Chiral hierarchical structure hydroxyapatite nanosheet / bacterial cellulose membrane and its preparation and application in elderly skin wound dressing

Abstract

The present application relates to the technical field of chiral materials and its application, especially relates to a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane and its preparation and application in old skin wound dressing. The chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane is prepared by the method of biomimetic mineralization through chiral hydroxyapatite nanosheet and bacterial cellulose. The chiral hydroxyapatite nanosheet grows on the bacterial cellulose membrane. The chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane adopts a simple molecular induced self-assembly strategy, the synthesis method is simple and controllable, introduces a unique chiral structure, expands new medical applications; when applied as a wound dressing, has low sensitivity and safety, regulates the immune microenvironment, effectively reduces the inflammatory response and promotes tissue regeneration, realizes the multi-stage precise regulation of the wound healing process.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chiral materials and their applications, and particularly relates to a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane and a preparation method and application thereof in old skin wound dressings. BACKGROUND

[0002] With age, the clinical features of skin are a gradual thinning, drying and loss of elasticity of the dermis. At the same time, senescent cells accumulate with age, secreting factors such as senescence-associated secretory phenotype (SASP), thereby inhibiting macrophage-dependent clearance functions, leading to skin aging. Intrinsic skin aging is characterized by skin appendage dysfunction and cell features of prolonged cell cycle and decreased mitotic activity, especially in keratinocytes, leading to delayed wound healing. However, epidermal aging is associated with dysregulated lipid metabolism, manifested as impaired permeability barrier function and delayed recovery after external injury. Delayed wound healing increases the risk of local infection and systemic complications, while imposing a significant socio-economic burden on affected individuals and the healthcare system. Currently, the molecular mechanisms between skin aging and impaired wound healing are not clear, which limits the development of effective therapeutic interventions for age-related wound healing disorders.

[0003] To date, an important class of biomaterials, including hydrogels, nanofibrous membranes and bacterial cellulose (BC) fibrous membranes, have been used for wound healing management through a dual mechanism of modulating inflammatory responses and promoting wound tissue regeneration. Bacterial cellulose (BC) fibrous membranes have special mechanical strength, excellent water retention and inherent biocompatibility, and are a very promising functional wound dressing that can control drug delivery to the wound bed while preventing microbial colonization. However, BC fibrous membranes lack antibacterial activity and cannot release bioactive compounds to effectively modulate inflammation.

[0004] Compared with young people, 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, which together contribute to the reduced efficiency of wound healing in the elderly. In addition, the immune microenvironment of aging skin is characterized by macrophage dysfunction, impaired phagocytic activity and increased neutrophil infiltration, indicating that the skin barrier is impaired and healing is slowed. Notably, recent studies have shown that macrophages involved in wound healing in aged skin often exhibit pro-inflammatory characteristics. As an inorganic biomaterial, hydroxyapatite (HAP) can mechanically enhance the structure of biopolymers and modulate the immune microenvironment, especially by activating macrophages and regulating their polarization. Considering that chirality is a fundamental feature of all scales in nature and life sciences, the inventors conducted research (see patent CN114105114B) and found that chiral-induced hydroxyapatite can promote bone bonding and regeneration. Emerging evidence suggests that chiral polymers, such as L-forms, have immunomodulatory properties that can help activate immune components and signaling pathways. However, the role of chiral polymers in immunomodulation has been little studied.

[0005] In summary, with age, skin aging features are apparent, such as thinning of the dermis, decreased elasticity, and accumulation of senescent cells and secreted factors that inhibit macrophage function, leading to skin aging and delayed wound healing. Epidermal aging is also associated with dysregulated lipid metabolism, and delayed wound healing poses risks and economic burdens, and its molecular mechanisms are unknown, which restricts effective treatment. Existing wound healing-related biomaterials, such as BC fibrous membranes, have advantages but lack antibacterial activity and effective inflammation modulation ability, and there is a lack of comprehensive and precise treatment options for senescent skin wounds, without fully considering the effects of senescent cells. In terms of material preparation, existing hydroxyapatite materials have a single structure and lack chiral order, limiting their application in biomedical and other fields, and there is also little research on the immunomodulatory properties of chiral structures. SUMMARY

[0006] Based on the shortcomings of the prior art described above, the purpose of the present application is to provide a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane and its preparation and application in elderly skin wound dressings. The present application aims to explore the changes in the immune microenvironment of elderly wounds and the characteristics of wound healing, and to develop a chiral wound dressing that can effectively promote the healing of aging wounds and immune regulation. The chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane provided by the present application is synthesized by a chiral glutamic acid molecule-induced self-assembly strategy. The biological safety 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 application also uses NOD-Prkdc scid Il2rg nullConstruct a skin defect model in a severe immunodeficient mouse (NPSG), and after targeted knockout of T / B / NK cells, observe the effect of innate immune cells in biological (such as a mouse) mediated wound healing.

[0007] Based on the above purpose, the present application provides the following technical solutions.

[0008] One of the technical solutions of the present application provides a chiral multi-level structure hydroxyapatite nanoplatelet / bacterial cellulose membrane, which is prepared by a biomimetic mineralization method from chiral hydroxyapatite nanoplatelets and bacterial cellulose (BC). The chiral hydroxyapatite nanoplatelets are prepared from a simulated body fluid (SBF) and chiral glutamic acid. The chiral glutamic acid (GA) acts as a structure directing agent and an asymmetric inducer, so that calcium ions and phosphate ions in the simulated body fluid (SBF) gradually form a crystal nucleus of a calcium-phosphorus compound, and finally form an asymmetric crystal (chiral hydroxyapatite nanoplatelets, CMHAP) structure. The chiral hydroxyapatite nanoplatelets grow on the bacterial cellulose membrane.

