Silk-based double-layer composite guided bone regeneration film and preparation method thereof

Abstract

The invention relates to the technical field of biological material processing, in particular to a preparation method of a silk-based double-layer composite guided bone regeneration film, which comprises the following steps: S1, degumming, washing and drying wet silkworm cocoon slices to obtain degummed fibroin fibers; s2, dissolving the degummed silk fibroin with a lithium bromide solution, and dialyzing to obtain a silk fibroin aqueous solution; s3, putting the degummed fibroin fiber obtained in the step S1 into an aqueous solution, and performing high-speed shearing treatment to obtain a micro-nano fibroin fiber homogeneous solution; and S4, pouring the homogeneous solution on the surface of the high-toughness silk-based smooth film, and drying to form a micro-nano fiber layer. Freeze-drying / spinning is replaced with a high-speed shearing method, the process is shortened, sewage discharge is reduced, and high-salt dissolution and long-term dialysis are not needed; the bonding layer is subjected to silk fibroin solution and HFIP dual treatment, and the problem that the porous layer falls off in a body fluid environment is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomaterial processing, and more particularly, to a silk-based double-layer composite guided bone regeneration membrane and a preparation method thereof. BACKGROUND

[0002] Guided bone regeneration (GBR) technology is a common treatment method in oral repair and orthopedic reconstruction surgery, and the guided bone regeneration membrane plays two key roles in the process of orthopedic reconstruction:

[0003] Barrier effect: isolates the soft tissue from the bone defect area, prevents epithelial and connective tissue invasion, and supports the osteogenesis space for the bone defect area;

[0004] Osteogenesis promotion: promotes bone regeneration and acts as a scaffold for bone cell migration.

[0005] An ideal GBR membrane should have high toughness to prevent damage to soft tissue, and also have high strength so that the GBR membrane can still provide good support in a thin state, and should also have good biocompatibility and biodegradability, and the degradation products are non-toxic and have no side effects.

[0006] The currently used GBR membranes in clinical practice can be divided into two categories: non-degradable membranes, such as metal titanium membranes, and degradable membranes, such as collagen membranes;

[0007] Among them, the metal titanium membrane has high strength, but has very low ductility and easily causes mucosal irritation, and is difficult to degrade;

[0008] Commercial collagen membranes, such as Geistlich Bio-Gide and ZH-Bio have good osteogenesis promotion, but their mechanical properties are poor, the support effect is weak, and the degradation period is much shorter than the bone regeneration period, and early collapse often occurs after implantation in the bone defect area.

[0009] In view of the many defects of the above existing commercial membranes, many studies have designed and manufactured new guided bone regeneration membranes from the perspectives of raw material selection, material modification, and morphology design, such as mixing chitosan, graphene, and calcium silicate to prepare a double-layer membrane with enhanced mechanical properties, a dense pearl layer, and a porous layer, which has excellent antibacterial properties; titanium carbide MXene is bridged with silk fibroin through hydrogen bonding;

[0010] As patent publication CN119139541A, this invention prepares a high-strength MXene composite film by integrating sequentially bridged roll-to-roll auxiliary blade coating strategy arrangement assembly, with a tensile strength of 755 MPa, and good bone formation promoting effect. However, compared with thin films based on natural biological materials, thin films enhanced with materials such as graphene and MXene often perform poorly in terms of flexibility and sufficient degradability.

[0011] Traditional single-layer GBR thin films can only meet the barrier and support effects, and cannot meet the bone formation promoting function of clinical application. Therefore, double-layer or multi-layer asymmetric thin films have become one of the development trends of GBR thin films. Among them, the most widely used in clinical application at present is the double-layer asymmetric thin film. Commercial GBR films have a typical double-layer asymmetric structure, in which the dense layer faces the soft tissue, and the dense structure can effectively prevent fibroblasts from invading, and has good barrier effect, and the porous layer faces the bone defect area, and can promote osteoblast adhesion, proliferation and differentiation.

[0012] The preparation methods of the porous layer mainly include freeze-drying and electrospinning methods, among which the freeze-drying method is disclosed in patent publications CN118490900A, CN117695449A and CN117547659A, and the electrospinning method is disclosed in patent publications CN118029056A, CN117860964A and CN115737938A.

