Multi-layer biological barrier membrane with antibacterial and osteogenesis promoting functions as well as preparation method and application of multi-layer biological barrier membrane

Abstract

The invention provides a multilayer biological barrier membrane with antibacterial and osteogenesis promoting functions as well as a preparation method and application thereof. The multilayer biological barrier membrane comprises a barrier antibacterial layer with a nanofiber membrane structure as an outer layer, a micron-sized criss-cross support as a mechanical property layer as a middle layer and an inner bone induction layer with a porous degradable structure. Wherein the barrier antibacterial layer is an electrostatic spinning film, comprises a photocurable natural polymer matrix, a thermoplastic polyester material and an antibacterial functional metal component, and plays an antibacterial function; the mechanical property layer is prepared from a 3D printing thermoplastic polyester material and provides tensile mechanical strength and a supporting effect; the bone induction layer is composed of a photocurable natural polymer matrix doped with bioactive nanoparticles and promotes growth of bone tissue. The multilayer biological barrier membrane provided by the invention has antibacterial and osteogenesis promoting functions, and has excellent mechanical strength, thereby promoting bone tissue regeneration and repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and particularly relates to a multilayer biological barrier membrane with antibacterial and osteogenic properties, its preparation method, and its application. Background Technology

[0002] Periodontal disease is an inflammatory condition that can lead to tooth loss, damaging the tissues that support the teeth. The ultimate goal of periodontal treatment is to protect and maintain tooth health by restoring the original morphology, structure, and function of the damaged periodontal supporting tissues. Alveolar bone repair is one of the most important aspects of this treatment. Guided bone regeneration (GBR) has been widely used in the treatment of periodontal bone defects, and bio-barrier membranes are commonly used therapeutic materials. Bio-barrier membrane materials can effectively prevent fibrous connective tissue from entering the defect area while creating a space conducive to the growth of blood vessels and osteoblasts, thereby promoting the regeneration of damaged tissue. However, the application of biodegradable membranes also has some drawbacks, such as weak mechanical strength, insufficient duration of action, and insufficient bone-inducing activity. For example, the clinically commonly used Bio-Gide barrier membrane material is less stable in terms of space maintenance compared to non-absorbable barrier membranes, which may lead to membrane collapse, thereby reducing the protective effect of the barrier membrane and affecting tissue growth and treatment outcomes. Therefore, the development of novel bio-barrier membrane materials with degradability, space maintenance ability, and bone-inducing activity is of significant research value.

[0003] Biological barrier membranes are often designed with a bilayer structure, with a loose inner layer to induce angiogenesis and bone tissue formation, and a dense outer layer to block external cells from invading the bone defect area. Electrospinning technology can prepare barrier membrane materials that guide bone regeneration, similar to ECMs. However, bilayer fibrous membranes prepared by electrospinning may suffer from unstable mechanical properties and susceptibility to external influences. The unstable mechanical properties of the barrier membrane material may lead to insufficient space maintenance, potentially causing the membrane to collapse during treatment, thereby reducing its protective effect and affecting tissue growth and treatment outcomes. 3D printing technology can use various materials to manufacture barrier membranes in different forms, thus producing customized materials suitable for individual patient conditions. Researchers have used electrostatic direct-write 3D printing and electrospinning techniques to prepare a barrier membrane material with a bilayer structure, including a loose and porous electrostatic direct-write 3D-printed layer to support and promote bone tissue growth, and a dense and solid electrospun layer to resist interference from non-osteoblasts (Acta Biomaterialia, 2020, 117: 83-99). The barrier membrane with this double-layer structure exhibits enhanced mechanical properties, but the two layers are mainly physically stacked together, making them prone to separation. Meanwhile, the antibacterial and osteogenic functions produced by releasing copper ions through copper-doped mesoporous silicon nanoparticles are not very effective (the antibacterial rate does not exceed 60% in 24 hours). Summary of the Invention

[0004] This invention provides a multilayered biological barrier membrane with antibacterial and osteogenic properties, its preparation method, and its application. It is a multilayered biological barrier membrane that can achieve barrier and antibacterial effects from the outside to the inside, as well as good space maintenance performance, osteoinductive activity, good mechanical strength, and structural integrity. It is expected to become an ideal alveolar bone repair material.