[0009] The 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. The SBF can be used as a simulated environment for coating preparation, and can deposit a calcium-phosphorus coating on the surface of the material, thereby simulating the mineralization process of bone tissue in the body. The prepared coating has good bioactivity and biocompatibility.

[0010] The second technical solution of the present application provides a preparation method of a chiral multi-level structure hydroxyapatite nanoplatelet / bacterial cellulose membrane, which comprises the following steps:

[0011] S1. Chiral glutamic acid, SBF, Tris and hydrochloric acid are sequentially added to deionized water to obtain a mixed solution;

[0012] S2. The bacterial cellulose membrane (BC) is sequentially cleaned with deionized water and anhydrous ethanol, and then the bacterial cellulose membrane (BC) is placed in the mixed solution prepared in step S1 to perform a biomimetic mineralization reaction;

[0013] S3. After the biomimetic mineralization reaction is completed, the bacterial cellulose membrane (BC) with chiral hydroxyapatite (BC@CMHAP) is taken out, cleaned and dried to obtain the chiral multi-level structure hydroxyapatite nanoplatelet / bacterial cellulose membrane.

[0014] Further, the chiral glutamic acid in step S1 is selected from any one of L-glutamic acid, D-glutamic acid or racemic glutamic acid; the concentration of the chiral glutamic acid in the mixed solution is 0.1-1 mM, preferably 0.2 mM; the concentration of calcium ions (Ca 2+ ) contained in the mixed solution is 0.1-1 mM, preferably 0.26 mM; and the concentration of phosphate ions (PO4 3- ) contained in the mixed solution is 1-10 mM, preferably 5 mM.

[0015] Further, the ratio of the use amount of the bacterial cellulose film (BC) to the mixed solution in step S2 is (0.0001-0.001) g:(10-100) mL, preferably 0.0005 g:20 mL; and the pH value of the solution is controlled within 7.2-7.4 during the reaction. The time of the biomimetic mineralization reaction in step S2 is 12-48 h.

[0016] Further, the drying time in step S3 is 2-48 h, and the temperature is 20-60℃; preferably, the time is 12-24 h, and the temperature is 37℃.

[0017] The third technical solution of the present application provides an application of the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose film as a wound dressing.

[0018] Further, the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose film has good biological safety, has a significant healing-promoting effect on the wound of the elderly, effectively reduces the inflammatory response and promotes tissue regeneration by regulating the immune microenvironment.

[0019] The fourth technical solution of the present application provides a wound dressing, and the effective component of the wound dressing is a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose film.

[0020] Further, the wound dressing is a dressing for the wound of the elderly skin.

[0021] Further, the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose film is applied to the skin and directly adheres to the wound of the skin.

[0022] In summary, the technical scheme of the present application is to design the chiral multi-level structure hydroxyapatite (CMHAP) nanosheet on the bacterial cellulose (BC) fiber membrane through a simple chiral glutamic acid (GA) mediated self-assembly method. The BC@CMHAP composite materials synthesized by L-glutamic acid (left-handed glutamic acid), D-glutamic acid (right-handed glutamic acid) and R-glutamic acid (racemic-glutamic acid) are systematically named as L-CHB, D-CHB and R-CHB (wherein, the prefixes L, D and R represent different chiralities, CH represents CMHAP, and B represents BC). In order to further explore the regeneration potential of chiral biomaterials in old skin, the inventors selected the old mouse full-thickness dorsal skin damage model to carry out experiments, and simultaneously evaluated the immune regulation effect of the material, and the results showed that the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane not only can closely adhere to the wound to form effective coverage, but also can significantly reduce the recruitment of neutrophils, and exhibits good wound care effect. In addition, L-CHB promotes the polarization of M1 macrophages to M2 phenotype, thereby reducing inflammation and enhancing tissue repair. In order to identify the specific immune cells involved, the inventors used NPSG mice for research, and the results showed that M2 macrophages gathered at the wound and secreted anti-inflammatory cytokines. These observation results show that the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane provides a protective barrier, regulates macrophage polarization, and jointly promotes wound healing.

[0023] Compared with the prior art, the present application has at least the following advantages:

[0024] (1) Unique chiral structure is introduced: most of the hydroxyapatite materials obtained in the prior art do not have chirality, while the present scheme successfully grows the chiral multi-level structure hydroxyapatite nanosheet on the surface of the bacterial cellulose membrane in situ through a simple chiral glutamic acid molecule induced self-assembly strategy, and prepares a hydroxyapatite / bacterial cellulose membrane with chiral structure, which fills the gap in this aspect in the prior art and expands new possibilities for the application of hydroxyapatite materials in the field of biomedicine;

[0025] (2) The synthesis method is simple and controllable: a simple molecular induced self-assembly strategy is adopted, a complex is formed by coordination of chiral glutamic acid molecules and calcium ions, 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 to control the nucleation and crystal growth rate, so as to realize the formation of asymmetric crystal structure. This method is relatively simple to operate, and the structure and performance of the material can be accurately controlled;

[0026] (3) Targeted solution to the problem of wound healing in aging skin: Existing technologies lack comprehensive and targeted treatment plans in the research on wound healing in aging skin. However, the present invention uses the prepared chiral membrane as a wound healing material. Its low allergenicity and safety have been confirmed through in vitro and in vivo experiments, indicating that the L-CHB membrane has a good healing effect on wounds in the elderly, providing an effective solution to this clinical problem;

[0027] (4) Multi-stage regulation of wound healing process: The L-CHB membrane in this protocol plays a role in different stages of wound healing. In the early inflammatory response stage, it can effectively reduce 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, it can reduce wound fibrosis and promote tissue regeneration, thus achieving multi-stage precise regulation of the wound healing process. Attached Figure Description

[0028] Figure 1 The chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes (BC@CMHAP membranes) provided in Examples 1-3 and Comparative Example 1, namely L-CHB, D-CHB, R-CHB, and Ach-HB respectively, are described below. Figures 2~8 Scanning electron microscope images (same as above);

