Bioabsorbable silk membrane for guiding bone regeneration as well as preparation method and application of bioabsorbable silk membrane

Abstract

The invention discloses a bio-absorbable silk membrane for guiding bone regeneration as well as a preparation method and application thereof. The silk membrane is designed based on a bionic structure, degummed silk fibers are utilized to form a continuous force-bearing fiber skeleton, and a fiber network is infiltrated and cured by a recombinant silk protein solution, so that a fiber-matrix composite structure is constructed. An asymmetric structure with a compact layer and a porous layer is formed after freeze-drying, and then formation of a beta-folded crystal region is induced through annealing, so that the membrane material obtains enhanced interface bonding and overall mechanical properties. The mechanical retention capacity of the obtained membrane material in the traction, tearing and degradation processes is obviously superior to that of a conventional collagen guided bone regeneration membrane, and the membrane material can bear tissue pressure, suture traction and micro-motion environment and maintain barrier stability in the early stage of bone defect healing. According to the technical route for realizing mechanical enhancement by means of structural construction in the single-component silk system, a new design strategy is provided for application of the absorbable implant material in a mechanical unstable regeneration environment.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a bioabsorbable silk membrane for guiding bone regeneration, its preparation method, and its application. Background Technology

[0002] In guided bone regeneration (GBR), the GBR membrane constructs the space required for bone defect healing and serves as a structural boundary between soft and hard tissues in the early postoperative stage, preventing fibroblast invasion and guiding osteoblast regeneration. Currently, this boundary faces numerous challenges in the clinical setting. During alveolar bone regeneration, the membrane's stress state is closely related to physiological activities, primarily including soft tissue pressure, suture fixation traction, and transient mechanical effects transmitted during functional movements. Such loading is easily generated when the defect site lacks internal support and the stability of the regeneration space rests entirely on the membrane. Furthermore, during the degradation and absorption process, maintaining the stability of the membrane structure and function throughout the mechanically vulnerable period is crucial for bone defect repair.

[0003] Collagen-based bone regeneration membranes (GBRs) remain the clinical standard for treating bone defects due to their excellent biocompatibility and biodegradability. However, during bone repair, the loss of this hierarchical structure limits their initial strength, accelerates degradation and absorption, and reduces their ability to withstand soft tissue compression or suture traction. Synthetic polyesters, such as polylactic acid (PLA), polycaprolactone (PCL), and polylactic-glycolic acid (PLGA) copolymers, have high hardness, but their brittleness and acidic degradation can impair the mechanical retention of GBRs. While absorbable metals can increase the hardness of GBRs and provide sufficient support, they are prone to premature loss of mechanical properties with the release of alkalization and degradation gases, and degradation products may also induce inflammation. Furthermore, natural polymers, such as chitosan, cellulose, silk fibroin, and alginate, and even multi-component composite GBR materials, while offering good biological characteristics, have mechanical limitations that cannot meet the mechanical performance requirements of early osteogenic processes.

[0004] Natural collagen soft tissue possesses excellent mechanical properties, primarily derived from the synergistic effect of its hierarchical structure rather than the complexity of its components. In these tissues, high-strength collagen fibers are embedded within a soft matrix composed of elastin and proteoglycans. This hierarchical arrangement of elements coordinates and distributes load, the compliant matrix allows for long-distance tensile force transmission, and fiber sliding and delayed breakage release stored elastic energy and alleviate stress concentration at defects. This structure endows soft tissue with high strength, high toughness, and low defect sensitivity. In contrast, commercially available GBR collagen membranes lack this hierarchical structure and therefore cannot exhibit these mechanical properties. Similar principles can be observed in natural materials such as bamboo and spider silk, where unique mechanical functions are achieved through simple multi-level structures. Inspired by these phenomena, we attempted to replicate the structural logic of this connective tissue while retaining a single-component system. Because regenerated silk fibroin possesses molecular consistency, biocompatibility, and the ability to enhance fiber networks through tunable secondary structures, it provides a controllable platform for constructing viscous structures that enhance interfacial integrity and overall mechanical properties.

[0005] Currently, some technical solutions have been proposed to address the above-mentioned problems in the research on guiding bone regeneration barrier membranes, but certain limitations still exist.

[0006] Chinese invention patent CN 110882424 A discloses an alloy guided bone regeneration barrier membrane containing aluminum, zinc, manganese, silver, calcium, titanium dioxide, and Mg. In this GBR membrane, aluminum and zinc constitute half the composition. Although reliable support properties can be imparted to the GBR membrane by adjusting the alloy composition, its mechanical properties are still limited by the active metals themselves. Especially under long-term load, the alloy GBR membrane is prone to degradation and failure. Furthermore, when the various metal ions (Al³⁺, Zn²⁺, Mn²⁺, Ag⁺) released during degradation reach a certain concentration, they can have toxic effects on surrounding cells (fibroblasts, osteoblasts, and immune cells), inhibiting cell proliferation and metabolism, and even leading to cell death, thus hindering tissue healing and integration.

