolation layer located in the central region of the cellulose chromatography paper; a bacterial cellulose membrane (BC) (MICP antibacterial functional layer) located on the side of the cellulose chromatography paper near the wound and loaded on the inner side of the wax isolation layer, constituting the anode region; a reducing material coating located on the side of the chromatography paper away from the wound and located on the outer side of the wax isolation layer, constituting the cathode region; and side carbon conductive layers are also provided on both sides of the chromatography paper.

[0012] Preferably, the forward and reverse separation paper-based microbial fuel cell assembly includes:

[0013] A cellulose chromatography paper measuring 10×10cm;

[0014] A wax isolation layer located in the central area of ​​the chromatography paper, formed by printing and high-temperature melting and penetration;

[0015] A 3D-printed bacterial cellulose membrane located on the side of the chromatography paper closest to the wound and loaded inside the wax isolation layer constitutes the anode region.

[0016] The reducing agent coating located on the side of the chromatography paper away from the wound and outside the wax isolation layer constitutes the cathode region;

[0017] The carbon adhesive conductive layer located on both the front and back edges and sides of the chromatography paper has a width of 2-5 mm. The carbon adhesive on the side closer to the wound forms a conductive ring around the edge of the cellulose chromatography paper, and the carbon adhesive on the side away from the wound forms a conductive ring around the reducing agent coating and draws an X-shaped pattern in the center of the cathode area. The carbon adhesive on the side connects the conductive rings on the front and back to form a three-dimensional conductive circuit.

[0018] The outer breathable protective layer is a medical polyurethane transparent dressing, a hydrocolloid transparent dressing, or a silicone-based breathable protective dressing.

[0019] Preferably, the outer breathable protective layer is a medical polyurethane transparent dressing.

[0020] The bacterial cellulose membrane is formed by 3D printing. The thickness of the bacterial cellulose membrane is 0.3-1.0 mm, and the internal structure has a grid-like microstructure with pore size of 50-100 μm, which promotes uniform spore attachment and uniform mineralization reaction.

[0021] The reducing agent coating is a mixture of manganese dioxide, PEDOT:PSS, DMSO and deionized water. The reducing agent coating needs to be coated and dried in 3-5 cycles to form a stable reaction layer.

[0022] As a preferred option, the amounts of each component are 350 mg MnO2, 2 mL PEDOT:PSS, 100 μL DMSO, and 5 mL deionized water.

[0023] The MICP antibacterial functional layer is a bacterial cellulose membrane loaded with Bacillus pasteurellii spores, calcium chloride, urea, graphene oxide, PEDOT:PSS and DMSO.

[0024] The resistivity of the carbon adhesive conductive layer is less than 10 Ω·cm.

[0025] Preferably, the MICP antibacterial functional layer is a 3D-printed bacterial cellulose membrane loaded with Bacillus pasteurii spores, calcium chloride, urea, and graphene oxide. It can achieve a repair function through microbial-induced calcium carbonate precipitation and achieve natural antibacterial effects through substances secreted by the bacterial strain. The Bacillus pasteurii is *S. pasteurii*, which enhances the antibacterial effect of the wound by synergistically reacting its secreted antibacterial substances with the alkaline environment generated by the reaction of MICP, unlike Bacillus subtilis dressings which only have a single antibacterial function. The forward and reverse separated paper-based microbial fuel cell component can provide micro-electrical stimulation through bacterial metabolic electricity generation. The MICP antibacterial functional layer achieves dynamic wound closure through microbial-induced CaCO3 precipitation, and can adapt to changes in the pH of the wound exudate to adjust the mineralization process, enhancing wound sealing. The graphene oxide concentration is 2-5 mg / mL, which can adsorb CaCO3. 2+ Improve MICP deposition efficiency and reduce internal resistance of paper-based MFC modules.

[0026] The *Pasteurella* species is *Sporosarcina pasteurii*, preferably ATCC11859.

[0027] Furthermore, the loading concentration of the *Pasteurella multocida* spores is 10. 8 -10 9 CFU / cm².

[0028] The preparation method of the microbial mineralization self-generating antibacterial wound dressing of the present invention includes the following steps:

[0029] (1) Preparation of 3D-printed bacterial cellulose membranes:

[0030] After activation, *Bacillus pasteurellii* was induced to produce sporulation. Bacterial cellulose was mixed with sodium alginate to prepare a 3D-printable bio-ink. The bio-ink was then printed into a bacterial cellulose membrane using a 3D bioprinter. The printed bacterial cellulose membrane was first immersed in a solution containing PEDOT:PSS and DMSO, and then immersed in a mixed solution containing CaCl2, urea, and graphene oxide. The BC membrane was then removed, and a suspension of *Bacillus pasteurellii* spores was sprayed onto its surface. The membrane was then air-dried for later use.

[0031] (2) Assembly of front- and back-reverse separated paper-based MFC components:

[0032] Using cellulose chromatography paper as a substrate, wax is added to the central region of the chromatography paper until it completely melts and penetrates the paper, then cooled and solidifies to form a wax isolation layer. The 3D-printed bacterial cellulose membrane prepared in step (1) is then attached to the inner region of the isolation layer near the wound. On the outer region of the isolation layer away from the wound, a reducing agent coating is applied using a coating-drying cycle process. Conductive carbon adhesive is used to draw conductive layers on the edges and sides of both sides of the chromatography paper.

