Silk fibroin-based biomimetic artificial tympanic membrane with gradient structure and its preparation method

Abstract

This invention discloses a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure and its preparation method, comprising the following steps: encapsulating and dissolving an antibacterial drug in an organic solvent and then adding it dropwise to a silk fibroin solution; casting the film and inducing the formation of a β-sheet structure with methanol to obtain an outer dense layer; electrospinning the silk fibroin solution to obtain oriented silk fibroin fiber felt, and cross-linking it in an ethanol solution to obtain an intermediate fiber layer; mixing the silk fibroin solution with a pore-forming agent and casting it onto the intermediate fiber layer; covering the intermediate fiber layer with the outer dense layer; after pre-freezing and lyophilizing, immersing the membrane in deionized water to dissolve the pore-forming agent to form an inner porous layer; then placing it in a PBS buffer containing growth factors and shaking treatment; and finally drying to obtain the silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure. This invention's three-layer biomimetic artificial tympanic membrane possesses antibacterial and regenerative functions, while also exhibiting excellent mechanical properties, meeting the requirements of a biomimetic tympanic membrane.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure and its preparation method. Background Technology

[0002] Tympanic membrane perforation is a common ENT disease that can lead to hearing loss, otitis media, cholesteatoma, and other problems. Repairing a perforated tympanic membrane primarily involves using the scaffold provided by the placed repair material to allow existing cells to migrate and spread, thus promoting healing, improving hearing, restoring the middle ear environment, and reducing the risk of infection. The tympanic membrane consists of three layers: the outer epithelium, composed of stratified squamous epithelium formed by keratinocytes, which is continuous with the skin of the external auditory canal; the middle elastic fiber layer, composed of collagen and fibroblasts, which gives the tympanic membrane its elasticity, stability, and tension; and the inner layer, composed of non-keratinized mucosal epithelium, which is continuous with the tympanic cavity mucosa.

[0003] Tympanic membrane repair materials can be divided into autologous tissue materials and tissue-engineered materials, and the surgical procedures differ depending on the material. Autologous tissue transplantation involves open or endoscopic surgery to cover or embed the patient's own temporalis fascia, perichondrium, cartilage, and adipose tissue at the tympanic membrane perforation site, serving as a scaffold for epithelial cells to crawl and cover it. Since the material is derived from the patient's own body, it has excellent biocompatibility and virtually no rejection reaction. However, it also has many drawbacks, such as secondary trauma, limitations in source and size, and uneven healing quality. With the development of biomaterials science, the focus has shifted to bioabsorbable and bioactive artificial tympanic membrane materials. Existing artificial tympanic membrane materials (such as gelatin sponge, silicone membrane, and some absorbable polymers) mainly have the following defects: (1) Simple structure: Most of them are homogeneous films or sponges, which cannot simultaneously simulate the three-layer gradient structure and mechanical properties of the outer epithelial layer, the middle fibrous layer and the inner mucosa layer of the tympanic membrane, resulting in poor mechanical properties after healing and easy re-perforation; (2) Simple function: They only have physical isolation and scaffolding functions, lack the ability to actively resist inflammation, resist infection and promote cell differentiation, and have a high risk of postoperative infection and poor healing; (3) Poor operability: They lack appropriate rigidity and are difficult to place and fit under minimally invasive otoscope.

[0004] Silk fibroin is a unique natural protein extracted from silkworm silk. It possesses excellent biocompatibility, biodegradability, absorbability, low immunogenicity, and adjustable mechanical properties, and has been widely used in tissue engineering. Researchers have shown that tympanic membrane keratinocytes can grow on silk fibroin scaffolds, and this biomaterial supports the growth and proliferation of keratinocytes. Patent CN101879098A discloses an artificial tympanic membrane using silk fibroin and its manufacturing method. This involves desalting and drying a solution of silk fibroin or a silk fibroin complex obtained after removing sericin from silkworm cocoons or silk fibers to form a silk fibroin membrane, which stimulates the regeneration of a perforated tympanic membrane caused by disease or sudden accident. However, it lacks a biomimetic scaffold structure and cannot simulate the microstructure and mechanical properties of the tympanic membrane; the degradation rate and tissue regeneration rate are also difficult to match. Patent CN115245601A discloses a tympanic membrane repair material and its preparation method. It uses graphene oxide to form a stable first tympanic membrane repair material by combining it with hyaluronic acid and collagen. Then, it mixes it with silk fibroin, gelatin and organic solvent to form a second tympanic membrane repair material. However, it is only a mixture of solutions of various components (graphene oxide, silk fibroin, gelatin, tannic acid, etc.) and does not have a structure with different components, fiber arrangement or physicochemical properties in the outer, middle and inner layers. It does not have a biomimetic three-layer structure and cannot meet the requirements of biomimetic scaffolds.