[0007] Chinese invention patent CN 116672513 A discloses a composite material GBR membrane, which is cast under a bacterial cellulose membrane and cured as a mixed solution of polylactic acid, polycaprolactone, surfactants (ethylene oxide-propylene oxide-ethylene oxide triblock copolymer or polyethylene glycol-propylene glycol-ethylene glycol triblock copolymer), and carbon materials. This method uses a composite material of synthetic polymers, carbon nanospheres, and bacterial cellulose for bone repair. This composite material can provide certain mechanical strength during the bone healing cycle and provides antibacterial and reactive oxygen species removal during the repair process. However, the drawbacks of this method are that the degradation products of the introduced surfactants have unknown toxicity, acidic degradation environments may accelerate material degradation, and the carbon nanospheres are difficult to degrade and may remain indefinitely.

[0008] Chinese invention patent CN114249982B discloses a method for preparing high-strength, high-modulus silk material. The method involves adding fibroin nanofibers to a fibroin protein solution to obtain a mixture of fibroin protein solution and fibroin nanofibers. The mixture is then dialyzed and dried at low temperature. The resulting silk material exhibits high strength, high modulus, good biocompatibility, and biodegradability. Furthermore, the fibroin nanofibers are uniformly distributed within the material. This method, based on the principle of homogeneous reinforcement, uses natural fibroin nanofibers added to a fibroin protein solution to obtain silk material. While retaining the strength and modulus advantages of natural fibroin nanofibers, it has limitations in resisting tearing and inhibiting crack propagation because the size reduction leads to increased defects and limits its crack bridging ability. This invention aims to achieve self-reinforcement through a degummed long silk fiber network. Its complete β-fold crystal structure and fiber entanglement directly bear the main tensile load, significantly enhancing the fracture strength of the composite material. The long fibers dissipate fracture energy through a bridging-pull-out mechanism, significantly improving the fracture toughness of the composite material. Furthermore, it constructs a porous structure where the silk fiber network and fibroin matrix work synergistically. In addition, the DSF–RSF composite system of this invention constructs a continuous load transfer path through solution infiltration, freeze-drying, and structure induction, which differs from the mechanism of mixing silk nanofibers and fibroin solutions.

[0009] Chinese invention patent CN115998957A discloses a self-reinforcing structure formed by alternating layers of regenerated silk fibroin powder and commercially available silk fabric and densifying them in a hot-pressing mold. This material is mainly used to prepare silk medical bone screws or bone fixation plates for fracture fixation, with the focus on achieving high overall mechanical strength and implantability. This approach involves forming a thick bulk structure through a densification process, with the material's reinforcement primarily relying on the alternating arrangement of fabric and powder layers. However, such hot-pressed plates differ from this invention in terms of structural type, formation mechanism, stress response mode, and target application. This invention aims to obtain a membrane-like, flexible, and interfacially functional absorbable barrier material, requiring the formation of a continuous fiber bundle-matrix synergistic structure at a relatively low thickness, and the formation of heterogeneous pore structures during construction to meet the differentiated functional requirements of soft and hard tissue interfaces. The bulk material formed by hot-pressing densification does not possess such membrane-like structural characteristics and is difficult to form controllable bifacial surfaces within a thick plate. Furthermore, the degummed fiber network and regenerated silk fibroin composite system of the present invention constructs a continuous load transfer path through steps such as solution infiltration, freeze-drying, and structural induction. This differs from the fabric layering reinforcement mechanism relied upon by hot-pressed plates, thus forming another type of structural system suitable for guiding bone regeneration.

[0010] Building upon traditional GBR membranes, some researchers have attempted to provide reliable mechanical properties while maintaining bioactivity by employing bioactive metals, biodegradable synthetic polymers, and natural polymers. However, these approaches have failed to effectively address issues such as rapid degradation and insufficient mechanical properties. Furthermore, during long-term use, external loads can cause the support structure to collapse, resulting in an inability to provide a stable osteogenic space. Therefore, although these improved technologies can enhance some aspects of GBR membrane performance, they do not fundamentally resolve the issues of insufficient mechanical properties and long-term stability.

[0011] Previous studies have only mentioned improving the mechanical properties of GBR barrier membranes through component diversification and mechanical densification. However, current research has overlooked the fact that even a single component can be enhanced and toughened using a gentler approach by controlling changes in hierarchical structure. Summary of the Invention

[0012] To address the problems existing in the prior art, the present invention aims to provide a bioresorbable silk membrane for guiding bone regeneration. This all-silk membrane is composed of degummed silk fibers and regenerated silk fibroin. The degummed silk fibers form a continuous fiber network as a fiber skeleton. The regenerated silk fibroin fills the gaps in the fiber network to form a continuous matrix phase. Furthermore, the regenerated silk fibroin is annealed to induce the formation of β-sheet crystal regions, which combine with the fiber network through the interface to form a fiber-matrix composite structure. This composite structure is an asymmetric structure with a dense layer and a porous layer.

