WO2024077418A1 - 生物活性多级结构引导组织再生膜及其制备方法 - Google Patents

生物活性多级结构引导组织再生膜及其制备方法 Download PDF

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WO2024077418A1
WO2024077418A1 PCT/CN2022/124150 CN2022124150W WO2024077418A1 WO 2024077418 A1 WO2024077418 A1 WO 2024077418A1 CN 2022124150 W CN2022124150 W CN 2022124150W WO 2024077418 A1 WO2024077418 A1 WO 2024077418A1
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polymer material
magnesium
organic polymer
layer
membrane
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PCT/CN2022/124150
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French (fr)
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张卫
赖毓霄
张原驰
聂杨逸
李龙
秦岭
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深圳先进技术研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/02—Inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/06—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials

Definitions

  • the invention belongs to the technical field of biomedical materials, and in particular relates to a bioactive multi-level structure guided tissue regeneration membrane and a preparation method thereof.
  • GTR Guided Tissue Regeneration
  • GTR membranes should not only play a good barrier role, but also promote the adhesion, proliferation and bone tissue regeneration of osteoblasts.
  • GTR membranes can also be used for wound dressings, corneas, and as diaphragms, they can also prevent postoperative muscle and peritoneal adhesions and other clinical applications.
  • Non-degradable membranes such as titanium membranes, silicone membranes and polytetrafluoroethylene membranes, can maintain the regeneration space under the membrane for a long time, but they need to be removed by a second operation, which brings pain and the risk of a second operation to the patient. If they are retained in situ, complications such as bacterial infection may occur, and they are prone to cause soft tissue flap rupture and early exposure of the membrane, affecting the amount of new bone formation.
  • Degradable absorbable membranes include natural polymer material membranes, such as collagen membranes, chitosan membranes, silk, etc., as well as synthetic material membranes, such as polyurethane and polylactic acid membranes.
  • degradable absorbable membranes have gradually shown great advantages in clinical practice.
  • membrane materials can also be self-degraded or absorbed by the body in the later stage of repair, which is better adapted to clinical application needs.
  • the excellent performance of the degradable absorbable GTR membrane material also needs to ensure that the material has good adhesion to cells and the ability to guide bone regeneration in vivo and in vitro.
  • the degradable absorbable membrane currently used in clinical practice still has some defects and shortcomings.
  • the most commonly used degradable absorbable membrane in clinical practice is collagen membrane, which has good cell affinity and a certain ability to guide tissue regeneration, but its mechanical properties are poor, its texture is soft, it is not easy to operate and shape, and it degrades quickly, and the regeneration space of the tissue under the membrane is difficult to maintain. Complications such as membrane collapse and displacement may also occur.
  • Polylactic acid-based membranes have certain mechanical strength and adjustable degradability, but pure polylactic acid membranes will produce a large amount of acidic degradation products during the degradation process, which can easily lead to nonspecific inflammation.
  • polyester-based degradable absorbable membrane materials also have important problems such as poor surface adhesion to cells and poor activity in guiding bone tissue regeneration.
  • Composite membranes that have the advantages of polyester materials and natural polymer materials and overcome their shortcomings still need to be further developed and applied in clinical practice.
  • magnesium has good biocompatibility, degradability and anti-infection ability.
  • the magnesium ions produced during the degradation process can effectively activate bone cells, promote the proliferation and differentiation of osteoblasts, and promote the generation of a large number of osteoblasts around the material, showing a high mineralization attachment rate and an increase in bone quality, effectively promoting bone repair at the implant site.
  • the degradation products of magnesium metal are weakly alkaline, which affects the pH value of the microenvironment of the implant site, can effectively resist infection at the implant site, prevent the occurrence of local inflammation, and stabilize the microenvironment of the implant site tissue.
  • the ideal GTR membrane structure has one side that is dense and smooth, which is used to prevent connective tissue from entering the bone defect area; the other side is loose and porous, which is conducive to the attachment and growth of bone cells and the stability of blood clots. Therefore, a GTR membrane with a double layer and a multi-level structure with different pores is more conducive to its function.
  • the membrane material can play a barrier role to separate the bone from the surrounding soft tissue.
  • the void structure can be conducive to the adhesion and proliferation of osteoblasts, and is conducive to the transportation of nutrients, the formation of vascular networks, etc., which is more conducive to promoting bone tissue regeneration from a structural perspective.
  • the most common methods for preparing GTR membranes are solution method, electrospinning method and phase transformation method.
  • the main methods at present can only prepare GTR membranes with a single structure and cannot be used to prepare multi-level structures.
  • the GTR membrane prepared by the solution method is usually a dense structure and cannot form a porous layer.
