Double-layer artificial periosteum and preparation method thereof

Abstract

This invention provides a double-layer artificial bone membrane and its preparation method. The preparation method includes the following steps: a tissue membrane with a natural rough surface and a smooth surface is sequentially subjected to defatting, virus inactivation, and decellularization to obtain a pretreated tissue membrane and a biomimetic fiber layer; the pretreated tissue membrane is sequentially subjected to freeze-drying and liquid nitrogen grinding to obtain decellularized matrix microparticles; the decellularized matrix microparticles are mixed with a solvent and homogenized under high pressure to obtain a decellularized matrix gel solution; the decellularized matrix gel solution is mixed with cuttlebone powder and magnesium oxide powder to obtain a germinal layer gel solution; the germinal layer gel solution is spread evenly on the rough surface of the biomimetic fiber layer, and sequentially subjected to freeze-drying, vacuum drying, and roller pressing to obtain a double-layer artificial bone membrane. The double-layered artificial periosteum obtained by the above method can achieve physical barrier by utilizing the fibrous layer. Moreover, the magnesium ions released by magnesium oxide in the germinal layer can regulate macrophage polarization, reduce inflammation, and induce angiogenesis. Cuttlebone provides calcium source and active ingredients. The two work together to construct a regenerative microenvironment that can actively regulate inflammation, promote angiogenesis and osteogenic formation, thereby achieving efficient repair of bone defects.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a double-layer artificial bone membrane and its preparation method. Background Technology

[0002] Bone defects caused by trauma, infection, or tumors remain a key challenge in modern medicine, with approximately 5-10% of patients experiencing delayed healing or nonunion, often requiring bone grafting. The osteogenic microenvironment is crucial for bone regeneration, composed of the extracellular matrix (ECM), signaling molecules, and the dynamic interactions of various cells and tissues (such as nerve, blood vessel, and immune cells). Effective tissue engineering requires the development of materials that can assist bone repair and regulate this complex microenvironment to support regeneration. The periosteum, as a connective tissue capsule covering more than 80% of the bone surface, plays a central role in the early stages of bone repair, responsible for over 70% of bone formation. The periosteum is divided into a fibrous layer and a germinal layer. The superficial fibrous layer, composed of interwoven collagen fibers and fewer cells, primarily acts as a physical barrier, maintaining the stability of the inner microenvironment and providing a suitable environment for osteocyte growth, differentiation, and metabolism, indirectly supporting normal bone physiological function. The germinal layer (also known as the osteogenic layer), adjacent to the bone surface, is rich in blood vessels and cells, possessing significant osteogenic capacity and serving as a key functional region for bone growth, repair, and metabolism.

[0003] In the bone repair process, an ideal artificial periosteum should possess dual functions: on the one hand, it should mimic the fibrous layer, acting as a physical barrier to block the invasion of exogenous healing-related cells (such as fibroblasts) from surrounding soft tissues; on the other hand, it should mimic the germinal layer, providing a microenvironment that supports bone regeneration, regulating inflammation, inducing angiogenesis, and promoting new bone formation. However, currently available clinical artificial periosteum are relatively simple in structure and function, making it difficult to simultaneously meet both performance requirements.

[0004] Therefore, there is an urgent need to provide a double-layer artificial bone membrane and its preparation method. Summary of the Invention

[0005] This invention provides a double-layer artificial periosteum and its preparation method, which can solve the problem that traditional artificial periosteum cannot simultaneously provide both physical barrier and osteogenic promotion functions.

[0006] In a first aspect, the present invention provides a method for preparing a double-layered artificial periosteum, the method comprising the following steps: (1) Tissue membranes with natural rough and smooth surfaces are subjected to defatting, virus inactivation and decellularization treatment in sequence to obtain pretreated tissue membranes and biomimetic fiber layers; wherein, the biomimetic fiber layer includes a rough surface and a dense surface; (2) The pretreated tissue membrane was freeze-dried and ground with liquid nitrogen in sequence to obtain decellularized matrix microparticles. The decellularized matrix microparticles were mixed with solvent and homogenized under high pressure to obtain a decellularized matrix gel solution. (3) The decellularized matrix gel solution is mixed with cuttlebone powder and magnesium oxide powder to obtain a germinal layer gel solution; (4) The hair growth layer gel solution is spread on the rough surface of the biomimetic fiber layer, and then freeze-dried, vacuum-dried and rolled in sequence to obtain the double-layer artificial bone membrane.