[0005] Therefore, it is urgent to develop a new type of artificial tympanic membrane that is structurally biomimetic, functionally active, and easy to operate. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure and its preparation method. The outer dense layer is designed as an antibacterial and airtight barrier layer, the middle fiber layer as a mechanical conduction and directional guidance layer, and the inner porous layer as a tissue regeneration and vitality layer. It has a three-layer biomimetic structure and possesses antibacterial properties and excellent mechanical properties, thus meeting the requirements of a biomimetic tympanic membrane.

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0008] S1. After dissolving the antibacterial drug in an organic solvent, it is added dropwise to the first silk fibroin solution, then cast onto a petri dish, dried to form a homogeneous film, and the silk fibroin is induced to form a stable β-sheet structure by methanol vapor to obtain the outer dense layer.

[0009] S2. Electrospinning the second silk fibroin solution with a high-speed rotating drum as the receiver, to obtain silk fibroin fiber felt oriented along the rotation direction of the drum, which is then soaked in an ethanol solution for cross-linking and dried to serve as the intermediate fiber layer.

[0010] S3. After mixing the third silk fibroin solution with the pore-forming agent, pour it onto the flat intermediate fiber layer to form an inner blank layer on one side of the intermediate fiber layer.

[0011] S4. The outer dense layer is covered on the surface of the middle fiber layer away from the inner blank layer. After pre-freezing and freeze-drying, it is immersed in deionized water. The inner blank layer dissolves the pore-forming agent to form an inner porous layer. Then it is placed in PBS buffer containing growth factors and shaken. After drying, the silk fibroin-based biomimetic artificial tympanic membrane with gradient structure is obtained.

[0012] This invention features a three-layer biomimetic artificial tympanic membrane structure: an outer dense layer, a middle fibrous layer, and an inner porous layer. This structure possesses antibacterial properties and excellent mechanical characteristics, meeting the requirements of a biomimetic tympanic membrane. Specifically:

[0013] The outer dense layer serves as an antibacterial and airtight barrier layer. Through casting and methanol treatment, a smooth and dense layer is formed. This dense structure effectively isolates external bacteria and moisture, creating a stable environment for inner layer repair. Antibacterial drugs are encapsulated in an organic solvent and then added to a silk fibroin solution, which better disperses the hydrophobic drugs. Subsequent methanol vapor-induced cross-linking "locks" the drugs within a silk fibroin network that forms a stable β-sheet crystalline structure. This not only achieves long-term sustained release of the drugs but also enhances the mechanical properties and water resistance of the film, providing the first line of defense for the implant and effectively preventing postoperative otitis media.

[0014] The intermediate fiber layer, serving as a mechanical conduction and directional guidance layer, is prepared using electrospinning and a high-speed rotating roller to create a highly oriented silk fibroin nanofiber felt. This precisely mimics the radial collagen fiber arrangement of the natural tympanic membrane's intermediate layer, a key structural feature for achieving its high sound transmission efficiency. The oriented fiber structure endows this layer with extremely high radial tensile strength and appropriate flexibility, effectively transmitting sound wave vibrations and acting as the "skeleton" of the artificial tympanic membrane. The topological structure of the nanofibers guides fibroblasts and other cells to grow along the fiber direction, promoting the formation of orderly regenerative tissue and accelerating healing. As the intermediate layer of the three-layer biomimetic structure, it connects and supports the inner and outer layers, ensuring the integrity of the overall structure.

[0015] The inner porous layer serves as a tissue regeneration and bioactive layer. A high-porosity, sponge-like structure is constructed in situ on the surface of the intermediate layer using freeze-drying combined with porogen leaching. The interconnected pores facilitate the exchange of nutrients and metabolic waste. The high porosity provides a three-dimensional space for the migration, proliferation, and vascularization of residual epithelial cells and mesenchymal cells in the tympanic membrane, allowing new tissue to remain permanently within the material, achieving biointegration rather than mere encapsulation. After freeze-drying, the entire structure is immersed in a solution containing growth factors. The large specific surface area of ​​the porous layer enables efficient adsorption, achieving the loading and sustained release of bioactive factors. This sustained release of growth factors actively stimulates cell proliferation and differentiation, accelerating the closure of tympanic membrane perforations and functional reconstruction.