[0029] Figure 2 XRD patterns of the chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1;

[0030] Figure 3 UV-Vis diffuse reflectance (DRUV-Vis) and circular dichroism (DRCD) spectra of the bacterial cellulose membrane (BC), the chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1;

[0031] Figure 4 Fourier transform infrared spectra of bacterial cellulose membranes (BC), chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1;

[0032] Figure 5 Stress-strain curves of the chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1;

[0033] Figure 6 Tensile strength of bacterial cellulose membranes (BC), chiral hierarchical hydroxyapatite nanosheets / bacterial cellulose membranes provided in Examples 1-3 and Comparative Example 1;

[0034] Figure 7The maximum elongation of the chiral hierarchical structured hydroxyapatite nanosheet / bacterial cellulose membrane provided for the bacterial cellulose membrane (BC), examples 1-3 and comparative example 1;

[0035] Figure 8 The contact angle of the chiral hierarchical structured hydroxyapatite nanosheet / bacterial cellulose membrane provided for the bacterial cellulose membrane (BC), examples 1-3 and comparative example 1 to characterize the hydrophilic and hydrophobic properties;

[0036] Figure 9 The experimental results of (A) HE staining, (B) Masson staining and (C) CD45 immunohistochemical staining of the NPSG mice subcutaneously buried with materials on the 3rd, 7th and 14th days in Example 5; BC in the figure represents the bacterial cellulose membrane treatment group, L-CHB, D-CHB, R-CHB and Ach-HB are respectively the chiral hierarchical structured hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1;

[0037] Figure 10 The HE staining results of important organs of NPSG mice after subcutaneous embedding of different materials for 2 weeks in Example 5; BC in the figure represents the bacterial cellulose membrane treatment group, L-CHB, D-CHB, R-CHB and Ach-HB are respectively the chiral hierarchical structured hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1;

[0038] Figure 11 The flowchart of the experiment in Example 6;

[0039] Figure 12 In Example 6: (A) wound healing photos of different materials for skin wounds of old C57 mice at different time points, (B) wound healing trajectory graph of old C57 mice at different time points in different material groups, (C) wound closure rate statistics graph of old C57 mice in different material groups; L-CHB, D-CHB, R-CHB and Ach-HB in the figure are respectively the chiral hierarchical structured hydroxyapatite nanosheet / bacterial cellulose membrane treatment groups provided in Examples 1-3 and Comparative Example 1, and the following Figures 13~17 The control group is the Control group without treatment on the wound, and the following Figures 13~17 ;

[0040] Figure 13 The wound HE staining graph of the five groups of old C57 mice in Example 6, and the MASSON staining graph on the 14th day;

[0041] Figure 14 The immunohistochemical staining of neutrophil-related ly6G of the five groups of old C57 mice on the first day and the third day in Example 6, and the immunohistochemical staining of NF-κB of the five groups of old C57 mice on the first day;

[0042] Figure 15 (A) five groups of old C57 mice in Example 6 on the first day of M1 macrophage related index of immunohistochemical staining, (B) five groups of old C57 mice in Example 6 on the third day of M2 macrophage related immunohistochemical staining;

[0043] Figure 16 (A) five groups of NPSG mice in Example 7 wound healing chart, (B) five groups of NPSG mice in Example 7 wound HE and MASSON staining chart on the 7th day and the 14th day;

[0044] Figure 17 (A) five groups of NPSG mice in Example 7 wound HE staining chart, (B) five groups of NPSG mice in Example 7 wound NF-κB immunohistochemical staining, (C) five groups of NPSG mice in Example 7 on the 14th day of wound Sirius red and its polarized light scanning pictures. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in conjunction with specific embodiments. It should be pointed out that the following embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0046] All raw materials of the present application have no special restrictions on their sources, and if no specific manufacturer is 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.

[0047] SBF is prepared according to the conventional operation of those skilled in the art (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 ISO23317:2014 Implants for surgery — In vitro evaluation for apatite - forming ability of implant materials).

[0048] Sodium chloride (NaCl, 99.5%), sodium bicarbonate (NaHCO3, 99.8%), potassium chloride (KCl, 90%), dipotassium phosphate (K2HPO4), magnesium chloride hexahydrate (MgCl2-6H2O, 99.5%), sodium sulfate (Na2SO4, 98%), and calcium chloride (CaCl2, 99.8%) were from National Medicine 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. Ultrapure water (18.2 MΩ / cm) was used throughout the process, which was generated by a Heal Force SMART ultrapure water system. Sirius red staining solution was purchased from Nanjing Sunbeiqia Biotechnology Co., Ltd. (Sbjbio), model number BP-DL030; Masson staining solution was purchased from Morphisto, Germany, model number MASSON GOLDNER 12043.00500.

[0049] The tissue sample preparation and staining experiments (including HE staining, MASSON staining, Sirius red staining, and immunohistochemical staining) involved in the examples are described as follows.

[0050] (I) Paraffin section preparation

[0051] (1) The fresh tissue was immersed in formalin solution for 12 h;

[0052] (2) The tissue was placed in an automatic dehydrator for dehydration;

[0053] (3) The tissue was taken out and embedded with a embedding machine;

[0054] (4) After the paraffin solidified, it was placed in a-20℃ refrigerator for 15 min;

[0055] (5) The wax block was mounted on the fixing device of the microtome, the knife holder was mounted with a sectioning knife, the knife edge was at an angle of 5° with the surface of the wax block, the wax block and the knife were adjusted to the appropriate position, and the section thickness scale was adjusted to 4 μm;

[0056] (6) The rotary microtome was used, the left hand controlled the progress, and the right hand rotated at a constant speed. The wax block was roughly trimmed to the complete tissue morphology, the left hand stopped the progress, and the right hand continued to rotate. The right hand used a pair of tweezers to flatten the complete section, and the left hand used a brush to pull the right hand out of the water bath. The right hand used a pair of tweezers to flatten the section in a 42℃ water bath, and the left hand used a glass slide to pull the tissue out of the water bath. The tissue was inserted into a sectioning oven at 65℃.