[0013] Both methods can realize large-scale industrial production, but the porous scaffold layer obtained by the freeze-drying method is easy to degrade, and the electrospinning method has low production efficiency, high requirements for production equipment, high energy consumption, and the processes of the above two methods are complex, such as the preparation of silk fibroin freeze-dried scaffolds requires four steps of degumming, dissolution, dialysis and freeze-drying; The preparation of electrospun fiber scaffold requires five steps of degumming, dissolution, dialysis, spinning solution preparation and electrospinning. The dissolution process needs to use expensive high-salt solution, and the dialysis process consumes a large amount of water resources and produces a large amount of industrial wastewater, which takes several days. Therefore, both methods have the significant defects of long time consumption and high cost in actual industrial production.

[0014] In summary, the development and design of a double-layer guided bone regeneration film based on natural biological materials with high strength and high toughness, complete biodegradability, non-toxic side effects of degradation products, simple production process and low cost can solve the multiple problems existing in the current commercial GBR film, and has important clinical application significance and commercial value.

[0015] Therefore, we propose a silk-based double-layer composite guided bone regeneration film and its preparation method to solve the above problems. SUMMARY

[0016] To overcome the above-mentioned defects of the prior art, embodiments of the present application provide a silk-based double-layer composite guided bone regeneration membrane and a preparation method thereof to solve the problems raised in the above background.

[0017] To achieve the above object, the present application provides the following technical scheme: a preparation method of a silk-based double-layer composite guided bone regeneration membrane, comprising the following steps:

[0018] Step S1: degumming, washing and drying the wet cocoon flake to obtain degummed silk fibroin fibers;

[0019] Step S2: dissolving the degummed silk fibroin fibers with a lithium bromide solution and dialyzing to obtain a silk fibroin aqueous solution;

[0020] Step S3: placing the degummed silk fibroin fibers obtained in step S1 in an aqueous solution and treating with high-speed shearing to obtain a homogeneous solution of micro-nano silk fibers;

[0021] Step S4: pouring the homogeneous solution onto the surface of a high-toughness silk-based smooth film and drying to form a micro-nano fiber layer;

[0022] Step S5: pouring the silk fibroin aqueous solution onto the micro-nano fiber layer and drying to form an adhesive layer;

[0023] Step S6: pouring hexafluoroisopropanol (HFIP) onto the adhesive layer and volatilizing and drying;

[0024] Step S7: ultraviolet sterilization to obtain a double-layer composite membrane.

[0025] In a preferred embodiment, in step S1, the degumming is performed using a mixed solution of sodium dodecyl sulfate and sodium carbonate at 80°C for 6 minutes.

[0026] In a preferred embodiment, in step S3, the high-speed shearing speed is 10000 rpm-50000 rpm and the treatment time is 1-10 hours.

[0027] In a preferred embodiment, in step S4, the thickness of the micro-nano fiber layer is controlled to be 50 μm-1000 μm by adjusting the concentration and volume of the homogeneous solution.

[0028] In a preferred embodiment, in step S4, the high-toughness silk-based smooth film used is a high-toughness high-strength silk protein base film.

[0029] In a preferred embodiment, the silk-based double-layer composite guided bone regeneration membrane prepared by the method comprises:

[0030] a high-toughness silk protein base film;

[0031] a loose porous layer formed by silk protein micro-nano fibers;

[0032] Two layers are combined by a silk fibroin adhesive layer to form a double-layer film with asymmetric structure.

[0033] In a preferred embodiment, the loose porous layer comprises a scaffold of micro-sized fibers and nano-sized fiber-filled pores.

[0034] In a preferred embodiment, the total thickness of the double-layer film is 100-300 μm, and the thickness ratio of the dense layer is 10%-30%.

[0035] Technical effects and advantages of the present application:

[0036] 1. The present application replaces lyophilization / spinning with high-speed shearing, shortens the process, reduces sewage discharge, and does not require high-salt dissolution and long-term dialysis; the adhesive layer is treated with silk fibroin solution+HFIP, which solves the problem of shedding of the porous layer in a body fluid environment.

