Double-layer guided bone regeneration membrane and preparation method thereof

Abstract

The invention provides a double-layer guided bone regeneration membrane and a preparation method thereof, and relates to the technical field of bone regeneration, the double-layer guided bone regeneration membrane comprises a compact layer and a porous layer which are stacked up and down, the compact layer is prepared by crosslinking and modifying soluble eggshell membrane protein (SEP), epigallocatechin gallate (EGCG) and carboxymethyl chitosan (CMC) through genipin, has a compact nonporous structure and is used for blocking soft tissue cell migration; the porous layer is obtained by crosslinking hydroxyapatite modified by 3-aminopropyltriethoxysilane (APTES) and carboxymethyl chitosan (CMC) through genipin, has a three-dimensional porous structure and is used for promoting bone cell growth and bone regeneration. The double-layer guided bone regeneration membrane disclosed by the invention has good tensile strength, physical barrier function, antibacterial property and degradation controllability, and realizes a safe and effective osteogenesis promoting effect under irradiation of near and far infrared light of 808nm and power of 1W.

Description

Technical Field

[0001] This application belongs to the field of bone regeneration technology, specifically relating to a double-layer guided bone regeneration membrane and its preparation method, which is particularly suitable for bone defect repair in dental implant restoration. Background Technology

[0002] With the increasing demand for oral health, dental implants are widely used due to their excellent function and aesthetics. However, the implant restoration process often faces the challenge of insufficient physiological or pathological bone volume, which greatly limits the success rate of implant restoration and the long-term stability of the implant. Guided Bone Regeneration (GBR) technology, as a key means to solve this problem, focuses on using a biological barrier membrane to isolate soft tissues (such as fibroblasts), creating a stable space for osteocyte proliferation and osteogenic formation.

[0003] Currently, clinically used GBR membranes are mainly divided into two categories: non-absorbable membranes (such as polytetrafluoroethylene membranes and titanium membranes) and absorbable membranes (such as collagen membranes, chitosan membranes, and polylactic acid membranes). While non-absorbable membranes possess excellent space-maintaining capabilities and cell barrier effects, they require a second surgery for removal, increasing patient suffering and the risk of infection. Absorbable membranes, although avoiding a second surgery, suffer from insufficient mechanical strength, a porous structure, and an inability to effectively prevent fibroblast invasion of the bone defect area. Furthermore, their degradation rate is too rapid to match the bone regeneration process, and their antibacterial effects are suboptimal. Therefore, developing novel absorbable GBR membranes that combine good mechanical properties, a suitable degradation rate, effective antibacterial activity, and biocompatibility is of significant clinical importance. Summary of the Invention

[0004] The purpose of this application is to provide a double-layer guided bone regeneration membrane and its preparation method, aiming to solve the technical problems of existing absorbable guided bone regeneration membranes having weak mechanical strength, fast degradation rate and poor antibacterial effect.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides a double-layer guided bone regeneration membrane, comprising a dense layer and a porous layer stacked on top of each other. The dense layer is formed by cross-linking soluble eggshell membrane protein, epigallocatechin gallate, and carboxymethyl chitosan with genipin. The porous layer is formed by cross-linking 3-aminopropyltriethoxysilane-modified hydroxyapatite and carboxymethyl chitosan with genipin.

[0006] In one or more embodiments of this application, the 3-aminopropyltriethoxysilane-modified hydroxyapatite is any one of 3-aminopropyltriethoxysilane-modified hydroxyapatite nanorods, 3-aminopropyltriethoxysilane-modified hydroxyapatite nanowires, or 3-aminopropyltriethoxysilane-modified hydroxyapatite nanospheres.

[0007] In one or more embodiments of this application, the double-layer guided bone regeneration membrane has a photothermal effect, and under irradiation with 808nm near-infrared light and 1W power, the temperature rises to 41±1℃ in a humid environment.

[0008] Secondly, this application also provides a method for preparing a double-layer guided bone regeneration membrane, comprising the following steps: (1) Preparation of dense layer of guiding bone regeneration membrane: Epigallocatechin gallate and soluble eggshell membrane protein were dissolved in deionized water, carboxymethyl chitosan was added and stirred to form a uniform solution. Genipin was then added to carry out cross-linking reaction. The cross-linked solution was poured into a mold and dried in an oven to obtain dense layer of guiding bone regeneration membrane. (2) Preparation of a double-layer guided bone regeneration membrane: 3-aminopropyltriethoxysilane modified hydroxyapatite and carboxymethyl chitosan are uniformly mixed and genipin is added for cross-linking reaction to form a porous layer precursor solution. The porous layer precursor solution is cast onto the dense layer obtained in step (1) and then freeze-dried to obtain a double-layer guided bone regeneration membrane.

[0009] In one or more embodiments of this application, in step (1), the mass ratio of epigallocatechin gallate, soluble eggshell membrane protein and carboxymethyl chitosan is 2:(1~4):8.

[0010] In one or more embodiments of this application, in step (1), the mass of carboxymethyl chitosan is 1.5 to 2% of the volume of deionized water.

[0011] In one or more embodiments of this application, in step (2), the mass ratio of 3-aminopropyltriethoxysilane-modified hydroxyapatite to carboxymethyl chitosan is (1~8):8.