[0016] Furthermore, in S1, the antibacterial drug is selected from one or more of levofloxacin, dexamethasone, gentamicin, penicillin G, amoxicillin, and metronidazole. Preferably, the organic solvent is selected from one or more of dimethyl sulfoxide, methanol, ethanol, acetone, chloroform, and dichloromethane.

[0017] Furthermore, in S1, the drying conditions are: humidity 60-70%, temperature 25-37℃;

[0018] The methanol vapor induction time is 10-120 min.

[0019] Furthermore, in S2, the parameters of the electrospinning are: roller speed 2000-3000 rpm, voltage 10-20 kV, receiving distance 10-20 cm, and solution propulsion rate 0.1-2.0 mL / h.

[0020] Furthermore, in S2, the soaking and cross-linking time is 1-6 hours.

[0021] Furthermore, in S3, the pore-forming agent is selected from one or more of sodium chloride, lactose, mannitol, sucrose, sodium bicarbonate, and sorbitol;

[0022] The mass ratio of the pore-forming agent to silk fibroin is (5-10):1.

[0023] Furthermore, in S4, the pre-freezing temperature is -80±5℃, and the time is 0.5-3h;

[0024] The freeze-drying temperature is -30℃ to -50℃, and the time is 24-72h.

[0025] Furthermore, in S4, the growth factor is selected from one or more of bFGF, EGF, FGF, VEGF, BMP, and EGF;

[0026] The concentration of growth factors in the PBS buffer is 5-15 μg / mL;

[0027] The oscillation treatment is performed at a temperature of 2-6℃ for 2-6 hours.

[0028] Furthermore, in S4, the drying conditions are 37±2℃ and the time is 10-30℃.

[0029] Furthermore, in S1-S3, the concentrations of the first silk fibroin solution, the second silk fibroin solution, and the third silk fibroin solution are independently selected from 1-40 wt%.

[0030] Furthermore, S4 also includes: sterilization treatment using gamma rays at a dose of 25-45 kGy.

[0031] The second aspect of the present invention provides a silk fibroin-based biomimetic tympanic membrane with a gradient structure prepared by the preparation method described in the first aspect.

[0032] The beneficial effects of this invention are:

[0033] This invention actively defends against infection through an outer dense layer, whose dense structure effectively isolates external bacteria and moisture, creating a stable environment for inner layer repair. The directional fiber structure of the middle fibrous layer endows this layer with extremely high radial tensile strength and appropriate flexibility, serving as the "skeleton" of the artificial tympanic membrane. The high porosity of the inner porous layer provides three-dimensional space for the migration, proliferation, and vascularization of epithelial cells and mesenchymal cells, enabling new tissue to remain permanently within the material and achieve biointegration rather than mere encapsulation. The "sandwich" three-layer biomimetic gradient structure highly mimics the natural tympanic membrane, facilitating directional cell ingrowth and functional tissue regeneration.

[0034] The artificial tympanic membrane drug delivery system of this invention simultaneously achieves anti-infection and promotes regeneration, has active repair effects, and significantly improves healing quality and success rate. Attached Figure Description

[0035] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a SEM image of the dense layer on the outer side of the artificial tympanic membrane obtained in Example 1 of the present invention. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0039] S1. After dissolving the antibacterial drug in an organic solvent, it is added dropwise to the first silk fibroin solution, then cast onto a petri dish, dried to form a homogeneous film, and the silk fibroin is induced to form a stable β-sheet structure by methanol vapor to obtain the outer dense layer.

[0040] S2. Electrospinning the second silk fibroin solution with a high-speed rotating drum as the receiver, to obtain silk fibroin fiber felt oriented along the rotation direction of the drum, which is then soaked in an ethanol solution for cross-linking and dried to serve as the intermediate fiber layer.

[0041] S3. After mixing the third silk fibroin solution with the pore-forming agent, pour it onto the flat intermediate fiber layer to form an inner blank layer on one side of the intermediate fiber layer.

[0042] S4. The outer dense layer is placed over the surface of the middle fiber layer away from the inner blank layer. After pre-freezing and lyophilization, it is immersed in deionized water. The inner blank layer dissolves the pore-forming agent to form an inner porous layer. Then, it is placed in PBS buffer containing growth factors and shaken. After drying, the silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure is obtained. This embodiment sets up a three-layer biomimetic artificial tympanic membrane structure with an outer dense layer, a middle fiber layer, and an inner porous layer, which has antibacterial properties and excellent mechanical properties, meeting the requirements of a biomimetic tympanic membrane.