[0007] Preferably, in step (1), the tissue membrane is peritoneal tissue, pericardial tissue, or submucosal tissue of the small intestine.

[0008] Preferably, in step (1), sodium bicarbonate is used to degrease the tissue membrane; preferably, the mass ratio of sodium bicarbonate to tissue membrane is 1:(0.2~1).

[0009] Preferably, in step (1), the tissue membrane is inactivated by peracetic acid solution.

[0010] Preferably, in step (1), the concentration of the peracetic acid solution is 0.1~0.4wt%, and the treatment time is 0.5~2.0h.

[0011] More preferably, in step (1), the mass-to-volume ratio of peracetic acid solution to tissue membrane is 1:(3~10)g / mL.

[0012] Preferably, in step (1), the tissue membrane is subjected to virus inactivation treatment by sequentially using Triton solution and DNase enzyme solution.

[0013] Preferably, in step (1), the concentration of the Triton solution is 0.1~2wt%, and the treatment time is 2~12h.

[0014] More preferably, in step (1), the concentration of the DNase enzyme solution is 2~50U / mL, and the treatment time is 2~12h.

[0015] Preferably, in step (1), the mass-to-volume ratio of Triton solution and DNase enzyme solution to tissue membrane is 1:(3~10)g / mL.

[0016] Preferably, in step (2), the freeze-drying process includes pre-freezing and vacuum drying; wherein the temperature of the pre-freezing process is -20 to -80°C and the time is 1 to 6 hours; the temperature of the vacuum drying process is -3 to -20°C and the time is 12 to 72 hours.

[0017] Preferably, the solvent is physiological saline or PBS buffer, and the mass concentration of the gel solution is 5-20 wt%.

[0018] Preferably, the particle size of the decellularized matrix microparticles does not exceed 100 μm; the particle size of the cuttlebone powder is 50~500 μm.

[0019] Preferably, in step (3), the mass ratio of decellularized matrix microparticles to cuttlebone powder in the germinal layer gel solution is 1:(0.5~2), and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:(0.01~0.05).

[0020] Preferably, in step (4), the thickness of the hair growth layer gel solution is 1~5mm.

[0021] Preferably, in step (4), the freeze-drying temperature is -20~-80℃ and the time is 1~6h; the vacuum drying temperature is -3~-20℃ and the time is 12~72h.

[0022] Secondly, the present invention also provides a double-layer artificial bone membrane, which is prepared by the method for preparing a double-layer artificial bone membrane as described in any one of the first aspects above.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects: In this invention, a naturally occurring tissue membrane with both a rough and a dense layer is first pretreated to form a fibrous layer. The dense surface effectively blocks soft tissue invasion, acting as a physical barrier, while the rough surface provides a substrate for subsequent bonding. Next, the pretreated tissue membrane is processed using low-temperature grinding and high-pressure homogenization to form a gel solution. This gel solution is then combined with cuttlebone powder (which has hemostatic and osteogenic properties) and magnesium oxide (which has anti-inflammatory and angiogenic properties) to prepare a functionalized germinal layer gel. Finally, the germinal layer gel is coated onto the rough surface of the biomimetic fibrous layer, and the two layers are freeze-dried and rolled to tightly bond them together, forming an integrated bilayer artificial bone membrane. The bilayer artificial bone membrane obtained by the above preparation method can utilize the fibrous layer to achieve physical barrier properties. Furthermore, the magnesium ions released from the magnesium oxide in the germinal layer can regulate macrophage polarization, reduce inflammation, and induce angiogenesis. Cuttlebone provides a calcium source and active ingredients. The two work synergistically to construct a regenerative microenvironment that can actively regulate inflammation, promote angiogenesis, and promote osteogenic processes, thereby achieving highly efficient repair of bone defects. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1This is a scanning electron microscope image of the dense surface of the biomimetic fiber layer provided in Embodiment 1 of the present invention; Figure 2 This is a scanning electron microscope image of the rough surface of the biomimetic fiber layer provided in Embodiment 1 of the present invention; Figure 3 This is a scanning electron microscope image of the dense surface of the biomimetic fiber layer provided in Embodiment 2 of the present invention; Figure 4 This is a scanning electron microscope image of the rough surface of the biomimetic fiber layer provided in Embodiment 2 of the present invention.

[0026] Figure 5 This is a macroscopic view of one side of the biomimetic fiber layer in the double-layer artificial bone membrane provided in Embodiment 1 of the present invention.