[0057] (ii) HE staining

[0058] (1) The section after baking for 2h was taken out from the oven at 65°C;

[0059] (2) Sequentially dipped into xylene I-xylene II-xylene III, each for 5 min;

[0060] (3) Sequentially dipped into 100% alcohol-100% alcohol-85% alcohol-75% alcohol, each for 3 min, gradient de-waxing, and rinsed with running water for 3 min;

[0061] (4) Dipped into hematoxylin staining solution for 5 min, and rinsed with running water for 2 min (if it is a frozen tissue section, directly start from step 4 after fixation);

[0062] (5) Dipped into 0.5% hydrochloric acid alcohol differentiation solution for 3 s, and then taken out, and rinsed with running water for 3 min;

[0063] (6) Dipped into 95% alcohol for 1 min;

[0064] (7) Dipped into eosin staining solution for 30 s;

[0065] (8) Sequentially dipped into 80% alcohol-85% alcohol-95% alcohol-95% alcohol-100% alcohol-100% alcohol, each for 1 min, gradient dehydration;

[0066] (9) Sequentially dipped into xylene I-xylene II-xylene III, each for 2 min;

[0067] (10) The section was taken out, neutral gum was added dropwise, and the cover glass was mounted. The mounted section was placed under a microscope, and the observed results were recorded and photographed.

[0068] (iii) Immunohistochemical staining (IHC)

[0069] (1) The sample was routinely de-waxed to water;

[0070] (2) High pressure repair: the repair solution was preheated in a high pressure pot, after boiling, the glass placed on the plastic staining rack was put into the repair solution, the tissue must be completely covered, after the pressure limiting valve rotated and sprayed, the timing was started for 2.5 min, at the same time, the electromagnetic oven was adjusted from high fire to medium fire, after the timing was completed, the heat source was removed, and the cold water was cooled to room temperature, attention should be paid not to inject cold water into the high pressure pot;

[0071] (3) PBS washing 3-5 times, each for 2 min (Tween 20 was added in PBS);

[0072] (4) 3% hydrogen peroxide, incubated at room temperature for 10 min;

[0073] (5) Distilled water cleaning, PAP stroke circle (circle when 2-3mm distance from the tissue) ;

[0074] (6) PBS cleaning 3-5 times, each 2min (PBS adding 1% Tween 20) ;

[0075] (7) Drop a suitable amount of primary antibody, 37°C 1h (when adding primary antibody, the water on the slice needs to be shaken clean, pay attention to not dry piece) ;

[0076] (8) PBS cleaning 3-5 times, each 2min (PBS adding Tween 20) ;

[0077] (9) Drop a suitable amount of one-step detection system (PV-8000D, 37°C 30min, when adding secondary antibody, the water on the slice needs to be shaken clean, pay attention to not dry piece) ;

[0078] (10) PBS cleaning 3-5 times, each 2min;

[0079] (11) DAB (ZLI-9018) color development after secondary antibody, present use, 6-8min, no need to control color development under the microscope, tap water termination and rinse 3-5min;

[0080] (12) Hematoxylin restain, avoid restain color too dark (according to hematoxylin new and old adjust time) ;

[0081] (13) Tap water gentle rinse;

[0082] (14) Hydrochloric acid alcohol differentiation (according to hematoxylin restain situation flexible control time) ;

[0083] (15) According to the situation, can choose to use EDTA pH8.0 repair liquid return blue 3-5min, running water rinse 3-5min;

[0084] (16) Gradual gradient alcohol dehydration, xylene transparency, neutral gum mounting. Through observing the intensity, range and distribution of staining, analyze the expression difference of antigen in tissue.

[0085] (Four) safranin staining (for collagen fiber staining)

[0086] (1) Paraffin section deparaffinization to water: put the section into xylene I, xylene II for 10min respectively, then put into anhydrous ethanol, 95% ethanol solution, 85% ethanol solution, 75% ethanol solution for 5min respectively, and then put into distilled water for a few seconds;

[0087] (2) After washing, the section is placed on the workbench, and 1-2 drops of sky red dye are completely covered on the tissue surface, and the drop dyeing is 1 hour;

[0088] (3) Wash with running water for 5 min, and Mayer hematoxylin dyeing solution for 8-10 min;

[0089] (4) Wash with running water for 10 min, and then dehydrate and transparentize after absorbing the surface water, and gum seal.

[0090] (Five) MASSON staining

[0091] (1) The sample is routinely deparaffinized to water;

[0092] (2) MORPHISTO hematoxylin solution for 6-8 minutes;

[0093] (3) Wash with tap water for 10 minutes, and if over-dyed, differentiate with hydrochloric acid alcohol;

[0094] (4) Use Goldner I solution of eosin Y acid complex red liquid for 3 min. Wash with distilled water for a few seconds, dry, and observe under a microscope;

[0095] (5) Phosphomolybdic acid-orange reagent Goldner II solution for 3 minutes, and distilled water for a few seconds;

[0096] (6) Green Goldner III solution for 3 minutes, and distilled water for a few seconds;

[0097] (7) Dehydrate with gradient alcohol, xylene transparent, and neutral gum sealing.

[0098] Example 1:

[0099] Preparation of chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane (L-CHB).

