Chiral multi-stage structure hydroxyapatite nanosheet / bacterial cellulose membrane, preparation thereof and application of chiral multi-stage structure hydroxyapatite nanosheet / bacterial cellulose membrane in elderly skin wound dressing

Abstract

The invention relates to the technical field of chiral materials and application thereof, in particular to a chiral multi-stage structure hydroxyapatite nanosheet / bacterial cellulose membrane, preparation thereof and application of the chiral multi-stage structure hydroxyapatite nanosheet / bacterial cellulose membrane in senile skin wound dressing. The chiral multi-stage structure hydroxyapatite nanosheet / bacterial cellulose membrane is prepared from chiral hydroxyapatite nanosheets and bacterial cellulose through a biological mineralization method. The chiral hydroxyapatite nanosheet grows on the bacterial cellulose membrane. According to the chiral multilevel structure hydroxyapatite nanosheet / bacterial cellulose membrane, a simple molecular induced self-assembly strategy is adopted, the synthesis method is simple and controllable, a unique chiral structure is introduced, and new medical application is expanded; when being used as a wound dressing, the hydrogel has low sensitivity and safety, effectively reduces inflammatory response and promotes tissue regeneration by regulating and controlling an immune microenvironment, and realizes multi-stage accurate regulation and control of a wound healing process.

Description

Technical Field

[0001] The present invention relates to the technical field of chiral materials and their applications, and in particular to a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane, its preparation and application in wound dressings for elderly skin. Background Art

[0002] With the increase of age, the clinical characteristics of the skin are that the dermis gradually thins, becomes dry, and its elasticity decreases. At the same time, senescent cells accumulate with age and secrete factors such as the senescence-associated secretory phenotype (SASP), thereby inhibiting the macrophage-dependent clearance function and leading to skin aging. Intrinsic skin aging is manifested as dysfunction of skin appendages and cell characteristics of extended cell cycle and decreased mitotic activity, especially in keratinocytes, resulting in delayed wound healing. However, epidermal aging is related to lipid metabolism disorders, manifested as impaired permeability barrier function and delayed recovery after external injury. Delayed wound healing increases the risk of local infection and systemic complications, and at the same time brings a significant socio-economic burden to the affected individuals and the healthcare system. At present, the molecular mechanism between skin aging and impaired wound healing is still unclear, which limits the development of effective therapeutic interventions for age-related wound healing disorders.

[0003] So far, an important class of biomaterials, including hydrogels, nanofiber membranes and bacterial cellulose (BC) fiber membranes, has been used in wound healing management through the dual mechanisms of regulating the inflammatory response and promoting wound tissue regeneration. Bacterial cellulose (BC) fiber membranes have special mechanical strength, excellent water retention and inherent biocompatibility, and are a promising functional wound dressing that can control the delivery of drugs to the wound bed while preventing microbial colonization. However, the BC fiber membrane lacks antibacterial activity and cannot release bioactive compounds to effectively regulate inflammation.

[0004] Compared with the young, the reduction of Langerhans cells, keratinocytes and fibroblasts in the elderly leads to impaired pathogen clearance, delayed re-epithelialization and weakened extracellular matrix reorganization ability, jointly resulting in reduced wound healing efficiency in the elderly. In addition, the aging skin immune microenvironment is characterized by macrophage dysfunction, impaired phagocytic activity and increased neutrophil infiltration, indicating impaired skin barrier and slowed healing. Notably, recent studies have shown that macrophages involved in the wound healing of aging skin tend to exhibit pro-inflammatory characteristics. As an inorganic biomaterial, hydroxyapatite (HAP) can mechanically strengthen the structure of biopolymers and regulate the immune microenvironment, especially by activating macrophages and regulating their polarization. Considering that chirality is a fundamental feature at all scales in nature and life sciences, the inventors conducted research (see Patent CN114105114B for details) and found that chirality-induced hydroxyapatite can promote bone bonding and regeneration. Emerging evidence suggests that chiral polymers, such as the L-form, have immunomodulatory properties and may contribute to activating immune components and signaling pathways. However, the role of chiral polymers in immunomodulation has been rarely studied.

[0005] In summary, with aging, obvious skin aging characteristics such as thinning of the dermis and decreased elasticity occur. The accumulation of senescent cells and secreted factors inhibit macrophage function, leading to skin aging and delayed wound healing. Epidermal aging is also associated with dysregulation of lipid metabolism. Delayed wound healing brings risks and economic burdens, and its molecular mechanism is unclear, restricting effective treatment. Existing wound healing-related biomaterials, such as BC fiber membranes, although having advantages, lack antibacterial activity and effective inflammatory regulation ability, and also lack a comprehensive and precise treatment plan for aging skin wounds, without fully considering the impact of senescent cells. In terms of material preparation, existing hydroxyapatite materials have a single structure and lack chiral ordered arrangement, restricting their application in biomedical and other fields, and there is also less research on the immunomodulation of chiral-structured polymers. Summary of the Invention

[0006] Based on the above-mentioned disadvantages existing in the prior art, the object of the present invention is to provide a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane and its preparation and application in dressings for elderly skin wounds. This application aims to explore the changes in the immune microenvironment and wound healing characteristics of elderly wounds, and develop a chiral wound dressing that can effectively promote the healing of aging wounds and immunomodulation. A chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane provided by the present invention is synthesized by a chiral glutamate molecule-induced self-assembly strategy. The biosecurity of the material is verified by in vivo experiments. The chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane has a wound healing promoting effect on elderly wounds. In addition, the present invention also uses NOD-Prkdc scid Il2rg nullA skin defect model was constructed in severely immunodeficient mice (NPSG). After targeted knockout of T / B / NK cells, the role of innate immune cells in wound healing mediated by organisms (such as mice) was observed.

[0007] Based on the above objectives, the present invention provides the following technical solutions.