[0033] (3) Component integration and sterilization:

[0034] The assembled MFC components are covered with a medical polyurethane transparent dressing as an outer breathable protective layer and then sterilized; after sterilization, they are packaged and refrigerated.

[0035] In step (3), the conductive carbon adhesive is used to draw a conductive layer on the edges and sides of both sides of the chromatography paper, including:

[0036] (1) On the side near the wound: Draw a 2-5 mm wide annular conductive layer around the edge of the BC membrane;

[0037] (2) On the side away from the wound: Draw a ring-shaped conductive layer with a width of 2-5 mm around the reducing material area, and draw an X-shaped pattern in the center of the cathode area; drawing an X-shaped pattern in the center of the cathode area can better guide electrons to the cathode; compared with more complex patterns, the X-shaped pattern is more convenient.

[0038] (3) Both sides: Connect the conductive rings on both sides with carbon glue to form a closed circuit.

[0039] The application of the microbial mineralization self-generating antibacterial wound dressing described in this invention in the preparation of chronic wound care materials or drugs.

[0040] The chronic wounds include diabetic ulcers, pressure ulcers, and burn wounds.

[0041] This invention is the first to integrate microbial induced mineralization (MICP) technology with paper-based biofuel cell (MFC) technology into wound dressings. It is the first to fuse MIP and paper-based MFC technologies, using *S. pasteurii* to induce the formation of a dynamic mineralization membrane from CaCO3. This membrane responds to changes in the pH of wound exudate, achieving adaptive wound closure and continuously releasing calcium ions to aid tissue regeneration, overcoming the limitations of traditional dressings that only offer dual functions of 'antibacterial + electrical stimulation'. A composite structure is constructed using low-cost cellulose chromatography paper as a carrier, consisting of an outer breathable protective layer, a forward-reverse separated paper-based MFC component, and a 3D-printed bacterial cellulose MICP functional layer. The MICP functional layer utilizes a 3D-printed bacterial cellulose membrane with a mesh pore size of 50-100 nm, effectively promoting the uniform attachment of *Bacillus pasteurellii* spores and ensuring the uniformity of the mineralization reaction. This membrane is loaded with *Bacillus pasteurellii* spores, CaCl2, urea, graphene oxide, PEDOT:PSS, and DMSO, and can be activated in situ by wound exudate. On one hand, it deposits a CaCO3 mineralization membrane in situ on the wound surface through the MICP reaction to seal the wound and promote tissue regeneration; on the other hand, it utilizes lipopeptides secreted by the strain... Organic acids achieve broad-spectrum natural antibacterial effects; the paper-based MFC component uses cellulose chromatography paper as a substrate, forming a central isolation zone through high-temperature molten wax, and setting an anode (bacterial cellulose membrane) and a cathode (reducing substance coating) on ​​both sides respectively. The reducing substance in the cathode area needs to undergo 3-5 coating-drying cycles to form a stable reaction layer; a 2-5mm wide carbon adhesive conductive layer is set around the perimeter and sides of the component, forming a three-dimensional low-resistance conductive circuit around the edges and connecting the sides, realizing continuous micro-electric stimulation driven by bacterial metabolism, effectively promoting angiogenesis and epithelial migration. This invention achieves a breakthrough in realizing the three-in-one synergistic function of "mineralization repair - natural antibacterial - self-generating power stimulation", solving key problems such as the single function of traditional dressings, the dependence of electrical stimulation dressings on external power sources, and insufficient mechanical strength of hydrogels. The 3D-printed micro-mesh structure makes the spore distribution more uniform and the mineralization more uniform, and has the advantages of excellent biocompatibility, high adhesion, low cost and biodegradability.

[0042] This invention integrates three core functions—microbial mineralization repair, self-generated micro-electrical stimulation, and natural antibacterial properties—to achieve active wound repair. Microbial mineralization induces calcium carbonate precipitation to seal the wound; self-electrical stimulation relies on bacterial metabolic electricity generation from a forward-reverse separated paper-based MFC component; and antibacterial properties are achieved synergistically through in-situ secretion of substances by bacterial strains. This invention innovatively replaces traditional hydrogels with 3D-printed bacterial cellulose membranes as the carrier for the MCP functional layer. The forward-reverse separated MFC structure, combined with a three-dimensional carbon gel conductive network, solves key problems such as the single function of traditional dressings, reliance on external power sources for electrical stimulation dressings, and insufficient mechanical strength of hydrogels. After the dressing adheres to the wound, exudate activates *Pasteurella multocida* spores in the 3D-printed bacterial cellulose membrane. On one hand, through the MCP reaction, a CaCO3 mineralization membrane is formed in situ on the wound surface, promoting tissue regeneration; on the other hand, lipopeptides and organic acids are secreted to achieve broad-spectrum antibacterial effects. Simultaneously, electrons generated by bacterial metabolism form a stable microcurrent through the three-dimensional carbon gel conductive network, continuously providing micro-electrical stimulation and accelerating angiogenesis and epithelial migration. This invention achieves a synergistic treatment integrating "mineralization repair - natural antibacterial - self-generated stimulation" without the need for an external power source, making it particularly suitable for intelligent care of chronic, difficult-to-heal wounds such as diabetic foot ulcers, pressure ulcers, and burns.