[0043] In a preferred embodiment, in S1, the antibacterial drug is selected from one or more of levofloxacin, dexamethasone, gentamicin, penicillin G, amoxicillin, and metronidazole; the organic solvent is selected from one or more of dimethyl sulfoxide, methanol, ethanol, acetone, chloroform, and dichloromethane; the drying conditions are: humidity 60-70%, temperature 25-37℃; and the methanol vapor induction time is 10-120 min.

[0044] In a preferred embodiment, in S2, the parameters of the electrospinning are: roller speed 2000-3000 rpm, voltage 10-20 kV, receiving distance 10-20 cm, solution propulsion rate 0.1-2.0 mL / h; and the soaking crosslinking time is 1-6 h.

[0045] In a preferred embodiment, in S3, the pore-forming agent is selected from one or more of sodium chloride, lactose, mannitol, sucrose, sodium bicarbonate, and sorbitol; the mass ratio of the pore-forming agent to silk fibroin is (5-10):1.

[0046] In a preferred embodiment, in step S4, the pre-freezing temperature is -80±5℃ and the time is 0.5-3h; the freeze-drying temperature is -30℃ to -50℃ and the time is 24-72h; the growth factor is selected from one or more of bFGF, EGF, FGF, VEGF, BMP, and EGF; the concentration of the growth factor in the PBS buffer is 5-15 μg / mL; the shaking treatment temperature is 2-6℃ and the time is 2-6h; and the drying conditions are 37±2℃ and the time is 10-30℃.

[0047] In a preferred embodiment, in S1-S3, the concentrations of the first silk fibroin solution, the second silk fibroin solution, and the third silk fibroin solution are independently selected from 1-40 wt%; after S4, the method further includes sterilization treatment with γ-rays at a dose of 25-45 kGy.

[0048] Another embodiment provides a silk fibroin-based biomimetic tympanic membrane with a gradient structure prepared by the preparation method described in the above embodiments.

[0049] Example 1

[0050] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0051] S1: Preparation of silk fibroin raw materials

[0052] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 6 wt% for later use.

[0053] S2: Preparation and Assembly of the Three-Layer Structure

[0054] (1) Outer dense layer: Dissolve 1 g of levofloxacin powder in 0.2 mL of dimethyl sulfoxide, and then slowly add it dropwise to the silk fibroin solution. Stir magnetically for 2 hours to ensure that the final concentration of levofloxacin is 1.0 mg / mL. Then, cast the mixed solution onto a smooth culture dish and dry it in a constant temperature and humidity chamber at 60% relative humidity and 25℃ for 24 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 30 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0055] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 1.0 mL / h, a voltage of 15 kV between the spinneret and the receiver, and a receiving distance of 15 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed to 2500 rpm, the silk fibroin nanofibers were highly oriented along the roller rotation direction (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was then placed in an ethanol solution and soaked for crosslinking for 2 hours, then thoroughly washed with deionized water and dried for later use.

[0056] (3) Inner porous layer: Take the silk fibroin solution and add pre-frozen and crushed sodium chloride particles as a pore-forming agent. The mass ratio of the pore-forming agent to the silk fibroin is 9:1. Stir evenly. Spread the middle fiber layer flat and pour the above silk fibroin-sodium chloride mixture evenly on its surface.

[0057] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 3 hours, and then dried in a freeze dryer at -30°C for 48 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the sodium chloride particles, forming a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing bFGF (10 μg / mL), gently shaken and loaded at 4°C for 4 hours, and then dried in a 37°C oven for 20 hours to obtain the biomimetic artificial tympanic membrane.

[0058] S3: Sterilization

[0059] Terminal sterilization was performed using 25 kGy gamma rays.

[0060] Example 2

[0061] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0062] S1: Preparation of silk fibroin raw materials

[0063] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 1 wt% for later use.

[0064] S2: Preparation and Assembly of the Three-Layer Structure

[0065] (1) Outer dense layer: Dissolve 1 g of dexamethasone powder in 0.2 mL of methanol, then slowly add it dropwise to the silk fibroin solution and stir magnetically for 0.5 hours to ensure the final concentration of dexamethasone is 1.0 mg / mL. Then cast the mixture onto a smooth petri dish and dry it in a constant temperature and humidity chamber at 37°C and 60% relative humidity for 20 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 10 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0066] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 0.1 mL / h, a voltage of 10 kV between the spinneret and the receiver, and a receiving distance of 10 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed to 2000 rpm, the silk fibroin nanofibers were highly oriented along the roller rotation direction (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was placed in an ethanol solution and soaked for crosslinking for 1 hour, then thoroughly washed with deionized water and dried for later use.