[0037] 2. Micro-nano fiber synergistically constructs the porous layer: micro-sized fibers form the main scaffold, and nano-sized fibers fill the pores, with a porosity of 85±5%, a pore size distribution of 20 μm-150 μm, and cell migration; the dense layer provides tensile strength, the porous layer improves flexibility, and avoids tearing of the membrane during surgery

[0038] 3. The present application uses a high-toughness high-strength silk-based composite film as a smooth dense layer, which improves the spatial support capacity of the GBR membrane, while maintaining the flexibility of the film, facilitating surgical operation, and reducing the irritation to the patient's soft tissue wound.

[0039] 4. The raw material used in the present application is renewable silk, with a cost of only 1 / 3 of that of collagen membrane, and without the need for special equipment throughout the process, making it suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The preparation process of the double-layer composite guided bone regeneration film in the embodiments of the present application;

[0041] Figure 2 The morphology of the double-layer composite guided bone regeneration film RGF-1 in Example 1 of the present application;

[0042] Figure 3 The morphology of the double-layer guided bone regeneration film RGF-2 in Example 2 of the present application;

[0043] Figure 4 The cell compatibility test results of the double-layer guided bone regeneration films RGF-1 and RGF-2 in the embodiments of the present application;

[0044] Figure 5 The barrier function test results of the double-layer guided bone regeneration films RGF-1 and RGF-2 in the embodiments of the present application;

[0045] Figure 6 Figure for the in vitro study of the double-layered guided bone regeneration membrane RGF-1 and RGF-2 in promoting bone regeneration in the embodiments of the present application;

[0046] Figure 7 Figure for the in vivo study of the double-layered guided bone regeneration membrane RGF-1 and RGF-2 in promoting bone regeneration in the embodiments of the present application. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0048] Reference Figures 1-7

[0049] Embodiment 1:

[0050] 1 g of cocoon was weighed, the cocoon was torn into thin pieces, and then soaked in pure water for 30 minutes. Then, the cocoon was put into 500 mL of a mixed degumming solution of SDS (0.25%, w / v) and sodium carbonate (0.25%, w / v), and degumming treatment was performed on the cocoon under water bath heating (80°C) for 6-8 minutes. The degumming process was accompanied by vigorous stirring to accelerate the dissolution of sericin.

[0051] The degummed silk fibroin fibers were washed in hot water and pure water for multiple times, and then placed in a 55°C oven for drying. The SDS is sodium dodecyl sulfate.

[0052] The dried silk fibroin fibers were put into 10 mol / L LiBr and vortexed for 1 hour for dissolution. The solution was transferred into a dialysis bag with a molecular cut-off of 6-8 kDa for water dialysis for 3 days, and the dialysis solution was replaced every 12 hours. kDa is the protein molecular weight, kilodalton.

[0053] After dialysis, the protein solution was centrifuged at high speed for 1 hour to remove insoluble impurities, and then freeze-dried to obtain high molecular weight regenerated silk fibroin.

[0054] 2 parts of high molecular weight regenerated silk fibroin and 1 part of genipin powder were weighed into a test tube, and then HFIP was added to rotate and mix to obtain a uniform mixed solution.

[0055] The water-washed degummed silk fibroin fibers were placed in an aqueous solution and subjected to high-speed shearing at 32,000 rpm for 240 minutes to obtain a fiber homogenate of micron and nanometer fibers mixed.

[0056] First, the fiber homogenate is evenly poured on the surface of the high-strength and high-toughness film, and is left to dry;

[0057] Then, the high-molecular-weight regenerated silk fibroin / genipin / HFIP solution is evenly poured on the surface of the fiber layer, and is left to volatilize (the preparation process is shown in Figure 1 , and the obtained double-layer film is sterilized by ultraviolet irradiation to obtain a bone regeneration guiding film, as shown in the accompanying drawings Figure 2 , (regenerated silk fibroin / genipin / fiber, hereinafter referred to as RGF for short).

[0058] Example 2:

[0059] 1 g of cocoon is weighed, the cocoon is torn into thin pieces, and after being soaked in pure water for 30 minutes, it is put into 500 ml of SDS (0.25%, w / v) + sodium carbonate (0.25%, w / v) mixed degumming solution, and degumming treatment is carried out on the silkworm cocoon under the condition of water bath heating (80°C) for 6-8 minutes. The degumming process is accompanied by vigorous stirring to accelerate the dissolution of silk glue protein.

[0060] The degummed silk fibroin is washed in hot water and pure water for several times and is placed in a 55°C oven for drying.