[0008] One of the technical solutions of the present invention provides a chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane, which is prepared by a biomineralization method from chiral hydroxyapatite nanosheets and bacterial cellulose (BC). The chiral hydroxyapatite nanosheets are prepared from simulated body fluid (SBF) and chiral glutamic acid. The chiral glutamic acid (GA) serves as a structure-directing agent and an asymmetric inducer, enabling calcium ions and phosphate ions in the simulated body fluid (SBF) to gradually form crystal nuclei of calcium phosphate compounds and finally form an asymmetric crystal (chiral mesostructured hydroxyapatite nanoplatelets, CMHAP) structure. The chiral hydroxyapatite nanosheets grow on the bacterial cellulose membrane.

[0009] SBF is a solution with an ion concentration similar to that of human plasma and is commonly used to evaluate the bioactivity of biomaterials in vitro. SBF can serve as a simulated environment for coating preparation to deposit a calcium phosphate coating on the material surface, simulating the mineralization process of bone tissue in vivo. The prepared coating has good bioactivity and biocompatibility.

[0010] Another technical solution of the present invention provides a method for preparing a chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane, and the preparation method includes the following steps: S1. Chiral glutamic acid, SBF, Tris, and hydrochloric acid are sequentially added to deionized water to obtain a mixed solution; S2. The bacterial cellulose membrane (BC) is washed clean with deionized water and absolute ethanol in sequence, and then the bacterial cellulose membrane (BC) is placed in the freshly prepared mixed solution in step S1 for a biomineralization reaction; S3. After the biomineralization reaction ends, the bacterial cellulose membrane with chiral hydroxyapatite (BC@CMHAP) is taken out, washed clean and dried to obtain the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane.

[0011] Furthermore, in step S1, the chiral glutamic acid is selected from any one of L-glutamic acid, D-glutamic acid, or racemic glutamic acid; the concentration of chiral glutamic acid in the mixed solution is 0.1 - 1 mM, preferably 0.2 mM; the calcium ions (Ca 2+)has a concentration of 0.1 to 1 mM, preferably 0.26 mM; the contained phosphate ions (PO 4 3- ) has a concentration of 1 to 10 mM, preferably 5 mM.

[0012] Furthermore, the dosage ratio of the bacterial cellulose membrane (BC) to the mixed solution in step S2 is (0.0001 - 0.001) g : (10 - 100) mL, preferably 0.0005 g : 20 mL; the pH value of the solution during the reaction is controlled within 7.2 - 7.4. The time for the biomineralization reaction in step S2 is 12 - 48 h.

[0013] Furthermore, the drying time in step S3 is 2 - 48 h, and the temperature is 20 - 60 °C; preferably, the time is 12 - 24 h and the temperature is 37 °C.

[0014] The third technical solution of the present invention provides an application of a chiral multi - level structure hydroxyapatite nanosheet / bacterial cellulose membrane as a wound dressing.

[0015] Furthermore, the chiral multi - level structure hydroxyapatite nanosheet / bacterial cellulose membrane has good biosafety, has a significant wound - healing effect on elderly wounds, and effectively reduces the inflammatory response and promotes tissue regeneration by regulating the immune microenvironment.

[0016] The fourth technical solution of the present invention provides a wound dressing, and the active ingredient of the wound dressing is: a chiral multi - level structure hydroxyapatite nanosheet / bacterial cellulose membrane.

[0017] Furthermore, the wound dressing is a dressing for elderly skin wounds.

[0018] Furthermore, the chiral multi - level structure hydroxyapatite nanosheet / bacterial cellulose membrane is applied to the skin and directly adheres to the skin wound.

[0019] In summary, the technical solution of the present invention designs chiral hierarchical hydroxyapatite (CMHAP) nanosheets onto bacterial cellulose (BC) fiber membranes through a simple chiral glutamic acid (GA)-mediated self-assembly method. The BC@CMHAP composites synthesized from L-glutamic acid (L - glutamate), D-glutamic acid (D - glutamate), and R-glutamic acid (racemic - glutamic acid) are systematically named L-CHB, D-CHB, and R-CHB respectively (where the prefixes L, D, and R represent different chirality, CH represents CMHAP, and B represents BC). To deeply explore the regeneration potential of chiral biomaterials in aged skin, the inventors selected a full - thickness dorsal skin injury model of aged mice to conduct experiments and simultaneously evaluate the immunomodulatory effects of the materials. The results showed that the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane could not only closely adhere to the wound to form an effective coverage but also significantly reduce the recruitment of neutrophils, demonstrating good wound care effects. In addition, L-CHB promoted the polarization of M1 macrophages to the M2 phenotype, thereby reducing inflammation and enhancing tissue repair. To identify the specific immune cells involved, the inventors used NPSG mice for research, and the results showed that M2 macrophages aggregated at the wound surface and secreted anti - inflammatory cytokines. These observations indicate that the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane provides a protective barrier, regulates macrophage polarization, and jointly promotes wound healing.

[0020] Compared with the prior art, the present application has at least the following advantages: (1) Introduction of a unique chiral structure: Most of the hydroxyapatite materials obtained in the prior art do not have chirality. However, in this solution, through a simple chiral glutamic acid molecule - induced self - assembly strategy, chiral hierarchical hydroxyapatite nanosheets are successfully grown in situ on the surface of bacterial cellulose membranes, preparing hydroxyapatite / bacterial cellulose membranes with chiral structures, filling the gap in this aspect of the prior art, and expanding new possibilities for the application of hydroxyapatite materials in biomedical and other fields; (2) Simple and controllable synthesis method: A simple molecular - induced self - assembly strategy is adopted. Chiral glutamic acid molecules coordinate with calcium ions to form complexes. Chiral glutamic acid is used as a structure - directing agent and an asymmetric inducer, and the pH value of the reaction system is adjusted by Tris - hydrochloric acid buffer solution to control the nucleation and crystal growth rates, ultimately achieving the formation of an asymmetric crystal structure. This method is relatively simple to operate and can precisely regulate the structure and properties of the materials; (3) Targetedly solve the problem of wound healing in aging skin: In the existing technology, there is a lack of a comprehensive and targeted treatment plan for the research on wound healing in aging skin. In this invention, the prepared chiral film is used as a wound healing material, and its low sensitivity and safety are confirmed through in vitro and in vivo experiments, indicating that the L-CHB film has a good effect on promoting the healing of elderly wounds, providing an effective solution to this clinical problem; (4) Multistage regulation of the wound healing process: The L-CHB film in this solution plays a role at different stages of wound healing. In the early inflammatory response stage, it can effectively reduce the pro-inflammatory cytokines in the wound surface, reduce the aggregation of neutrophils and M1 macrophages, and regulate the immune microenvironment; in the later tissue repair stage, it can reduce wound fibrosis and promote tissue regeneration, achieving precise multistage regulation of the wound healing process. Description of the Drawings