[0027] Figure 6 This is a macroscopic view of one side of the germinal layer in the double-layer artificial periosteum provided in Embodiment 1 of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] This invention provides a method for preparing a double-layer artificial periosteum, which includes the following steps: (1) Tissue membranes with natural rough and smooth surfaces are subjected to defatting, virus inactivation and decellularization treatment in sequence to obtain pretreated tissue membranes and biomimetic fiber layers; wherein, the biomimetic fiber layer includes a rough surface and a dense surface; (2) The pretreated tissue membrane was freeze-dried and ground with liquid nitrogen in sequence to obtain decellularized matrix microparticles. The decellularized matrix microparticles were mixed with solvent and homogenized under high pressure to obtain a decellularized matrix gel solution. (3) The decellularized matrix gel solution is mixed with cuttlebone powder and magnesium oxide powder to obtain a germinal layer gel solution; (4) The hair growth layer gel solution is spread on the rough surface of the biomimetic fiber layer, and then freeze-dried, vacuum-dried and rolled in sequence to obtain the double-layer artificial bone membrane.

[0030] In the above-described embodiment, a naturally occurring tissue membrane with a rough and dense layer structure is first pretreated to form a fibrous layer. The dense surface of this fibrous layer effectively blocks soft tissue invasion, acting as a physical barrier, while the rough surface provides a substrate for subsequent bonding. Next, the pretreated tissue membrane is processed using low-temperature grinding and high-pressure homogenization to form a gel solution. This gel solution is then combined with cuttlebone powder (which has hemostatic and osteogenic properties) and magnesium oxide (which has anti-inflammatory and angiogenic properties) to prepare a functionalized germinal layer gel. Finally, the germinal layer gel is coated onto the rough surface of the fibrous layer, and the two layers are freeze-dried and rolled to tightly bond them together, forming an integrated bilayer artificial bone membrane. The bilayer artificial bone membrane obtained by the above preparation method can utilize the fibrous layer to achieve physical barrier properties. Furthermore, the magnesium ions released from the magnesium oxide in the germinal layer can regulate macrophage polarization, reduce inflammation, and induce angiogenesis. Cuttlebone provides a calcium source and active ingredients. The two work synergistically to construct a regenerative microenvironment that can actively regulate inflammation, promote angiogenesis, and promote osteogenic processes, thereby achieving highly efficient repair of bone defects.

[0031] According to some preferred embodiments, in step (1), the tissue membrane is peritoneal tissue, pericardial tissue, or submucosal tissue of the small intestine.

[0032] According to some preferred embodiments, in step (1), sodium bicarbonate is used to degrease the tissue membrane; the mass ratio of sodium bicarbonate to the tissue membrane is 1:(0.2~1) (for example, it can be 1:0.2, 1:0.3, 1:0.5, 1:0.8 or 1:1); peracetic acid solution is used to inactivate the virus in the tissue membrane; the concentration of the peracetic acid solution is 0.1~0.4wt% (for example, it can be 0.1wt%, 0.2wt%, 0.3wt% or 0.4wt%), and the treatment time is 0.5~2.0h (for example, it can be 0.5h, 0.8h, 1.0h, 1.5h, 1.8h or 2.0h); the mass-volume ratio of peracetic acid solution to the tissue membrane is 1:(3~10)g / mL (for example, it can be 1:3g / mL, 1:5g / mL, 1:8g / mL or 1:10g / mL).

[0033] In this embodiment of the invention, fresh peritoneum, pericardium, and submucosal tissue of the small intestine (derived from at least one of human, pig, cattle, sheep, horse, donkey, and monkey) are first gently hand-washed with sodium bicarbonate in a certain proportion to effectively remove fat and serous membrane from the rough surface layer, while preserving the natural fibrous structure and mechanical integrity of the tissue to the greatest extent. Subsequently, the tissue is soaked in peracetic acid solution to efficiently inactivate potential viruses, significantly improving the safety of the biomaterial. Peracetic acid also causes minimal damage to the extracellular matrix, which is beneficial for maintaining the biological activity of the tissue structure. After treatment with sodium bicarbonate and peracetic acid solution, the tissue is washed with physiological saline or PBS buffer to remove residual sodium bicarbonate and peracetic acid.