[0043] The working principle of this invention is as follows: When the dressing is applied to the wound, moisture and nutrients in the wound exudate permeate into the 3D-printed bacterial cellulose membrane, activating the attached Bacillus pasteurellium spores. Because the 3D-printed bacterial cellulose membrane has a mesh pore structure with a diameter of 50-100 nm, the spores are distributed more evenly within the membrane, ensuring uniform microbial activation.

[0044] The activated strain, on the one hand, catalyzes the hydrolysis of urea in the exudate to generate NH4 through urease catalysis. + and CO3 2- It is worth noting that, in addition to enhancing the mechanical properties of the membrane, the graphene oxide loaded in the 3D-printed bacterial cellulose membrane can specifically adsorb Ca loaded within the membrane due to its negative surface charge. 2+ , making Ca 2+ More evenly mixed with CO3 2- This combination promotes the formation of a uniformly distributed CaCO3 mineralization film on the wound surface. In particular, when wound infection leads to an increase in the pH of the exudate, the urease activity of S. pasteurii will naturally increase, further accelerating the formation of the mineralization film, achieving adaptive wound closure, and continuously releasing calcium ions to promote cell proliferation. On the other hand, the multi-component antibacterial substances secreted by the strain (such as lipopeptides, aminoglycoside antibiotics, organic acids, etc.) diffuse into the wound surface through the microporous structure of the bacterial cellulose membrane, directly acting on the bacteria and destroying their cell membranes to achieve broad-spectrum antibacterial activity.

[0045] In terms of electrical stimulation, electrons generated by the bacterial strain's metabolism can be accelerated through the high conductivity of graphene oxide and PEDOT:PSS. These electrons rapidly converge in the carbon conductive layer via the graphene oxide and PEDOT:PSS in the bacterial cellulose membrane, forming an efficient transmission pathway. Due to the use of a forward-reverse separated MFC structure, the 3D-printed bacterial cellulose membrane near the wound acts as the anode, while the reducing agent coating away from the wound acts as the cathode. A 2-5mm wide carbon conductive layer forms a three-dimensional low-resistance conductive loop around the paper and on both sides, significantly shortening the electron transport path and reducing internal resistance. In particular, the X-shaped carbon pattern at the center of the cathode increases the contact area with the reducing agent. Combined with the stable reducing agent layer formed by 3-5 coating-drying cycles, this ensures the high efficiency and stability of electron transmission. The resulting microcurrent continuously stimulates the local electric field at the wound site, accelerating angiogenesis and epithelial cell migration. Simultaneously, changes in the MFC's voltage can reflect bacterial activity and wound healing status in real time, providing dynamic monitoring data for clinical assessment.

[0046] This invention uses *Bacillus pasteurellii* instead of the conventional *Bacillus subtilis*, due to fundamental differences in their electrogenic mechanisms and antibacterial substances. *Bacillus pasteurellii* is a urease-positive bacterium, its core function being to generate electrons by decomposing urea to form an electric current; while *Bacillus subtilis*'s electrogenic pathway does not depend on urease, and the antibacterial active substances secreted by the two also differ significantly. This invention focuses MICP (microbial-induced calcium carbonate deposition) technology on the medical field, with applications in environmental remediation and building reinforcement that are significantly different from existing methods. In medical settings, the treatment principle utilizes the biomineralization properties of MICP to regulate the local microenvironment, rather than traditional chemical drug interventions or physical therapy methods.

[0047] This invention effectively solves the problem of the single wound healing promotion mechanism in existing technologies. This invention adds minerals produced by microorganisms as an auxiliary agent. The material prepared by this invention produces calcium carbonate through urease-producing bacteria, thereby promoting wound healing; it also produces antibacterial substances to inhibit pathogen growth and prevent infection; and it can generate electric current through metabolism, promoting wound healing through electrical stimulation.

[0048] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0049] High functional integration and significant synergistic repair effect: This invention achieves a breakthrough in realizing the synergistic function of "mineralization repair - natural antibacterial - self-generating stimulation", solving the problem of the single function of traditional dressings. The 50-100nm grid structure of the 3D printed bacterial cellulose membrane makes the spore distribution more uniform and the mineralization more uniform, ensuring the stable performance of MICP function; the positive and negative separated MFC structure combined with the three-dimensional carbon gel conductive network (width 2-5mm) greatly improves the power generation efficiency, effectively overcoming the defects of existing bioelectric stimulation dressings that "can only stimulate and require additional antibacterial / repair methods".

[0050] The advantages of 3D printing structure are significant: Compared with traditional hydrogels, 3D printed bacterial cellulose membranes have superior mechanical strength and adhesion, making them particularly suitable for wound care in easily movable areas such as joints and feet; the 50-100nm mesh pore size not only promotes uniform spore attachment but also ensures uniform mineralization reaction, avoiding problems of local over-mineralization or under-mineralization; at the same time, this structure is conducive to the absorption and transport of wound exudate, maintaining a suitable moist environment.