[0013] Furthermore, the fiber network forming the fiber skeleton creates a continuous mechanical load-bearing path inside the membrane, which is used to bear the main load under suture tension or local stress concentration conditions.

[0014] Furthermore, the dense layer forms one side of the silk membrane to block soft tissue cell infiltration; the porous layer forms the other side of the silk membrane to promote osteoblast-associated cell adhesion.

[0015] Furthermore, annealing is performed using methanol, which induces the formation of β-folded crystal regions that enhance inter-fiber bonding and the tear and tensile strength of the membrane material.

[0016] Another object of the present invention is to provide a method for preparing the above-mentioned silk membrane, the method comprising the following steps: 1) Provide degummed silk fibers and construct a fiber network; Specifically: S1, degumming the silkworm cocoons, then washing them with deionized water more than ten times until the water is clear and there is no obvious foam to remove the sericin, and drying them to obtain dry degummed silk. Furthermore, the silkworm cocoons are placed in a degumming solution and boiled for degumming; the boiling time is 30-45 minutes; the mass ratio of silkworm cocoons to degumming solution is 1:50; the degumming solution is a Na2CO3 solution with an alkali concentration of 0.05 wt%. S2, Dissolve degummed silk in a good solvent to obtain a homogeneous regenerated silk fibroin solution; Furthermore, LiBr solution was used as a good solvent for degummed silk; the concentration of LiBr solution was 9.3 mol / L, the mass ratio of degummed silk to volume of LiBr solution was 1 g: 5 mL, the dissolution temperature was 60 ℃, and the dissolution time was 6 h. S3, Place the regenerated silk fibroin solution in a dialysis bag with a molecular weight cutoff of 3500, and then place the dialysis bag in deionized water for full dialysis to remove LiBr and small molecule impurities; Furthermore, the dialysis temperature is 4℃, and the dialysis time is 3 days. On the first day, the water is changed every 1 to 2 hours; on the second day, the water is changed every 2 to 6 hours; and on the third day, the water is changed every 6 to 12 hours. S4. The dialyzed regenerated silk fibroin solution is centrifuged to remove insoluble impurities, resulting in a pure regenerated silk fibroin solution. Furthermore, the centrifugation process was carried out at a speed of 12,000–18,000 rpm for 15–30 minutes, and repeated 3–5 times. S5, Take a portion of the pure regenerated silk fibroin solution and add it to a square dish, then evaporate and dry it to obtain a dense evaporated and dried regenerated silk fibroin membrane. Furthermore, the RSF solution evaporated and dried had a concentration of 6 w / v%, a volume of 3 mL, an evaporation and drying temperature of 40℃, and the square dish was made of polystyrene with dimensions of 6×6×5.5 cm. S6. After boiling the silkworm cocoons in deionized water, manually open them to form sheet-like fiber layers. After multiple layers are stacked, a loosely wrapped sheet-like structure is formed. Pull the silk fibers evenly along the perimeter to distribute them evenly. Then fix it in a square tray for degumming, washing and drying to obtain a uniformly distributed, isotropic degummed silk fiber support that retains the internal microstructure of natural fibroin. Furthermore, the boiling time for the silkworm cocoons is 30 minutes. The square tray is made of stainless steel and has dimensions of 35×35×3cm. The evenly cut and stretched silk fiber sheets are placed in the square tray and fixed to the tray with metal clips to prevent damage to the original structure of the fiber network during degumming or washing. Then, the cut and stretched DSF support is degummed, washed, and dried according to S1.

[0017] 2) Add the regenerated silk fibroin solution and allow it to soak the fiber network to form a composite wet film; Specifically: S7. At room temperature, regenerated silk fibroin is mixed in solution with the degummed silk fiber network sheet placed on the thermally conductive substrate, allowing it to be fully impregnated and mixed. Furthermore, the concentration of regenerated silk fibroin is 2-6 w / v%, the volume is 2.5 mL, the mass of the degummed silk fiber network membrane is 0.1 g, the size is 6×6 cm, and the thermally conductive substrate is a copper plate with a thickness of 0.5 mm and a length and width of 10×10 cm.

[0018] 3) After freezing the composite wet membrane, the air contact surface is lightly scraped off, and after freeze-drying, a dry membrane with an asymmetric pore structure is obtained. Specifically: S8. A thermally conductive copper sheet containing an impregnation mixture is placed on two pre-cooled iron blocks at different temperatures for two-stage temperature-controlled freezing to create pores. Then, a blade pre-cooled with liquid nitrogen is used to gently scrape the air contact surface to fully expose the pore size. Finally, after freeze-drying, an asymmetrical pore size structure is achieved, specifically a regenerated silk fibroin framework film with large pores on one side and small pores on the other. Furthermore, the iron block, measuring 20×15×3 cm in length, width, and height, was pre-cooled in a -80 ℃ refrigerator to achieve temperatures ranging from -60 to -50 ℃ and from -5 to 0 ℃, respectively. It was then placed on a hollow foam board, with liquid nitrogen added underneath to control the iron block's temperature. A thermally conductive copper sheet containing the impregnation mixture was placed on the -60 to -50 ℃ iron block. After observing the surface of the impregnation mixture at the copper sheet turn white, it was immediately transferred to the -5 to 0 ℃ iron block for complete freezing. The freeze dryer was then set to a vacuum of 1 Pa and a temperature of -80 ℃ for one day of freeze-drying.