[0067] (3) Inner porous layer: Take silk fibroin solution, add pre-frozen and crushed lactose particles as a pore-forming agent, the mass ratio of pore-forming agent to silk fibroin is 5:1, and stir evenly. Spread the middle fiber layer flat, and pour the above silk fibroin-lactose mixture evenly on its surface.

[0068] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 0.5 hours, and then dried in a freeze dryer at -35°C for 24 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the lactose particles and form a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing EGF (5 μg / mL), gently shaken and loaded at 4°C for 4 hours, and then dried in an oven at 37°C for 10 hours to obtain the biomimetic artificial tympanic membrane.

[0069] S3: Sterilization

[0070] Terminal sterilization was performed using 30 kGy gamma rays.

[0071] Example 3

[0072] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0073] S1: Preparation of silk fibroin raw materials

[0074] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 10 wt% for later use.

[0075] S2: Preparation and Assembly of the Three-Layer Structure

[0076] (1) Outer dense layer: Dissolve 1 g of gentamicin powder in 0.2 mL of ethanol, then slowly add it dropwise to the silk fibroin solution and stir magnetically for 2 hours to ensure a final concentration of gentamicin of 1.0 mg / mL. Then cast the mixture onto a smooth culture dish and dry it in a constant temperature and humidity chamber at 70% relative humidity and 25°C for 25 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 60 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0077] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 1.5 mL / h, a voltage of 15 kV between the spinneret and the receiver, and a receiving distance of 15 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed to 2750 rpm, the silk fibroin nanofibers were highly oriented along the roller rotation direction (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was then placed in an ethanol solution and soaked for crosslinking for 3 hours, then thoroughly washed with deionized water and dried for later use.

[0078] (3) Inner porous layer: Take the silk fibroin solution and add pre-frozen and crushed mannitol particles as a pore-forming agent. The mass ratio of the pore-forming agent to the silk fibroin is 7:1. Stir evenly. Spread the middle fiber layer flat and pour the above silk fibroin-mannitol mixture evenly on its surface.

[0079] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 2 hours, and then dried in a freeze dryer at -40°C for 48 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the mannitol particles, forming a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing FGF (12 μg / mL), gently shaken and loaded at 4°C for 4 hours, and then dried in a 37°C oven for 15 hours to obtain the biomimetic artificial tympanic membrane.

[0080] S3: Sterilization

[0081] Terminal sterilization was performed using 35 kGy gamma rays.

[0082] Example 4

[0083] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0084] S1: Preparation of silk fibroin raw materials

[0085] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 15 wt% for later use.

[0086] S2: Preparation and Assembly of the Three-Layer Structure

[0087] (1) Outer dense layer: Dissolve 1 g of penicillin G powder in 0.2 mL of acetone, then slowly add it dropwise to the silk fibroin solution and stir magnetically for 0.5 hours to ensure a final concentration of penicillin G of 1.0 mg / mL. Then cast the mixture onto a smooth culture dish and dry it in a constant temperature and humidity chamber at 60% relative humidity and 25°C for 25 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 10 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0088] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 1.5 mL / h, a voltage of 18 kV between the spinneret and the receiver, and a receiving distance of 18 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed to 2500 rpm, the silk fibroin nanofibers were highly oriented along the direction of roller rotation (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was then placed in an ethanol solution and soaked for crosslinking for 4 hours, then thoroughly washed with deionized water and dried for later use.

[0089] (3) Inner porous layer: Take silk fibroin solution, add pre-frozen and crushed sucrose particles as a pore-forming agent, the mass ratio of pore-forming agent to silk fibroin is 5:1, and stir evenly. Spread the middle fiber layer flat, and pour the above silk fibroin-sucrose mixture evenly on its surface.

[0090] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 2.5 hours, and then dried in a freeze dryer at -45°C for 72 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the sucrose particles and form a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing BMP (10 μg / mL), gently shaken and loaded at 4°C for 4 hours, and then dried in an oven at 37°C for 20 hours to obtain the biomimetic artificial tympanic membrane.