[0005] To achieve the above and other related objectives, the first aspect of the present invention provides a multilayer biobarrier membrane with antibacterial and osteogenic properties, which is composed of three layers: an outer layer is a barrier antibacterial layer X with a nanofiber membrane structure, which is composed of an electrospun membrane doped with antibacterial functional metal components; a middle layer is a micron-scale crisscross scaffold as a mechanical property layer Y, which is composed of N layers of 3D printed microfiber scaffolds; and an inner bone-inducing layer Z with a porous and biodegradable structure, which is composed of a porous matrix doped with bioactive nanoparticles.

[0006] A method for preparing a multilayer biological barrier membrane includes the following steps: Step S1: Prepare the slurry for electrospinning; Step S2: Using an electrospinning machine, prepare an electrospun membrane from the slurry obtained in step S1 according to a specified procedure; obtain the barrier antibacterial layer X; Step S3: Using a 3D printer, melt extrusion printing of polyester material is performed according to a specified program, with the barrier antibacterial layer X as the receiving platform; mechanical property layer Y is obtained; Step S4: The melt-printed thermoplastic polyester material is bonded to the barrier antibacterial layer X, which serves as a receiving platform, at high temperature to form a double-layer structure XY; Step S5: Disperse the bioactive nanoparticles evenly in water to obtain a suspension, dissolve the photocurable natural polymer matrix in the suspension to obtain the desired slurry; pour the slurry onto the bilayer structure XY, and irradiate with ultraviolet light to crosslink the bone-inducing layer Z and the barrier antibacterial layer X to form a trilayer structure XYZ.

[0007] Application of a multilayered bio-barrier membrane with antibacterial and osteogenic properties in bone defect repair.

[0008] As described above, the multilayer biological barrier membrane with antibacterial and osteogenic properties, its preparation method, and its application provided by the present invention have the following beneficial effects: (1) The present invention provides a multi-layered bio-barrier membrane with antibacterial and osteogenic properties, its preparation method and application. The multi-layered structure provides antibacterial, support and bone repair effects respectively. The multi-functional biodegradable barrier membrane is prepared by electrospinning technology, 3D printing technology and photocrosslinking. Based on the above barrier membrane, it is expected to obtain better alveolar bone repair effect.

[0009] (2) The present invention provides a multilayer biological barrier membrane with antibacterial and osteogenic properties, its preparation method and application, by dissolving natural polymer matrix and antibacterial components in an organic solvent and mixing them evenly to obtain a barrier antibacterial layer slurry; and by using electrospinning technology to prepare the above slurry into a membrane as a barrier antibacterial layer; the barrier antibacterial layer has good fibroblast compatibility during co-culture with cells and has a broad-spectrum antibacterial effect against bacteria.

[0010] (3) The present invention provides a multilayer biological barrier membrane with antibacterial and osteogenic properties, its preparation method and application, wherein thermoplastic polyester material is heated and dissolved in a 3D printing barrel to obtain a mechanical property layer slurry; and the above slurry is prepared into a scaffold using the above barrier antibacterial layer as a receiving platform by melt extrusion 3D printing technology, which serves as the mechanical property layer; the addition of the above mechanical property layer can effectively improve the tensile mechanical strength of the barrier membrane, and can effectively improve the structural stability of the barrier membrane during alveolar bone repair.