[0051] Three-dimensional conductive network enhances electrical performance: The positive and negative separated MFC structure, combined with the carbon adhesive conductive layer surrounding the edges and connecting sides, forms a three-dimensional low-resistance conductive loop. Compared with the traditional planar conductive structure, the three-dimensional conductive loop significantly shortens the electron transport path, reduces the internal resistance of the component, and improves electron transport efficiency and power density stability. The X-shaped carbon adhesive pattern in the center of the cathode area, combined with the 3-5 coating-drying cycle process, ensures the stability of the reducing material layer and the high efficiency of the electrochemical reaction.

[0052] High safety due to wound isolation of reducing agent: This invention employs a front-and-back separated MFC structure design, completely isolating the reducing agent coating on the side away from the wound. This ensures that the reducing agent (containing manganese dioxide and other components) does not directly contact wound tissue, avoiding potential chemical irritation or adverse reactions. Simultaneously, efficient electron transfer is achieved through a carbon conductive layer, ensuring the electrical performance of the MFC component while significantly improving the dressing's safety. This structural design is a key innovation of this invention, resolving the safety hazard of potential adverse effects on the wound from reducing agents in traditional electrical stimulation dressings.

[0053] Safe, environmentally friendly, and cost-controllable: It relies on wound exudate to activate functional components in situ, without the need for external addition of enzymes, antibiotics, or other substances; the lipopeptides and organic acids secreted by Bacillus pasteurellium have no risk of drug resistance; all materials are biocompatible, biodegradable natural or synthetic materials with no heavy metal residues; the core materials (cellulose chromatography paper, CaCl2, etc.) are inexpensive, and the preparation process uses mature technologies such as 3D printing and screen printing, requiring no complex equipment and suitable for large-scale production.

[0054] Wide adaptability and high clinical application value: The size can be customized according to the size of the wound, and it is suitable for a variety of chronic and difficult-to-heal wounds such as diabetic foot ulcers, pressure ulcers, and burns; it does not depend on external power sources, making it particularly suitable for primary healthcare and home care scenarios; the electrical generation performance of MFC can indirectly reflect the wound healing status, providing real-time assessment data for clinical use; after being sterilized with ethylene oxide, the dressing can be stably stored for 12 months, meeting the needs of long-term care. Attached Figure Description

[0055] Figure 1 is a schematic diagram of the structure of the wound dressing of the present invention; the markings in the figure are: 1 polyurethane protective layer; 2 outer carbon conductive layer (outermost upper layer); 3 reducing substance; 4 side carbon conductive layer; 5 chromatography paper / wax isolation layer; 6 MICP antibacterial functional layer (3D printed bacterial cellulose membrane containing Bacillus pasteurellii spores, CaCl2, urea, GO, which is the functional core layer); 7 carbon conductive layer (innermost lower layer).

[0056] Figure 2 is a flowchart of the synergistic mechanism of microbial mineralization, self-generated power and broad-spectrum antibacterial activity of the present invention.

[0057] Figure 3 shows the inhibition zone test results of Bacillus pasteurellii and Bacillus subtilis in Example 2 of the present invention;

[0058] Figure 4 shows the performance test results of the paper-based MFC component in Embodiment 3 of the present invention;

[0059] Figure 5 shows the XRD analysis results of the dressing in Example 4 of the present invention;

[0060] Figure 6 This is a schematic diagram of the antibacterial substance secreted by Bacillus pasteurellum used in this invention. Detailed Implementation

[0061] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0062] Example 1

[0063] I. Core Components and Parameters

[0064] The wound dressing of this invention has a multi-layer composite structure (size can be customized, standard size 10×10cm), and the components and parameters are as follows:

[0065] (I) Outer breathable protective layer: The material is medical polyurethane transparent dressing, which has breathable, waterproof and stain-resistant functions, and can effectively isolate external pollution and maintain the stability of the wound microenvironment.

[0066] (II) Reverse-phase separated paper-based microbial fuel cell module (self-generating core):

[0067] 1. Substrate: A 10×10cm cellulose chromatography paper, 0.34mm thick, with an inherent flow rate of 130mm / 30min;

[0068] 2. Wax isolation layer: Located in the central area of ​​the chromatography paper, it is an isolation band formed by printing and high-temperature melting and penetration, ensuring that the paraffin wax completely penetrates the chromatography paper (preferably with a uniform wax layer visible on the back of the chromatography paper). After curing, it forms a sealed isolation band that separates the anode and cathode areas.

[0069] 3. Anode Zone: Located on the side of the chromatography paper closest to the wound, inside the wax isolation layer, is a 3D-printed bacterial cellulose membrane. This membrane is 3D printed and has a mesh-like structure with pore sizes of 50-100 μm, which can effectively promote the uniform attachment of Bacillus pasteurellii spores and the uniform mineralization reaction; the membrane is loaded with Bacillus pasteurellii spores (concentration 10). 8 -10 9 CFU / cm 2 ), calcium chloride (0.5 mol / L), urea (0.5 M), and graphene oxide (2 mg / mL).