[0019] 4) Anneal the dry film to induce the regenerated silk fibroin to form β-sheet crystalline regions and obtain a composite scaffold; Specifically: S9 was then soaked in methanol to improve water stability, and then solvent-exchanged with deionized water to remove methanol. It was then freeze-dried again to obtain a regenerated silk fibroin composite freeze-dried scaffold reinforced with degummed silk fiber network. Furthermore, anhydrous methanol was used for soaking for 15 minutes. After soaking, the methanol was removed by solvent exchange with deionized water. The deionized water was changed every 2 hours during the day and left overnight. This process was repeated more than 10 times. Then, the small hole was placed with the hole facing down and the container was frozen again at -80 °C. After that, it was taken out and freeze-dried. The specific freezing process was carried out according to S8. S10, the evaporated and dried regenerated silk fibroin membrane is evenly covered on one side of the small holes of the pre-wetted composite scaffold and quickly pressed and smoothed. Taking advantage of the poor water stability of the evaporated and dried silk fibroin membrane, the silk fibroin membrane and the composite porous scaffold are automatically combined. Finally, the DRSF membrane with Janus structure is obtained by evaporation and drying. Furthermore, the temperature on the drying plate was set to 40 ℃, and the drying time was 1 day.

[0020] 5) The DRSF membrane obtained in step 4) is dried and then annealed again to obtain a bioabsorbable silk membrane for guiding bone regeneration. Specifically: S11, The DRSF membrane is treated with methanol and exchanged with solvent, and then evaporated and dried again to obtain a bioabsorbable silk membrane that guides bone regeneration.

[0021] Furthermore, methanol treatment and solvent exchange are carried out according to S9, with the drying temperature at 40 °C and the drying time at 1 day.

[0022] Another object of the present invention is to provide the application of the silk membrane prepared by the above preparation method in the preparation of medical devices for alveolar bone defect repair, bone defect filling or guided bone regeneration surgery.

[0023] This invention constructs a hierarchical, all-silk DRSF membrane by combining degummed silk fibers with regenerated silk fibroin. This achieves a single-component silk fibroin system, where embedded silk fibroin fibers form a continuous load-bearing pathway, while the regenerated silk fibroin matrix enhances the interfacial bonding between the bonded fibers through β-sheet assembly. A two-step manufacturing process yields a GBR membrane with a flexible Janus structure suitable for clinical applications. This biomimetic soft tissue structure produces a porous membrane with higher tensile strength, significantly improved resistance to local deformation, and enhanced resistance to suture traction. Its mechanical retention during degradation is significantly higher than commercially available collagen membranes and single-component regenerated silk fibroin membranes, supporting stable performance during the early healing phase. The membrane exhibits good cell compatibility, promotes osteogenic differentiation, and provides an effective barrier function, preventing fibroblast infiltration.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1) In this invention, degummed silk fibers are combined with regenerated silk fibroin to construct a whole-silk DRSF membrane with a hierarchical structure, realizing a single-component silk fibroin system. After methanol treatment, the embedded fibers form a continuous load-bearing path, while the regenerated silk fibroin enhances the adhesion between fibers through β-sheet crystal regions. The load transfer of the fibers is strengthened through the interfacial shear between the degummed silk fibers and the regenerated silk fibroin, thus solidifying the structural-mechanical relationship.

[0025] 2) In this invention, the degummed silk network membrane is thoroughly mixed with regenerated silk fibroin in the form of an aqueous solution, and then freeze-dried and annealed in methanol to achieve a self-reinforcing effect. Utilizing the poor water stability of the evaporated silk fibroin membrane, it automatically binds by absorbing interfacial water from the pore side of the freeze-dried scaffold. Simultaneously, the capillary action of the pores facilitates precise clogging of the silk fibroin membrane after moisture absorption and mechanical interlocking with the scaffold, ensuring its barrier function and interfacial bonding. The macroporous side retains its directional macroporous structure. This directional porous structure provides a beneficial environment for directional cell growth, new bone tissue formation, and blood vessel growth, promoting bone tissue repair.

[0026] 3) This invention utilizes a two-step directional freeze-casting process combined with freeze-drying to form a freeze-dried scaffold film. Under a scanning electron microscope, an asymmetrical pore structure can be clearly observed on both sides, namely, one side with large noodle-shaped pores and the other side with a honeycomb-like small pore structure. Through this two-step manufacturing process, a Janus structure with morphological flexibility suitable for clinical processing is obtained.

[0027] 4) The mechanical properties and biodegradability of the composite material of the present invention are controllable: by adjusting the content of degummed silk fibers and the methanol annealing time, the mechanical properties and biodegradability can be controlled to match the osteogenic cycle.