[0091] S3: Sterilization

[0092] Terminal sterilization was performed using 35 kGy gamma rays.

[0093] Example 5

[0094] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0095] S1: Preparation of silk fibroin raw materials

[0096] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 20 wt% for later use.

[0097] S2: Preparation and Assembly of the Three-Layer Structure

[0098] (1) Outer dense layer: Dissolve 1 g of amoxicillin powder in 0.2 mL of chloroform, then slowly add it dropwise to the silk fibroin solution and stir magnetically for 5 hours to ensure a final concentration of amoxicillin of 1.0 mg / mL. Then cast the mixture onto a smooth petri dish and dry it in a constant temperature and humidity chamber at 65% relative humidity and 25℃ for 30 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 90 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0099] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 2.0 mL / h, a voltage of 18 kV between the spinneret and the receiver, and a receiving distance of 18 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed to 2800 rpm, the silk fibroin nanofibers were highly oriented along the roller rotation direction (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was then placed in an ethanol solution and soaked for crosslinking for 6 hours, then thoroughly washed with deionized water and dried for later use.

[0100] (3) Inner porous layer: Take silk fibroin solution, add pre-frozen and crushed sodium bicarbonate particles as a pore-forming agent, the mass ratio of pore-forming agent to silk fibroin is 5:1, and stir evenly. Spread the middle fiber layer flat, and pour the above silk fibroin-sodium bicarbonate mixture evenly on its surface.

[0101] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 3 hours, and then dried in a freeze dryer at -50°C for 72 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the sodium bicarbonate particles, forming a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing VEGF (10 μg / mL), gently shaken and loaded at 4°C for 6 hours, and then dried in a 37°C oven for 30 hours to obtain the biomimetic artificial tympanic membrane.

[0102] S3: Sterilization

[0103] Terminal sterilization was performed using 40 kGy gamma rays.

[0104] Example 6

[0105] This embodiment relates to a method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, comprising the following steps:

[0106] S1: Preparation of silk fibroin raw materials

[0107] High-purity silk fibroin was extracted from silkworm cocoons, purified to obtain a silk fibroin solution, and adjusted to the required concentration of 40 wt% for later use.

[0108] S2: Preparation and Assembly of the Three-Layer Structure

[0109] (1) Outer dense layer: Dissolve 1 g of metronidazole powder in 0.2 mL of dichloromethane, and then slowly add it dropwise to the silk fibroin solution. Stir magnetically for 6 hours to ensure that the final concentration of metronidazole is 1.0 mg / mL. Then, cast the mixed solution onto a smooth petri dish and dry it in a constant temperature and humidity chamber at 65% relative humidity and 37℃ for 30 hours to form a homogeneous film. The dried film is then treated in methanol vapor for 120 minutes to induce the silk fibroin to form a stable β-sheet structure and complete the cross-linking.

[0110] (2) Intermediate fiber layer: The silk fibroin solution was electrospun with a solution feed rate of 2.0 mL / h, a voltage of 20 kV between the spinneret and the receiver, and a receiving distance of 20 cm. A high-speed rotating metal roller was used as the receiver. By controlling the roller speed at 3000 rpm, the silk fibroin nanofibers were highly oriented along the direction of roller rotation (simulating the radial direction of the tympanic membrane). The oriented fiber mat was collected by controlling the oriented alignment of the fibers through the rotating roller. It was then placed in an ethanol solution and soaked for crosslinking for 6 hours, then thoroughly washed with deionized water and dried for later use.

[0111] (3) Inner porous layer: Take silk fibroin solution, add pre-frozen and crushed sorbitol particles as a pore-forming agent, the mass ratio of pore-forming agent to silk fibroin is 10:1, and stir evenly. Spread the middle fiber layer flat, and pour the above silk fibroin-sorbitol mixture evenly on its surface.

[0112] (4) Assembly and drug loading: The outer dense layer was carefully placed over the middle fibrous layer to form a "sandwich" structure. The entire "sandwich" structure, along with the mold, was pre-frozen in an ultra-low temperature freezer at -80°C for 3 hours, and then dried in a freeze dryer at -40°C for 72 hours. After freeze-drying, the structure was immersed in deionized water to completely dissolve the sorbitol particles, forming a highly porous inner layer. Subsequently, the structure was immersed in PBS buffer containing EGF (15 μg / mL), gently shaken and loaded at 4°C for 6 hours, and then dried in a 37°C oven for 30 hours to obtain the biomimetic artificial tympanic membrane.