[0070] 4. Cathode Zone: The reducing agent coating located on the side of the chromatography paper away from the wound and outside the wax isolation layer needs to be coated and dried 3-5 times to form a stable reaction layer. The composition of the reducing agent coating is as follows: Preparation of reducing agent coating: Weigh 350mg MnO2, add 2mL PEDOT:PSS (1.4wt%, Shanghai Titan Scientific Co., Ltd.), 100uL DMSO, and 5mL deionized water;

[0071] (III) Carbon conductive layer: 4mm wide, located on both the edges and sides of the chromatography paper, including:

[0072] 1. Carbon adhesive near the wound forms a conductive ring around the 3D-printed bacterial cellulose membrane;

[0073] 2. The carbon adhesive on the side away from the wound forms a conductive ring around the reducing agent coating, and an X-shaped pattern is drawn in the center of the cathode area to increase the effective reaction area;

[0074] 3. The carbon adhesive on both sides connects the conductive rings on both sides to form a three-dimensional low-resistance conductive circuit with a resistivity of less than 10 Ω·cm;

[0075] (iv) MICP antibacterial functional layer: namely the 3D printed bacterial cellulose membrane mentioned above, which has the following functional characteristics:

[0076] 1. The 50-100nm mesh aperture formed by 3D printing makes the distribution of Bacillus pasteurellii spores more uniform, ensuring that the MICP reaction proceeds uniformly;

[0077] 2. The uniform dispersion of GO in the BC membrane not only enhances the membrane's mechanical properties, but its surface negative charge can also specifically adsorb Ca. 2+ It assists the efficient execution of the MICP reaction; at the same time, its excellent conductivity can optimize the electron transport path, reduce the internal resistance of paper-based MFC components, and realize the bridging and synergy of MICP and MFC functions.

[0078] 3. The thickness of the mineralized film can be controlled by adjusting the CaCl2 concentration (0.2-0.4 mol / L);

[0079] 4. After activation, the strain generates CO3 through urease-catalyzed hydrolysis of urea. 2- , with Ca 2+ It combines to form a CaCO3 mineralized film to seal the wound, while secreting lipopeptides, organic acids and other substances to achieve natural broad-spectrum antibacterial effects;

[0080] In addition to secreting lipopeptides, aminoglycoside antibiotics, and organic acids, 5.S. pasteurii also raises the local pH of the wound to 9-10 through the MICP reaction, inhibiting acid-sensitive pathogens such as Staphylococcus aureus, and achieving a passive-active synergistic effect of 'physical precipitation blocking + chemical antibacterial'.

[0081] 6. The MICP antibacterial functional layer has pH-responsive properties: When the pH of wound exudate increases (such as pH>7.5 during infection), it can directly enhance the urease activity of S. pasteurii and accelerate CaCO3 precipitation to strengthen wound closure; when the pH returns to normal, the urease activity is simultaneously reduced to avoid over-mineralization.

[0082] II. Specific Preparation Method

[0083] (I) Preparation of 3D-printed bacterial cellulose membranes:

[0084] 1. Pasteurella multocida (ATCC 11859) was inoculated into LB medium and activated by incubation at 37°C in a shaker for 24 hours;

[0085] 2. Transfer to Schaeffer-Fulton sporulation medium (formulation: 0.8% nutrient broth, 0.05% yeast extract, 1% glucose, 10% manganese sulfate, pH=7.2), and incubate at 30℃ for 60h to induce sporulation;

[0086] 3. After microscopic examination confirms sporulation, collect the spores by centrifugation, wash three times with sterile physiological saline, and resuspend at OD. 600 =1.0;

[0087] 4. Bacterial cellulose (BC, lyophilized powder, Xidian Experiment) and sodium alginate (SA) were mixed at a mass ratio of 3:7 to prepare a 3D printable bio-ink. The specific process was as follows: 0.86 g of bacterial cellulose (BC) and 2.0 g of sodium alginate (SA) were weighed and added to 100 mL of sterile deionized water to prepare a mixed solution with a total solids content of approximately 2.86% (w / v). The solution was dispersed using an ultrasonic mixer for 15 minutes, with a 30-second pause every 3 minutes to avoid overheating. After ultrasonic mixing, a uniform bio-ink was obtained. A Voladora 3D printer (brand / manufacturer: Tumaker, SL, Spain) was used, compatible with a layer-by-layer syringe extrusion 3D printing system. The printing parameters were: nozzle diameter 0.8 mm, printing speed 4 mm / s. After printing, the scaffold was immersed in a 0.5% (w / v) CaCl2 solution for 30 minutes for cross-linking and curing.

[0088] 5. Use a 3D bioprinter to print bio-ink into bacterial cellulose membranes (BC membranes) that are 4.7×4.7cm in size, 1mm thick, and have a mesh pore size of 50-100μm.

[0089] 6. The printed BC membrane was incubated in a 2 mL PEDOT:PSS and 100 μL DMSO mixed solution at 30 °C and 200 rpm for 2 hours. After incubation, the surface was rinsed with deionized water. Then, it was immersed in a mixed solution containing 0.5 M CaCl2, 0.5 M urea and 2 mg / mL graphene oxide (Suqian Nakaite New Material Technology Co., Ltd., Nct-go02) for 2 hours to ensure that the functional material was uniformly loaded into the BC network.