[0012] In one or more embodiments of this application, it further includes: Preparation of soluble eggshell membrane protein: Fresh eggshells were collected, soaked in an aqueous acetic acid solution, and the eggshells and membranes were separated. The membranes were collected, washed with deionized water, and freeze-dried. The freeze-dried membranes were placed in an aqueous sodium hydroxide solution and placed in a water bath at 40°C. The pH of the solution was adjusted to 8 with acetic acid, and trypsin was added. The enzymes were enzymatically hydrolyzed in a water bath at 37°C for 4 hours. The water bath temperature was then adjusted to 90°C and maintained for 10 minutes to inactivate the enzymes. After centrifugation, the upper light yellow liquid was obtained, which was then freeze-dried to obtain soluble eggshell membrane protein.

[0013] In one or more embodiments of this application, it further includes: Preparation of 3-aminopropyltriethoxysilane-modified hydroxyapatite: Oleic acid, deionized water, and methanol were mixed and stirred. Then, sodium hydroxide solution, calcium chloride solution, and sodium dihydrogen phosphate dihydrate solution were added sequentially, stirring for 5 min after each addition. The mixture was placed in a polytetrafluoroethylene reactor and hydrothermally heated to 180℃ for 10 h. After centrifugation, the mixture was washed with ethanol and deionized water and lyophilized to obtain hydroxyapatite nanowires. Hydroxyapatite nanowires were dissolved in cyclohexane to form a 1% solution, and then an equal volume of 10% 3-aminopropyltriethoxysilane ethanol solution was added. Finally, 1% deionized water relative to the total solution volume was added, and the mixture was stirred overnight. After centrifugation, the nanowires were washed with ethanol and deionized water to obtain 3-aminopropyltriethoxysilane modified hydroxyapatite nanowires.

[0014] Based on the above technical solution, the double-layer guided bone regeneration membrane and its preparation method of this application have at least the following beneficial technical effects: The bilayer guided bone regeneration membrane of this application possesses good tensile strength and antibacterial properties. The dense layer forms a physical barrier through its dense, non-porous structure, effectively preventing soft tissues such as fibroblasts from invading the bone defect area. Simultaneously, the porous layer provides the pore structure required for bone cell growth, achieving the dual functions of spatial selective barrier and regeneration guidance. Furthermore, the degradation rate of the bilayer guided bone regeneration membrane of this application shows a high degree of matching with the bone regeneration progress, with moderate and controllable degradation performance. Under irradiation with 808nm near-far-infrared light at a power of 1W, it can achieve a safe and effective osteopromoting effect.

[0015] The preparation method of this application utilizes soluble eggshell membrane protein (SEP) in the dense layer of the bone regeneration membrane. SEP, rich in amino and amide groups, aids wound healing and promotes cross-linking of the dense layer. Epigallocatechin gallate (EGCG), a natural polyphenol, possesses multiple phenolic hydroxyl groups and exhibits significant inhibitory effects on common periodontal pathogens, disrupting bacterial membranes and inhibiting metabolic enzymes. Simultaneously, genipin, a cross-linking agent, is used to cross-link SEP and EGCG, significantly improving the membrane's mechanical strength and barrier durability, and reducing the risk of premature degradation. The dense layer is predominantly blue-black, as shown in the image. Figure 3 As shown, it exhibits a photothermal effect. The porous layer of the bilayer guided bone regeneration membrane uses hydroxyapatite nanowires (HAp NWs) modified with 3-aminopropyltriethoxysilane (APTES), which can interweave to form a three-dimensional (3D) porous structure, exhibiting excellent self-supporting properties, as well as good hydrophilicity and flexibility. The introduction of carboxymethyl chitosan (CMC) can further improve the membrane's bioactivity and osteogenic induction capacity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a diagram illustrating the genipin crosslinking reaction mechanism of the guided bone regeneration membrane described in this application, in which... Figure 1 a is a diagram of the Schiff base reaction mechanism initiated by genipin. Figure 1 b is a diagram of the amidation reaction mechanism initiated by genipin.

[0018] Figure 2 This is a schematic diagram of the cross-linking reaction of the guided bone regeneration membrane of this application, wherein... Figure 2 a is a schematic diagram of the cross-linking reaction of the dense layer of the bone regeneration membrane. Figure 2 b is a schematic diagram of the cross-linking reaction of the porous layer guiding the bone regeneration membrane.

[0019] Figure 3 These are photographs of the guided bone regeneration membranes prepared in Examples 3 and 8 of this application, wherein... Figure 3 a is a photograph of the dense layer of the guided bone regeneration membrane prepared in Example 3. Figure 3 b is a photograph of the double-layer guided bone regeneration membrane prepared in Example 8.

[0020] Figure 4 These are tensile property diagrams of the dense layers of the guided bone regeneration membrane prepared with different amounts of SEP in Examples 2 to 5 of this application, wherein... Figure 4 a is the stress-strain curve. Figure 4 b is a statistical graph of tensile strength.

[0021] Figure 5 This is a physicochemical characterization diagram of the APTES-modified HAp NWs prepared in Example 6 of this application, wherein, Figure 5 a is a scanning electron microscope image of APTES-modified HAp NWs. Figure 5 b represents the X-ray photoelectron spectrum of APTES-modified HAp NWs. Figure 5 c is the Fourier transform infrared spectrum of APTES-modified HAp NWs. Figure 5 d is the X-ray diffraction pattern of APTES-modified HAp NWs.