[0011] (4) The present invention provides a multilayer biological barrier membrane with antibacterial and osteogenic properties, its preparation method, and its application. A polymer matrix and osteogenic components are dissolved in water and mixed uniformly to obtain a bone-inducing layer slurry. This slurry is then infused onto a double-layer membrane consisting of a barrier antibacterial layer and a mechanical property layer, and photocrosslinked to form the desired bone-inducing layer of the biological barrier membrane. The three-layer structure can be stably bonded together through photocrosslinking. The aforementioned bone-inducing layer exhibits good compatibility with bone marrow mesenchymal stem cells during co-culture with cells and significantly promotes osteogenic differentiation of stem cells. Attached Figure Description

[0012] Figure 1 Microscopic image of a three-layer bio-barrier scaffold material.

[0013] Figure 2 The image shows the cell viability results of a three-layer composite scaffold doped with zinc oxide nanoparticles of different concentrations.

[0014] Figure 3 The image shows the cell viability results of three-layer composite scaffolds doped with different concentrations of lithium magnesium silicate.

[0015] Figure 4 The figure shows the alkaline phosphatase activity results of bone marrow mesenchymal stem cells cultured for 7 days on a three-layer composite scaffold doped with different concentrations of magnesium lithium silicate.

[0016] Figure 5 Figure showing the antibacterial performance of three-layer composite scaffolds doped with different concentrations of zinc oxide nanoparticles.

[0017] Figure 6 The images show the results of imaging examinations of composite scaffolds with different components in the repair of alveolar bone defects in rats.

[0018] Figure 7 Histological staining results of composite scaffolds with different components in the repair of alveolar bone defects in rats. Detailed Implementation

[0019] The first aspect of this invention provides a multilayer biobarrier membrane with antibacterial and osteogenic properties, comprising an outer barrier antibacterial layer with a nanofiber membrane structure; a middle layer with a micron-scale crisscrossing scaffold as a mechanical property layer; and an inner bone-inducing layer with a porous and biodegradable structure. The barrier antibacterial layer is fabricated by electrospinning, the mechanical property layer by 3D printing, and the bone-inducing layer by casting.

[0020] Figure 1 These are microscopic images of a three-layered bio-barrier scaffold material. Image a is a scanning electron microscope (SEM) image of the scaffold's antibacterial barrier layer; image b is a SEM image of the double-layered scaffold after the antibacterial barrier layer and the mechanical performance layer are combined; and image c is a SEM image of the three-layered composite scaffold.

[0021] A second aspect of this invention provides a method for preparing a multilayer bio-induced barrier membrane, comprising the following steps: 1) Dissolve the natural polymer matrix and antibacterial components in an organic solvent, mix them evenly to obtain a barrier antibacterial layer slurry; and use electrospinning technology to prepare the above slurry into a film as a barrier antibacterial layer; 2) Thermoplastic polyester material is heated and melted in a 3D printing barrel to obtain a mechanical property layer slurry; and the above slurry is prepared into a scaffold using the above barrier antibacterial layer as a receiving platform by melt extrusion 3D printing technology, which serves as the mechanical property layer; 3) Dissolve the natural polymer matrix and osteogenic components in water and mix them evenly to obtain a bone-inducing layer slurry; pour the above slurry onto the double membrane of the barrier antibacterial layer and the mechanical property layer, and perform photocrosslinking to form the bone-inducing layer of the desired biological barrier membrane.

[0022] A third aspect of the present invention provides the application of the above-mentioned multilayer biological barrier membrane with antibacterial and osteogenic properties in bone defect repair.

[0023] The multilayer bio-barrier membrane of the present invention is a multifunctional oral repair tissue engineering scaffold, or it can be an integrated antibacterial and repair barrier membrane.

[0024] The multilayer biological barrier membrane of the present invention has a core-skin structure, wherein the barrier antibacterial layer and the bone-inducing layer encapsulate the mechanical property layer, forming a sandwich-like structure.

[0025] In the scaffold of the multilayer biobarrier membrane of the present invention, the thermoplastic polyester material includes, but is not limited to, polylactic acid (PLA) (CAS No. 26680-10-4), polycaprolactone (PCL) (CAS No. 24980-41-4), thermoplastic polyurethane (PU) (CAS No. 51852-81-4), polyetheretherketone (PEEK) (CAS No. 29658-26-2), etc.