[0028] 5) The all-silk GBR membrane prepared by this invention can be used in the field of bone tissue repair, especially as a barrier membrane for repairing bone defects. This medical silk GBR membrane has excellent biocompatibility, can promote the growth and development of bone cells, and can be degraded into amino acids and small peptides in the human body. After being absorbed by the human body, it can be removed without secondary surgery. At the same time, the cost is very low, which can greatly reduce the cost of medical implants.

[0029] 6) This preparation method is simple to operate, has low energy consumption and low cost. The single component means that the construction of the membrane material does not require the design of complex interface chemistry, does not involve heterogeneous interfaces and complex components, and does not bring additional regulatory complexity, making it suitable for industrial production. Attached Figure Description

[0030] Figure 1 The microstructure features of the all-filament DRSF freeze-dried scaffold in Example 1; Figure 2 The structural design and material properties of the all-silk DRSF-guided bone regeneration barrier membrane in Example 1; Figure 3 The molecular conformation and mechanical properties of DRSF membranes; Figure 4 The tensile properties of DRSF-guided bone regeneration barrier membranes with different ratios of degummed silk and regenerated silk fibroin in Example 1 and Comparative Examples 1, 2, 3, and 4 under dry conditions. Figure 5Study on the wet tensile properties of DRSF-guided bone regeneration barrier membranes with different ratios of degummed silk and regenerated silk fibroin in Example 1 and Comparative Examples 1, 2, 3, and 4; Figure 6 For suture retention and tear resistance; Figure 7 The blood compatibility of DRSF-guided bone regeneration barrier membrane was studied for different ratios of degummed silk and regenerated silk fibroin in Examples 1 and 2, 3, and 4. Figure 8 Cell compatibility, osteoblast adhesion, and barrier properties of Example 1 and commercial products. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. Example

[0032] A degummed silk fiber network measuring 6×6 cm and weighing 0.1 g was impregnated and mixed with 2.5 mL of 4 w / v% regenerated silk fibroin solution on a thermally conductive copper plate. The copper sheet containing the impregnated mixture was placed on an iron block pre-cooled at -50°C. After the surface of the mixture in contact with the copper block rapidly froze and turned white, it was quickly transferred to an iron block frozen at -5°C until it was completely frozen. Then, the air contact surface was gently scraped with a blade pre-cooled by liquid nitrogen to fully expose the pores. The completely frozen mixture was placed in a freeze dryer at -80 ℃ and the vacuum degree was 1 Pa.

[0033] The freeze-dried degummed fiber-silk fibroin scaffold was treated in anhydrous methanol for 15 min, rinsed with deionized water to remove methanol, and then freeze-dried again to obtain a composite freeze-dried scaffold with good water stability and a solid content ratio of degummed silk fiber to regenerated silk fibroin of 2:2.

[0034] The regenerated silk fibroin membrane, which was evaporated and dried at 40°C, was attached to the small holes of a pre-wetted composite scaffold. After drying at 40°C again, it was treated with anhydrous methanol for 15 min, rinsed with deionized water, and dried at 40°C to obtain Example 1 (named: DRSF (2:2)).

[0035] Comparative Example 1 The procedure was essentially the same as step 1, except that the concentration of the regenerated silk fibroin solution was 2 w / v%, resulting in a composite freeze-dried scaffold with good water stability and a solid content ratio of degummed silk fiber to regenerated silk fibroin of 2:1. The silk fibroin membrane, dried at 40°C, was attached to the pores of the pre-wetted composite scaffold. After drying at 40°C, it was treated again with anhydrous methanol for 15 min, rinsed with deionized water, and dried at room temperature to obtain Comparative Example 1 (named: DRSF (2:1)).

[0036] Comparative Example 2 The procedure was essentially the same as step 1, except that the concentration of the regenerated silk fibroin solution was 6 w / v, resulting in a composite freeze-dried scaffold with good water stability and a solid content ratio of degummed silk fiber to regenerated silk fibroin of 2:3. The regenerated silk fibroin membrane, dried at 40°C, was attached to the pores of the pre-wetted composite scaffold. After drying at 40°C, it was treated again with anhydrous methanol for 15 min, rinsed with deionized water, and dried at room temperature to obtain Comparative Example 2 (named: DRSF (2:3)).

[0037] Comparative Example 3 A 6×6 cm degummed silk fiber network membrane with a mass of 0.1 g was mixed with 2.5 mL of deionized water on a thermally conductive copper plate. The copper sheet carrying the mixture was placed on an iron block pre-cooled at -50 ℃. After the surface of the mixture in contact with the copper block rapidly froze and turned white, it was transferred to an iron block frozen at -5 ℃ until it was completely frozen. Then, the air contact surface was gently scraped with an ice knife pre-cooled by liquid nitrogen to fully expose the pores. The completely frozen mixture was placed in a freeze dryer at -80°C and the vacuum degree was 1 Pa.