[0022] Figure 6 This is a tensile property diagram of the guided bone regeneration membranes prepared with different amounts of APTES-modified HAp NWs added in Example 7 of this application. Figure 6 a is the stress-strain curve. Figure 6b is a statistical graph of tensile strength.

[0023] Figure 7 These are characterization and tensile property diagrams of the CES / CH bilayer guided bone regeneration membrane prepared in Example 8 of this application, wherein... Figure 7 a is a scanning electron microscope image of a cross-section of the double-layered guided bone regeneration membrane. Figure 7 b is a scanning electron microscope image of the dense layer of the double-layered guided bone regeneration membrane. Figure 7 c is a scanning electron microscope image of the porous layer of the double-layered guided bone regeneration membrane. Figure 7 d is the stress-strain curve of the double-layered guided bone regeneration membrane.

[0024] Figure 8 This is an in vitro degradation performance diagram of the double-layer guided bone regeneration membrane prepared in Example 8 of this application.

[0025] Figure 9 These are photothermal images of the bilayer guided bone regeneration membrane prepared in Example 8 of this application under different environments and power conditions, wherein... Figure 9 a shows the photothermal curves of the double-layer guided bone regeneration membrane under different powers in a dry environment. Figure 9 b is the photothermal curve of the double-layer guided bone regeneration membrane under different powers in a humid environment.

[0026] Figure 10 This is a diagram showing the antibacterial properties of the double-layer guided bone regeneration membrane prepared in Example 8 of this application.

[0027] Figure 11 This is a cytotoxicity diagram of the bilayer guided bone regeneration membrane prepared in Example 8 of this application, wherein... Figure 11 a is the L929 cytotoxicity graph. Figure 11 b is a graph showing the cytotoxicity of MC3T3-E1 cells.

[0028] Figure 12 This is a test diagram of the healing performance of the double-layer guided bone regeneration membrane prepared in Example 8 of this application on skull defects. Detailed Implementation

[0029] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0030] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0031] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0032] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0033] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0034] This application provides a method for preparing a double-layer guided bone regeneration membrane, comprising the following steps: (1) Preparation of soluble eggshell membrane protein (SEP).

[0035] Fresh eggshells were collected and soaked in a 4% acetic acid aqueous solution for 20 minutes. The eggshells and membranes were then manually separated, the membranes were collected, washed with deionized water, and freeze-dried. The freeze-dried eggshell membranes were placed in a 4% sodium hydroxide aqueous solution and incubated at 40°C for 30 minutes. Subsequently, the pH of the solution was adjusted to 8 with acetic acid, trypsin was added, and the solution was incubated at 37°C for 4 hours. The water bath temperature was then increased to 90°C and maintained for 10 minutes to inactivate the enzyme. The supernatant pale yellow liquid was obtained by centrifugation and freeze-dried to obtain SEP (Self-Extracted Polysaccharide).

[0036] (2) Preparation of hydroxyapatite nanowires (HAp NWs) modified by 3-aminopropyltriethoxysilane (APTES).

[0037] 9.35 g oleic acid, 13.5 g deionized water, and 4.75 g methanol were mixed and stirred for 10 min. Then, 15 g sodium hydroxide solution (1.05 g sodium hydroxide), 12 g calcium chloride solution (0.333 g calcium chloride), and 18 g sodium dihydrogen phosphate dihydrate solution (0.94 g sodium dihydrogen phosphate dihydrate) were added sequentially, and stirred for 5 min for each. The mixture was then placed in a polytetrafluoroethylene reactor and hydrothermally heated at 180 °C for 10 h. After centrifugation, the mixture was washed with ethanol and deionized water and lyophilized to obtain HAp NWs. HAp NWs were dissolved in cyclohexane to form a 1% solution, and then an equal volume of 10% silanizing reagent APTES ethanol solution was added. Finally, 1% of the total solution volume of deionized water was added, and the mixture was stirred overnight. After centrifugation, the mixture was washed with ethanol and deionized water to obtain APTES-modified HAp NWs (APTES-HAp).

[0038] (3) Preparation of the dense layer of the bone regeneration membrane.

[0039] Epigallocatechin gallate (EGCG) and soluble eggshell membrane protein (SEP) were dissolved in deionized water, and then 1.5-2% carboxymethyl chitosan (CMC) was added and stirred to form a homogeneous solution. Then, 0.1% genipin was added for cross-linking. The cross-linked solution was poured into a mold and dried in an oven at 37°C to obtain a dense layer of guiding bone regeneration membrane.

[0040] (4) Preparation of double-layer guided bone regeneration membrane.

[0041] 3-Aminopropyltriethoxysilane-modified hydroxyapatite nanowires (APTES-HAp) and carboxymethyl chitosan (CMC) were uniformly mixed and cross-linked with genipin to form a porous layer precursor solution. This porous layer precursor solution was cast onto the dense layer of a guided bone regeneration membrane, followed by freeze-drying to obtain a bilayer guided bone regeneration membrane. The dense layer has a dense, non-porous surface, which serves to block fibroblast migration. The porous layer has a porous structure, which supports osteoblast adhesion and growth.