[0034] According to some preferred embodiments, in step (1), the tissue membrane is subjected to virus inactivation treatment sequentially using Triton solution and DNase enzyme solution; the concentration of Triton solution is 0.1~2wt% (e.g., 0.1wt%, 0.5wt%, 0.8wt%, 1.0wt%, 1.2wt%, 1.5wt%, 1.8wt%, or 2wt%), and the treatment time is 2~12h (e.g., 2h, 5h, 8h, 10h, or 12h); the concentration of DNase enzyme solution is 2~50U / mL (e.g., ...). The concentrations are 2 U / mL, 5 U / mL, 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 35 U / mL, 40 U / mL, 45 U / mL, or 50 U / mL, and the treatment time is 2 to 12 hours (e.g., 2 hours, 5 hours, 8 hours, 10 hours, or 12 hours). The mass-to-volume ratio of Triton solution and DNase enzyme solution to tissue membrane is 1:(3 to 10) g / mL (e.g., 1:3 g / mL, 1:5 g / mL, 1:8 g / mL, or 1:10 g / mL).

[0035] In this embodiment of the invention, after virus inactivation treatment, the tissue membrane undergoes a stepwise decellularization process. First, the tissue membrane is shaken with Triton solution to efficiently dissolve and remove cell membranes and intracellular components, while preserving the natural structure and fiber network of the extracellular matrix. Subsequently, DNase enzyme solution is used for further shaking treatment to thoroughly remove nucleic acid substances that may trigger an immune response. Through the above distribution treatment, deep decellularization is achieved while maximizing the protection of the integrity and biomechanical properties of the extracellular matrix, which is beneficial to ensuring good biocompatibility of the biomimetic fiber layer.

[0036] According to some preferred embodiments, in step (2), the freeze-drying process includes a pre-freezing process and a vacuum drying process; wherein, the temperature of the pre-freezing process is -20~-80℃ (for example, it can be -20℃, -50℃ or -80℃), and the time is 1~6h (for example, it can be 1h, 3h, 5h or 6h); the temperature of the vacuum drying process is -3~-20℃ (for example, it can be -3℃, -5℃, -10℃, -15℃ or -20℃), and the time is 12~72h (for example, it can be 12h, 24h, 36h or 72h); the solvent is physiological saline or PBS buffer, and the mass concentration of the gel solution is 5~20wt% (for example, it can be 5wt%, 10wt%, 15wt% or 20wt%).

[0037] According to some preferred embodiments, the particle size of the decellularized matrix microparticles does not exceed 100 μm; the particle size of the cuttlebone powder is 50~500 μm (for example, it can be 50 μm, 100 μm, 150 μm, 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm or 500 μm).

[0038] In this embodiment of the invention, the pretreated tissue membrane is freeze-dried, then ground using a liquid nitrogen grinder, and sieved using a vibrating sieve to obtain decellularized matrix microparticles with a particle size ≤100µm. Decellularized matrix microparticles of this particle size not only help maintain the biological activity and structural integrity of the cell matrix, but also facilitate uniform dispersion with other components. Then, physiological saline or PBS buffer is added, and the mixture is homogenized using a high-pressure homogenizer to form a gel solution of a certain concentration. This treatment method not only avoids the damage to the decellularized matrix caused by traditional enzymatic extraction processes, but also ensures the biological activity of the natural decellularized matrix.

[0039] According to some preferred embodiments, in step (3), the mass ratio of decellularized matrix microparticles to cuttlebone powder in the germinal layer gel solution is 1:(0.5~2) (for example, it can be 1:0.5, 1:0.8, 1:1, 1:1.5 or 1:2), and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:(0.01~0.05) (for example, it can be 1:0.01, 1:0.02, 1:0.03, 1:0.04 or 1:0.05).

[0040] In this embodiment of the invention, pharmaceutical-grade cuttlebone is pulverized and sieved to obtain powder with a particle size of 50µm to 500µm. This particle size range ensures a large specific surface area to facilitate the release of active ingredients while allowing for uniform dispersion in the gel and maintaining a suitable viscosity. Subsequently, the cuttlebone powder, magnesium oxide powder, and decellularized matrix gel solution are mixed in a specific ratio to form a functionalized germinal layer gel solution. The decellularized matrix gel serves as a three-dimensional scaffold and functional carrier, providing a biomimetic adhesion environment for cells and ensuring stable loading and controllable release of active ingredients. Cuttlebone primarily acts as a biological calcium source and structural guiding matrix, directly promoting osteogenic formation. Magnesium oxide optimizes the repair microenvironment by regulating immune responses and promoting angiogenesis. Through multi-level synergistic effects, these three components jointly accelerate the repair and regeneration of bone defects. Furthermore, precise control of the cuttlebone and magnesium oxide content can optimize repair performance. Experiments have shown that if the cuttlebone powder content is too low, the calcium source and bioactivity provided are insufficient, which is not conducive to the rapid formation of callus; if the content is too high, it is easy to aggregate in the gel and detach due to weak binding after combining with the tissue membrane. Similarly, if the magnesium oxide content is too high, the rapidly released magnesium ions may produce certain cytotoxicity; if the content is too low, it is insufficient to effectively regulate the inflammatory microenvironment and induce sufficient angiogenesis.