[0113] S3: Sterilization

[0114] Terminal sterilization was performed using 45 kGy gamma rays.

[0115] Comparative Example 1

[0116] The difference between this comparative example and Example 1 is that in step S2 (1), the silk fibroin solution is directly cast onto a smooth culture dish without adding levofloxacin solution, while other steps and parameters remain unchanged.

[0117] Comparative Example 2

[0118] The difference between this comparative example and Example 1 is that the soaking and shaking steps with PBS buffer containing bFGF (10 μg / mL) are omitted in step S2 (4), while other steps and parameters remain unchanged.

[0119] Comparative Example 3

[0120] The difference between this comparative example and Example 1 is that the inner porous layer preparation step S2 (3) is omitted, and the step of dissolving sodium chloride in deionized water in step (4) is also omitted. Other steps and parameters remain unchanged, and an artificial tympanic membrane containing an outer dense layer and an intermediate fiber layer is prepared.

[0121] Comparative Example 4

[0122] The difference between this comparative example and Example 1 is that step S2 (2) of preparing the intermediate fiber layer is omitted, and in step (3) the silk fibroin-sodium chloride mixture is poured onto the outer dense layer. Other steps and parameters remain unchanged, and an artificial tympanic membrane containing an outer dense layer and an inner porous layer is prepared.

[0123] Comparative Example 5

[0124] The difference between this comparative example and Example 1 is that step S2 (1) outer dense layer preparation step is omitted, and after pouring in step (3), the process directly proceeds to the pre-freezing step in step (4). Other steps and parameters remain unchanged, and an artificial tympanic membrane containing an intermediate fiber layer and an inner porous layer is prepared.

[0125] Test Example 1: Antibacterial Performance Experiment

[0126] The silk fibroin-based biomimetic artificial tympanic membranes (containing antibacterial drugs with the side facing outwards) of Examples 1-6 and Comparative Examples 1-2 were respectively attached to agar plates coated with bacterial solution. After culturing for 24 hours, the diameter of the inhibition zone was measured. The experimental results are shown in Table 1.

[0127] Table 1

[0128]

[0129] As shown in Table 1, the artificial tympanic membranes loaded with antibacterial drugs in Examples 1-6 and Comparative Example 2 have antibacterial properties, while the artificial tympanic membrane in Comparative Example 1 without antibacterial drugs has no antibacterial properties.

[0130] Test Example 2: Cell Proliferation Promotion Experiment

[0131] The silk fibroin-based biomimetic artificial tympanic membranes of Examples 1-6 and Comparative Examples 1-2 were co-cultured with fibroblast (NIH-3T3) cells. The cell proliferation rate was quantified by CCK-8 staining, and the results are shown in Table 2.

[0132] Table 2

[0133]

[0134] As shown in Table 2, the artificial tympanic membranes loaded with growth factors in Examples 1-6 and Comparative Example 1 can promote cell proliferation.

[0135] Test Example 3: Scanning Electron Microscope

[0136] The silk fibroin-based biomimetic artificial tympanic membrane sample from Example 1 was cut with a sharp blade. The sample, with the observation surface facing upwards, was firmly adhered to the sample stage using conductive adhesive. The cross-section sample needed to be fixed upright. An approximately 10 nm thick gold-palladium alloy conductive layer was uniformly deposited onto the sample surface using an ion sputtering apparatus to eliminate charge accumulation. After gold sputtering, the sample was observed and photographed using a scanning electron microscope. The results are shown below. Figure 1 As can be seen, the outer dense layer of the artificial tympanic membrane presents a dense fibrous network, providing a stable outer structure.

[0137] Test Example 4: Mechanical Property Test

[0138] The silk fibroin-based biomimetic artificial tympanic membranes from Examples 1-6 and Comparative Examples 3-5 were cut into standard dumbbell or rectangular strips. Tensile tests were performed using a universal testing machine, and stress-strain curves were recorded. Tensile strength and elongation at break were calculated to evaluate the strength and toughness of the materials. The results are shown in Table 3.

[0139] Table 3

[0140]

[0141] As shown in Table 3, the intermediate fiber layer is an essential layer for maintaining the bionic tympanic membrane's mechanical structure. Comparative Example 4, due to the lack of an intermediate fiber layer, has a much lower tensile strength than the other groups.