[0021] Figure 1 are the scanning electron microscope images of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1 (BC@CMHAP membranes, namely L-CHB, D-CHB, R-CHB, Ach-HB respectively, hereinafter Figures 2 - 8 the same); Figure 2 are the XRD spectra of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 3 are the ultraviolet-visible diffuse reflection (DRUV-Vis) and circular dichroism (DRCD) spectra of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 4 are the Fourier transform infrared spectra of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 5 are the stress-strain curves of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 6 are the tensile strengths of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 7 are the maximum elongation rates of bacterial cellulose membrane (BC), chiral multistructured hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1 to 3 and Comparative Example 1; Figure 8The contact angles of the bacterial cellulose membrane (BC), the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1 were measured to characterize their hydrophilicity and hydrophobicity; Figure 9 The results of (A) HE staining, (B) Masson staining, and (C) CD45 immunohistochemical staining of the subcutaneous implanted materials in NPSG mice on the 3rd, 7th, and 14th days in Example 5 are shown; in the figures, BC represents the bacterial cellulose membrane treatment group, and L-CHB, D-CHB, R-CHB, and Ach-HB represent the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1, respectively; Figure 10 The HE staining results of the major organs of NPSG mice after subcutaneous implantation of different materials for 2 weeks in Example 5 are shown; in the figures, BC represents the bacterial cellulose membrane treatment group, and L-CHB, D-CHB, R-CHB, and Ach-HB represent the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1, respectively; Figure 11 The flow chart of the experiment in Example 6 is shown; Figure 12 In Example 6: (A) Photographs of the wound healing of different materials applied to the skin wounds of aged C57 mice at different time points, (B) Wound healing trajectory graphs of aged C57 mice in different material groups at different time points, (C) Statistical graphs of the wound closure rates of aged C57 mice in different material groups; in the figures, L-CHB, D-CHB, R-CHB, and Ach-HB represent the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1, respectively. The same applies hereinafter Figures 13 - 17 The same applies hereinafter; in the figures, the control group is the Control group with only the wound surface untreated. The same applies hereinafter Figures 13 - 17 The same applies; Figure 13 The HE staining images of the wounds of five groups of aged C57 mice and the MASSON staining images on the 14th day in Example 6 are shown; Figure 14 The immunohistochemical staining of neutrophils-related ly6G in five groups of aged C57 mice on the first and third days and the immunohistochemical staining of NF-κB in five groups of aged C57 mice on the first day in Example 6 are shown; Figure 15 In Example 6: (A) Immunohistochemical staining of M1 macrophage-related indicators in five groups of aged C57 mice on the 1st day, (B) Immunohistochemical staining of M2 macrophages-related in five groups of aged C57 mice on the 3rd day; Figure 16For the wound healing diagrams of five groups of NPSG mice in Example 7 (A), and the HE and MASSON staining diagrams of the wounds of five groups of NPSG mice on the 7th and 14th days (B); Figure 17 For the HE staining diagrams of the wounds of five groups of NPSG mice in Example 7 (A), the immunohistochemical staining of NF-κB in the wounds of five groups of NPSG mice (B), and the scanning pictures of Sirius red and its polarized light of the wounds of five groups of NPSG mice on the 14th day (C). Detailed implementation manners

[0022] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made. These all belong to the protection scope of the present invention.

[0023] For all raw materials of the present invention, there is no special limitation on their sources. If the specific manufacturers are not indicated, it means that they can be purchased on the market or prepared according to the conventional methods well-known to those skilled in the art.

[0024] SBF is prepared by those skilled in the art according to the conventional operations (such as 《ASTM F2129 - 07(2014) Standard Test Method for In vitro Evaluation of the Ability of Metallic Medical Implant Materials to Release Calcium Phosphate in a Simulated Body Fluid (SBF)》 or ISO 23317:2014 《Implants for surgery — In vitro evaluation for apatite - forming ability of implant materials》).

[0025] Sodium chloride (NaCl, 99.5%), sodium bicarbonate (NaHCO 3 , 99.8%), potassium chloride (KCl, 90%), dipotassium phosphate (K 2 HPO 4 ), magnesium chloride hexahydrate (MgCl 2 ·6H 2 O, 99.5%), sodium sulfate (Na 2 SO4 , 98%) and calcium chloride (CaCl 2 , 99.8%) were from Sinopharm Chemical Reagent Co., Ltd. D-, L-, and racemic glutamic acid (99.9%) were purchased from Tansoole Co., Ltd. Hydrochloric acid (99.9%) and Tris(hydroxymethyl)aminomethane (CAS: 77 - 86 - 1, 99.8%) were purchased from J&K Chemical Technology. Bacterial cellulose membrane BC was provided by Guilin Qihong Technology Co., Ltd. All reagents were used as supplied without further purification. Ultra-pure water (18.2 MΩ / cm) was used throughout the process and was generated by a Heal Force SMART ultra-pure water system. Sirius red staining solution was purchased from Nanjing Senbeijia Biotechnology Co., Ltd. (Sbjbio), model number BP-DL030; Masson staining solution was purchased from Morphisto, Germany, model number MASSON GOLDNER 12043.00500.

[0026] The preparation of tissue samples and staining experiments (including HE staining, MASSON staining, Sirius red staining, and immunohistochemical staining) involved multiple times in the examples are as follows.