[0041] According to some preferred embodiments, in step (4), the thickness of the hair growth layer gel solution is 1~5mm (e.g., it can be 1mm, 2mm, 3mm, 4mm or 5mm); the freeze-drying temperature is -20~-80℃ (e.g., it can be -20℃, -50℃ or -80℃), and the time is 1~6h (e.g., it can be 1h, 2h, 3h, 4h, 5h or 6h); the vacuum drying temperature is -3~-20℃ (e.g., it can be -3℃, -5℃, 10℃, -15℃ or -20℃), and the time is 12~72h (e.g., it can be 12h, 24h, 36h or 72h).

[0042] In this embodiment of the invention, the rough surface of the biomimetic fiber layer is laid flat in a freeze-drying tray, and the prepared germinal layer gel solution is spread flat on the rough surface, ensuring a thickness of 1-5 mm. Subsequently, freeze-drying and vacuum drying are performed sequentially to allow the two layers to initially bond and solidify. Finally, roller pressing further strengthens the interlayer bonding force, thereby obtaining an integrated double-layer artificial bone membrane. This composite method fully utilizes the anchoring effect of the rough surface of the biomimetic fiber layer, combined with the physical densification effect of roller pressing, to form a stable and tight bond between the biomimetic fiber layer and the germinal layer gel solution. Thus, in the final double-layer artificial bone membrane, the dense surface of the biomimetic fiber layer effectively acts as a barrier, while the germinal layer provides a microenvironment that regulates inflammation, promotes angiogenesis and osteogenic activity. The two layers complement each other structurally and functionally, jointly promoting the efficient repair of bone defects.

[0043] This invention also provides a double-layer artificial bone membrane, prepared using the preparation method described in any one of the first aspects above.

[0044] To more clearly illustrate the technical solution and advantages of the present invention, the following describes in detail a double-layer artificial bone membrane and its preparation method through several embodiments.

[0045] Example 1: (1) Take fresh porcine peritoneum tissue, add sodium bicarbonate at a ratio of 1:0.5, manually rub the rough layer to remove surface fat and serous membrane, and wash with physiological saline to remove sodium bicarbonate; then add the treated tissue to a 0.2wt% peracetic acid solution at a ratio of 1:5 g / mL and soak for 0.5 h, and wash with physiological saline to remove peracetic acid; add the treated tissue to a 1wt% Triton solution at a ratio of 1:5 g / mL and shake for 4 h, and wash with physiological saline to remove Triton; then add to a 40U / mL DNase enzyme solution at a ratio of 1:5 g / mL, shake for 6 h to completely remove cell components, and wash with physiological saline to remove DNase enzyme, to obtain pretreated tissue membrane and biomimetic fiber layer; (2) The pretreated tissue membrane was freeze-dried at -20℃ for 3h, then vacuum-dried at -3℃ for 24h. The dried tissue membrane was then ground with liquid nitrogen and sieved with a vibrating screen to obtain decellularized matrix microparticles with a particle size ≤100µm. The decellularized matrix microparticles were then mixed with physiological saline in a certain proportion to form a suspension with a mass fraction of 10%. Subsequently, the suspension was homogenized and pulverized with a high-pressure homogenizer to form a decellularized matrix gel solution. (3) Mix the decellularized matrix gel solution, cuttlebone powder (particle size 50~500μm) and magnesium oxide powder to obtain a germinal layer gel solution; wherein, in the germinal layer gel solution, the mass ratio of decellularized matrix microparticles to cuttlebone powder is 1:1, and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:0.02; (4) Spread the hair growth layer gel solution on the rough surface of the biomimetic fiber layer with a thickness of 3 mm. Then freeze-dry it at -20℃ for 6 h, vacuum dry it at -3℃ for 48 h, and finally roll it (pressure 3 MPa, holding time 60 s) to obtain a double-layer artificial bone membrane.