[0061] The dried silk fibroin is placed in 10 mol / L LiBr and is vortexed for 1 hour to dissolve, and the solution is transferred into a dialysis bag with a molecular cut-off of 6-8 kDa for water dialysis for 3 days, and the dialysate is replaced every 12 hours.

[0062] After dialysis, the protein solution is centrifuged at high speed for 1 hour to remove insoluble impurities, and a high-molecular-weight regenerated silk fibroin aqueous solution is obtained.

[0063] The water-washed degummed silk fibroin is placed in an aqueous solution and is subjected to high-speed shearing at 32,000 rpm for 240 minutes to obtain a fiber homogenate mixed with micron and nanometer fibers.

[0064] First, the fiber homogenate is evenly poured on the surface of the high-strength and high-toughness film, and is left to dry;

[0065] Then, the high-molecular-weight regenerated silk fibroin aqueous solution is evenly poured on the surface of the fiber layer, and is left to dry;

[0066] Finally, HFIP is poured on the surface of the fiber layer, and is left to volatilize (the preparation process is shown in Figure 1 , and the obtained double-layer film is sterilized by ultraviolet irradiation to obtain a bone regeneration guiding film, as shown in the accompanying drawings Figure 3 .

[0067] Test method

[0068] The surface and cross-sectional morphology of the double-layer guided bone regeneration membranes RGF-1 and RGF-2 obtained in Example 1 and Example 2 were observed using an Apreo S LoVac field emission scanning electron microscope. In particular, the Apreo S LoVac is a hot field emission scanning electron microscope.

[0069] As shown in the accompanying drawings Figure 2 and the accompanying drawings Figure 3 The guided bone regeneration membranes obtained in Example 1 and Example 2 exhibit a typical double-layer structure, i.e., a smooth dense layer and a loose porous layer, respectively.

[0070] The double-layer guided bone regeneration membranes RGF-1 and RGF-2 obtained in the above examples were cut into the size of a 12-well plate, sterilized by ultraviolet irradiation, and then laid in the plate.

[0071] After cell counting, rat bone marrow mesenchymal stem cells (rBMSCs) were uniformly inoculated on the protein membrane, and after co-culturing for 24 hours, a live and dead staining kit (Beyotime, China) was used for detection, as shown in the accompanying drawings Figure 4 A;

[0072] After co-culturing the extracts of different RGF membranes with rat bone marrow mesenchymal stem cells (rBMSCs) in a 96-well plate for 24 hours, 48 hours, and 72 hours, a cell counting kit (cell Counting Kit-8, Biosharp, China) was used for detection and calculation of cell viability, as shown in the accompanying drawings Figure 4 B;

[0073] After rat bone marrow mesenchymal stem cells (rBMSCs) were uniformly inoculated on the protein membrane and co-cultured for 24 hours, the cells were stained with FITC-Phalloidin (Solarbio, China) to observe the cell morphology, as shown in the accompanying drawings Figure 4 C.

[0074] The results show that both RGF-1 and RGF-2 membranes have no cytotoxicity and can promote cell proliferation, adhesion, and migration.

[0075] The dense layer of the double-layer guided bone regeneration membrane RGF in the present application was used to replace the protein membrane of the original Transwell chamber, and fibroblasts were digested, counted, and uniformly inoculated in the chamber.

[0076] After 24 hours, the cells that passed through the chamber were stained with crystal violet (Solarbio, China), observed under a microscope, and photographed.

[0077] The number of cells was measured using Image J, as shown in the accompanying drawingsFigure 5 .

[0078] The results show that the dense layer of RGF membrane effectively inhibits fibroblast migration, thus creating a stable microenvironment conducive to bone regeneration.

[0079] After 7 days of culture with different RGF membrane extracts, rat bone marrow mesenchymal stem cells (rBMSCs) were stained using an ALP kit (Beyotime, China) as described in the instructions, as shown in the figure Figure 6 A.

[0080] After 14 days of culture with different RGF membrane extracts, rat bone marrow mesenchymal stem cells (rBMSCs) were stained using 0.2% Cynthia Red S (Solarbio, China) for ARS staining as described in the instructions, as shown in the figure Figure 6 B.

[0081] The results show that both RGF membranes can promote the osteogenic differentiation and mineralization of rBMSCs, with RGF-2 showing a more significant osteogenic effect.