  • the nanofiber membrane is prepared by electrospinning. Due to the small fiber fineness, the membrane material prepared by electrospinning has a high specific surface area and porosity, and cannot prepare a dense structure layer.
  • Patent document 1 discloses a method for preparing a three-layer guided tissue regeneration membrane, which is prepared by electrospinning a polymer solution with different proportions of hydroxyapatite. Although it is a multi-layer structure, it still lacks a dense layer structure.
  • Patent document 2 discloses a method for preparing a double-layer regeneration membrane, including a dense layer and a porous layer, the porous layer is prepared by electrospinning, and can be composited with bioactive materials.
  • Patent document 3 uses electrospinning to prepare an absorbable membrane of composite inorganic particles, but the patent requires that the particle size of the composite inorganic particles does not exceed 100nm.
  • the electrospinning nozzle is usually within 1 ⁇ m, the spinning solution is required to be a good solution or the particles dispersed therein are nanometer-level, and the spinning diameter is usually within 1 ⁇ m.
  • the electrospinning method is not universal. Therefore, it is necessary to develop new technical means to prepare a multi-level structured degradable and absorbable GTR membrane with composite magnesium-containing micro-nano particles.
  • Patent Document 1 Three-layer guided tissue regeneration membrane with gradient, CN101584885B
  • Patent document 2 Bone tissue regeneration guiding membrane and preparation method thereof, CN 112190771 A
  • Patent Document 3 A functionally integrated absorbable guided tissue regeneration membrane and its preparation method, CN 111494720A
  • the present invention aims to provide a bioactive multi-level structure guided tissue regeneration membrane and a preparation method thereof.
  • the present invention focuses on the demand for new guided tissue regeneration membranes in the clinical treatment of bone defect repair, develops new bioactive guided tissue regeneration membrane materials, and prepares bioactive multi-level structure membranes through innovative material design and process optimization, which are more conducive to exerting the functions of tissue barrier, cell adhesion and promoting bone repair, thereby achieving more effective bone defect repair.
  • the first aspect of the present invention provides a bioactive multi-level structure guided tissue regeneration membrane, which includes a dense smooth layer and a loose porous layer.
  • the dense smooth layer is made of an organic polymer material
  • the loose porous layer is made of an organic polymer material, a hydrophilic natural polymer material and magnesium-containing micro-nanoparticles.
  • the organic polymer material and the hydrophilic natural polymer material are biodegradable polymers.
  • the organic polymer material is selected from at least one of medical grade polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyurethane, poly- ⁇ -hydroxybutyric acid (PHB), PLA modified material, PCL modified material and PLGA modified material;
  • the hydrophilic natural polymer material is selected from at least one of medical grade collagen, gelatin, hyaluronic acid, carboxymethyl cellulose, chitosan, silk fibroin and sodium alginate;
  • the magnesium-containing micro-nano particles are selected from at least one of magnesium metal particles, magnesium calcium particles, magnesium zinc particles, magnesium silicide particles, magnesium boride particles, magnesium hydride particles and magnesium oxide particles;
  • the particle size of the magnesium-containing micro-nano particles is 10 nm to 100 ⁇ m;
  • the particle size of the magnesium-containing micro-nano particles is 1 ⁇ m to 100 ⁇ m;
  • the particle size of the magnesium-containing micro-nano particles is 50-80 ⁇ m.
  • the mass percentage of the magnesium-containing micro-nano particles in the loose porous layer is 1% to 20%;
  • the mass ratio of the organic polymer material to the hydrophilic natural polymer material in the loose porous layer is (10-19): (1-10).
  • the thickness of the dense smooth layer is 0.5 mm to 2 mm, and the thickness of the loose porous layer is 1 mm to 5 mm.
  • the second aspect of the present invention provides a method for preparing the bioactive multi-level structure guided tissue regeneration membrane, comprising the following steps:
  • a loose porous layer is prepared on the dense smooth layer by using thermally induced phase separation technology.
  • the steps of preparing a dense and smooth layer by the solution casting method include: dissolving an organic polymer material in an organic solvent to obtain an organic polymer solution; casting the organic polymer solution into a clean and flat mold, placing it in a vacuum drying oven to evacuate the air bubbles therein, and then drying it at room temperature or under heating conditions until the organic solvent is completely volatilized to obtain a dense and smooth layer.
  • the organic solvent is one or a mixed solvent of dichloromethane, chloroform, dimethyl carbonate, diethyl carbonate, 1,4-dioxane, tetrahydrofuran and dimethylformamide;
  • the mass percentage concentration of the organic polymer solution is 10% to 30%;
  • the vacuuming time is 2 to 5 minutes, and the vacuuming is performed to a vacuum degree of 0 to 100 Pa.