[0046] Example 2: (1) Take fresh bovine pericardium tissue, add sodium bicarbonate at a ratio of 1:0.5, manually rub the rough layer to remove surface fat and serous membrane, and wash with physiological saline to remove sodium bicarbonate; then add the treated tissue to a 0.3wt% peracetic acid solution at a ratio of 1:5 g / mL and soak for 1 h, and wash with physiological saline to remove peracetic acid; add the treated tissue to a 2wt% Triton solution at a ratio of 1:5 g / mL and shake for 6 h, and wash with physiological saline to remove Triton; then add to a 50U / mL DNase enzyme solution at a ratio of 1:5 g / mL, shake for 12 h to completely remove cell components, and wash with physiological saline to remove DNase enzyme, to obtain pretreated tissue membrane and biomimetic fiber layer; (2) The pretreated tissue membrane was freeze-dried at -30℃ for 4h, then vacuum-dried at -3℃ for 50h, and the dried tissue membrane was ground with liquid nitrogen grinder and sieved with vibrating screen to obtain decellularized matrix microparticles with a particle size ≤100µm. The decellularized matrix microparticles were mixed with physiological saline in a certain proportion to form a suspension with a mass fraction of 15%, and then homogenized and pulverized with high pressure homogenizer to form a decellularized matrix gel solution. (3) Mix the decellularized matrix gel solution, cuttlebone powder (particle size 50~500μm) and magnesium oxide powder to obtain a germinal layer gel solution; wherein, in the germinal layer gel solution, the mass ratio of decellularized matrix microparticles to cuttlebone powder is 1:2, and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:0.02; (4) Spread the hair growth layer gel solution on the rough surface of the biomimetic fiber layer with a thickness of 4 mm. Then freeze-dry it at -30℃ for 4 h, then vacuum dry it at -3℃ for 48 h, and finally roll-press it (pressure 3 MPa, holding time 60 s) to obtain a double-layer artificial bone membrane.

[0047] Example 3: (1) Take fresh submucosal tissue of pig small intestine, add sodium bicarbonate at a ratio of 1:0.2, manually rub the rough layer to remove surface fat and serous membrane, and wash with physiological saline to remove sodium bicarbonate; then add the treated tissue to a 0.1wt% peracetic acid solution at a ratio of 1:3 g / mL and soak for 0.5 h, and wash with physiological saline to remove peracetic acid; add the treated tissue to a 0.1wt% Triton solution at a ratio of 1:3 g / mL and shake for 2 h, and wash with physiological saline to remove Triton; then add to a 10U / mL DNase enzyme solution at a ratio of 1:3 g / mL, shake for 3 h to completely remove cellular components, and wash with physiological saline to remove DNase enzyme, to obtain pretreated tissue membrane and biomimetic fiber layer; (2) The pretreated tissue membrane was freeze-dried at -20℃ for 2h, and then vacuum-dried at -10℃ for 24h. The dried tissue membrane was then ground with liquid nitrogen and sieved with a vibrating screen to obtain decellularized matrix microparticles with a particle size ≤100µm. The decellularized matrix microparticles were then mixed with physiological saline in a certain proportion to form a suspension with a mass fraction of 20%. Subsequently, the suspension was homogenized and pulverized with a high-pressure homogenizer to form a decellularized matrix gel solution. (3) Mix the decellularized matrix gel solution, cuttlebone powder (particle size 50~500μm) and magnesium oxide powder to obtain a germinal layer gel solution; wherein, in the germinal layer gel solution, the mass ratio of decellularized matrix microparticles to cuttlebone powder is 1:2, and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:0.02; (4) Spread the hair growth layer gel solution on the rough surface of the biomimetic fiber layer with a thickness of 2 mm. Then freeze-dry it at -20℃ for 2 h, then vacuum dry it at -10℃ for 24 h, and finally roll-press it (pressure 3 MPa, holding time 60 s) to obtain a double-layer artificial bone membrane.

[0048] Example 4: Example 4 is basically the same as Example 1, except that in step (3), the mass ratio of decellularized matrix microparticles to cuttlebone powder in the germinal layer gel solution is 1:3.

[0049] Example 5: Example 5 is basically the same as Example 1, except that in step (3), the mass ratio of cuttlebone powder to magnesium oxide powder in the germinal layer gel solution is 1:0.1.