[0027] (I) Preparation of paraffin sections (1) Immerse fresh tissue in formalin solution for 12 h for fixation; (2) Dehydrate the tissue in an automatic dehydrator; (3) Take out the tissue and embed it in paraffin using an embedding machine; (4) After the paraffin solidifies, place it in a -20 °C refrigerator and freeze for 15 min; (5) Mount the wax block on the fixing device of the microtome, install the microtome knife on the knife holder, with the blade at a 5° angle to the surface of the wax block, adjust the wax block and the blade to the appropriate position, and adjust the section thickness scale to 4 μm; (6) Use a rotary microtome. Control the progress with the left hand and rotate evenly with the right hand. Coarsely trim the wax block until the tissue morphology is complete. Stop the progress with the left hand and continue to rotate with the right hand for fine trimming until it is flat. Hold a writing brush in the left hand, pull the complete section turned out by the right hand flat. Use forceps in the right hand to spread the section into a 42 °C water bath until it is flat. Scoop out the tissue from the water bath with a glass slide, insert it into the slide rack, and place it in a 65 °C oven.

[0028] (II) HE staining (1) Take out the sections baked for 2 h from the 65 °C oven; (2) Immerse them successively in xylene I - xylene II - xylene III, each for 5 min; (3)Immerse successively in 100% alcohol - 100% alcohol - 85% alcohol - 75% alcohol, 3 min each, for gradient dewaxing, and rinse with running water for 3 min; (4)Immerse in hematoxylin staining solution for 5 min, and rinse with running water for 2 min (if it is a frozen tissue section, start directly from step 4 after fixation); (5)Immerse in 0.5% hydrochloric acid alcohol differentiation solution for 3 s, then take out and rinse with running water for 3 min; (6)Immerse in 95% alcohol for 1 min; (7)Immerse in eosin staining solution for 30 s; (8)Immerse successively in 80% alcohol - 85% alcohol - 95% alcohol - 95% alcohol - 100% alcohol - 100% alcohol, 1 min each, for gradient dehydration; (9)Immerse successively in xylene I - xylene II - xylene III, 2 min each; (10)Take out the section, drop neutral gum, and cover with a coverslip. Place the covered section under a microscope, record and photograph the observed results.

[0029] (III)Immunohistochemistry (IHC) (1)Conventionally dewax the sample to water; (2)High - pressure repair: Preheat the repair solution in a pressure cooker. After boiling, put the slides on a plastic staining rack into the repair solution, ensuring complete coverage of the tissue. Start timing when the pressure - limiting valve rotates and jets for 2.5 min. While timing, turn the induction cooker from high heat to medium heat. After timing ends, remove from the heat source and cool with cold water to room temperature, taking care not to pour cold water into the pressure cooker; (3)Wash with PBS 3 - 5 times, 2 min each time (add Tween 20 to PBS); (4)Incubate with 3% hydrogen peroxide at room temperature for 10 min; (5)Wash clean with distilled water, and draw a circle with a PAP pen (keep a distance of 2 - 3 mm from the tissue when drawing the circle); (6)Wash with PBS 3 - 5 times, 2 min each time (add 1% Tween 20 to PBS); (7)Drop an appropriate amount of primary antibody, incubate at 37°C for 1 h (when adding the primary antibody, shake off the water on the section, taking care not to let the section dry); (8)Wash with PBS 3 - 5 times, 2 min each time (add Tween 20 to PBS); (9)Drop an appropriate amount of one - step detection system (PV - 8000D), incubate at 37°C for 30 min (when adding the secondary antibody, shake off the water on the section, taking care not to let the section dry); Wash with PBS 3 - 5 times, 2 min each time; (11) After the secondary antibody, develop color with DAB (ZLI - 9018), prepare and use immediately, for 6 - 8 min. There is no need to control the color development under the microscope. Terminate with tap water and rinse for 3 - 5 min; (12) Counterstain with hematoxylin. Be careful not to over - counterstain (adjust the time according to the freshness of hematoxylin); (13) Gently rinse with tap water; (14) Differentiate with hydrochloric acid alcohol (flexibly control the time according to the counterstaining situation with hematoxylin); (15) According to the situation, you can choose to use the previously used EDTA pH8.0 repair solution to bluing for 3 - 5 min, and rinse with running water for 3 - 5 min; (16) Dehydrate with alcohol in a step - by - step gradient, clear with xylene, and mount with neutral gum. Analyze the expression differences of antigens in tissues by observing the staining intensity, range, and distribution, etc.

[0030] (IV) Sirius red staining (for collagen fiber staining) (1) Deparaffinize the paraffin sections to water: Immerse the sections in xylene I and xylene II for 10 min each in turn, then immerse them in absolute ethanol, 95% ethanol solution, 85% ethanol solution, and 75% ethanol solution for 5 min each in turn, and then rinse the sections with distilled water for several seconds; (2) Place the rinsed sections on the workbench, cover the tissue surface completely with 1 - 2 drops of Sirius red staining solution, and stain by dropping for 1 hour; (3) Rinse with running water for 5 min, stain the cell nuclei with Mayer hematoxylin staining solution for 8 - 10 min; (4) Rinse with running water for 10 min, dry the surface moisture, and then dehydrate and clear routinely, and seal with gum.

[0031] (V) MASSON staining (1) Routinely deparaffinize the samples to water; (2) Stain the nuclei with MORPHISTO hematoxylin solution for 6 - 8 minutes; (3) Wash with tap water for 10 min. If over - stained, differentiate with hydrochloric acid alcohol; (4) Use Ponceau acid fuchsin solution Goldner I solution for 3 min. Wash with distilled water for several seconds, shake dry, and observe under the microscope; (5) Phosphomolybdic acid - orange reagent Goldner II solution for 3 minutes, wash with distilled water for several seconds; (6) Green Goldner III solution for 3 minutes, wash with distilled water for several seconds; (7) Dehydrate with alcohol in a step - by - step gradient, clear with xylene, and seal with neutral gum.