[0026] In one specific embodiment, the thermoplastic polyester material is selected from at least one of polylactic acid, polycaprolactone, thermoplastic polyurethane, and polyetheretherketone.

[0027] Preferably, the thermoplastic polyester material is polycaprolactone.

[0028] The multilayer bio-barrier membrane of the present invention includes, but is not limited to, hyaluronic acid (CAS No. 9004-61-9), silk fibroin (CAS No. 96690-41-4), methacrylamide gelatin (GelMA), methacrylamide hyaluronic acid (HAMA), sodium alginate (CAS No. 9005-38-3), methacrylamide silk fibroin (SilMA), etc.

[0029] In one specific embodiment, the polymer matrix is ​​selected from at least one of hyaluronic acid, silk fibroin, methacrylamide gelatin, methacrylamide hyaluronic acid, sodium alginate, and methacrylamide silk fibroin.

[0030] Preferably, the polymer matrix is ​​methacrylamide gelatin.

[0031] The multilayer bio-barrier membrane of the present invention includes, but is not limited to, nano-clay (lithium saponite / lap) (CAS No. 37220-90-9), bioactive glass nanoparticles, hydroxyapatite nanoparticles (CAS No. 1306-06-5), etc.

[0032] In one specific embodiment, the nano-clay is selected from at least one of lithium magnesium silicate, aluminum magnesium silicate, halloysite, and montmorillonite.

[0033] In a further embodiment, the nano-clay is lithium magnesium silicate (lithium saponite) (CAS No. 37220-90-9).

[0034] The multilayer biological barrier membrane of the present invention includes, but is not limited to, zinc oxide (CAS No. 1314-13-2), magnesium oxide (CAS No. 1309-48-4), copper oxide (CAS No. 1317-38-0), etc., as its antibacterial functional metal components.

[0035] In one specific embodiment, the antibacterial functional metal component is selected from at least one of zinc oxide, magnesium oxide, and copper oxide.

[0036] Preferably, the antibacterial functional metal component is nano-zinc oxide particles.

[0037] The multilayer biological barrier membrane of the present invention comprises a barrier antibacterial layer in which zinc oxide nanoparticles are uniformly distributed, and a bone-inducing layer in which lithium magnesium silicate is uniformly distributed. This results in a multilayer biological barrier membrane with an inner layer that forms bone tissue and an outer layer that is antibacterial.

[0038] A second aspect of the present invention provides a method for preparing a multilayer bio-induced barrier membrane with antibacterial and osteogenic properties, comprising the following steps: 1) The preparation of the barrier antibacterial layer slurry is selected from any of the following steps: 11) Dissolve thermoplastic polyester material and natural polymer matrix in hexafluoroisopropanol (HFIP), mix evenly, and obtain a barrier antibacterial layer slurry; 12) Dissolve thermoplastic polyester material and natural polymer matrix in hexafluoroisopropanol, mix evenly to obtain a barrier antibacterial layer mixed solution, add ultrasonically dispersed nano metal oxide particles, resuspend to form a suspension, and obtain a barrier antibacterial layer slurry. 2) The preparation of the bone-inducing layer slurry is selected from any of the following steps: 21) The natural polymer matrix is ​​dissolved in water by heating and mixed evenly to obtain the bone induction layer slurry; 22) The natural polymer matrix is ​​heated and dissolved in water, mixed evenly to obtain a bone-inducing layer mixed solution, and lithium saponite after ultrasonic dispersion is added, and the solution is resuspended to obtain a suspension to obtain a bone-inducing layer slurry. 3) The preparation steps of the mechanical property layer slurry are as follows: thermoplastic polyester material is placed in the barrel of the 3D printer and melted at high temperature to obtain the mechanical property layer slurry; 4) The barrier antibacterial layer slurry obtained in step 1) is used to prepare the required barrier membrane using an electrospinning machine.