[0100] This example grows chiral multi-level structure hydroxyapatite (CMHAP) nanosheets on the surface of a bacterial cellulose membrane (BC) in situ through a simple chiral glutamic acid (GA) molecule-induced self-assembly strategy, which specifically includes the following steps:

[0101] S1. L-glutamic acid, SBF (simulated body fluid), Tris, and hydrochloric acid are sequentially added to deionized water to obtain a mixed solution, which is ready for use; in the mixed solution obtained in this step, the concentration of L-glutamic acid is 0.2 mM; the concentration of calcium ions (Ca 2+ ) is 0.26 mM, and the concentration of phosphate ions (PO4 3- ) is 5 mM; the total volume is 20 mL, and the pH is 7.2-7.4.

[0102] In this step, chiral L-glutamic acid is a bioactive material that can coordinate with calcium ions to form a complex, so chiral L-glutamic acid is selected as a structure-directing agent and an asymmetric induction agent. The main components of SBF include calcium ions (Ca 2+ ) and phosphate ions (PO4 3- ). When a bioactive material is soaked in SBF, the surface of the material interacts with the ions in the solution. The active groups on the surface of the material adsorb calcium ions in the solution, and then the calcium ions attract phosphate ions, gradually forming the crystal nucleus of calcium phosphate compounds. Over time, the crystal nucleus grows, and eventually forms a hydroxyapatite layer. This process is called "biomineralization", which needs to be carried out within a certain pH range, generally 7.2-7.4. In this embodiment, Tris-hydrochloric acid is used as an acid-base regulator to adjust the pH value of the reaction system and control the nucleation and crystal growth rate of the reaction system, and finally realize the formation of an asymmetric crystal (chiral HAP nanosheet, CMHAP) structure.

[0103] S2. Clean the bacterial cellulose membrane (BC) with deionized water and anhydrous ethanol in turn, then put the bacterial cellulose membrane (BC) into the mixed solution prepared in step S1, and carry out biomineralization at 37℃ for 24 h.

[0104] In this step, the dosage ratio of bacterial cellulose membrane (BC) to mixed solution is 0.0005 g:20 mL, and the pH value of the solution during the reaction is controlled within 7.2-7.4.

[0105] S3. After the biomineralization reaction is completed, the bacterial cellulose membrane (BC) with chiral hydroxyapatite grown on it is taken out, washed with deionized water, and then placed in a vacuum drying oven and dried at 37℃ for 12 h. The obtained chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane is named L-CHB. That is, the L-CHB and the BC@CMHAP represent the same substance.

[0106] Example 2:

[0107] Preparation of chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane (D-CHB).

[0108] This embodiment is the same as Example 1, except that L-glutamic acid is replaced by D-glutamic acid.

[0109] Example 3:

[0110] Preparation of chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane (R-CHB).

[0111] This example is the same as Example 1, except that L-glutamic acid is replaced by racemic-glutamic acid.

[0112] Comparative Example 1:

[0113] Preparation of hydroxyapatite nanosheet / bacterial cellulose membrane without adding chiral molecules (Ach-HB).

[0114] This comparative example is compared with Example 1, without adding chiral molecules, and the obtained hydroxyapatite nanosheet / bacterial cellulose membrane is named Ach-HB.

[0115] Example 4: Mechanical property characterization of the material.

[0116] This example characterizes the mechanical properties of the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membranes prepared in Examples 1-3 and Comparative Example 1 above. The relevant tests include the following.

[0117] 4.1 Scanning electron microscopy.

[0118] Scanning electron microscopy is a routine operation familiar to those skilled in the art, and the specific experimental method can be operated according to the instrument manual, and will not be described here.

[0119] The chiral multi-level structure hydroxyapatite nanosheet structure in the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane is denoted as CMHAP.

[0120] Experimental results: As shown in Figure 1 , it can be seen that the BC membrane presents a reticular porous structure composed of nanofibers, and the CMHAP presents a flower-like structure of assembled nanosheets distributed on the surface of the BC fiber membrane. On the L-CHB membrane, the L-CMHAP flower is 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 from the surface of the BC fiber. The left-handed helical accumulation of the nanosheets is observed, representing the tertiary level of chirality. The enlarged scanning electron microscopy image shows that the chirality of the CMHAP nanosheet is secondary. Based on the inventors' previous research (Chemistry of Materials, 2021, 34(1):53-62.), it can be considered that the misplacement of atoms also leads to the initial horizontal chirality.

[0121] Similarly, the right-handed helical arrangement of nanosheets can be observed in D-CMHAP flowers on D-CHB films, which is considered as tertiary chirality. The magnified SEM images show the wave-like twisted pattern of nanosheets. The right-handed twisted structure appeared in nanosheets is considered as secondary chirality of D-CMHAP flowers. The primary chirality result is also right-handed (Chemistry of Materials, 2021, 34(1): 53-62.). Ach-CMHAP and R-CMHAP flower on Ach-HB and R-CHB films, respectively, indicating that the nanosheets are randomly arranged, irregular in structure, and thus do not have chiral multilayer structures.

[0122] 4.2 XRD spectra.

[0123] The crystal structures of BC, Ach-HB, D-CHB, L-CHB and R-CHB films were further analyzed by wide-angle X-ray diffractometer (XRD). XRD detection is a routine operation familiar to those skilled in the art, which can be operated according to the instrument manual, and the specific experimental method is not described in detail here.

[0124] The XRD spectra of Ach-HB, D-CHB, L-CHB and R-CHB films and BC substrates are shown in Figure 2 Although there is strong BC substrate intensity in the XRD spectra of Ach-HB, D-CHB, L-CHB and R-CHB films, the hexagonal phase characteristic reflection of HAP with lattice parameters a = b = 9.4166 Å and c = 6.8745 Å and space group P63 / m can still be identified [JCPDS file 09-0432]. In addition, the corresponding powders were also detected. At the same time, certain steamed bun peaks were also found in the low angle region, indicating that the hydroxyapatite powder contains some components with lower crystallinity.