[0032] Example 1: Preparation of Chiral Hierarchical Hydroxyapatite Nanosheet / Bacterial Cellulose Membrane (L-CHB)

[0033] In this example, chiral hierarchical hydroxyapatite (CMHAP) nanosheets were in-situ grown on the surface of bacterial cellulose membrane (BC) through a simple chiral glutamic acid (GA) molecule-induced self-assembly strategy, which specifically includes the following steps: S1. L-glutamic acid, SBF (simulated body fluid), Tris, and hydrochloric acid were successively added to deionized water to obtain a mixed solution for standby; in the mixed solution obtained in this step, the concentration of L-glutamic acid was 0.2 mM; the concentration of calcium ions (Ca 2+ )was 0.26 mM, and the concentration of phosphate ions (PO 4 3- )was 5 mM; the total volume was 20 mL, and the pH was 7.2 - 7.4.

[0034] In this step, chiral L-glutamic acid is a bioactive material that can coordinate with calcium ions to form a complex. Therefore, chiral L-glutamic acid was selected as the structure-directing agent and asymmetric inducer. The main components of SBF include calcium ions (Ca 2+ )and phosphate ions (PO 4 3- ). When a bioactive material is immersed in SBF, the surface of the material will interact with the ions in the solution. The active groups on the material surface will adsorb calcium ions in the solution, and then the calcium ions will attract phosphate ions, gradually forming crystal nuclei of calcium phosphate compounds. Over time, the crystal nuclei continue to grow and finally form a hydroxyapatite layer. This process is called "biomineralization", and this process needs to be carried out within a certain pH range, generally 7.2 - 7.4. In this example, Tris-hydrochloric acid was used as the acid-base regulator to adjust the pH value of the reaction system and thus control the nucleation and crystal growth rate of the reaction system, ultimately achieving the formation of an asymmetric crystal (chiral HAP nanosheet, CMHAP) structure.

[0035] S2. The bacterial cellulose membrane (BC) was successively cleaned with deionized water and absolute ethanol, and then the bacterial cellulose membrane (BC) was placed in the mixed solution prepared in step S1 and subjected to biomineralization at 37 °C for 24 h.

[0036] In this step, the dosage ratio of the bacterial cellulose membrane (BC) to the mixed solution was 0.0005 g : 20 mL, and the pH value of the solution during the reaction was controlled within 7.2 - 7.4.

[0037] After the biomineralization reaction is completed, the bacterial cellulose membrane (BC@CMHAP) with chiral hydroxyapatite grown on it is taken out, washed clean with deionized water, and then placed in a vacuum drying oven and dried at 37 °C for 12 h for standby. The obtained chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane is named L-CHB. That is, the L-CHB and BC@CMHAP represent the same substance.

[0038] Example 2: Preparation of chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane (D-CHB).

[0039] This example is the same as Example 1 except that L-glutamic acid is replaced by D-glutamic acid.

[0040] Example 3: Preparation of chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane (R-CHB).

[0041] This example is the same as Example 1 except that L-glutamic acid is replaced by racemic-glutamic acid.

[0042] Comparative Example 1: Preparation of hydroxyapatite nanosheet / bacterial cellulose membrane (Ach-HB) without adding chiral molecules.

[0043] Compared with Example 1, this comparative example does not add chiral molecules, and the obtained hydroxyapatite nanosheet / bacterial cellulose membrane is named Ach-HB.

[0044] Example 4: Mechanical property characterization of materials.

[0045] This example characterized the mechanical properties of the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membranes prepared in Examples 1 to 3 and Comparative Example 1 above, and the related tests included the following content.

[0046] 4.1 Scanning electron microscopy.

[0047] Scanning electron microscopy is a conventional operation familiar to those skilled in the art and can be operated according to the instrument's instructions. The specific experimental methods will not be elaborated here.

[0048] The chiral hierarchical hydroxyapatite nanosheet structure in the chiral hierarchical hydroxyapatite nanosheet / bacterial cellulose membrane is denoted as CMHAP.

[0049] Experimental results: As Figure 1As shown, it can be seen that the BC film presents a network porous structure composed of nanofibers, while CMHAP presents a flower-like structure of assembled nanosheets distributed on the surface of the BC fiber film. On the L-CHB film, the L-CMHAP flowers are densely arranged by nanosheets with a width of 50 - 200 nm, a thickness of 10 - 20 nm, and a height of 0.1 - 1 µm grown on the surface of BC fibers. The left-handed helical packing of the nanosheets was observed, representing the tertiary level of chirality. The magnified scanning electron microscope image shows that the chirality of the CMHAP nanosheets is secondary chirality. Based on the inventor's previous research (Chemistry of Materials, 2021, 34(1):53 - 62.), it can be considered that the dislocation of atoms also leads to the initial level of chirality.

[0050] Similarly, in the D-CMHAP flowers on the D-CHB film, the right-handed helical arrangement of the nanosheets can be observed, which is considered to be the tertiary chirality. The magnified scanning electron microscope image shows a wavy distortion pattern presented by the nanosheets. The right-handed twisted structure appearing in the nanosheets is considered to be the secondary chirality of the D-CMHAP flowers. The primary chirality result is also right-handed (Chemistry of Materials, 2021, 34(1): 53 - 62.). Ach-CMHAP and R-CMHAP bloom on the Ach-HB and R-CHB films respectively, indicating that the nanosheets are randomly arranged with an irregular structure and thus do not have a chiral multilayer structure.

[0051] 4.2 XRD patterns.

[0052] The crystal structures of BC, Ach-HB, D-CHB, L-CHB, and R-CHB films were further analyzed using a wide-angle X-ray diffractometer (XRD). XRD detection is a conventional operation familiar to those skilled in the art and can be operated according to the instrument's instruction manual, so the specific experimental methods will not be elaborated here.