[0038] The freeze-dried degummed silk fiber scaffold was treated in anhydrous methanol for 15 min, rinsed with deionized water to remove methanol, and then freeze-dried again to obtain a methanol-treated degummed silk fiber network.

[0039] The regenerated silk fibroin membrane dried at 40℃ was attached to a pre-wetted contact copper plate. After drying at 40℃, it was treated again with anhydrous methanol for 15 min, rinsed with deionized water, and dried at room temperature to obtain Comparative Example 3 (named: DSF).

[0040] Comparative Example 4 A frame measuring 60×60×5 mm was formed by attaching silicone to a thermally conductive copper sheet. 5 mL of a 4 w / v% regenerated silk fibroin solution was added inside the frame. The copper sheet containing the regenerated silk fibroin solution was placed on an iron block pre-cooled to -50 ℃. After the contact surface between the solution and the copper block rapidly froze and turned white, it was transferred to an iron block frozen to -5 ℃ until it was completely frozen. Then, the air contact surface was gently scraped with an ice knife pre-cooled by liquid nitrogen to fully expose the pores. The completely frozen mixture was placed in a freeze dryer at -80 ℃ and the vacuum degree was 1 Pa.

[0041] The freeze-dried silk fibroin scaffold was treated in anhydrous methanol for 15 min, rinsed with deionized water to remove methanol, and then freeze-dried again to obtain the methanol-treated freeze-dried silk fibroin scaffold.

[0042] The regenerated silk fibroin membrane dried at 40℃ was attached to one of the pre-wetted holes. After drying at 40℃, it was treated again with anhydrous methanol for 15 min, rinsed with deionized water, and dried at 40℃ to obtain Comparative Example 4 (named: RSF).

[0043] The mechanical properties of the products obtained in Example 1 and Comparative Examples 1-4 were tested under the same conditions in both dry and hydrated states. The results are shown in Table 1 and Table 2, respectively.

[0044] Table 1: Statistical Analysis of Mechanical Properties in Dry Conditions

[0045] Table 2: Statistics of mechanical properties under hydration state

[0046] As shown in Tables 1 and 2, the mechanical properties of the DRSF membrane prepared in Example 1 of this invention, under dry conditions, monotonically increase with the increase of regenerated silk fibroin content, and are significantly improved compared to single-component DSF or RSF membranes. The tensile strength (4.52 MPa) and fracture energy (1.70 MJ / m³) under hydration are the highest among all samples, far superior to single-component DSF or RSF membranes. This indicates that hydration enhances the flexibility of the regenerated silk fibroin molecular chains, overcoming the inherent rigidity of regenerated silk fibroin as a binding continuous phase under dry conditions. Through greater interfacial shear deformation, the load is gradually transferred to the fibers, reducing stress concentration at the interface and preventing premature fiber debonding. Optimal synergy between interfacial bonding and load transfer is achieved. Excessive degummed silk fibers or insufficient regenerated silk fibroin matrix disrupts the continuity of the matrix, weakening interfacial stress transfer and energy dissipation capabilities. Through precise control of the spatial combination and interfacial bonding of rigid and flexible components, efficient division of labor is achieved in "fiber load-bearing, matrix force transmission, and interfacial coordination," ultimately breaking through the performance limits of single materials.

[0047] Verification Example 1: Characterization of Structure and Morphology When a regenerated silk fibroin solution is injected into a degummed silk fiber network scaffold, the regenerated silk fibroin occupies fiber gaps similar to the conforming matrix in natural connective tissue, achieving a soft tissue-like structure. Example 1 produced a porous DRSF composite membrane exhibiting a two-sided asymmetric pore size structure, with an average pore size of approximately 9 μm on the contact thermally conductive copper sheet. Figure 1 ac), while the silk fibroin matrix forms continuous pore walls that bind adjacent fibers ( Figure 1 The resulting DRSF composite material exhibits interconnected pores. Microscopically, degummed silk fibers are embedded within the pore walls derived from regenerated silk fibroin, establishing bonding and mechanical continuity throughout the entire fiber network scaffold. Figure 2 After methanol annealing, an additional layer of regenerated silk fibroin was attached to the pore side and then dried, resulting in an asymmetric membrane structure. The coated surface formed a dense, non-porous barrier, while the uncoated surface maintained an interconnected microporous morphology with an average pore size of approximately 75 μm. Figure 2 g). Scanning electron microscopy imaging confirmed the presence of a dense barrier surface that restricts soft tissue penetration and a porous surface that maintains characteristics conducive to osteoblast attachment, migration, and differentiation. Figure 2 The final Janus-structured DRSF membrane exhibits geometric adaptability and mechanical compliance. When formed into a curved shape, it can withstand a 2 g load and is securely fixed by suturing to a curved alveolar bone model, demonstrating surgical stability and compatibility with clinically relevant anatomical geometry. Figure 2 a, 2i-j).