[0042] Among them, the cross-linking reaction mechanism of epigallocatechin gallate (EGCG), soluble eggshell membrane protein (SEP), and carboxymethyl chitosan (CMC) with genipin is as follows: Figure 1 and Figure 2 As shown in Figure a, the crosslinking mechanism of 3-aminopropyltriethoxysilane-modified hydroxyapatite nanowires (APTES-HAp) with carboxymethyl chitosan (CMC) and genipin is as follows: Figure 1 and Figure 2 As shown in b.

[0043] Figure 1 This is a diagram illustrating the cross-linking reaction mechanism initiated by genipin. Genipin can undergo two reactions with amino-containing substances: Figure 1a represents a Schiff base reaction, where the amino group nucleophilically attacks the olefinic carbon at the C3 position of genipin to initiate ring opening, forming an imine intermediate. Subsequently, a Schiff base ring-closing reaction is performed to generate a blue fluorescent heterocyclic cross-linked structure. Figure 1 b is an amidation reaction, in which the amino group undergoes a nucleophilic substitution reaction with the genipin ester group to form an amide bond, further enhancing the crosslinking density.

[0044] Figure 2 A schematic diagram illustrating the cross-linking reaction guiding the bone regeneration membrane compact layer. Carboxymethyl chitosan (CMC) and soluble eggshell membrane protein (SEP) both contain amino groups, which can covalently cross-link with genipin to form cross-linked networks including CMC-CMC, CMC-SEP, and SEP-SEP. Furthermore, epigallocatechin gallate (EGCG) is rich in phenolic hydroxyl groups, which can form hydrogen bonds with the peptide carbonyl and amino groups of SEP, and also with the carboxyl, hydroxyl, and amino groups of CMC.

[0045] Figure 2 b is a schematic diagram of the cross-linking reaction guiding the porous layer of the bone regeneration membrane. Both carboxymethyl chitosan (CMC) and 3-aminopropyltriethoxysilane-modified hydroxyapatite nanowires (APTES-HAp) contain amino groups, which can covalently cross-link with genipin to form CMC-CMC and CMC-APTES-HAp cross-linked networks. Simultaneously, the carboxyl groups of CMC can electrostatically attract the amino groups of APTES-HAp.

[0046] The technical solution of this application will be described below with reference to the embodiments.

[0047] Example 1 Preparation of SEP.

[0048] Fresh eggshells were collected and soaked in a 4% acetic acid aqueous solution for 20 minutes. The eggshells and membranes were then manually separated, the membranes were collected, washed with deionized water, and freeze-dried. The freeze-dried eggshell membranes were placed in a 4% sodium hydroxide aqueous solution at a mass ratio of 1:100 and incubated at 40°C for 30 minutes. Subsequently, the pH of the solution was adjusted to 8 with glacial acetic acid (purity ≥99.5%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and trypsin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was added. The solution was then incubated at 37°C for 4 hours. The water bath temperature was then increased to 90°C and maintained for 10 minutes to inactivate the enzyme. The resulting pale yellow liquid was obtained by centrifugation and freeze-dried to obtain SEP (Self-Extracted Protein).

[0049] Example 2

[0050] Preparation of the CE dense layer of the bone regeneration membrane.

[0051] Weigh 100 mg EGCG (98% purity, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and 400 mg CMC (substitution degree ≥80%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and dissolve them in 20 ml of deionized water to form solution A. Then weigh 20 mg genipin (98% purity, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and dissolve it fully in solution A to form a precursor solution. Dry the precursor solution in an oven at 37 °C to obtain a dense CE layer.

[0052] Example 3 Preparation of the CE-0.25S dense layer of the bone regeneration membrane.

[0053] Weigh 100mg EGCG, 50mg SEP and 400mg CMC and dissolve them in 20ml of deionized water to form solution A. Then weigh 20mg genipin and dissolve it completely in solution A to form a precursor solution. Dry the precursor solution in an oven at 37℃ to obtain a dense CE-0.25S layer.

[0054] Example 4 Preparation of the CE-0.5S dense layer of the bone regeneration membrane.

[0055] Weigh 100mg EGCG, 100mg SEP and 400mg CMC and dissolve them in 20ml of deionized water to form solution A. Then weigh 20mg genipin and dissolve it completely in solution A to form a precursor solution. Dry the precursor solution in an oven at 37℃ to obtain a dense CE-0.5S layer.

[0056] Example 5 Preparation of the CE-1S dense layer of the bone regeneration membrane.

[0057] Weigh 100mg EGCG, 200mg SEP and 400mg CMC and dissolve them in 20ml of deionized water to form solution A. Then weigh 20mg genipin and dissolve it completely in solution A to form a precursor solution. Dry the precursor solution in an oven at 37℃ to obtain a dense CE-1S layer.

[0058] The tensile properties of the dense layer of the guided bone regeneration membrane prepared with different amounts of SEP in Examples 2 to 5 were studied.

[0059] The dense layer of the guide bone regeneration membrane was removed from the mold. Both ends of the guide bone regeneration membrane were clamped on a tensile testing machine, ensuring the membrane hung naturally. A tensile test was conducted at a tensile speed of 1 mm / min to test its tensile properties. The results are as follows: Figure 4 As shown. Figure 4 These are tensile property diagrams of the dense layers of guided bone regeneration membranes prepared with different amounts of SEP in Examples 2 to 5 of this application, wherein... Figure 4 a is the stress-strain curve. Figure 4 b is a statistical graph of tensile strength.