[0053] The XRD patterns of Ach-HB, D-CHB, L-CHB, and R-CHB films and the BC substrate are as Figure 2 shown: Although there is a strong BC substrate intensity in the XRD patterns of Ach-HB, D-CHB, L-CHB, and R-CHB films, the hexagonal phase characteristic reflections of HAP with a space group of P63 / m and lattice parameters of a = b = 9.4166 Å and c = 6.8745 Å can still be identified [JCPDS file 09 - 0432]. In addition, the corresponding powders were also detected. At the same time, some broad peaks were also found in the low-angle region, indicating that the hydroxyapatite powder contains some components with lower crystallinity.

[0054] 4.3 Ultraviolet-visible diffuse reflectance (DRUV-Vis) and circular dichroism (DRCD) spectra.

[0055] Since the BC, Ach-HB, D-CHB, L-CHB, and R-CHB films are opaque, their chirality was unambiguously detected based on optical activity (OA) using diffuse reflectance ultraviolet-visible (DRUV-Vis) and circular dichroism (DRCD) spectra. Ultraviolet-visible diffuse reflectance (DRUV-Vis) and circular dichroism (DRCD) spectra are routine operations familiar to those skilled in the art and can be operated according to the instrument's instructions. The specific experimental methods will not be elaborated here.

[0056] The experimental results are as Figure 3 shown: The DRCD of the white and black backgrounds shows approximately complete absorption-based OA (AOA) and scattering-based OA (SOA) and AOA because almost all visible light is reflected by the white light and absorbed by the black backplane, respectively. It is known that the distance between semiconductor nanounits aggregated in a chiral manner is less than the Bohr exciton radius, and an asymmetric field is induced over the entire aggregate by the delocalization of the excitation, resulting in AOA based on electronic transitions. Based on mλ = Pn_avg, SOA occurs at multiple integral wavelengths, where m is an integer, n_avg is the average refractive index, and P is the pitch length of the chiral medium. The left-handed structure is more inclined to absorb right-handed circularly polarized light and reflect left-handed light, resulting in negative AOA and SOA signals for DRCD measurements.

[0057] As Figure 3 shown, the L-CHB and D-CHB films show mirror image signals at 240 - 800 nm. Taking L-CHB as an example, the ultraviolet-visible spectrum has a broadband in the range of 240 - 800 nm. The CD spectrum has two strong peaks at 240 - 350 nm and 350 - 800 nm, and the strongest peaks are at 285 nm and 650 nm, respectively. According to the detection mechanism, the OA of the L-CHB film includes AOA and SOA because the BC substrate has a white background. The mirror image DRCD spectrum of the D-CHB film shows the opposite chirality.

[0058] The DRCD spectrum of the BC substrate confirms that the optical activities OAs of the L-CHB and D-CHB films originate from the hierarchical chiral structure rather than the BC substrate. The R-CHB film morphology is similar to the corresponding CMHAP flower but has no chirality.

[0059] 4.4 Fourier transform infrared spectroscopy.

[0060] Fourier transform infrared spectroscopy is a routine operation familiar to those skilled in the art and can be operated according to the instrument's instructions. The specific experimental methods will not be elaborated here.

[0061] FTIR confirmed the reliability of BC, Ach-HB, D-CHB, L-CHB, and R-CHB. As Figure 4 shown, the 961 cm -1 band reflected qvl PO 4 3- , and the 1125 cm -1 band reflected qvl PO 4 3- . The weak intensities of hydroxyl stretching and vibration were 3426 cm -1 and 637 cm -1 , respectively. The 874 and 1420 - 1470 cm -1 bands were for b-type carbonate apatite, where carbonate replaced the phosphate position in the hydroxyapatite lattice. The FTIR spectrum showed that in the 2270 - 2457 cm -1 region, there were at least three weak to strong stretching vibrations of protons directly connected to P-H of phosphorus, indicating three structural positions or three conformations of the phosphate group.

[0062] 4.5 Stress-strain curves, tensile strength, and maximum elongation.

[0063] Mechanical properties are one of the main factors affecting the performance of tissue engineering wound repair materials. In this example, the stress-strain behavior, tensile strength, and maximum elongation of BC, Ach-HB, D-CHB, L-CHB, and R-CHB membranes were studied. In this example, the detection was carried out in a universal material testing machine. In the tensile test, the testing machine fixed both ends of the specimen and then gradually applied a tensile force at a constant speed until the specimen broke. During this process, the testing machine recorded the applied force and the elongation of the specimen in real time, thus obtaining the stress-strain curve. The specific experiment can be operated according to the instrument's instruction manual, which is a conventional measurement method in this field and will not be elaborated here.

[0064] The experimental results are respectively as Figures 5 - 7 shown. Ach-HB, D-CHB, L-CHB, and R-CHB showed similar stress-strain and tensile strength, and their strength was 2 - 3 times that of pure BC, proving that the mineralized chiral hydroxyapatite coating could effectively improve the strength and toughness of the BC membrane simultaneously.

[0065] 4.6 Hydrophilicity and hydrophobicity.

[0066] The hydrophilicity and hydrophobicity of the material were measured by a contact angle measuring instrument. The specific experiment can be operated according to the instrument's instruction manual, which is a conventional measurement method in this field and will not be elaborated here.

[0067] The experimental results are as Figure 8As shown, it can be seen that after BC passes through the chiral hydroxyapatite coating, due to the high hydrophilicity of BC itself, its hydrophilicity remains basically unchanged. The contact angles of BC, Ach-HB, D-CHB, L-CHB, and R-CHB films are 17.751°, 16.943°, 16.834°, 16.662°, and 16.529°, respectively. This lays a solid foundation for biocompatibility and biological applications.

[0068] Based on the above several material characterizations, this example confirms the successful preparation of each group of materials and their good mechanical properties, laying a foundation for their application in biomaterials.

[0069] Biological safety assessment of the membrane materials in Example 5.