[0142] Test Example 5

[0143] The biomimetic tympanic membranes obtained in Example 1 and Comparative Examples 1-5 were placed in 24-well plates, one membrane per well. After soaking in PBS overnight, the membranes were replaced with complete culture medium and pre-wetted for 2 h. Mouse fibroblasts (L929) were then added to the surface of the biomimetic tympanic membrane at a density of 5 × 10⁴ cells per well and cultured at 37°C and 5% CO₂. The cell adhesion rate (%) was detected after 4 h and the results are shown in Table 4.

[0144] Table 4

[0145]

[0146] As shown in Table 4, the biomimetic tympanic membrane loaded with cell growth factors (Example 1) facilitates cell adhesion compared to the biomimetic tympanic membrane without growth factors (Comparative Example 2). Furthermore, the inner porous layer serves as the main site for cell adhesion and migration, promoting the three-dimensional growth and spread of cells. The biomimetic tympanic membrane lacking the inner layer (Comparative Example 3) is not conducive to cell adhesion.

[0147] Test Example 6: Animal Model Validation (In vivo Functional Evaluation)

[0148] Fifty SD rats were selected, and a standard-sized (3.4-3.6 mm) tympanic membrane perforation was created in the ear of each rat. Ten rats were divided into groups, with one group serving as a blank control group and the other four groups using artificial tympanic membranes from Example 1, Comparative Example 3, Comparative Example 4, and Comparative Example 5, respectively, to repair the perforated tympanic membrane. The closure of the perforation was observed periodically (1, 2, 4, and 8 weeks) using an otoscope. The repair rate was the proportion of rats with closed perforations. The results are shown in Table 5.

[0149] Table 5

[0150]

[0151] As shown in Table 5, the repair rate of the bionic tympanic membrane with complete 3-layer structure (Example 1) is much higher than that of the group with missing structure (Comparative Example 3, Comparative Example 4 and Comparative Example 5), proving that the 3-layer structure is indispensable.

[0152] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure, characterized in that, Includes the following steps: S1. After dissolving the antibacterial drug in an organic solvent, it is added dropwise to the first silk fibroin solution, then cast onto a petri dish, dried to form a homogeneous film, and the silk fibroin is induced to form a stable β-sheet structure by methanol vapor to obtain the outer dense layer. S2. Electrospinning the second silk fibroin solution with a high-speed rotating drum as the receiver, to obtain silk fibroin fiber felt oriented along the rotation direction of the drum, which is then soaked in an ethanol solution for cross-linking and dried to serve as the intermediate fiber layer. S3. After mixing the third silk fibroin solution with the pore-forming agent, pour it onto the flat intermediate fiber layer to form an inner blank layer on one side of the intermediate fiber layer. S4. The outer dense layer is covered on the surface of the middle fiber layer away from the inner blank layer. After pre-freezing and freeze-drying, it is immersed in deionized water. The inner blank layer dissolves the pore-forming agent to form an inner porous layer. Then it is placed in PBS buffer containing growth factors and shaken. After drying, the silk fibroin-based biomimetic artificial tympanic membrane with gradient structure is obtained.

2. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S1, the antibacterial drug is selected from one or more of levofloxacin, dexamethasone, gentamicin, penicillin G, amoxicillin, and metronidazole.

3. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S1, the drying conditions are: humidity 60-70%, temperature 25-37℃; The methanol vapor induction time is 10-120 min.

4. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S2, the parameters of the electrospinning are: roller speed 2000-3000 rpm, voltage 10-20 kV, receiving distance 10-20 cm, and solution propulsion rate 0.1-2.0 mL / h.

5. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S2, the soaking and cross-linking time is 1-6 hours.

6. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S3, the pore-forming agent is selected from one or more of sodium chloride, lactose, mannitol, sucrose, sodium bicarbonate, and sorbitol; The mass ratio of the pore-forming agent to silk fibroin is (5-10):

1.

7. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In step S4, the pre-freezing temperature is -80±5℃, and the time is 0.5-3h; The freeze-drying temperature is -30℃ to -50℃, and the time is 24-72h.

8. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, In S4, the growth factor is selected from one or more of bFGF, EGF, FGF, VEGF, BMP, and EGF; The concentration of growth factors in the PBS buffer is 5-15 μg / mL; The oscillation treatment is performed at a temperature of 2-6℃ for 2-6 hours.

9. The method for preparing a silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure as described in claim 1, characterized in that, S4 also includes: sterilization treatment using gamma rays at a dose of 25-45 kGy.

10. A silk fibroin-based biomimetic artificial tympanic membrane with a gradient structure prepared by the preparation method according to any one of claims 1-9.

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