  • the step of preparing the loose porous layer by the thermally induced phase separation technology comprises:
  • the organic polymer solution and the hydrophilic natural polymer solution are mixed, and the magnesium-containing micro-nano particle powder is added thereto, and stirred evenly to obtain a mixed solution;
  • the mixed solution is added to the surface of the dense smooth layer, allowed to stand for a certain period of time to allow the interface to fuse, rapidly frozen for phase separation, and then the organic solvent is removed by extraction or freeze-drying to form a loose porous layer on the dense smooth layer.
  • the extraction method for removing the organic solvent comprises the following steps: adding an extractant after phase separation, placing the sample with the extractant at 4°C, replacing the extractant after 12 hours and continuing the extraction, extracting multiple times to ensure that the organic solvent is extracted, and finally vacuum drying;
  • the extractant is a non-benign solvent for the organic polymer material and the hydrophilic natural polymer material;
  • the extractant is selected from one or a mixed solution of methanol, ethanol, n-hexane, ether, ethyl acetate and acetone;
  • the freeze-drying method for removing the organic solvent comprises the following steps: after phase separation, placing the sample in a freeze dryer and freeze-drying it at -40 to -60°C for more than 48 hours to ensure that all the solvent is completely removed.
  • the organic solvent used for the organic polymer material is one or a mixed solvent of dichloromethane, chloroform, dimethyl carbonate, diethyl carbonate, 1,4-dioxane and tetrahydrofuran;
  • the organic solvent used for the hydrophilic natural polymer material is hexafluoroisopropanol or trifluoroethanol;
  • the mass percentage concentration of the organic polymer solution is 10% to 30%.
  • the standing time is 3 to 10 minutes
  • the temperature of the rapid freezing is -20°C to -80°C, and the time is 1 to 2 hours.
  • the third aspect of the present invention provides the use of the bioactive multi-level structure guided tissue regeneration membrane in the preparation of tissue repair products.
  • tissue repair includes bone defect repair and periodontal defect repair.
  • the bioactive multi-level structure guided tissue regeneration membrane of the present invention has properties such as a double-layer structure, different porosities, and different hydrophilicity and hydrophobicity.
  • One side is a dense and smooth layer composed of organic polymers, and the other side is a loose porous layer composed of organic polymer materials, hydrophilic natural polymer materials, and magnesium-containing micro-nano particle composite materials.
  • the multi-level structure guided tissue regeneration membrane has good mechanical properties, promotes cell adhesion and proliferation, and promotes osteovascularization.
  • the double-layer structure overcomes the shortcomings of the mechanical properties of a single material, has suitable mechanical strength and elastic modulus, and is more conducive to playing a supporting and barrier role in the process of inducing tissue regeneration.
  • the loose porous layer composed of the composite material has better cell adhesion, which is more conducive to cell adhesion and proliferation, and the sustained release of bioactive magnesium ions can more effectively promote the osteogenic differentiation of cells and the formation of new blood vessels, so it can more effectively promote the formation of new tissues.
  • the dense and smooth layer of the present invention uses organic polymer materials to facilitate the preparation of the dense and smooth layer by solution casting method.
  • the preparation of the bioactive multi-level structure guided tissue regeneration membrane of the present invention adopts a process technology combining solution casting and thermally induced phase separation, so that the interface of the double-layer membrane structure is tightly combined and is not easy to split, fracture, collapse or shrink during use.
  • this method compared with methods such as electrospinning, this method has a wider range of material adaptability and can be applied to different types of polymer materials and composite micro-nano particles of different sizes.
  • Figure 1 Schematic diagram of the design of the double-layer multi-level structure guided tissue regeneration membrane.
  • Figure 2 Schematic diagram of the preparation of a double-layer multi-level structure guided tissue regeneration membrane.
  • FIG. 1 Study on the osteogenic activity of different membrane materials in vitro: A: In vitro mineralized calcium nodule staining; B: Mineralization quantitative test results.
  • the present invention provides a bioactive multi-level structure guided tissue regeneration membrane with good mechanical properties, cell adhesion and proliferation promotion, and osteovascularization activity.
  • the guided tissue regeneration membrane has a multi-level structure, including a double-layer structure, different porosities, different hydrophilicity and hydrophobicity and other properties.
  • the bioactive multi-level structure guided tissue regeneration membrane of the present invention includes a dense smooth layer and a loose porous layer, the dense smooth layer is made of an organic polymer material, the loose porous layer is made of an organic polymer material, a hydrophilic natural polymer material and magnesium-containing micro-nano particles, and the organic polymer material and the hydrophilic natural polymer material are biodegradable polymers.