[0048] Verification Example 2: Correlation Analysis of Structure and Mechanics The DRSF membrane integrates a hierarchical structure that directly determines its mechanical behavior under hydration conditions relevant to the early stages of osteogenic development. Example 1 confirmed through Fourier transform infrared spectroscopy analysis that the regenerated silk fibroin matrix exhibited abundant β-sheet formation after methanol treatment. Figure 3 (ac). This combination produces a composite structure in which slowly degrading, highly crystalline degummed silk fibers are embedded in a bonded RSF matrix, the secondary structure and intermolecular stacking of which can be adjusted through an annealing process. This design is similar to the structural logic of collagen soft tissue, in which a robust fibrous skeleton and a flexible matrix work together to support fiber rearrangement, sliding, and load transfer.

[0049] Mechanical tests showed that DRSF membranes exhibited different stress characteristics in dry and hydrated states. Dry tensile tests in Example 1 demonstrated that DRSF outperformed RSF and commercial Bio-Gide (Girdle Collagen GBR membrane) in terms of elastic modulus, strength, and fracture work, and its performance monotonically increased with increasing regenerated silk fibroin content. Figure 4Compared to Comparative Example 4, Example 1 showed very limited dry-state strength of the RSF due to its limited load-transfer capacity. Despite containing collagen, Bio-Gide lacked a continuous phase load-bearing pathway under dry load. The limited mobility of the dry-state RSF restricted matrix deformation, and load transfer was primarily determined by fiber stiffness and fracture resistance. Failure mainly involved fiber breakage and minimal interfacial slip, indicating that dry-state mechanics reflects the inherent rigidity of the degummed silk fiber network. Hydrated tensile tests of Example 1 and Comparative Examples 1, 2, 3, and 4 demonstrate that water molecules, acting as plasticizers in the wet state, enhance the flexibility of the regenerated silk fibroin molecular chains, enabling progressive fiber bonding and controlling interfacial shear transfer. Under these conditions, the mechanical behavior no longer monotonically increases with increasing silk fibroin content, primarily due to the dilution of the fiber reinforcement effect and the shift in failure modes. Conversely, tensile strength and fracture work reached significant maximum values ​​in a 2:2 composition of degummed silk and regenerated silk fibroin. Figure 5 Combinations with insufficient fiber content exhibit premature matrix yielding, while compositions with high fiber content lack sufficient matrix continuity to sustain interfacial shear. A suitable composition provides the most effective balance between fiber stiffness, matrix cohesion, and interfacial shear transfer. Under hydration conditions, RSF itself is easily deformable but lacks sufficient strength. Bio-Gide exhibits a low hydration modulus and early failure, consistent with its spongy collagenous structure, which softens extensively in water and fails to maintain a continuous load-bearing pathway. Figure 3 df) Enzymatic degradation affected matrix quality but preserved the structural continuity of the composite material. The degummed silk fibers degraded much more slowly than the regenerated silk fibroin, maintaining the primary load-bearing framework in the early stages associated with guided bone regeneration. The remaining regenerated silk fibroin maintained tight connectivity and retained sufficient interfacial friction to sustain continuous fiber bonding. In Example 1, compared to the Bio-Gide commercial group, this DRSF membrane retained significantly higher initial strength and Young's modulus ( ) over the corresponding degradation time. Figure 3 Bio-Gide exhibits rapid mechanical degradation due to the lack of a continuous fibrous structure and its sensitivity to enzymatic cleavage. When normalized to an equal residual mass fraction, DRSF maintains several times the residual strength, confirming that mechanical preservation stems from the integrity of the hierarchical structure, rather than simply mass retention.

[0050] Tear tests showed that the DRSF generated higher peak forces and greater displacements before fracture, while the Bio-Gide failed under significantly lower loads, and the RSF exhibited negligible forces. These results indicate that the DRSF maintains a greater stress redistribution around the suture path and delays crack initiation during edge loading.

[0051] Suture pull-out tests further highlighted these differences; in clinically relevant suture dimensions, DRSF consistently demonstrated higher maximum suture force than Bio-Gide, while RSF failed immediately after loading. Figure 6 The enhanced bridging ability of DRSF stems from its continuous degummed silk fiber skeleton providing an uninterrupted load-bearing path, and the stabilization of interfacial shear transfer through a viscous regenerated silk fibroin matrix. Bio-Gide, composed of a porous collagen scaffold without a continuous fiber arrangement, has limited capacity for early tearing and stress distribution. These differences reflect the different structural origins of resistance to localized stress. Pre-cracked tensile testing revealed further differences in deformation behavior. Intact DRSF exhibited high strength and large strain-to-failure, and even pre-cracked specimens maintained significant load-bearing capacity, indicating that the composite supports bridging across the crack zone and allows for progressive fiber bonding. Figure 6 Bio-Gide exhibited a significant strength decrease after pre-cracking and failed at low strain, consistent with its limited ability to maintain stress redistribution at the crack tip. RSF lacked sufficient structural reinforcement to support the bridge or control deformation in either case. The behavior observed in DRSF conformed to a hierarchical mechanism in which crystalline fibers provide long-range load transfer, while the hydrated matrix provides the interfacial cohesion required to stabilize deformation near the crack surface. This hierarchical structure design makes DRSF films superior to Bio-Gide in mechanical properties. Figure 2 k).