[0082] Rat bone marrow mesenchymal stem cells (rBMSCs) were directly co-cultured with RGF-1 and RGF-2, respectively, and RNA lysate was collected.

[0083] Total RNA was extracted according to the instructions of the RNA extraction kit (Promega, China).

[0084] cDNA synthesis was performed according to the instructions of the All-in-One-First-Strand cDNA synthesis SuperMix for qPCR (One-step gDNA Removal) kit (TransGen Biotech, China), with special attention to the fact that All-in-One First-Strand cDNA Synthesis SuperMix for qPCR is a kit for reverse transcription reactions that can efficiently synthesize cDNA from total RNA or mRNA and remove residual genomic DNA in the RNA template.

[0085] Amplification was performed using the qRT-PCR SuperMix kit (Transgen Biotech, China), and detection was performed using a PCR system (Thermofisher, USA), with the internal reference gene Gapdh as a control. The data were arranged and analyzed using the 2-ΔΔCt method, as described in the instructions and as shown in the figure Figure 6C, wherein qRT-PCR SuperMix is a premix for one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the 2-ΔΔCt method is a simple method for analyzing the relative change of gene expression in real-time quantitative PCR experiments.

[0086] The primer sequences used in the qRT-PCR are shown in Table 1. The results show that both RGF membranes can up-regulate early and late osteogenic genes, and RGF-2 shows more significant osteogenic effects.

[0087] Table 1

[0088]

[0089] The animal experiment was approved by Tianjin Medical University (TMUaMEC 2024039).

[0090] Six-week-old rats were purchased and acclimated for one week. All rats were anesthetized with isoflurane gas, and then a longitudinal incision was made along the sagittal line of the skull to fully expose the sagittal suture, bilateral skull, part of the frontal bone, and occipital bone.

[0091] A 5mm defect was created on both sides of the skull using a trephine, and then RGF-1 and RGF-2 were placed on the surface, respectively.

[0092] Finally, the periosteum and skin were sutured in turn. After 4 weeks, the skulls of the rats in each group were taken and each bone tissue was scanned using micro-CT (Bruker, Germany). The results are shown in the attached figures of the specification Figure 7 A.

[0093] The data were reconstructed using CTAn software to obtain bone mineral density (BMD), bone volume / total volume (BV / TV), trabecular number (Tb.N), and trabecular separation (Tb.Sp) parameters, as shown in the attached figures of the specification Figure 7 B.

[0094] The results show that the amount of new bone formation in the RGF-1 and RGF-2 treated groups is significantly higher than that in the blank control group, and the maturity of the new bone is higher than that in the blank control group.

[0095] Among them, RGF-2 shows unique advantages in promoting bone regeneration in vivo, with the largest new bone formation area, the fastest bone regeneration progress, and the most mature bone tissue development.

[0096] It should be noted that, Figure 2 The morphology of the double-layer guided bone regeneration membrane RGF-1 in Example 1 of the present application is shown in the attached figures of the specification:

[0097] Macroscopic photograph, scale bar: 1cm (A);

[0098] Scanning electron microscope image of the RGF-1 cross section, scale bar: 50 μm (B);

[0099] Scanning electron microscope image of the surface of the RGF-1 loose porous layer, scale bar: 5 μm (C).

[0100] Figure 3 The morphology of the double-layer guided bone regeneration film RGF-2 in Embodiment 2 of the present invention is shown below:

[0101] Macroscopic photograph, scale bar: 1cm (A);

[0102] Scanning electron microscope image of the RGF-2 cross section, scale bar: 50 μm (B);

[0103] Scanning electron microscope image of the surface of the loose porous layer of RGF-2, scale bar: 5 μm (C).

[0104] Figure 4 The following are the cell compatibility test results of the bilayer guided bone regeneration films RGF-1 and RGF-2 in this embodiment of the invention:

[0105] (A) Live / dead staining of rBMSCs co-cultured with RGF membrane rough surface for 24h, green fluorescence (Calcein-AM staining for live cells) and red fluorescence (propidium iodide, PI staining for dead cells), scale bar: 200μm.

[0106] (B) Cell viability of rBMSCs after co-culturing with extracts of different RGF membranes for 1, 2 and 3 days.

[0107] (C) Cell morphology of rBMSCs after direct co-culture with RGF-1 and RGF-2 for 24 hours (phalloidin-stained cytoskeleton), scale bar: 200 μm.