[0125] 4.3 UV-Vis diffuse reflectance (DRUV-Vis) and circular dichroism (DRCD) spectra.

[0126] Since BC, Ach-HB, D-CHB, L-CHB and R-CHB films are opaque, their chirality was clearly detected based on optical activity (OA) using diffuse reflectance ultraviolet-visible (DRUV-Vis) and circular dichroism (DRCD) spectra. UV-Vis diffuse reflectance (DRUV-Vis) and circular dichroism (DRCD) spectra are routine operations familiar to those skilled in the art, which can be operated according to the instrument manual, and the specific experimental method is not described in detail here.

[0127] The experimental results are shown in Figure 3As shown: DRCDs with white and black backgrounds display approximately absorption-based OA (AOA) and scattering-based OA (SOA) and AOA, since almost all visible light is reflected by white light and absorbed by the black backplane, respectively. It is known that the distance between semiconductor nanounits aggregated in a chiral manner is smaller than the Bohr exciton radius, inducing an asymmetric field throughout the aggregate through excited delocalization, leading to AOA based on electronic transitions. SOA occurs at multiple integration wavelengths based on mλ = Pn_avg, where m is an integer, n_avg is the average refractive index, and P is the pitch length of the chiral medium. Left-handed structures tend to absorb right-handed circularly polarized light and reflect left-handed light, resulting in negative AOA and SOA signals for DRCD measurements.

[0128] like Figure 3 As shown, L-CHB and D-CHB films exhibit mirror signals in the 240–800 nm range. Taking L-CHB as an example, the UV-Vis spectrum shows a broadband pattern in the 240–800 nm range. The CD spectrum has two strong peaks at 240–350 nm and 350–800 nm, with the strongest peaks at 285 nm and 650 nm, respectively. According to the detection mechanism, the OA of the L-CHB film includes both AOA and SOA, since the BC substrate has a white background. The mirror DRCD spectrum of the D-CHB film shows opposite chirality.

[0129] DRCD spectroscopy of the BC substrate confirmed that the optically active OAs of the L-CHB and D-CHB films originated from the hierarchical chiral structure, rather than the BC substrate. The R-CHB film morphology was similar to the corresponding CMHAP flower, but lacked chirality.

[0130] 4.4 Fourier transform infrared spectrum.

[0131] Fourier transform infrared spectroscopy is a routine operation familiar to those skilled in the art, and can be performed according to the instrument's instruction manual. Specific experimental methods will not be elaborated upon here.

[0132] FTIR confirmed the reliability of BC, Ach-HB, D-CHB, L-CHB, and R-CHB. Figure 4 As shown, 961 cm -1 Band reflection qvl PO4 3- 1125 cm -1 Band reflection qvl PO4 3- The weak tensile and vibrational strengths of the hydroxyl group are 3426 cm⁻¹. -1 and 637 cm -1 874 and 1420~1470 cm -1The band is b-type carbonate apatite, carbonate replaces the phosphate position in the hydroxyapatite lattice. FTIR spectrum shows that there are at least three weak to strong stretching vibrations of protons directly connected to phosphorus vP-H, indicating that there are three structural positions or three conformations of phosphate groups. -1 Within the region, there are at least three weak to strong stretching vibrations of protons directly connected to phosphorus vP-H, indicating that there are three structural positions or three conformations of phosphate groups.

[0133] 4.5 Stress-strain curve, tensile strength and maximum elongation.

[0134] Mechanical properties are one of the main factors affecting the performance of tissue engineering wound repair materials. This embodiment studies the stress-strain behavior, tensile strength and maximum elongation of BC, Ach-HB, D-CHB, L-CHB and R-CHB films. This embodiment is detected in a universal material testing machine. In the tensile test, the tester fixes the two ends of the sample, and then gradually applies tension at a constant speed until the sample breaks. In this process, the tester records the applied force and the elongation of the sample in real time, thereby obtaining the stress-strain curve. The specific experiment can be operated according to the instruction manual of the instrument, which is a conventional measurement means in the art and will not be described here.

[0135] The experimental results are shown in Figures 5~7 Ach-HB, D-CHB, L-CHB and R-CHB show similar stress-strain and tensile strength, and their strength is 2-3 times that of pure BC, proving that the mineralized chiral hydroxyapatite coating can effectively improve the strength and toughness of the BC film.

[0136] 4.6 Hydrophilicity and hydrophobicity.

[0137] The hydrophilicity and hydrophobicity of the material are measured by a contact angle measuring instrument. The specific experiment can be operated according to the instruction manual of the instrument, which is a conventional measurement means in the art and will not be described here.

[0138] The experimental results are shown in Figure 8 It can be seen that after the BC is coated with chiral hydroxyapatite, the hydrophilicity of the BC remains basically unchanged due to the high hydrophilicity of the BC itself. 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 application.

[0139] Based on the above several material characterizations, this embodiment proves the successful preparation of each group of materials and good mechanical properties, which lays a foundation for its application in biomaterials.

[0140] Example 5 Evaluation of the biological safety of the film material.

[0141] The severe immunodeficient mouse NPSG mouse used in this embodiment was purchased from Phenotek Biotechnology (Shanghai) Co., Ltd. The severe immunodeficient mouse NPSG mouse NOD-Prkdc scid Il2rg null The NPSG mouse (scid means severe combined immunodeficiency, null means knockout of IL2rg) model shows inherent immunodeficiency characterized by reduced complement system and reduced dendritic cell function. Importantly, the genetic modification involves knocking out the Prkdc gene of these mice, resulting in the absence of T lymphocytes and B lymphocytes, ultimately leading to severe combined immunodeficiency. This broad immunodeficiency has a negative impact on both cellular and humoral immunity, highlighting the key 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 deficiency of the immune system, most notably causing a sharp decrease in natural killer (NK) cell activity. The loss of NK cell function emphasizes the critical impact of the IL2rg gene on overall immune function in these mouse models. Therefore, the NPSG mouse is severely immunodeficient due to Prkdc and IL2rg gene knockout, can reduce immune interference, observe potential infection risk, and is suitable for membrane material biosafety evaluation.