  • the organic polymer is selected from at least one of medical grade polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyurethane, poly- ⁇ -hydroxybutyric acid (PHB), PLA modified materials, PCL modified materials and PLGA modified materials.
  • the hydrophilic natural polymer is selected from at least one of medical grade collagen, gelatin, hyaluronic acid, carboxymethyl cellulose, chitosan, silk fibroin, and sodium alginate.
  • the magnesium-containing micro-nano particles are selected from at least one of magnesium metal particles, magnesium calcium particles, magnesium zinc particles, magnesium silicide particles, magnesium boride particles, magnesium hydride particles, and magnesium oxide particles, and the particle size is 10 nm to 100 ⁇ m.
  • the mass percentage of the magnesium-containing micro-nano particles is 1% to 20%, and the mass ratio of the organic polymer material to the hydrophilic natural polymer material is (10-19): (1-10).
  • the particle size of the magnesium-containing micro-nano particles is 1 ⁇ m to 100 ⁇ m, more preferably 50 to 80 ⁇ m.
  • the organic polymer is selected from polylactic acid-co-glycolic acid (PLGA), the hydrophilic natural polymer is selected from gelatin, and the magnesium-containing micro-nanoparticles are selected from magnesium metal particles.
  • PLGA polylactic acid-co-glycolic acid
  • the hydrophilic natural polymer is selected from gelatin
  • the magnesium-containing micro-nanoparticles are selected from magnesium metal particles.
  • the mass ratio of the organic polymer material to the hydrophilic natural polymer material is 10:1.
  • the mass percentage of the magnesium-containing micro-nano particles is 1% to 5%.
  • a multi-level structure guided tissue regeneration membrane was prepared by solution casting combined with thermally induced phase separation.
  • the double-layer structure was prepared by a layer-by-layer molding process, and the layers could be tightly fused. According to the subsequent method of removing the diluent in the thermally induced phase separation method, it can be divided into extraction method and freeze-drying method.
  • the design schematic diagram of the multi-level structure membrane is shown in Figure 1, and its preparation steps are shown in Figure 2.
  • the organic polymer is dissolved in an organic solvent, heated and stirred to fully dissolve it to form a transparent solution, and an organic polymer solution with a mass fraction of 10% to 30% is prepared; in a specific embodiment, the organic solvent used is one or a mixed solvent of dichloromethane, chloroform, dimethyl carbonate, diethyl carbonate, 1,4-dioxane, tetrahydrofuran, and dimethylformamide.
  • the organic polymer solution is cast into a clean and flat glass mold, placed in a vacuum drying oven and evacuated for 2 to 5 minutes to remove the bubbles therein, the vacuum degree can be 0 to 100 Pa, and then continued to dry at room temperature or under heating conditions until the organic solvent is completely evaporated to obtain a dense and smooth layer, and then the subsequent loose porous layer is prepared.
  • the thickness of the dense smooth layer can be 0.5 mm to 2 mm, or can be prepared according to actual needs.
  • the organic polymer is dissolved in an organic solvent, heated and stirred to fully dissolve it, and an organic polymer solution with a mass fraction of 10% to 30% is prepared; in a specific embodiment, the diluent organic solvent used is one or a mixed solvent of dichloromethane, chloroform, dimethyl carbonate, diethyl carbonate, 1,4-dioxane, and tetrahydrofuran.
  • the hydrophilic natural polymer is dissolved in the organic solvent hexafluoroisopropanol or trifluoroethanol, stirred to fully dissolve it, and the corresponding solution is prepared according to the solubility of different polymers.
  • the organic polymer solution and the hydrophilic natural polymer solution are mixed, and the magnesium-containing micro-nano particle powder is added and stirred evenly to obtain a mixed solution.
  • the sample with the extractant is placed in a 4°C refrigerator, and the liquid is changed after 12 hours to continue extraction, 2 to 4 times to ensure that all the diluent organic solvents are extracted, and finally, vacuum drying is performed to obtain a double-layer multi-level structure membrane.
  • the organic polymer is dissolved in an organic solvent, heated and stirred to fully dissolve it, and an organic polymer solution with a mass fraction of 10% to 30% is prepared; in a specific embodiment, the diluent organic solvent used is one or a mixed solvent of dichloromethane, chloroform, dimethyl carbonate, diethyl carbonate, 1,4-dioxane, and tetrahydrofuran.
  • the hydrophilic natural polymer is dissolved in the organic solvent hexafluoroisopropanol or trifluoroethanol, stirred to fully dissolve it, and the corresponding solution is prepared according to the solubility of different polymers.