[0039] 5) The mechanical property layer slurry obtained in step 3) is used to 3D print the required scaffold using the barrier membrane prepared in step 4) as the receiving plate.

[0040] 6) The bone-inducing layer slurry obtained in step 2) is injected into the scaffold prepared in step 5) and then photocrosslinked and cured.

[0041] In steps 11), 12), 21), or 22) above, the natural polymer matrix is ​​selected from at least one of hyaluronic acid, silk fibroin, methacrylamide gelatin, methacrylamide hyaluronic acid, sodium alginate, and methacrylamide silk fibroin.

[0042] Preferably, the natural polymer matrix is ​​methacrylamide gelatin.

[0043] In a further embodiment, the methacrylamide gelatin is a solution of 3-7% methacrylamide gelatin, preferably a solution of 5% methacrylamide gelatin.

[0044] In steps 11), 12), or 3) above, the thermoplastic polyester material includes, but is not limited to, polylactic acid, polycaprolactone, thermoplastic polyurethane, polyetheretherketone, etc.

[0045] Preferably, the thermoplastic polyester material is polycaprolactone.

[0046] In a further embodiment of 11) or 12), the weight ratio of the methacrylamide gelatin to polycaprolactone is 1-10:1-10, specifically 1-5:1-5, 1-5:5-10, 5-10:1-5, preferably 1-5:1-5.

[0047] In one specific embodiment, in step 21) or 22) above, the hydrogel is an aqueous solution of 5-10% methacrylamide gelatin, preferably an aqueous solution of 8% methacrylamide gelatin.

[0048] In step 11) or 12) above, after the hydrogel is dissolved in water, the mass concentration of the polymer matrix is ​​5-15% (w / v), preferably 7-14% (w / v).

[0049] In step 21) or 22) above, the natural polymer matrix, after dissolving in water, has a mass concentration of 2-10% (w / v), specifically such as 4-10% (w / v) or 2%. 8% (w / v), 4-8% (w / v), preferably 4-8% (w / v).

[0050] In step 21) or 22) above, the dissolution temperature of the methacrylamide gelatin in water is 30-50°C, preferably 35-45°C; the dissolution time is 25-35 min, preferably 30 min. This allows the methacrylamide gelatin to dissolve in water to obtain an aqueous solution of methacrylamide gelatin.

[0051] In step 11) or 12) above, the antibacterial functional metal component is selected from at least one of zinc oxide, magnesium oxide, and copper oxide.

[0052] In one specific embodiment, the antibacterial functional metal component is nano-zinc oxide particles.

[0053] In step 11) or 12) above, after the antibacterial functional metal component is dispersed in the solution, the mass concentration of the antibacterial functional metal component is 0-4% (w / v), preferably 2% (w / v).

[0054] In step 21) or 22) above, the bioactive nanoparticles are selected from at least one of nanoclay, bioactive glass nanoparticles, and hydroxyapatite nanoparticles.

[0055] Preferably, the bioactive nanoparticles are lithium magnesium silicate nanoclay.

[0056] In step 21) or 22 above, the magnesium lithium silicate nanoclay, after being dispersed in the solution, has a mass concentration of 0-2% (w / v), preferably 1% (w / v).

[0057] In steps 11), 12), 21), or 22) above, a photoinitiator is also added to the slurry. The photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) (CAS No. 85073-19-4), which enables photocurable polymer matrix to undergo photocrosslinking.

[0058] In steps 11), 12), 21), or 22) above, the mass concentration of the photoinitiator is 0.1-0.5%, specifically 0.1-0.25%, 0.25-0.5%, preferably 0.25%.

[0059] In step 6) above, the wavelength of the irradiation light for photocrosslinking is 365-405nm; the irradiation time is 10-150s, preferably 120s.