[0090] 7. Remove the BC membrane and spray the surface with a suspension of Bacillus pasteurellii spores (OD). 600 =1.0), 100 microliters, air dry for later use.

[0091] (II) Assembly of front-and-back separated paper-based MFC components:

[0092] 1. Cut a 5×5cm piece of cellulose chromatography paper (Beijing Saifulaibo Technology Co., Ltd., item number 3030-861) as a substrate;

[0093] 2. In the central area of ​​the chromatography paper, use a wax printer to draw a square area with a side length of about 4.7cm. Then place it on a hot plate and heat it until the wax is completely melted and penetrates the paper (temperature 80-100℃, time 5-10min). Cool and solidify to form a wax isolation layer.

[0094] 3. Precisely attach the 3D-printed BC membrane prepared in step (I) to the inner region of the isolation layer near the wound;

[0095] 4. Apply the reducing agent coating to the outer area of ​​the isolation layer away from the wound using a coating-drying cycle process: add 100 microliters each time, apply it to the cathode area with a metal scraper, and air dry at room temperature for 30 minutes; repeat 5 times; after the last air drying, place the component in a 40°C oven to dry for 1 hour to ensure that the reducing agent is completely cured.

[0096] 5. Apply conductive carbon adhesive to the edges and sides of both sides of the chromatography paper to create a conductive layer:

[0097] (1) On the side near the wound: Draw a 4mm wide annular conductive layer around the edge of the chromatography paper on one side of the BC membrane;

[0098] (2) The side away from the wound: Draw a 4mm wide annular conductive layer around the edge of the chromatography paper on the side of the reducing substance, and draw an X-shaped pattern in the center of the cathode area;

[0099] (3) Side of chromatography paper: The conductive rings on both sides are connected by carbon glue to form a closed circuit;

[0100] (III) Component Integration and Sterilization:

[0101] 1. Cover the assembled MFC component with a medical polyurethane transparent dressing (larger than the chromatography column to completely cover it) as an outer breathable protective layer;

[0102] 2. Place the assembled dressing in an ethylene oxide sterilizer for sterilization. The sterilization temperature is controlled at 37-40℃, the sterilization pressure is 0.05-0.1MPa, the ethylene oxide concentration is 600-800mg / L, and the sterilization duration is 6h.

[0103] 3. After sterilization, an analytical treatment is performed at a temperature of 37℃ for a time of ≥12h to ensure that the residual ethylene oxide in the dressing is ≤10μg / g, which meets the requirements of GB 18279 "Ethylene Oxide for Sterilization of Medical Devices".

[0104] 4. After sterilization, seal in a sterile aluminum-plastic bag and store at 4°C. Shelf life is 12 months.

[0105] Example 2

[0106] Validation and correlation analysis of antibacterial active substances in dressings

[0107] 1. Preparation of active substances: Bacillus pasteurellii (ATCC 11859) and Bacillus subtilis (MCCC 1A14617) were inoculated into LB medium and cultured in a shaker at 37°C for 20 h. OD 600At approximately 2°C (simulating the metabolic environment after in-situ activation of spores in the dressing), the supernatant (containing antibacterial substances such as lipopeptides, aminoglycoside antibiotics, and organic acids secreted by the strain) was collected by centrifugation.

[0108] 2. Preparation of test bacteria: Staphylococcus aureus (ATCC 6538) was inoculated into LB medium and incubated at 37°C for 18 hours, then diluted with sterile physiological saline to OD. 600 =0.1.

[0109] 3. Paper disc diffusion test: Prepare LB agar plates and spread Staphylococcus aureus culture evenly; take a 6 mm diameter sterile antimicrobial susceptibility test paper, add 25 μL of the above supernatant, place 4 discs on each plate, and incubate at 37℃ for 24 h.

[0110] 4. Results and Correlation Analysis: such as Figure 3 As shown, the average diameter of the inhibition zone of *Bacillus pasteurellii* against *Staphylococcus aureus* reached 18 mm, and the pH value around the inhibition zone was 9.2 ± 0.3, confirming that *Bacillus pasteurellii* can secrete effective antibacterial substances. Combined with the "spore in situ activation" mechanism in the dressing, it can be inferred that after the dressing adheres to the wound, the activated strains can produce antibacterial substances through the same metabolic pathway, achieving a wound-soothing effect. The average diameter of the inhibition zone of *Bacillus subtilis* was 11 mm, and there was no pH increase. It also proves that the *Bacillus pasteurellii* used in this invention has better effects. Targeted mass spectrometry analysis and primary mass spectrometry results show that its secreted substances include multiple subtypes of Surfactin, Fengycin, etc., achieving multifunctional antibacterial activity, such as... Figure 6 As shown.

[0111] Example 3

[0112] Performance testing of the microbial mineralization self-generating antibacterial wound dressing prepared in Example 1 of this invention:

[0113] 1. Comparative Example 1 uses a 2D structure (i.e., the anode and cathode regions are on the same side of the cellulose chromatography paper), specifically prepared as follows: The method of Example 1 is followed, except that:

[0114] Assembly of the front-reverse separated paper-based MFC assembly in step (II):

[0115] Paper base pretreatment: Cut cellulose chromatography paper to 6×6 cm, plan an anode area of ​​22×22 mm in the center area, plan a cathode area around the anode area, reserve coating positions, draw a 9 mm wide wax isolation wall with high temperature molten paraffin (ensure that the paraffin completely penetrates the chromatography paper), cool and cure at room temperature, and then air dry for later use.