[0142] In this embodiment, several membrane materials prepared in Embodiments 1-3 and Comparative Example 1 were respectively implanted subcutaneously in NPSG mice, and then HE staining, Masson staining and CD45 immunohistochemical staining were performed on the subcutaneous tissues of the mice on days 3, 7 and 14, respectively. The staining results are shown in Figure 9 It can be seen that the inflammatory response of the subcutaneous tissue of the mouse is small, there is no abnormal recruitment of immune cells, and there is no obvious difference between groups. This means that the membrane material has good biosafety, low sensitivity, and at least a constant effect on the surrounding tissue for 14 days.

[0143] Next, in this embodiment, HE staining was performed on the important organs of the mice, i.e. heart, liver, lung and kidney, at 14 days, and the results are shown in Figure 10 It can be seen that the important organ tissues have not undergone obvious changes, and the tissue structure is normal, indicating that the membrane material prepared in the present application has no visceral toxicity.

[0144] The results of this embodiment show that the membrane material provided in the present application has low sensitivity and safety.

[0145] Example 6: Construction of an aged C57BL / 6 mouse skin defect model to verify the effect of chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane on wound healing of aged skin in vivo.

[0146] This example used naturally aging C57BL / 6 mice to demonstrate the good healing effect of L-CHB film on the wounds of the elderly, and the process is shown in Figure 11 After the skin of the mouse dorsal surgical area was prepared, two circular full-thickness skin wounds (5 mm in diameter, one on each side) were established in each mouse, which were divided into five groups: Control, Ach-HB, L-CHB, R-CHB and D-CHB, and each group was covered with the corresponding material. Photographs were taken on postoperative days 0, 1, 3, 5, 7 and 14, and analyzed using Image J software. 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, School of Medicine, Shanghai Jiao Tong University.

[0147] As shown in Figure 12 (A), it can be seen that the wound covered with L-CHB film heals the fastest. This shows that L-CHB film can shorten the healing period, efficiently repair the wound and accelerate healing. As shown in Figure 12 (B) and Figure 12 (C), the results obtained by Image J software analysis clearly show the wound healing trajectory of the elderly C57 mice at different time points in different material groups, which is the same as the phenomenon observed in Figure 12 (A).

[0148] Next, in order to study the basic structure of the skin and cells, and to show the fibrous connective tissue in the tissue, especially the collagen fibers, this example performed HE staining (1, 3, 7, 14 days) and MASSON staining (14 days) on the skin samples of the wounds of the five groups of elderly C57 mice, and the results are shown in Figure 13 : After the wound was covered with L-CHB film, the infiltration of inflammatory cells at the wound site was significantly reduced, and epithelial keratinization, collagen generation and hair follicle regeneration were promoted.

[0149] Next, in order to study the immune activity and functional state of the skin wound, this example performed immunohistochemical detection of the distribution of the neutrophil surface specific antigen ly6G protein in the skin wounds of the five groups of elderly C57 mice on day 1 and day 3, and also performed immunohistochemical staining of the distribution of NF-κB protein on day 1, and the results are shown in Figure 14ly6G-positive neutrophils play a key role in immune defense. When the body is infected with pathogens such as bacteria and viruses, neutrophils are activated, and by detecting the level of ly6G, it can be determined whether the neutrophils are in an active immune response state and their ability to phagocytize and kill pathogens. Similarly, NF-κB is a key regulator of inflammatory response. When cells are stimulated by inflammation such as LPS, TNF-α, IL-1β, etc., NF-κB is activated and initiates a series of transcription of inflammation-related genes, such as genes encoding TNF-α, IL-1β, IL-6, etc. pro-inflammatory cytokines. The results show that the aggregation of neutrophils at the lower edge of the wound in the L-CHB membrane group is less severe, and the inflammatory response is weaker.

[0150] Next, this embodiment carries out immunohistochemical staining on the related indicators CD68, IL-1β, TNF-α of M1 type macrophages on the first day and the related indicators CD163, CD206 of M2 type macrophages on the third day of the five groups of old C57 mice, and the results are shown in Figure 15 M1 type macrophages mainly play a pro-inflammatory and immune killing role, and detecting related indicators 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 with pathogens, M1 type macrophages are activated, and the levels of these indicators increase, indicating that the immune system is actively responding to infection. M2 type macrophages have anti-inflammatory, tissue repair and immune regulation functions, and detecting indicators such as CD163 and CD206 can understand the body's anti-inflammatory and repair state. By detecting the indicators of the two types of macrophages, the body's immune balance state can be comprehensively evaluated, and it can be determined whether the immune function is in hyperactivity or hypofunction. It can be seen that in the early inflammatory response stage, L-CHB membrane can effectively reduce the pro-inflammatory cytokines of the wound, reduce the aggregation of neutrophils and M1 macrophages, and regulate the immune microenvironment; in the later tissue repair stage, L-CHB membrane can reduce the fibrosis of the wound and promote tissue regeneration.

[0151] Example 7: In vivo experiment: Construct a severe immunodeficient mouse (NPSG) skin defect model to study the mechanism of the membrane material and regulate innate immune cells.

[0152] In order to further understand the mechanism of different cells, the present application also selects NPSG mice (the same as in the above embodiment) for experiments, and targets to knock out T / B / NK cells, and uses ICR mice of the same age as a control to compare the effect of the material on wound healing in immunodeficient and normal immune microenvironment, and this study involves 45 NPSG mice.