[0070] The severely immunodeficient NPSG mice used in this example were purchased from Phenotek Biotechnology (Shanghai) Co., Ltd. The severely immunodeficient NPSG mice NOD-Prkdc scid Il2rg null (NPSG, scid indicates severe combined immunodeficiency, and null indicates the knockout of IL2rg) model exhibits inherent immunodeficiency, characterized by a weakened complement system and weakened dendritic cell function. Importantly, gene modification involves knocking out the Prkdc gene in these mice, resulting in the absence of T lymphocytes and B lymphocytes, ultimately leading to severe combined immunodeficiency. This extensive immunodeficiency has an adverse effect on both cellular and humoral immunity, highlighting the crucial role of these cell types in maintaining immune homeostasis. At the same time, the knockout of the interleukin-2 receptor gamma chain gene (IL2rg) further exacerbates the immune system deficiency, most notably resulting in a sharp reduction in natural killer (NK) cell activity. The loss of NK cell function emphasizes the key impact of the IL2rg gene on overall immune function in these mouse models. Therefore, NPSG mice are severely immunodeficient due to the knockout of Prkdc and IL2rg genes, can reduce immune interference, observe potential infection risks, and are suitable for the biological safety assessment of membrane materials.

[0071] In this example, several membrane materials prepared in Examples 1 to 3 and Comparative Example 1 above were respectively implanted subcutaneously into NPSG mice, and then the subcutaneous tissues of the mice were subjected to HE staining, Masson staining, and CD45 immunohistochemical staining on the 3rd, 7th, and 14th days respectively. The staining results are as Figure 9 shown. It can be seen that the inflammatory reaction in the subcutaneous tissues of the mice is small, there is no recruitment of abnormal immune cells, and there is no significant difference in comparison between groups. This means that the membrane materials have good biological safety and low sensitivity, and have a relatively constant impact on the surrounding tissues at least within 14 days.

[0072] Next, in this example, HE staining was performed on the important organs of the mice, namely the heart, liver, lungs, and kidneys, at 14 days. The results are as follows: Figure 10 As shown, it can be seen that the organizational structures of the important organs are normal and no obvious changes have occurred. The normal organizational structure indicates that the membrane material prepared in this application has no visceral toxicity.

[0073] The results of this example show that the membrane material provided in this application has low sensitivity and safety.

[0074] Example 6: A skin defect model of aged C57BL / 6 mice was constructed to verify the effect of chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes on the healing of aged skin wounds through in vivo experiments.

[0075] In this example, naturally aged C57BL / 6 mice were used to confirm the good wound healing effect of the L-CHB membrane on aged wounds. The process is as follows: Figure 11 As shown. After depilation of the surgical area on the back of the mice, two circular full-thickness skin wounds (5 mm in diameter, one on each side) were created on each mouse, and they were divided into 5 groups: Control, Ach-HB, L-CHB, R-CHB, and D-CHB. The corresponding materials were used to cover the wounds in each group. Photos were taken at 0, 1, 3, 5, 7, and 14 days after surgery, and Image J software was used for analysis. The skin samples of the wounds were collected for further analysis. The experimental procedures and operations were approved by the Animal Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.

[0076] As Figure 12 shown in (A) below, it can be seen that after the L-CHB membrane covered the wound, the wound healed the fastest. This indicates that the L-CHB membrane can shorten the healing cycle, efficiently repair the wound, and accelerate healing. As Figure 12 shown in (B) below and Figure 12 shown in (C) below, the results obtained by Image J software analysis clearly show the wound healing trajectories of aged C57 mice at different time points in different material groups, which is the same as the phenomenon observed in Figure 12 shown in (A) below.

[0077] Next, in order to study the basic structural conditions of the skin tissue and cells and show the fibrous connective tissue in the tissue, especially collagen fibers, HE staining (at 1, 3, 7, and 14 days) and MASSON staining (at day 14) were performed on the wound skin samples of the above five groups of aged C57 mice. The results are as follows: Figure 13 As shown: After the L-CHB membrane group covered the wound, the infiltration of inflammatory cells in the wound was significantly reduced, and epithelial keratinization, as well as collagen production and hair follicle regeneration, were promoted.

[0078] Next, in order to study the immune activity and functional status of skin wounds, in this example, immunohistochemical detection was performed on the distribution of the specific antigen ly6G protein on the surface of neutrophils in the skin wounds of five groups of elderly C57 mice on the 1st and 3rd days, and immunohistochemical staining was also performed on the distribution of NF-κB protein on the 1st day. The results are as Figure 14 shown. Neutrophils positive for ly6G play a key role in immune defense. When the body is infected by pathogens (such as bacterial and viral infections), neutrophils will be activated. By detecting the ly6G level, it can be judged whether neutrophils are in an active immune response state and their ability to phagocytose and kill pathogens. Similarly, NF-κB is a key regulator of the inflammatory response. When cells are stimulated by inflammation such as LPS, TNF-α, IL-1β, etc., NF-κB will be activated and initiate the transcription of a series of inflammation-related genes, such as genes encoding pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. The results show that: the degree of neutrophil aggregation at the lower edge of the wound in the L-CHB membrane group is relatively light, and the inflammatory response is weak.

[0079] Next, in this example, immunohistochemical staining was performed on the related indexes CD68, IL-1β, TNF-α of M1 macrophages and the related indexes CD163, CD206 of M2 macrophages in five groups of elderly C57 mice on the first day. The results are as Figure 15 shown. M1 macrophages mainly play a role in promoting inflammation and immune killing. Detecting its related indexes (such as IL-1β, TNF-α) can reflect the intensity of the body's inflammatory response and immune defense ability. When the body is infected by pathogens, M1 macrophages are activated, and the levels of these indexes increase, indicating that the immune system is actively responding to the infection. M2 macrophages have anti-inflammatory, tissue repair and immune regulatory functions. Detecting indexes such as CD163 and CD206 can understand the anti-inflammatory and repair status of the body. By detecting the indexes of the two types of macrophages, the immune balance state of the body can be comprehensively evaluated, and it can be judged whether the immune function is hyperactive or hypoactive. It can be seen that: in the early inflammatory response stage, the L-CHB membrane can effectively reduce the pro-inflammatory cytokines in the wound, reduce the aggregation of neutrophils and M1 macrophages, and regulate the immune microenvironment; in the later tissue repair stage, the L-CHB membrane can reduce wound fibrosis and promote tissue regeneration.