[0050] Comparative Example 1: Comparative Example 1 is basically the same as Example 1, except that in step (3), the decellularized matrix gel solution and cuttlebone powder (particle size of 50~500μm) are mixed to obtain a germinal layer gel solution; wherein, in the germinal layer gel solution, the mass ratio of decellularized matrix microparticles to cuttlebone powder is 1:1.

[0051] Comparative Example 2: Comparative Example 2 is basically the same as Example 1, except that in step (3), the decellularized matrix gel solution and magnesium oxide powder are mixed to obtain the germinal layer gel solution; wherein the mass ratio of decellularized matrix microparticles to magnesium oxide powder is 1:1.

[0052] The double-layer artificial bone membranes prepared in the examples and comparative examples were tested, and the test results are shown in Table 1. Among them, the DNA residue was tested according to the method specified in YY / T 1876-2023 "Tissue-engineered medical products - Part 25 Determination of DNA residue in animal-derived biomaterials: Fluorescent staining method".

[0053] Cytotoxicity assay: The extraction solution was prepared according to the method specified in GB / T 16886.12-2023 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials". Complete culture medium (89 vol% high glucose medium / 10 vol% fetal bovine serum / 1 vol% penicillin-dextrose antibody) was used as the extraction medium. After saturation with the extraction medium, the solution was extracted at a ratio of 3 cm⁻¹. 2 Extraction was performed at 1 × 10⁹ mL / mL, and the extraction time was 37 ± 1 °C for 72 ± 2 h. Mouse fibroblasts (L-929) were extracted at 1 × 10⁹ mL / mL. 4Cells were seeded per well in 96-well plates and cultured for 24 h. The culture medium was removed, and the cells were cultured for another 24 h using extraction buffer. Cytotoxicity was then performed according to the instructions of the CCK-8 kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0054] Alkaline phosphatase (ALP) assay: The prepared artificial bone membrane was cut into uniformly sized thin slices (8 mm in diameter) and placed in 6-well cell culture plates. Bone marrow mesenchymal stem cells (rBMSCs) were cultured at a density of 5 × 10⁻⁶ m² / g. 4 rBMSCs were seeded onto each well of a gel and, after 24 h of cell adhesion, were simultaneously cultured in basal low-glucose medium and osteogenic differentiation medium (5 mmol / L). -1 β-glycerophosphate sodium, 100 nmol / L -1 Dexamethasone, 50 μmol / L -1 (Ascorbic acid) Continue culturing for 14 days and test ALP activity.

[0055] Vascular endothelial growth factor (VEGF) expression assay: The extraction solution was prepared according to the method specified in GB / T 16886.12-2023 "Biological Evaluation of Medical Devices Part 12: Sample Preparation and Reference Materials". Complete culture medium (89 vol% high glucose medium / 10 vol% fetal bovine serum / 1 vol% penicillin-antibody) was used as the extraction medium. After the medium was saturated, the solution was extracted at a ratio of 3 cm⁻¹. 2 Extraction was performed at 37±1℃ for 72±2 h at a concentration of 2×10⁹ mL. 5 Endothelial cells (HUVECs) were seeded into 6-well culture plates and cultured for 24 hours. The cell culture medium was removed, and the cells were cultured with extraction buffer. After 24 hours of culture, the cell supernatant was collected, and the VEGF content was detected according to the instructions of the vascular endothelial growth factor (VEGF) ELISA kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0056] Table 1 Example 1 13.5±2.4 104±3.8 0.41±0.06 1660±136 Good morphology, no particle shedding Example 2 19.5±0.9 103±2.7 0.43±0.07 1557±127 Good morphology, no particle shedding Example 3 7.5±0.4 101±3.5 0.45±0.05 1697±174 Good morphology, no particle shedding Example 4 12.4±3.7 91±3.2 0.39±0.07 1586±159 Good morphology, with some particles falling off. Example 5 13.9±2.6 78±3.6 0.26±0.04 1010±138 Good morphology, no particle shedding Comparative Example 1 14.5±2.8 101±3.9 0.39±0.06 871±89 Good morphology, no particle shedding Comparative Example 2 11.8±3.2 98±2.5 0.30±0.04 1436±184 Good morphology, no particle shedding As shown in Table 1, the decellularized porcine peritoneum, pericardium, and small intestinal mucosa treated in Examples 1, 2, and 3 all effectively controlled the DNA content to below 50 ng / mg, meeting the requirement of less than 50 ng / mg for residual DNA in animal-derived medical devices. This proves that the method in this example can effectively remove residual DNA, and the biomimetic fiber layer has low immunogenicity. Compared with Comparative Examples 1-2, the introduction of cuttlebone and magnesium oxide in Examples 1-3 can effectively promote osteogenic and angiogenesis properties. As shown in Table 1, the periosteum prepared in Examples 1-3 showed no particle shedding, exhibiting good cell safety and osteogenic and angiogenesis properties. Compared with Examples 4 and 5, excessively high contents of cuttlebone and magnesium oxide can lead to problems such as poor morphology, shedding, and decreased cell viability. Furthermore, by Figures 1 to 4 As shown, the biomimetic fiber layers prepared in Examples 1 and 2 both possess a bifacial anisotropic structure with dense and rough surfaces. The dense layer facilitates barrier isolation from exogenous healing invasions, while the rough fiber structure promotes tight bonding with the biomimetic regrowth layer. Figure 5 , 6 As shown, the double-layer artificial bone membrane prepared in Example 1, after being rolled, allows the biomimetic fiber layer and the germinal layer to be tightly connected and form a whole, playing different roles in the tissue repair process.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a double-layered artificial periosteum, characterized by, The preparation method includes the following steps: (1) Tissue membranes with natural rough and smooth surfaces are subjected to defatting, virus inactivation and decellularization treatment in sequence to obtain pretreated tissue membranes and biomimetic fiber layers; wherein, the biomimetic fiber layer includes a rough surface and a dense surface; (2) The pretreated tissue membrane was freeze-dried and ground with liquid nitrogen in sequence to obtain decellularized matrix microparticles. The decellularized matrix microparticles were mixed with solvent and homogenized under high pressure to obtain a decellularized matrix gel solution. (3) The decellularized matrix gel solution is mixed with cuttlebone powder and magnesium oxide powder to obtain a germinal layer gel solution; (4) The hair growth layer gel solution is spread on the rough surface of the biomimetic fiber layer, and then freeze-dried, vacuum-dried and rolled in sequence to obtain the double-layer artificial bone membrane.