[0052] Verification Example 3: Evaluation of Biocompatibility and Cell Barrier Performance Cell compatibility of DRSF membranes was assessed using L929 fibroblasts and MC3T3-E1 osteoblasts. In Example 1, both cell types maintained high viability on the DRSF membrane at all time points, with values ​​comparable to the commercial control group and consistently higher than Bio-Gide (…). Figure 7 The results (ad) indicate that the membrane supports sustained cell attachment and proliferation, suggesting that its surface chemistry and hydration-mediated matrix cohesion provide a stable environment for cell activity. RSF maintains cell survival but exhibits a slower proliferation rate due to limited structural reinforcement. Hemolysis tests showed that DRSF had negligible erythrocyte lysis, similar to Bio-Gide and RSF, and significantly lower than the levels observed in the positive control. Figure 8 (ab) indicates that the membrane can meet the safety requirements for blood contact during surgery. The spreading and proliferation of MC3T3-E1 cells on DRSF gradually increased, indicating that the composite material surface can provide a good environment for adhesion and proliferation. Figure 7e). Bio-Gide supports less cell spreading, consistent with its softened hydration structure and reduced microstructural continuity. Barrier performance was assessed by fibroblast permeation assays on the asymmetric surface of the DRSF membrane; the dense surface of DRSF prevented cell permeation throughout culture, while the porous surface supported cell attachment but did not allow transmembrane migration. Figure 7 f). Bio-Gide exhibits partial permeability through its hydrated collagen network, consistent with its open porous morphology and limited structural stability. Figure 7 (g) These results confirm that the asymmetric structure of DRSF effectively restricts soft tissue invasion while maintaining the expected bioactive surface. Overall, DRSF provides a stable environment for cell adhesion and proliferation, preserves surfaces favorable for osteoblast adhesion, and maintains a barrier against fibroblast penetration, thereby meeting the biological and structural requirements for guiding bone regeneration.

Claims

1. A bioabsorbable silk membrane for guiding bone regeneration, characterized in that, The whole silk membrane is composed of degummed silk fibers and regenerated silk fibroin. The degummed silk fibers form a continuous fiber network as a fiber skeleton. The regenerated silk fibroin fills the gaps in the fiber network to form a continuous matrix phase. The regenerated silk fibroin is annealed to induce the formation of β-folded crystal regions, which combine with the fiber network through the interface to form a fiber-matrix composite structure. This composite structure is an asymmetric structure with a dense layer and a porous layer.

2. The bioabsorbable silk membrane for guiding bone regeneration as described in claim 1, characterized in that, The fiber network that forms the fiber skeleton creates a continuous mechanical load-bearing path inside the membrane, which is used to bear the main load under suture tension or local stress concentration conditions.

3. The bioabsorbable silk membrane for guiding bone regeneration as described in claim 1, characterized in that, The dense layer forms one side of the silk membrane and is used to block the infiltration of soft tissue cells; the porous layer forms the other side of the silk membrane and is used to promote the adhesion of osteoblast-associated cells.

4. The bioabsorbable silk membrane for guiding bone regeneration as described in claim 1, characterized in that, Annealing is performed with methanol, which induces the formation of β-folded crystal regions that enhance the interfiber bonding and the tear and tensile strength of the membrane.

5. A method for preparing the silk membrane according to any one of claims 1-4, characterized in that, The method includes the following steps: 1) Provide degummed silk fibers and construct a fiber network; 2) Add the regenerated silk fibroin solution and allow it to soak the fiber network to form a composite wet film; 3) The composite wet membrane is freeze-dried to obtain a dry membrane with an asymmetric pore structure; 4) Anneal the dry film to induce the regenerated silk fibroin to form β-sheet crystalline regions and obtain a composite scaffold; 5) A dense layer of silk fibroin is bonded to the pores of the composite scaffold obtained in step 4), and after drying, it is annealed again to obtain a bioabsorbable silk membrane that guides bone regeneration.

6. The method for preparing the silk membrane as described in claim 5, characterized in that, In step 2), the concentration of the regenerated silk fibroin solution is 2-6 w / v%, and the regenerated silk fibroin solution is impregnated into the fiber network by double-sided impregnation or single-sided partial impregnation to form DRSF freeze-dried scaffolds with different component contents.

7. The method for preparing the silk membrane as described in claim 5, characterized in that, In step 3), the freeze-drying process is to form the small and large pore surfaces of the film by unidirectional cooling or interfacial temperature difference control. Specifically, the thermally conductive substrate carrying the composite wet film is first brought into contact with a cold source of -60℃ to -50℃. After the contact surface is frozen, it is then transferred to a cold source of -5℃ to 0℃ to freeze it completely.

8. The use of the silk membrane according to any one of claims 1-7 in the preparation of medical devices for alveolar bone defect repair, bone defect filling or guided bone regeneration surgery.

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