[0080] Example 7 In vivo experiment: Construct a skin defect model of severely immunodeficient mice (NPSG) to study the mechanism of the membrane material and regulate innate immune cells.

[0081] To further understand the mechanisms of different cells involved, the present application also selected NPSG mice (the same as those in the above embodiments) for experiments, targeted knockout of T / B / NK cells, used age-matched ICR mice as controls, and compared the effects of materials on wound healing in immunodeficient and normal immune microenvironments. This study involved 45 NPSG mice.

[0082] In this example, the wound healing conditions of five groups of NPSG mice were photographed and compared. The results are as Figure 16 shown in (A) below. It can be seen that the healing of the skin wound surface of the mice in the L-CHB group was the best. HE and MASSON staining were performed on the wounds of the five groups of NPSG mice on the 7th and 14th days. The results are as Figure 16 shown in (B) below. It can be seen that: in the early stage, the inflammation of L-CHB was significantly the lightest. At 7 and 14 days in the later stage, it could be seen that the epidermis of the L-CHB wound became thinner and hair follicles formed, indicating the superior effect of L-CHB on skin wound healing.

[0083] Next, HE staining was performed on the wounds of the five groups of NPSG mice on the 1st and 3rd days. The results are as Figure 17 shown in (A) below. It can be seen that the tissue cell morphology of the L-CHB membrane group was good. Immunohistochemical staining of NF-κB on the wounds of the five groups of NPSG mice was performed. The results are as Figure 17 shown in (B) below. It can be seen that the L-CHB membrane could effectively reduce the pro-inflammatory cytokines in the wound surface. Sirius red staining and polarized light scanning were performed on the wounds of the five groups of NPSG mice on the 14th day. The results are as Figure 17 shown in (C) below. It can be seen that there were more hair follicles and the epidermis was thinner in the L-CHB membrane group, indicating good recovery.

[0084] In summary, in the examples of the present invention, first, a chiral multi-level structure hydroxyapatite (CMHAP) nanosheet was in-situ grown on the surface of a bacterial cellulose membrane through a simple chiral glutamic acid (GA) molecule-induced self-assembly strategy, and a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane material was prepared. The hydroxyapatite nanosheet / bacterial cellulose membranes finally made with L-glutamic acid, D-glutamic acid, and racemic-glutamic acid were named L-CHB, D-CHB, and R-CHB respectively. The scaffold synthesized without adding chiral molecules was named Ach-HB. Finally, the above-prepared membrane materials were used as wound healing materials (dressings).

[0085] Comprehensive material characterization confirmed the successful preparation of each group of materials and their good mechanical properties. In vitro and in vivo experiments confirmed the low sensitivity and safety of each group of materials. In vivo experiments confirmed that the L-CHB membrane had a significantly better wound-healing effect on senile wounds than other groups. Histological staining revealed that in the early inflammatory response stage, the L-CHB membrane effectively reduced bacterial attachment to the wound surface, inhibited the NF-κB pathway to exert an anti-inflammatory effect, reduced neutrophil aggregation, promoted macrophage polarization, and regulated the innate immune microenvironment; in the later tissue repair stage, the L-CHB membrane could reduce wound fibrosis and promote tissue regeneration. Further mechanism studies found that in the NPSG mouse model, the L-CHB membrane also demonstrated significant anti-inflammatory and tissue-healing abilities, suggesting that biomaterial-mediated wound healing requires the innate immune system.

[0086] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane, characterized in that: It is prepared by biomineralization of chiral hydroxyapatite nanosheets and bacterial cellulose; The chiral hydroxyapatite nanosheets are prepared from simulated body fluid and chiral glutamic acid; The chiral hydroxyapatite nanosheets grow on a bacterial cellulose membrane.

2. The method for preparing a chiral multi-level structured hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 1, characterized in that: The preparation method comprises the following steps: S1. chiral glutamic acid, simulated body fluid, tris(hydroxymethyl)aminomethane, and hydrochloric acid are sequentially added to deionized water to obtain a mixed solution; S2. Clean the bacterial cellulose membrane and then place it into the freshly prepared mixed solution in step S1 to perform a biomineralization reaction; S3. After the biomineralization reaction is completed, the bacterial cellulose membrane with the chiral hydroxyapatite grown thereon is taken out, cleaned and dried to obtain the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane.

3. The method for preparing a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 2, characterized in that: The chiral glutamate in step S1 is selected from any one of L-glutamate, D-glutamate or racemic glutamate.

4. The method for preparing a chiral multi-level structured hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 2, characterized in that: The concentration of chiral glutamate in the mixed solution of step S1 is 0.1-1 mM; the concentration of calcium ions contained in the mixed solution of step S1 is 0.1-1 mM; and the concentration of phosphate ions contained is 1-10 mM.

5. The method for preparing a chiral multi-level structured hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 2, characterized in that: The dosage ratio of the bacterial cellulose membrane to the mixed solution in step S2 is (0.0001~0.001) g: (10~100) mL.

6. The method for preparing a chiral multi-level structured hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 2, characterized in that: During the biomineralization reaction in step S2, the pH value of the solution is controlled within a range of 7.2 to 7.4, and the time for the biomineralization reaction in step S2 is 12 to 48 hours.

7. The method for preparing a chiral multi-level structured hydroxyapatite nanosheet / bacterial cellulose membrane according to claim 2, characterized in that: The drying time in step S3 is 12-24 h and the temperature is 20-60°C.

8. Application of chiral multi-level structured hydroxyapatite nanosheets / bacterial cellulose membrane in the preparation of wound dressings.

9. A wound dressing, characterized in that: The active ingredient of the wound dressing is the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane described in claim 1.

10. A wound dressing according to claim 9, characterized in that: The wound dressing is a dressing for elderly skin wounds.

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