2. The production method according to claim 1, characterized by, In step (1), the tissue membrane is peritoneal tissue, pericardial tissue, or submucosal tissue of the small intestine.

3. The preparation method according to claim 1, characterized in that, In step (1), sodium bicarbonate is used to degrease the tissue membrane; preferably, the mass ratio of sodium bicarbonate to tissue membrane is 1:(0.2~1).

4. The method of claim 1, wherein, In step (1), peracetic acid solution is used to inactivate the virus in the tissue membrane; Preferably, the concentration of the peracetic acid solution is 0.1~0.4wt%, and the treatment time is 0.5~2.0h; More preferably, the mass-to-volume ratio of peracetic acid solution to tissue membrane is 1:(3~10)g / mL.

5. The preparation method according to claim 1, characterized in that, In step (1), the tissue membrane was subjected to virus inactivation treatment by Triton solution and DNase enzyme solution in sequence; Preferably, the concentration of the Triton solution is 0.1~2wt%, and the treatment time is 2~12h; More preferably, the concentration of the DNase enzyme solution is 2-50 U / mL, and the treatment time is 2-12 h; and / or The mass-to-volume ratio of Triton solution and DNase enzyme solution to tissue membrane was 1:(3~10) g / mL.

6. The preparation method according to claim 1, characterized in that, In step (2), the freeze-drying process includes pre-freezing and vacuum drying; wherein the temperature of the pre-freezing process is -20 to -80°C and the time is 1 to 6 hours; the temperature of the vacuum drying process is -3 to -20°C and the time is 12 to 72 hours. and / or The solvent is physiological saline or PBS buffer, and the mass concentration of the gel solution is 5-20 wt%.

7. The preparation method according to claim 1, characterized in that, The particle size of the decellularized matrix microparticles does not exceed 100 μm; the particle size of the cuttlebone powder is 50~500 μm.

8. The method of claim 1, wherein, In step (3), the mass ratio of decellularized matrix microparticles to cuttlebone powder in the germinal layer gel solution is 1:(0.5~2), and the mass ratio of cuttlebone powder to magnesium oxide powder is 1:(0.01~0.05).

9. The method of claim 1, wherein, In step (4), the thickness of the hair growth layer gel solution is 1~5 mm; and / or The freeze-drying temperature is -20 to -80°C, and the time is 1 to 6 hours; the vacuum drying temperature is -3 to -20°C, and the time is 12 to 72 hours.

10. A bilayered artificial dura mater characterized by, It is prepared by any one of the preparation methods according to claims 1 to 9.

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