[0060] A third aspect of the present invention provides a multilayer biological barrier membrane with antibacterial and osteogenic properties, which is prepared by the above method.

[0061] The fourth aspect of the present invention provides the application of the above-mentioned multilayer biological barrier membrane with antibacterial and osteogenic properties in the preparation of a membrane that promotes both antibacterial and bone repair functions.

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0063] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0064] Example 1: Dense layer nanofibers were prepared using an electrospinning machine. PCL and GelMA (1:1, 10% w / v) were dissolved in hexafluoroisopropanol and mixed evenly to obtain a barrier antibacterial layer mixed solution. Ultrasonically dispersed nano-zinc oxide particles were added and resuspended to obtain a suspension. The barrier antibacterial layer slurry was then loaded into a syringe and assembled into an electrospinning machine. The applied high voltage was 15 kV, the feed rate was 1 mL / h, and the distance between the needle tip and the current collector was 12 cm to prepare a dense layer electrospun membrane.

[0065] The prepared dense-layer electrospun membrane is used as the receiving surface to receive loose-layer fibers, forming a double-layer fiber scaffold. The loose-layer scaffold is fabricated using 3D printing technology. PCL particles are filled into the loading chamber and heated to 70-80℃. Under computer control, a pre-designed movement path (fiber spacing of 400 μm, layup pattern of 0°~90°, 4 layers) is extruded through needles with a diameter of 100 μm, stacking the printed fibers layer by layer.

[0066] GelMA prepared in PBS was mixed with different concentrations of lap (1% and 2% w / v), and incubated with 0.25% w / v LAP. The mixture was stirred at 37°C in the dark for 10 h to prepare GelMA / lap hydrogels. The prepared hydrogels were then infused into a bilayer fiber scaffold and photocrosslinked under 405 nm visible blue light for 120 s.

[0067] Example 2: Bone marrow mesenchymal stem cells and fibroblasts were seeded onto electrospun membranes containing barrier antibacterial layers doped with different concentrations of zinc oxide (0%, 1%, 2%, 4%). Cell proliferation activity was measured using the CCK-8 assay on days 1, 3, and 5 of cell culture. Specific results are shown in [link to example]. Figure 2 . Figure 2 The images show the cell viability results of three-layer composite scaffolds doped with different concentrations of zinc oxide nanoparticles. Figure a shows the CCK-8 assay results using bone marrow mesenchymal stem cells; figure b shows the CCK-8 assay results using fibroblasts. Figure 2 It was found that, except for the 4% concentration group, all other groups showed no significant toxicity to either type of cell. This indicates that the tissue engineering scaffold exhibits good biocompatibility in the presence of low concentrations of nano-zinc oxide.

[0068] Bone marrow mesenchymal stem cells were seeded onto osteogenic induction layer hydrogels doped with different concentrations of lithium magnesium silicate (0%, 1%, and 2%). Cell proliferation activity was measured using the CCK-8 assay on days 1, 3, and 5 of cell culture. Specific results are detailed below. Figure 3 . Figure 3 This image shows the cell viability results of three-layer composite scaffolds doped with different concentrations of lithium magnesium silicate. The cells used were bone marrow mesenchymal stem cells, and cell viability was obtained using a CCK-8 assay. Figure 3 It was found that, except for the 2% concentration group, all other groups showed no significant toxicity to either type of cell. This indicates that the tissue engineering scaffold exhibits good biocompatibility in the presence of low concentrations of lithium magnesium silicate.

[0069] Example 3: Bone marrow mesenchymal stem cells were seeded onto composite scaffolds doped with different concentrations of lithium magnesium silicate (0%, 0.5%, 1%). The bone marrow mesenchymal stem cells were cultured to day 7, and alkaline phosphatase, an early marker of osteogenic development, was stained and quantitatively analyzed. Specific test results are shown below. Figure 4 . Figure 4 The images show the alkaline phosphatase activity results of bone marrow mesenchymal stem cells cultured on three-layer composite scaffolds doped with different concentrations of lithium magnesium silicate for 7 days. Figure a shows a microscopic photograph of alkaline phosphatase staining; figure b shows the results of alkaline phosphatase activity detection.