[0116] Preparation of reducing agent coating: Weigh 350 mg MnO2, add 5 mL deionized water and shake, then add 2 mL PEDOT:PSS and 100 μL DMSO, mix thoroughly and apply 5 layers to the cathode area, and air dry each layer at room temperature for 30 min after coating.

[0117] The preparation of the bacterial culture and the anode material were consistent with the 3D structure in Example 1. The bacterial cellulose membrane containing the functional components was loaded onto the anode region, covering the anode area.

[0118] 2. Comparative Example 2 uses the microbial mineralization self-generating antibacterial wound dressing from Example 1, except that Bacillus pasteurellii is replaced with Bacillus subtilis (Bacillus subtilis MCCC 1A14617).

[0119] 3. Add 100 μL of Bacillus pasteurellium bacterial solution and 100 μL of wound simulation solution (formulation: 0.3 g urea, 0.055 g CaCl2, 0.85 g NaCl, 58 μL lactic acid, add deionized water to make up to 50 mL, adjust pH to 7.4 to simulate the wound exudate environment in the early stage of infection) to the anode area of ​​the material prepared in Example 1 or Comparative Example 1 or 2. Use a multimeter to test the power generation performance.

[0120] Test results: such as Figure 4 As shown, the MFC prepared in Example 1 of this invention has a maximum voltage of 785mV, a maximum current of 101μA, and a maximum power density of 58.1μW / cm². The power generation time varies from 12 to 24 hours depending on the amount of simulated exudate replenished, with an average power density of 7-15μW / cm². This confirms that the paper-based MFC module possesses stable power generation capabilities and can support subsequent integration with MICP functions. In contrast, the 2D structure material prepared in Comparative Example 1 has a maximum voltage of 485mV, a maximum current of 80μA, and a maximum power density of 39μW / cm². Furthermore, the 2D structure inherently suffers from short power generation duration and poor later-stage stability. The Bacillus subtilis MFC prepared in Comparative Example 2 has a maximum voltage of 323mV, a maximum current of 40μA, and a maximum power density of 12μW / cm². This further demonstrates the advantages of the material constructed in this invention, which can be effectively applied to wound antibacterial and healing applications.

[0121] Compared with the horizontal structure, the "positive-negative separation three-dimensional MFC structure" (corresponding to the structure in Figure 1) constructed in Embodiment 1 of the present invention has significant advantages in biosafety, functional synergy and reaction balance through targeted structural design.

[0122] Biocompatibility: In horizontal structures, the cathode and anode areas are on the same plane, making it easy for reducing substances (such as MnO2) to diffuse into the wound with exudate, posing a risk of interfering with the wound healing microenvironment. This invention uses a wax isolation layer to position the cathode area (containing reducing substances) away from the wound, forming a three-dimensional separation with the anode area (a 3D-printed bacterial cellulose membrane close to the wound). This physically blocks the migration path of reducing substances to the wound, avoiding potential irritation of newly formed tissue by chemical substances and better meeting the biocompatibility requirements of medical dressings.

[0123] Functional synergy: The horizontal structure uses hydrogel to load functional components, and its random network easily leads to the formation of Bacillus pasteurellis spores and Ca2+. 2+ The uneven distribution of elements such as spores and graphene oxide leads to imbalances such as "excessive localized electricity generation with insufficient mineralization" or "excessive mineralization with limited electricity generation." The 3D-printed bacterial cellulose membrane of this invention possesses a precise 50-100nm mesh structure, enabling uniform anchoring of spores, CaCl2, urea, and graphene oxide. On one hand, the uniformly distributed spores ensure the stability of metabolic electricity generation; on the other hand, graphene oxide effectively anchors CaCl2, CaCl2, urea, and graphene oxide. 2+ The directional adsorption can simultaneously promote the uniformity of the MIP reaction, enabling the "electricity generation" and "mineralization" functions to be highly matched and synergistically performed in space.

[0124] Reaction uniformity: The horizontally structured annular carbon adhesive design tends to cause electrons to accumulate at the cathode edge and insufficient reaction in the central region, resulting in fluctuations in power generation efficiency. The present invention features an X-shaped carbon adhesive pattern at the center of the cathode region. Its radial structure guides electrons uniformly from the side carbon adhesive conductive layer to the entire cathode region, avoiding localized electron accumulation. Simultaneously, the X-shaped pattern and the grid structure of the 3D-printed BC film in the anode region form a three-dimensional correspondence (the uniformly distributed power generation points on the anode correspond to the uniformly distributed electron receiving points on the cathode), making the MFC power generation reaction more spatially balanced and providing a stable microcurrent output for "electric stimulation to promote healing."

[0125] Example 4

[0126] MICP Functional Verification

[0127] 1. Sample pretreatment: Take the paper-based MFC dressing prepared in Example 1 after 5 hours of power generation, scrape the bacterial cellulose membrane loaded on the anode area with a sterile spatula, gently rinse the surface twice with sterile physiological saline to remove non-specific adsorbents, and blot the surface moisture with filter paper for later use.