  • the organic polymer solution and the hydrophilic natural polymer solution are mixed, and the magnesium-containing micro-nano particle powder is added and stirred evenly to obtain a mixed solution.
  • the mixed solution is added to the surface of the dense and smooth layer obtained by the solution casting method, spread evenly, and allowed to stand for 3 to 10 minutes to allow the interface material of the dense and smooth layer to be slightly dissolved in the solution.
  • the sample is quickly frozen (-20°C to -80°C or liquid nitrogen), and after freezing, placed in a freeze dryer and freeze-dried at -40 to -60°C for more than 48 hours to ensure that all solvents are completely removed to obtain a double-layer multi-level structure membrane.
  • the thickness of the loose porous layer can be 1 mm to 5 mm, or the thickness can be prepared according to actual needs.
  • Preparation of double-layer PPMG Add PLGA polymer (3.0 g) to dichloromethane (20 mL), stir and dissolve to a clear and transparent solution, and prepare a 15 w/v% PLGA solution. Then cast 10 mL of the prepared PLGA solution into a clean and flat glass mold to form a uniform solution layer with a thickness of about 2 mm. Place the mold in a vacuum drying oven and evacuate for 2 minutes to remove the bubbles. Then continue to dry at room temperature until the organic solvent is completely volatilized to obtain a dense organic polymer film.
  • Preparation of double-layer membrane PPM Add PLGA polymer (3.0g) to dichloromethane (20mL), stir and dissolve until a clear and transparent solution is obtained to prepare a 15w/v% PLGA solution. Then cast 10mL of the prepared PLGA solution into a clean and flat glass mold to form a uniform solution layer with a thickness of about 2mm. Place the mold in a vacuum drying oven and evacuate for 2 minutes to remove the bubbles. Then continue to dry at room temperature until the organic solvent is completely volatilized to obtain a dense organic polymer membrane. Add magnesium metal particles (0.075g, particle size of 50-80 ⁇ m) to the PLGA solution (10mL), stir and disperse evenly.
  • Preparation of double-layer membrane PP Add PLGA polymer (3.0g) to dichloromethane (20mL), stir and dissolve until a clear and transparent solution is obtained to prepare a 15w/v% PLGA solution. Then cast 10mL of the prepared PLGA solution into a clean and flat glass mold to form a uniform solution layer with a thickness of about 2mm. Place the mold in a vacuum drying oven and evacuate for 2 minutes to remove the bubbles. Then continue to dry at room temperature until the organic solvent is completely volatilized to obtain a dense organic polymer film. Add the PLGA solution (10mL) to the surface of the already dried dense PLGA film in the above mold, spread evenly, and form a solution layer with a thickness of about 2mm.
  • PLGA polymer (3.0 g) was added to dichloromethane (20 mL), and the mixture was stirred and dissolved to a clear and transparent solution to prepare a 15 w/v% PLGA solution. Then 10 mL of the prepared PLGA solution was cast into a clean and flat glass mold to form a uniform solution layer with a thickness of about 2 mm. The mold was placed in a vacuum drying oven and evacuated for 2 minutes to remove the bubbles. Then, the mold was dried at room temperature until the organic solvent was completely volatilized to obtain a dense organic polymer membrane.
  • Preparation of porous membrane PM Add PLGA polymer (1.5g) to dichloromethane (10mL), stir and dissolve until a clear and transparent solution is obtained to prepare a 15w/v% PLGA solution. Add magnesium metal particles (0.075g) to the solution and stir and disperse evenly. Add the mixed solution to a clean and flat glass mold to form a uniform solution layer with a thickness of about 2mm. Place the mold in a vacuum drying oven and evacuate for 2 minutes to remove the bubbles. Then continue drying at room temperature until the organic solvent is completely volatilized to obtain a dense organic polymer membrane.
  • porous PMG membrane Preparation of porous PMG membrane: Add PLGA polymer (1.5g) to dichloromethane (10mL), stir thoroughly to dissolve into a clear and transparent solution, and prepare a 15w/v% PLGA solution. Add gelatin (0.15g) to hexafluoroisopropanol (1mL), heat to 50°C and stir thoroughly to dissolve into a clear and transparent solution, then add the solution to the above PLGA solution, stir thoroughly and evenly, then add magnesium metal particles (0.075g, particle size 50-80 ⁇ m), stir thoroughly and disperse evenly. Add the mixed solution to a clean and flat glass mold to form a uniform solution layer with a thickness of about 2mm, and then put the mold into liquid nitrogen for rapid freezing. Then put it in a freeze dryer and freeze-dry at -60°C for more than 48 hours to ensure that all solvents are completely removed to obtain a porous layer PMG membrane.