[0070] Depend on Figure 4 It can be seen that both 0.5% and 1% concentrations of magnesium lithium silicate have a certain inducing effect on osteogenic differentiation of bone marrow mesenchymal stem cells, and the promoting effect of 1% magnesium lithium silicate is more obvious.

[0071] Example 4: Composite scaffolds doped with different concentrations of nano-zinc oxide (0%, 1%, 2%, 4%) were co-cultured with *Escherichia coli* and *Staphylococcus aureus*. After 12 hours of incubation, the co-cultured bacterial solution was plated, and the colonies were counted after another 12 hours of plate incubation. Specific results are shown in [link to example]. Figure 5 . Figure 5Figures show the antibacterial performance of three-layer composite scaffolds doped with zinc oxide nanoparticles of different concentrations. Figure a shows the colony counts of *Escherichia coli* and *Staphylococcus aureus* on agar plates; figure b shows the colony counts of *Staphylococcus aureus*; and figure c shows the colony counts of *Escherichia coli*.

[0072] Depend on Figure 5 It can be seen that zinc oxide of different concentrations has a certain antibacterial effect, and the antibacterial effect has a concentration-dependent effect, with the higher the concentration, the stronger the antibacterial effect.

[0073] Example 5: SD rats were selected, and alveolar bone defects with a diameter of 3 mm were created in their maxillae. The rats were randomly divided into five groups, each implanted with a different material: Control group (modeling only), PG / P / G group (scaffold without particle doping), Zn@PG / P / G group (scaffold doped with nano-zinc oxide), Zn@PG / P / L@G group (scaffold doped with nano-zinc oxide and lithium magnesium silicate), and Bio-gide group (commercially available collagen membrane). Micro-CT analysis and histological staining were performed at weeks 8 and 12 post-surgery to evaluate the repair of the alveolar bone defects. Figure 6 This image shows the imaging results of composite scaffolds with different components in the repair of alveolar bone defects in rats. Image a shows the three-dimensional and two-dimensional reconstructed images from Micro-CT; image b shows the statistical graphs of bone defect spacing, bone density, and new bone volume fraction. Figure 7 The images show the histological staining results of composite scaffolds with different components in the repair of alveolar bone defects in rats, with H&E and Masson staining images respectively.

[0074] Depend on Figure 6 , Figure 7 As can be seen, at 8 and 12 weeks postoperatively, Micro-CT and histological analysis showed that the Zn@PG / P / L@G group had significantly better new bone mass and bone density than other scaffold material groups, and the material degradation and bone integration effects were the best, indicating that this composite scaffold material has significant advantages and application prospects in promoting alveolar bone defect repair.

[0075] This invention provides a multilayer biobarrier membrane with antibacterial and osteogenic properties, its preparation method, and its applications. The membrane comprises an outer barrier antibacterial layer with a nanofiber membrane structure, a middle layer with a micron-scale crisscrossing scaffold serving as a mechanical performance layer, and an inner bone-inducing layer with a porous and biodegradable structure. The barrier antibacterial layer is an electrospun membrane containing a photocurable natural polymer matrix, thermoplastic polyester material, and antibacterial functional metal components to exert its antibacterial function. The mechanical performance layer is prepared using 3D-printed thermoplastic polyester material, providing tensile strength and support. The bone-inducing layer consists of a photocurable natural polymer matrix doped with bioactive nanoparticles, promoting bone tissue growth. The multilayer biobarrier membrane provided by this invention combines antibacterial and osteogenic functions and possesses excellent mechanical strength, thereby promoting bone tissue regeneration and repair.