[0060] from Figure 4 It can be concluded that with the increase of SEP addition, the tensile properties of the guided bone regeneration membrane show a trend of first increasing and then decreasing, with the CE-0.25S group exhibiting the steepest curve and the highest stress peak. Figure 4 b indicates that the tensile strength of the CE-0.25S group was significantly higher than that of the CE group, with a statistically significant difference. Due to the dynamic mechanical stimulation in the oral cavity, such as chewing and swallowing, the high tensile strength membrane can better resist these external forces, maintain the membrane's spatial barrier function, and prevent fibrous tissue from invading the bone defect area. Subsequent embodiments used the method of Example 3 to prepare the dense layer of the guided bone regeneration membrane. Figure 3 As shown, Figure 3 Image a shows a photograph of the dense layer of the guided bone regeneration membrane prepared in Example 3. It can be seen that the dense layer is generally bluish-black.

[0061] Example 6 Preparation of APTES-modified HAp NWs.

[0062] 9.35 g of oleic acid (AR grade, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), 13.5 g of deionized water, and 4.75 g of methanol (purity ≥99.5%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) were weighed into a beaker and magnetically stirred for 10 min. Then, 15 g of sodium hydroxide solution (1.05 g sodium hydroxide), 12 g of calcium chloride solution (0.333 g calcium chloride), and 18 g of sodium dihydrogen phosphate dihydrate solution (0.94 g sodium dihydrogen phosphate dihydrate) were added sequentially. After each reagent was added, the mixture was manually stirred for 5 min to ensure thorough mixing. The mixture was transferred to a polytetrafluoroethylene-lined reactor and hydrothermally heated at 180℃ for 10 h. After the reaction was completed, the mixture was centrifuged, washed three times with anhydrous ethanol and deionized water, and then lyophilized to obtain HAp NWs.

[0063] Weigh 2g of lyophilized HAp NWs and disperse them in 200ml of cyclohexane (purity ≥99%, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.). Mix thoroughly to obtain solution A. Separately, measure 20ml of APTES (purity 98%, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) and dissolve it in 180ml of anhydrous ethanol to obtain an APTES ethanol solution. Slowly pour the APTES ethanol solution into solution A and mix with stirring. Then, add 4ml of deionized water to the mixture and continue stirring overnight. After the reaction is complete, centrifuge the mixture and collect the solid product. Wash the solid product with anhydrous ethanol and deionized water successively to obtain APTES-modified HAp NWs (APTES-HAp).

[0064] like Figure 5 As shown, Figure 5 The physicochemical characterization of the APTES-modified HAp NWs prepared in Example 6 of this application is shown, wherein, Figure 5 a is a scanning electron microscope image of APTES-modified HAp NWs. Figure 5 b represents the X-ray photoelectron spectrum of APTES-modified HAp NWs. Figure 5 c is the Fourier transform infrared spectrum of APTES-modified HAp NWs. Figure 5 d is the X-ray diffraction pattern of APTES-modified HAp NWs.

[0065] from Figure 5 As shown in Figure a, the microstructure of APTES-modified HAp NWs is linear, with the nanowires interwoven and well-dispersed, maintaining the original one-dimensional morphological characteristics of HAp NWs. X-ray photoelectron spectroscopy (XPS) was performed on the APTES-modified HAp NWs, as shown in Figure a. Figure 5 As shown in b. Figure 5 In addition to the O, Ca, and P elements naturally present in hydroxyapatite, characteristic peaks of Si 2p and N 1s were also observed in b, indicating that silicon and nitrogen elements from APTES were introduced into the surface of the modified material. Further analysis using Fourier transform infrared spectroscopy (FTIR) revealed... Figure 5 As shown in c. Figure 5 The spectrum of APTES-modified HAp NWs in c, in addition to retaining the characteristic peaks of hydroxyapatite, also showed characteristic absorption peaks for Si-O bonds (~470 cm⁻¹) and amino groups (-NH₂, 1629 cm⁻¹, 1525 cm⁻¹) corresponding to APTES. Figure 5 The elemental signals detected by XPS in b clearly indicate that APTES has been successfully grafted onto the HAp NWs surface through chemical bonding. Figure 5 The X-ray diffraction (XRD) results of d show that the APTES grafting modification did not change the crystal structure of hydroxyapatite, and the material still retains the hydroxyapatite phase.

[0066] Example 7 Preparation of guided bone regeneration membranes with different APTES-modified HAp NWs contents.

[0067] 50 mg, 100 mg, 200 mg, and 400 mg of APTES-modified HAp NWs were weighed and dissolved in 20 ml of deionized water to form solution A. Then, 400 mg of CMC was weighed and fully dissolved in solution A. Finally, 10 mg of genipin was added and sonicated for 5 min to form a homogeneous precursor solution. The solutions were dried in an oven at 37 °C to obtain guided bone regeneration membranes with different APTES-modified HAp NWs contents, which were named C-0.25AH, C-0.5AH, C-1AH, and C-2AH, respectively.

[0068] The tensile properties of guided bone regeneration membranes prepared with different amounts of APTES-modified HAp NWs were studied in Example 7.