  • gelatin film Gel Preparation of gelatin film Gel: Add gelatin (1.5 g) to hexafluoroisopropanol (10 mL), heat to 50°C, stir thoroughly and dissolve until a clear and transparent solution is obtained. Add the solution into a clean and flat glass mold to form a uniform solution layer with a thickness of about 2 mm, and then place the mold in liquid nitrogen for rapid freezing. Then place it in a freeze dryer and freeze-dry it at -60°C for more than 48 hours to ensure that all solvents are completely removed to obtain a gelatin film Gel.
  • Structural characteristics of membrane materials According to the national standards GB/T16886.18-2011 and GB/T16886.19-2011 for biological evaluation of medical devices, SEM was used to characterize the physical structure morphology of the stent surface and cross-section based on the established methods.
  • MC 3T3-E1 Normal osteoblasts (MC 3T3-E1) were inoculated on different membrane materials, including PP, PPM and PPMG, and cultured normally for 24 hours. Then the cells were stained with Calcein and the cell adhesion on the membrane material surface was observed under fluorescence microscopy.
  • MC 3T3-E1 Normal osteoblasts (MC 3T3-E1) were inoculated on different membrane materials, including PP and PPMG. After 3 days of normal culture, osteogenic induction agents were added. After 21 days of osteogenic induction, the cells were fixed with 10% neutral formalin and stained with 1% Alizarin red for 30 minutes, and then photographed under a microscope to observe the mineralized calcium nodules.
  • the PPMG membrane had the largest number of cavities formed by umbilical vein cells, while the PP membrane had the smallest number of cavities formed by umbilical vein cells, indicating that the ability of a simple hydrophobic organic polymer membrane to promote angiogenesis in umbilical vein cells was weak, while the PPMG membrane not only had good cell adhesion, but also could release magnesium ions, effectively promoting the angiogenesis activity of umbilical vein cells to form cavities.
  • the test results are shown in Figure 8.

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Abstract