[0128] 2. Operation of X-ray diffractometer:

[0129] Its phase composition was determined by X-ray diffraction.

[0130] 3. Results Analysis:

[0131] Qualitative: Through software analysis, such as Figure 5 As shown, the formation of CaCO3 was confirmed. This experiment was conducted in two phases.

[0132] Two curves were obtained, both of which showed the presence of CaCO3.

[0133] The above experiments demonstrate the presence of calcium carbonate in the dressing, further proving the effectiveness of the dressing's MIP function.

Claims

1. A microbial mineralization self-generating antibacterial wound dressing, characterized in that, It includes, from the outside to the inside, an outer breathable protective layer, an outer carbon adhesive conductive layer, a positive and negative separated paper-based microbial fuel cell assembly, and an inner carbon adhesive conductive layer; The forward and reverse separation paper-based microbial fuel cell assembly includes: cellulose chromatography paper and a wax isolation layer located in the central region of the cellulose chromatography paper; The bacterial cellulose membrane (BC) located on the side of the cellulose chromatography paper closest to the wound and loaded on the inner side of the wax isolation layer constitutes the anode region; the reducing agent coating located on the side of the chromatography paper away from the wound and on the outer side of the wax isolation layer constitutes the cathode region; and the side carbon adhesive conductive layers are also provided on both sides of the chromatography paper.

2. The microbial mineralization self-generating antibacterial wound dressing according to claim 1, characterized in that, The outer breathable protective layer is a medical polyurethane transparent dressing, a hydrocolloid transparent dressing, or a silicone-based breathable protective dressing.

3. The microbial mineralization self-generating antibacterial wound dressing according to claim 1, characterized in that, The bacterial cellulose membrane is formed by 3D printing. The thickness of the bacterial cellulose membrane is 0.3-1.0 mm, and the internal structure has a mesh-like microstructure with pore size of 50-100 μm.

4. The microbial mineralization self-generating antibacterial wound dressing according to claim 1, characterized in that, The reducing agent coating is a mixture of manganese dioxide, PEDOT:PSS, DMSO and deionized water. The reducing agent coating needs to go through 3-5 coating-drying cycles to form a stable reaction layer.

5. The microbial mineralization self-generating antibacterial wound dressing according to claim 1, characterized in that, The MICP antibacterial functional layer is a bacterial cellulose membrane loaded with Bacillus pasteurellii spores, calcium chloride, urea, graphene oxide, PEDOT:PSS and DMSO.

6. The microbial mineralization self-generating antibacterial wound dressing according to claim 5, characterized in that, The *Pasteurella* species is *Sporosarcina pasteurii*, preferably ATCC 11859.

7. A method for preparing the microbial mineralization self-generating antibacterial wound dressing according to claim 1, characterized in that, Includes the following steps: (1) Preparation of 3D-printed bacterial cellulose membranes: After activation, *Bacillus pasteurellii* was induced to produce sporulation. Bacterial cellulose was mixed with sodium alginate to prepare a 3D-printable bio-ink. The bio-ink was then printed into a bacterial cellulose membrane using a 3D bioprinter. The printed bacterial cellulose membrane (BC) was immersed in a mixed solution of PEDOT:PSS and DMSO, and then immersed in a mixed solution containing CaCl2, urea, and graphene oxide. The BC membrane was then removed, its surface was sprayed with a suspension of *Bacillus pasteurellii* spores, and air-dried for later use. (2) Assembly of front- and back-reverse separated paper-based MFC components: Using cellulose chromatography paper as a substrate, wax is added to the central region of the chromatography paper until it completely melts and penetrates the paper, then cooled and solidifies to form a wax isolation layer. The 3D-printed bacterial cellulose membrane prepared in step (1) is then attached to the inner region of the isolation layer near the wound. On the outer region of the isolation layer away from the wound, a reducing agent coating is applied using a coating-drying cycle process. Conductive carbon adhesive is used to draw conductive layers on the edges and sides of both sides of the chromatography paper. (3) Component integration and sterilization: The assembled MFC components are covered with a medical polyurethane transparent dressing as an outer breathable protective layer and then sterilized; after sterilization, they are packaged and refrigerated for storage.

8. The preparation method according to claim 7, characterized in that, Step (2) involves using conductive carbon adhesive to draw conductive layers on the edges and sides of both sides of the chromatography paper, including: (1) On the side near the wound: Draw a ring-shaped conductive layer with a width of 2-5 mm around the edge of the chromatography paper on one side of the BC membrane; (2) The side away from the wound: Draw a ring-shaped conductive layer with a width of 2-5 mm around the edge of the chromatography paper on the side of the reducing substance, and draw an X-shaped pattern in the center of the cathode area; (3) Side: Connect the conductive rings on both sides with carbon glue to form a closed circuit.

9. The use of the microbial mineralization self-generating antibacterial wound dressing of claim 1 in the preparation of chronic wound care materials or medicines.

10. The application according to claim 9, characterized in that, The chronic wounds include diabetic ulcers, pressure ulcers, and burn wounds.

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