[0069] Remove the guide bone regeneration membrane from the mold, clamp both ends of the membrane onto a tensile testing machine, ensuring the membrane hangs naturally, and conduct a tensile test at a tensile speed of 1 mm / min to test its tensile properties. The results are as follows. Figure 6 As shown. Figure 6 The tensile properties of guided bone regeneration membranes prepared with different amounts of APTES-modified HAp NWs added in Example 7 of this application are shown. Figure 6 a is the stress-strain curve. Figure 6 b is a statistical graph of tensile strength.

[0070] from Figure 6 As can be seen from a, with the increase of APTES-modified HAp NWs, the tensile properties of the porous layer of the guided bone regeneration membrane show a trend of first increasing and then decreasing. Figure 6 b shows that the C-1AH group has the best tensile strength and a significant statistical difference. In subsequent examples, the C-1AH group was used to prepare the double-layer guided bone regeneration membrane.

[0071] Example 8 Prepare a double-layer guided bone regeneration membrane CES / CH.

[0072] (1) Weigh 100mg EGCG, 50mg SEP and 400mg CMC and dissolve them in 20ml of deionized water to form solution A. Then weigh 20mg genipin and dissolve it in solution A to form a precursor solution. Pour the precursor solution into a 9×9cm square dish and dry it in an oven at 37℃ to obtain a dense layer.

[0073] (2) Weigh 200mg of APTES-modified HAp NWs and dissolve it in 20ml of deionized water to form solution B. Then weigh 400mg of CMC and dissolve it fully in solution B. Finally, add 10mg of genipin crosslinking agent and sonicate for 5min to form homogeneous precursor solution C.

[0074] (3) Cast the precursor solution C onto the dense layer, freeze it at -80℃ for 6 hours, and then vacuum dry it to obtain the double-layer guided bone regeneration membrane.

[0075] The microstructure and tensile properties of the CES / CH bilayer guided bone regeneration membrane were investigated. To test the mechanical properties of this bilayer membrane, the optimized upper and lower layer solution volume ratio and component concentration ratio from Example 8 were used to prepare the bilayer guided bone regeneration membrane in a tensile mold. The two ends of the membrane were fixed to the clamps of a tensile testing machine, ensuring the membrane was in a stress-free initial state of natural drooping, and a tensile test was conducted at a tensile speed of 1 mm / min. The results are as follows: Figure 7 As shown, Figure 7 The characterization and tensile properties of the double-layer guided bone regeneration membrane CES / CH prepared in Example 8 of this application are shown in the figure. Figure 7 a is a scanning electron microscope image of a cross-section of the double-layered guided bone regeneration membrane. Figure 7 b is a scanning electron microscope image of the dense layer of the double-layered guided bone regeneration membrane. Figure 7 c is a scanning electron microscope image of the porous layer of the double-layered guided bone regeneration membrane. Figure 7 d is the stress-strain curve of the double-layered guided bone regeneration membrane.

[0076] Figure 7 Figure a shows the cross-sectional morphology of the CES / CH bilayer guided bone regeneration membrane. The figure clearly shows a distinct layered structure: the upper layer is a dense layer, and the lower layer is a rough, porous layer. The two layers are well-bonded at the interface, with no obvious delamination or voids. Figure 7 b shows the microstructure of the upper dense layer: the surface of this layer is non-porous and the structure is dense, which can effectively block the migration of fibroblasts and ensure the integrity of the bone regeneration space. Figure 7 c corresponds to the lower rough and porous layer, whose surface exhibits a rich fibrous interwoven structure and porous characteristics. This rough and porous morphology is more conducive to cell adhesion and growth, and is adapted to the microenvironment requirements of bone tissue regeneration. Figure 7 The stress-strain curve results of d show that the stress of the double-layer guided bone regeneration membrane increases continuously with the increase of strain, and the final tensile strength can reach 30MPa. This mechanical property can meet the basic mechanical requirements of the guided bone regeneration membrane during intraoperative operation and postoperative process.

[0077] Study on the in vitro degradation performance of the double-layer guided bone regeneration membrane CES / CH.

[0078] Using the preparation method of Example 8, CES / CH circular samples of double-layered guided bone regeneration membranes with a diameter of 10 mm and uniform shape and size were prepared. Each sample was immersed in 10 ml of PBS solution, and lysozyme was added to the solution to achieve a final concentration of 1.5 μg / ml. The system was then placed in a shaker and incubated under simulated physiological conditions at 37°C and 120 rpm for degradation. Membrane samples were collected on days 1, 3, 7, 14, 21, and 28 after degradation incubation, and accurately weighed after freeze-drying (denoted as Wt). The initial weight of the membrane was taken as W0, and the degradation rate at each time point was calculated using the formula (W0 - Wt) / W0 × 100%. The results are as follows: Figure 8 As shown, Figure 8 The diagram shows the in vitro degradation performance of the bilayer guided bone regeneration membrane prepared in Example 8 of this application.

[0079] from Figure 8 The degradation curves show that in the initial stage of degradation (1-3 days), the degradation rate of the membrane increases rapidly, mainly corresponding to the initial swelling and release of components on the membrane surface. As time progresses, the degradation rate increases gradually, exhibiting a continuous and mild degradation characteristic. Analysis combined with the bone tissue regeneration cycle shows that the membrane's degradation rate is 33.84% at 28 days. It maintains structural integrity to function as a barrier in the early stages of tissue regeneration and gradually degrades with the regeneration process, without causing residual interference to newly formed tissue. Therefore, the bilayer guided bone regeneration membrane prepared in this application exhibits a high degree of matching between its degradation rate and the bone regeneration process, with moderate and controllable degradation performance.