本发明公开了生物活性多级结构引导组织再生膜及其制备方法，该生物活性多级结构引导组织再生膜包括致密光滑层和疏松多孔层，所述致密光滑层由有机高分子材料制成，所述疏松多孔层由有机高分子材料、亲水性天然高分子材料和含镁微纳颗粒制成，所述有机高分子材料和亲水性天然高分子材料为生物可降解聚合物。本发明采用溶液浇铸法制备致密光滑层，在致密光滑层上采用热致相分离技术制备疏松多孔层。本发明生物活性多级结构引导组织再生膜兼具良好力学性质、促细胞粘附增殖和促成骨成血管活性。采用溶液浇铸法和热致相分离法结合的工艺技术，使双层膜结构的界面紧密结合，使用中不易分裂断层和塌陷皱缩。同时，该方法具有更广泛的材料适应性。

Description

生物活性多级结构引导组织再生膜及其制备方法 技术领域
本发明属于生物医用材料技术领域，具体涉及生物活性多级结构引导组织再生膜及其制备方法。
背景技术
引导组织再生(Guided Tissue Regeneration，简称GTR)技术是临床上解决组织再生问题的最常用最有效的技术之一，它是将引导组织再生屏障膜置于软组织与需再生组织之间人为建立生物屏障、创造一个隔离空间，防止软组织中的成纤维细胞及上皮细胞长入缺损区，确保再生过程无成纤维细胞的干扰，最后实现缺损区完全的修复。GTR技术最早应用于临床口腔牙周及种植领域。近些年，随着生物医用材料的迅速发展，引导组织再生膜已广泛运用于口腔、颌面及骨科骨组织再生修复临床应用中。引导组织再生膜在GTR中发挥着重要的作用，理想的GTR膜不仅要能起到很好的屏障作用，同时还要能够促进成骨细胞的黏附、增殖和骨组织再生。随着研究的发展，GTR膜也可用于创面敷料、角膜，作为隔膜也可以防止术后的肌肉、腹膜黏连等临床应用。
GTR技术能否成功应用于临床的关键是屏障膜材料的选择，膜材料的类型及结构的设计是至关重要的。目前临床所用的膜材料主要有两类：包括不可降解膜和可降解吸收膜。不可降解膜，如钛膜、硅胶膜及聚四氟乙烯膜，可较长时间维持膜下的再生空间，但需二次手术取出，给患者带来痛苦和二次手术的风险。如果保留在原位，可能产生细菌感染等并发症，且易导致软组织瓣裂开、膜早期暴露，影响新骨生成量。可降解吸收膜，又包括天然高分子材料膜，如胶原膜、壳聚糖膜、蚕丝等，以及合成材料膜，如聚氨酯及聚乳酸类膜等。近年来可降解吸收膜在临床上逐渐显示出较大的优势，膜材料除了具有生物或组织相容性和良好的理化性能外，在修复后期还能自行降解或被机体吸收，具有更好的适应临床应用需求。
性能优秀的可降解吸收GTR膜材料除了具有生物或组织相容性和良好的理化性能外，还需要保证材料在体内外能对细胞有很好的粘附能力和引导骨再生的能力。目前临床所用的可降解吸收膜仍存在一些缺陷和不足。临床使用较多的可降解吸收膜是胶原膜，它具有良好的细胞亲和力和一定的引导组织再生能力，但其力学性能差，质地柔软，不易操作和塑形，且降解速度快，膜下组织的再生空间难以维持，还可能出现膜塌陷、移位等并发症。聚乳酸基 膜具有一定的力学强度及可调的降解性，但单纯的聚乳酸膜在降解过程中会产生大量的酸性降解产物，易导致非特异性炎症。同时，聚酯基可降解吸收膜材料还存在表面不易于细胞粘附和引导骨组织再生活性差的重要问题。兼具聚酯材料和天然高分子材料的优点并克服其缺点的复合材料膜仍然有待进一步研发应用于临床。
在促进骨再生活性材料研究方面，申请人研究发现镁具有良好的生物相容性、降解性及抗感染的能力。镁金属植入生物体内后，降解过程中产生的镁离子能有效活化骨细胞，促进成骨细胞的增殖及分化，促使材料周围生成大量成骨细胞，呈现高的矿化附着速率和骨质量的增加，有效促进植入部位的骨修复。同时金属镁的降解产物呈现弱碱性，影响植入部位微环境的pH值，可有效抵抗植入部位的感染，防止局部炎症的发生，稳定植入部位组织的微环境。对于GTR膜目前仍然缺乏具有良好促成骨生物活性的材料适用于临床应用。针对以上问题，研发新型的含镁复合材料引导组织再生的可降解吸收膜材料，满足良好的力学性能、有利于细胞粘附增殖的表面和促进骨再生的活性等综合性质，将具有重要的临床意义和应用前景。
在GTR膜的结构方面，研究表明理想的GTR膜结构一侧致密光滑，用于阻止结缔组织进入骨缺损区；另一侧疏松多孔，有利于骨细胞的攀附生长和凝血块的稳定。因而，具有双层及不同孔隙的多级结构GTR膜更有利于其发挥功能，一方面膜材料可发挥屏障作用，把骨与周围软组织分隔开，一方面空隙结构可以有利于成骨细胞粘附和增殖，并有利于营养的输送、血管网络的形成等，从而更有利于从结构方面促进骨组织再生。最常见的制备GTR膜的方式为溶液法、静电纺丝法和相转化法等。但是，目前主要的方法均只能制备单一结构的GTR膜，不能用于制备多级结构。溶液法制备的GTR膜通常为致密结构，不能形成多孔层。静电纺丝法制备纳米纤维膜，由于纤维细度小，静电纺丝法制备的膜材料具有高比表面积和孔隙率，不能制备致密结构层。