[0108] Figure 5 The diagram shows the barrier function test results of the bilayer guided bone regeneration films RGF-1 and RGF-2 in this embodiment of the invention.

[0109] (A) Transwell migration experiment image (left: blank control; right: smooth and dense film layer, i.e., the dense layer of RGF bilayer film);

[0110] (B) Crystal violet staining of migrating cells (left: blank control; right: smooth and dense thin film layer, i.e., the dense layer of the RGF bilayer membrane), scale bar: 200 μm; (F) Quantitative analysis results of migrating cells.

[0111] Figure 6Fig. 2 is a graph showing the results of in vitro bone regeneration promotion research of the double-layered guided bone regeneration membrane RGF-1 and RGF-2 in the embodiments of the present application,

[0112] (A) ALP (alkaline phosphatase) staining image, scale bar: 200 μm;

[0113] (B) ARS (alizarin red S) staining image. Scale bar: 200 μm;

[0114] (C) Alp, Col 1A1, BMP2, Runx2, Opn and Ocn gene expression levels.

[0115] Figure 7 Fig. 3 is a graph showing the results of in vivo bone regeneration promotion research of the double-layered guided bone regeneration membrane RGF-1 and RGF-2 in the embodiments of the present application.

[0116] (A) Micro-CT image of RGF-1 and RGF-2 implanted in the rat skull defect area for 4 weeks.

[0117] (B) BMD (bone mineral density), BV / TV (bone volume fraction), Tb.N (trabecular number) and Tb.Sp (trabecular separation) analysis results graph.

[0118] Finally, the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a silk-based bilayer composite guided bone regeneration film, characterized in that... ； Includes the following steps: Step S1: Degumming, washing, and drying the moistened silkworm cocoon sheets to obtain degummed silk fibroin fibers; Step S2: Dissolve the degummed silk fibroin fibers in lithium bromide solution and then dialyze to obtain an aqueous solution of silk fibroin. Step S3: Place the degummed silk fibroin fibers obtained in step S1 in an aqueous solution and perform high-speed shearing to obtain a homogeneous solution of micro and nano silk fibroin fibers. Step S4: The homogeneous solution is poured onto the surface of a high-toughness silk-based smooth film and dried to form a micro / nanofiber layer; Step S5: Cast the silk fibroin aqueous solution onto the micro / nanofiber layer and dry it to form an adhesive layer; Step S6: Cast hexafluoroisopropanol (HFIP) onto the adhesive layer and allow it to evaporate and dry; Step S7: Ultraviolet sterilization to obtain a double-layer composite membrane.

2. The method for preparing a silk-based bilayer composite guided bone regeneration film according to claim 1, characterized in that: In step S1, degumming is performed using a mixed solution of sodium dodecyl sulfate and sodium carbonate, treated at 80°C for 6 minutes.

3. The method for preparing a silk-based bilayer composite guided bone regeneration film according to claim 1, characterized in that: In step S3, the high-speed shearing speed is 10,000 rpm–50,000 rpm, and the processing time is 1–10 hours.

4. The method for preparing a silk-based bilayer composite guided bone regeneration film according to claim 1, characterized in that: In step S4, the thickness of the micro / nanofiber layer is controlled to be 50 μm–1000 μm by adjusting the concentration and volume of the homogenized solution.

5. The method for preparing a silk-based bilayer composite guided bone regeneration film according to claim 1, characterized in that: The high-toughness silk-based smooth film used in step S4 is a high-toughness, high-strength silk protein base film.

6. The silk-based bilayer composite guided bone regeneration film prepared according to any one of claims 1-5, characterized in that: include: A high-toughness silk protein basement membrane; A loose, porous layer formed by a layer of silk protein micro-nanofibers; The two layers are bonded together by a silk fibroin adhesive layer to form an asymmetrical bilayer membrane.

7. The silk-based bilayer composite guided bone regeneration film according to claim 6, characterized in that: The loose porous layer consists of a scaffold composed of micron-sized fibers and pores filled with nano-sized fibers.

8. The silk-based bilayer composite guided bone regeneration film according to claim 6, characterized in that: The total thickness of the bilayer film is 100–300 μm, with the dense layer accounting for 10%–30% of the thickness.

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