[0153] This embodiment photographs and compares the wound healing of the five groups of NPSG mice, and the results are shown in Figure 16As shown in FIG. 2 (A), it can be seen that the skin wound healing of the L-CHB group of mice is the best. The HE and MASSON staining results of the wounds of the five groups of NPSG mice on the 7th day and the 14th day are shown in FIG. 2 (B). Figure 16 As shown in FIG. 2 (B), it can be seen that: in the early stage, the inflammation of L-CHB is obviously the lightest, and in the later stage of 7 and 14 days, the epidermis of the L-CHB wound can be seen to be thin and hair follicles are formed, which shows the superior effect of L-CHB on skin wound healing.

[0154] Next, the HE staining results of the wounds of the five groups of NPSG mice on the 1st day and the 3rd day are shown in FIG. 3 (A). Figure 17 As shown in FIG. 3 (A), it can be seen that the cell morphology of the L-CHB film group is good. The immunohistochemical staining results of NF-κB of the wounds of the five groups of NPSG mice are shown in FIG. 3 (B). Figure 17 As shown in FIG. 3 (B), it can be seen that the L-CHB film can effectively reduce the pro-inflammatory cytokines of the wound. The polarized light scanning results of the Sirius red staining of the wounds of the five groups of NPSG mice on the 14th day are shown in FIG. 3 (C). Figure 17 As shown in FIG. 3 (C), it can be seen that the L-CHB film group has more hair follicles and thinner epidermis, and the recovery is good.

[0155] In summary, the embodiment of the present application first grows the chiral multi-level structure hydroxyapatite (CMHAP) nanosheet on the surface of the bacterial cellulose film in situ through a simple chiral glutamic acid (GA) molecule induced self-assembly strategy, and prepares a chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose film material. The hydroxyapatite nanosheet / bacterial cellulose film finally prepared by using left-handed glutamic acid, right-handed glutamic acid and racemic glutamic acid is named as L-CHB, D-CHB and R-CHB respectively. The scaffold synthesized without adding chiral molecules is named as Ach-HB. Finally, the film material prepared above is used as a wound healing material (dressing).

[0156] The material characterization confirms the successful preparation of each group of materials and good mechanical properties. In vivo and in vitro experiments confirm the low sensitivity and safety of each group of materials. In vivo experiments confirm that the L-CHB film has a more obvious pro-healing effect on the elderly wound than other groups. Histological staining finds that in the early inflammatory response stage, the L-CHB film can effectively reduce the adhesion of bacteria on the wound, inhibit the anti-inflammatory effect of the NF-κB pathway, reduce the aggregation of neutrophils, promote the polarization of macrophages, and regulate the innate immune microenvironment; in the later tissue repair stage, the L-CHB film can reduce the fibrosis of the wound and promote tissue regeneration. Further mechanism research finds that in the NPSG mouse model, the L-CHB film also exhibits significant anti-inflammatory and pro-tissue healing abilities, which indicates that the wound healing mediated by biomaterials requires the innate immune system.

[0157] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as a number of alternatives. It is not intended to limit the application to the precise form described.

Claims

1. The use of chiral hierarchical structure hydroxyapatite nanosheet / bacterial cellulose membrane in the preparation of wound dressings for regulating immune microenvironment, reducing inflammatory response and promoting tissue regeneration, characterized in that, The chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane is prepared by a biomimetic mineralization method from chiral hydroxyapatite nanosheets and bacterial cellulose; The chiral hydroxyapatite nanosheets are prepared from a simulated body fluid and chiral glutamic acid; The chiral hydroxyapatite nanosheets are grown on the bacterial cellulose membrane; The hydroxyapatite nanosheet / bacterial cellulose membrane is prepared by the following method: S1. Chiral glutamic acid, a simulated body fluid, tris(hydroxymethyl) aminomethane and hydrochloric acid are sequentially added into deionized water to obtain a mixed solution; S2. The bacterial cellulose membrane is cleaned, and then is placed into the mixed solution freshly prepared in step S1 to perform a biomimetic mineralization reaction; S3. After the biomimetic mineralization 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; In step S1, the chiral glutamic acid is L-glutamic acid; In step S1, the concentration of the chiral glutamic acid in the mixed solution is 0.1-1 mM; the concentration of calcium ions contained in the mixed solution in step S1 is 0.1-1 mM; and the concentration of phosphate ions contained is 1-10 mM; In step S2, the pH value of the solution during the biomimetic mineralization reaction is controlled within 7.2-7.4, and the biomimetic mineralization reaction in step S2 is performed for 12-48 hours. In the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane, the L-CMHAP flower is densely arranged by left-handed spiral stacked 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 the bacterial cellulose membrane.

2. The application according to claim 1, wherein the preparation method comprises the following steps: S1. Chiral glutamic acid, a simulated body fluid, tris(hydroxymethyl) aminomethane and hydrochloric acid are sequentially added into deionized water to obtain a mixed solution; S2. The bacterial cellulose membrane is cleaned, and then is placed into the mixed solution freshly prepared in step S1 to perform a biomimetic mineralization reaction; S3. After the biomimetic mineralization 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; In step S1, the chiral glutamic acid is L-glutamic acid; In step S1, the concentration of the chiral glutamic acid in the mixed solution is 0.1-1 mM; the concentration of calcium ions contained in the mixed solution in step S1 is 0.1-1 mM; and the concentration of phosphate ions contained is 1-10 mM; In step S2, the pH value of the solution during the biomimetic mineralization reaction is controlled within 7.2-7.4, and the biomimetic mineralization reaction in step S2 is performed for 12-48 hours.

3. The application according to claim 2, wherein the use ratio of the bacterial cellulose membrane to the mixed solution in step S2 is (0.0001-0.001) g to (10-100) mL.

4. The application according to claim 2, wherein the chiral multi-level structure hydroxyapatite nanosheet / bacterial cellulose membrane is used for the following purposes: ​ ​ The drying time in step S3 is 12-24 h at a temperature of 20-60°C.

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