[0080] Study on the photothermal properties of the double-layer guided bone regeneration membrane CES / CH.

[0081] Using the method of Example 8, CES / CH circular samples of double-layered guided bone regeneration membranes with a diameter of 10 mm and uniform shape and size were prepared. Near-infrared photothermal performance tests were conducted under dry and humid conditions. The samples were placed in a dry environment, and the dense layer region of the membrane was irradiated with 808 nm near-infrared lasers with powers of 0.5 W, 1 W, and 1.5 W. The sample temperature was recorded every 30 seconds using an infrared camera for 5 minutes. The corresponding results are as follows: Figure 9 As shown in Figure a. The sample was immersed in 1 ml of PBS solution. The dense layer region of the film was irradiated with 808 nm near-infrared lasers with power of 0.5 W, 1 W, and 1.5 W. The temperature was recorded every 30 seconds using an infrared camera for 10 minutes. The corresponding results are shown in Figure a. Figure 9 As shown in b.

[0082] from Figure 9As shown in Figure a, the temperature increases with irradiation time at different power levels, but the trends differ significantly: the 1.5W power group heats up the fastest, reaching 76.7℃ after 300s. The 1W power group has a moderate heating rate, stabilizing at 65℃ after 300s. The 0.5W power group heats up the slowest, reaching only 48℃ after 300s. Figure 9 b clearly shows that the thermal conduction effect of PBS reduces the overall temperature rise, but the power correlation is consistent with the dry state: the temperature of the 1.5W power group reaches 45.6℃ after 10 minutes. The 1W power group stabilizes at about 41℃. The 0.5W power group only rises to 35.3℃. The suitable photothermal temperature range for promoting bone regeneration is usually 40~45℃. At 1.5W power, the final temperature of both the dry and wet states exceeds this range, which can easily cause tissue thermal damage; at 0.5W power, the wet state temperature does not reach this range and cannot effectively stimulate the bone-promoting effect; at 1W power, although the dry state temperature (65℃) is relatively high, the wet state (physiological environment simulation) temperature stabilizes at about 41℃, which exactly matches the suitable temperature range for promoting bone regeneration, and the heating process is gradual with no risk of overheating. Therefore, 1W is the optimal near-infrared photothermal power for this double-layer guided bone regeneration membrane to achieve safe and effective bone promotion.

[0083] Study on the antibacterial properties of double-layer guided bone regeneration membrane.

[0084] Staphylococcus aureus and Escherichia coli, purchased from the Shanghai Biotechnology Preservation Center, were used to evaluate the in vitro antibacterial activity of the double-layer guided bone regeneration membrane CES / CH. A single colony was picked with an inoculation loop and inoculated into 6 ml of LB liquid medium, then incubated at 37°C with shaking for 12 h to prepare a bacterial suspension. The bacterial suspension was diluted with PBS solution, and 1 mL of the bacterial dilution (10⁻⁶ ppm) was used as the final concentration. 6 CFU / mL) and the membrane sample were added together into a 24-well plate and co-cultured for 24 h. The bacterial suspension was then diluted 100-fold with PBS, and 100 μL of the diluted solution was evenly spread onto the surface of solid agar medium. The plate was then inverted and incubated at 37°C for 24 h. Photos were taken after the incubation period. The results are shown in the figure. Figure 10 It can be seen that, compared with the control group, the double-layer guided bone regeneration membrane has good antibacterial activity against both Staphylococcus aureus and Escherichia coli.

[0085] A study on the cytotoxicity of a double-layered guided bone regeneration membrane.

[0086] Mouse fibroblasts L929 and mouse cranial anterior osteoblasts MC3T3-E1 were seeded at a density of 10,000 cells / well in 48-well plates and cultured for 24 h. Cytotoxicity was assessed using a co-culture method: Prepared CMC (the dense layer from Example 3 without EGCG and SEP), CES (the dense layer prepared according to the method in Example 3), and CES / CH (prepared according to the method in Example 8) were co-cultured with L929 cells in DMEM complete cell culture medium. Prepared CMC, CH (100 mg of APTES-modified HAp NWs and 200 mg of CMC were dissolved in 10 ml of deionized water, 10 mg of genipin crosslinking agent was added, and the mixture was sonicated for 5 min to form a homogeneous precursor solution, then freeze-dried to obtain CH material), and CES / CH were co-cultured with MC3T3-E1 cells in MEM complete cell culture medium. Cytotoxicity was determined using a CCK-8 assay kit, and the results are as follows: Figure 11 As shown, Figure 11 The diagram shows the cytotoxicity of the bilayer guided bone regeneration membrane prepared in Example 8 of this application, wherein... Figure 11 a is a graph showing the cytotoxicity of L929 cells, from... Figure 11 As can be seen, after co-culturing with L929 cells for 24h, 48h and 72h respectively, the cell viability of the CES / CH membrane prepared in this application can reach 80%. Figure 11 b is a graph showing the cytotoxicity of MC3T3-E1 cells. From Figure 11 As can be seen from b, after co-culturing with MC3T3-E1 cells for 24h, 48h and 72h respectively, the cell viability of the CES / CH membrane group was above 80%, which is within the safe range.

[0087] A study on the healing effect of a double-layered guided bone regeneration membrane on skull defects.