Claims

1. A multi-layered bio-barrier membrane with antibacterial and osteoconductive properties, characterized in that, The three-layer structure is combined, including: the outer layer is a barrier bacteriostatic layer X with nanofiber membrane structure, which is composed of electrospun film doped with antibacterial functional metal components; the middle layer is a micrometer level longitudinal and horizontal interlaced scaffold as a mechanical performance layer Y, which is composed of N layers of 3D printed microfiber scaffolds; and the inner layer is a bone induction layer Z with porous degradable structure, which is composed of a porous matrix doped with bioactive nanoparticles.

2. The multilayer biological barrier film according to claim 1, wherein, The electrospun film for preparing the barrier bacteriostatic layer X comprises an antibacterial functional metal component, a photocurable natural polymer matrix and a thermoplastic polyester material; wherein the mass concentration of the photocurable natural polymer matrix is 4-8% the mass concentration of the thermoplastic polyester material is 4-8%, and the functional metal component is 2 6% of the total mass of the gel matrix and the polyester material.

6. The composition according to claim 1, wherein the functional metal component is selected from the group consisting of silver, gold, copper, zinc, and mixtures thereof.

3. The multi-layer bioinductive barrier film of claim 2, wherein, The antibacterial functional metal component in the electrospun film slurry for preparing the barrier bacteriostatic layer X comprises at least one of zinc oxide, copper oxide and magnesium oxide; the photocurable natural polymer matrix comprises at least one of methacrylic acid acylated gelatin, methacrylic acid acylated hyaluronic acid and methacrylic acid acylated silk fibroin; and the thermoplastic polyester material comprises at least one of polylactic acid, polycaprolactone, thermoplastic polyurethane and polyether ether ketone.

4. The multilayer biological barrier film according to claim 1, wherein, The thermoplastic polyester material of the 3D printed microfiber scaffold for preparing the mechanical performance layer Y comprises at least one of polylactic acid, polycaprolactone, thermoplastic polyurethane and polyether ether ketone.

5. The multilayer biological barrier film according to claim 1, wherein, The inner layer bone induction layer Z comprises bioactive nanoparticles and a photocurable natural polymer matrix, wherein the bioactive nanoparticles comprise at least one of nano-clay, bioactive glass nanoparticles and hydroxyapatite nanoparticles; and the photocurable natural polymer matrix comprises at least one of methacrylic acid acylated gelatin, methacrylic acid acylated hyaluronic acid and methacrylic acid acylated silk fibroin.

6. A method of making the multilayered biological barrier film of any one of claims 1-5, characterized in that, The method comprises the following steps: Step S1: preparing a slurry for electrospinning; Step S2: preparing an electrospun film from the slurry obtained in step S1 by using an electrospinning machine according to a specified procedure; obtaining the barrier bacteriostatic layer X; Step S3: melting extrusion printing the polyester material by using a 3D printer according to a specified procedure, with the barrier bacteriostatic layer X as a receiving platform; obtaining the mechanical performance layer Y; Step S4: bonding the melt-printed thermoplastic polyester material to the barrier bacteriostatic layer X as the receiving platform by high temperature, to form a double-layer structure XY; Step S5: uniformly dispersing bioactive nanoparticles in water to obtain a suspension, dissolving a photocurable natural polymer matrix in the suspension to obtain a required slurry; and pouring the slurry on the double-layer structure XY, and cross-linking the bone induction layer Z and the barrier bacteriostatic layer X by ultraviolet light irradiation, to form a three-layer structure XYZ.

7. The method of multilayered biological barrier membrane according to claim 6, characterized in that, The method for preparing the electrospun slurry in step S1 comprises: dissolving the thermoplastic polyester material and the photocurable natural polymer matrix in hexafluoroisopropanol.

8. The method of claim 6, wherein the barrier X is prepared by, The spinning receiving distance is 12-15 cm; the constant flow rate of the slurry is 0.5-2 ml / h; and the spinning voltage is 8-15 kV.

9. Application of a multi-layer biological barrier film with antibacterial and osteogenic properties in the repair of bone defects.

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