[0088] The effect of a double-layer guided bone regeneration membrane on bone defect healing was tested using an SD rat skull defect model. The specific procedure is as follows: First, the experimental rats were anesthetized, and the hair on the top of their skulls was removed. The surgical area was then disinfected with povidone-iodine, and the skin on the top of the skull was incised layer by layer to create a circular bone defect with a diameter of 5 mm on each side of the skull. Five groups were set up in the experiment: a control group (no material was used on the bone defect area), a CES group, a CH group, a CES / CH group, and a CES / CH+NIR group (using a double-layer guided bone regeneration membrane and irradiation with 808nm near-infrared light). After covering the bone defect area of ​​the rats with the corresponding group's material, the surgical wound was sutured layer by layer. Rats in the CES / CH+NIR group received near-infrared light irradiation once a day, with each irradiation lasting 5 minutes.

[0089] The results are as follows Figure 12 As shown, compared with the control group, CES group, and CH group, the CES / CH+NIR group showed the best bone healing effect, and the bone healing effect improved with the extension of time.

[0090] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A double-layer guided bone regeneration membrane, characterized in that, It includes a dense layer and a porous layer stacked on top of each other. The dense layer is formed by cross-linking soluble eggshell membrane protein, epigallocatechin gallate, and carboxymethyl chitosan with genipin. The porous layer is formed by cross-linking 3-aminopropyltriethoxysilane-modified hydroxyapatite and carboxymethyl chitosan with genipin.

2. The double-layer guided bone regeneration membrane according to claim 1, characterized in that, The 3-aminopropyltriethoxysilane-modified hydroxyapatite is any one of 3-aminopropyltriethoxysilane-modified hydroxyapatite nanorods, 3-aminopropyltriethoxysilane-modified hydroxyapatite nanowires, or 3-aminopropyltriethoxysilane-modified hydroxyapatite nanospheres.

3. The double-layer guided bone regeneration membrane according to claim 1, characterized in that, The double-layered guided bone regeneration membrane has a photothermal effect, and under irradiation with 808nm near-infrared light and 1W power, the temperature rises to 41±1℃ in a humid environment.

4. A method for preparing a double-layer guided bone regeneration membrane, characterized in that, Includes the following steps: (1) Preparation of dense layer of guiding bone regeneration membrane: Epigallocatechin gallate and soluble eggshell membrane protein were dissolved in deionized water, carboxymethyl chitosan was added and stirred to form a uniform solution. Genipin was then added to carry out cross-linking reaction. The cross-linked solution was poured into a mold and dried in an oven to obtain dense layer of guiding bone regeneration membrane. (2) Preparation of double-layer guided bone regeneration membrane: 3-aminopropyltriethoxysilane modified hydroxyapatite and carboxymethyl chitosan are uniformly mixed and genipin is added for cross-linking reaction to form a porous layer precursor solution. The porous layer precursor solution is cast onto the dense layer obtained in step (1) and then freeze-dried to obtain the double-layer guided bone regeneration membrane.

5. The preparation method according to claim 4, characterized in that, In step (1), the mass ratio of epigallocatechin gallate, the soluble eggshell membrane protein and the carboxymethyl chitosan is 2:(1~4):

8.

6. The preparation method according to claim 4, characterized in that, In step (1), the mass of the carboxymethyl chitosan is 1.5 to 2% of the volume of deionized water.

7. The preparation method according to claim 4, characterized in that, In step (2), the mass ratio of the 3-aminopropyltriethoxysilane-modified hydroxyapatite to the carboxymethyl chitosan is (1~8):

8.

8. The preparation method according to claim 4, characterized in that, Also includes: Preparation of soluble eggshell membrane protein: Fresh eggshells were collected, soaked in an aqueous acetic acid solution, and the eggshells and membranes were separated. The membranes were collected, washed with deionized water, and freeze-dried. The freeze-dried membranes were placed in an aqueous sodium hydroxide solution and placed in a water bath at 40°C. The pH of the solution was adjusted to 8 with acetic acid, and trypsin was added. The enzymes were enzymatically hydrolyzed in a water bath at 37°C for 4 hours. The water bath temperature was then adjusted to 90°C and maintained for 10 minutes to inactivate the enzymes. After centrifugation, the upper light yellow liquid was obtained, which was then freeze-dried to obtain soluble eggshell membrane protein.

9. The preparation method according to claim 4, characterized in that, Also includes: Preparation of 3-aminopropyltriethoxysilane-modified hydroxyapatite: Oleic acid, deionized water, and methanol were mixed and stirred. Then, sodium hydroxide solution, calcium chloride solution, and sodium dihydrogen phosphate dihydrate solution were added sequentially, stirring for 5 min after each addition. The mixture was placed in a polytetrafluoroethylene reactor and hydrothermally heated to 180℃ for 10 h. After centrifugation, the mixture was washed with ethanol and deionized water and lyophilized to obtain hydroxyapatite nanowires. Hydroxyapatite nanowires were dissolved in cyclohexane to form a 1% solution, and then an equal volume of 10% 3-aminopropyltriethoxysilane ethanol solution was added. Finally, 1% deionized water relative to the total solution volume was added, and the mixture was stirred overnight. After centrifugation, the nanowires were washed with ethanol and deionized water to obtain 3-aminopropyltriethoxysilane modified hydroxyapatite nanowires.

Images