专利文献1公开了一种制备三层引导组织再生膜的方法，通过高分子溶液复合不同比例羟基磷灰石经过静电纺丝制备，虽然为多层结构仍然缺乏致密层结构。专利文献2公开了一种制备双层再生膜的方法，包括致密层和多孔层，多孔层由静电纺丝法制备，可复合生物活性材料。专利文献3采用静电纺丝法制备了复合无机颗粒的可吸收膜，但是专利要求所复合无机颗粒的粒径不超过100nm。由于静电纺丝的出丝喷嘴通常在1μm以内，要求纺丝液为良好的溶液或者分散在其中的颗粒为纳米级别，纺丝直径通常在1μm以内。对于镁或镁合金颗粒，由于其粉体性质非常活泼，商业化制备的镁或镁合金颗粒通常为微米级颗粒以保证生产 和使用的安全性。因而，静电纺丝法不具备普适性。因而需要研发新的技术手段制备复合含镁微纳颗粒的多级结构可降解吸收GTR膜。
专利文献1：具有梯度的三层引导组织再生膜，CN101584885B
专利文献2：骨组织再生引导膜及其制备方法，CN 112190771 A
专利文献3：一种功能一体化可吸收引导组织再生膜及其制备方法，CN 111494720A
发明内容
针对当前可降解吸收引导组织再生膜力学性能、细胞粘附性、降解时间及促成骨活性等性质不可控且不可兼备的难题，本发明旨在提供生物活性多级结构引导组织再生膜及其制备方法。
本发明围绕骨缺损修复临床治疗中对新型引导组织再生膜的需求，研发新型生物活性引导组织再生膜材料，通过创新的材料设计和工艺优化制备生物活性多级结构膜，更有利于发挥组织屏障、细胞粘附及促进成骨修复的功能，实现更有效的骨缺损修复。
本发明的具体技术方案如下：
本发明第一方面提供生物活性多级结构引导组织再生膜，所述生物活性多级结构引导组织再生膜包括致密光滑层和疏松多孔层，所述致密光滑层由有机高分子材料制成，所述疏松多孔层由有机高分子材料、亲水性天然高分子材料和含镁微纳颗粒制成，所述有机高分子材料和亲水性天然高分子材料为生物可降解聚合物。
进一步地，所述有机高分子材料选自医用级聚乳酸(PLA)、聚羟基乙酸(PGA)、聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚氨酯、聚β-羟基丁酸(PHB)、PLA改性材料、PCL改性材料和PLGA改性材料中的至少一种；
所述亲水性天然高分子材料选自医用级胶原、明胶、透明质酸、羧甲基纤维素、壳聚糖、丝素蛋白和海藻酸钠中的至少一种；
所述含镁微纳颗粒选自镁金属颗粒、镁钙颗粒、镁锌颗粒、硅化镁颗粒、硼化镁颗粒、氢化镁颗粒和氧化镁颗粒的至少一种；
优选地，所述含镁微纳颗粒的粒径为10nm～100μm；
优选地，所述含镁微纳颗粒的粒径为1μm～100μm；
优选地，所述含镁微纳颗粒的粒径为为50～80μm。
进一步地，所述含镁微纳颗粒占疏松多孔层的质量百分比为1％～20％；
所述疏松多孔层中有机高分子材料和亲水性天然高分子材料的质量比为(10-19)：(1-10)。
进一步地，所述致密光滑层的厚度为0.5mm～2mm，所述疏松多孔层的厚度为1mm～5mm。
本发明第二方面提供所述生物活性多级结构引导组织再生膜的制备方法，包括如下步骤：
(1)采用溶液浇铸法制备致密光滑层；
(2)在所述致密光滑层上，采用热致相分离技术制备疏松多孔层。
进一步地，所述溶液浇铸法制备致密光滑层的步骤包括：将有机高分子材料溶于有机溶剂中，获得有机高分子溶液；将有机高分子溶液浇铸到洁净平整的模具内，放入真空干燥箱中抽真空脱去其中的气泡，然后在室温或加热条件下干燥至有机溶剂完全挥发，得到致密光滑层。
进一步地，所述溶液浇铸法制备致密光滑层的步骤中，所述有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环、四氢呋喃和二甲基甲酰胺的一种或混合溶剂；
所述有机高分子溶液的质量百分比浓度为10％～30％；
所述抽真空的时间为2～5分钟，抽真空至真空度为0～100Pa。
进一步地，所述热致相分离技术制备疏松多孔层的步骤包括：
将有机高分子材料溶于有机溶剂中，获得有机高分子溶液；
将亲水性天然高分子材料溶于有机溶剂中，获得亲水性天然高分子溶液；
将有机高分子溶液和亲水性天然高分子溶液混合，向其中加入含镁微纳颗粒粉末，搅拌均匀，获得混合溶液；
将上述混合溶液加入到致密光滑层表面，静置一定时间使界面融合，快速冷冻进行相分离，然后通过萃取法或冻干法除去有机溶剂，即在所述致密光滑层上形成疏松多孔层。
进一步地，所述萃取法除去有机溶剂的步骤为：相分离后加入萃取剂，将加入萃取剂的样品置于4℃，12小时后更换萃取剂继续萃取，多次萃取确保有机溶剂被萃取出，最后真空干燥；
所述萃取剂为所述有机高分子材料和亲水性天然高分子材料的非良性溶剂；
优选地，所述萃取剂选自甲醇、乙醇、正己烷、乙醚、乙酸乙酯和丙酮中的一种或混合溶液；
所述冻干法除去有机溶剂的步骤为：相分离后将样品放入冻干机在-40～-60℃下冻干48 小时以上，确保所有溶剂被完全除去。
进一步地，所述热致相分离技术制备疏松多孔层的步骤中，所述有机高分子材料所用有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环和四氢呋喃的一种或混合溶剂；
所述亲水性天然高分子材料所用有机溶剂为六氟异丙醇或三氟乙醇；
优选地，所述有机高分子溶液的质量百分比浓度为10％～30％。
进一步地，所述静置的时间为3～10分钟；
所述快速冷冻的温度为-20℃～-80℃，时间为1～2小时。
本发明第三方面提供所述生物活性多级结构引导组织再生膜在制备组织修复用制品中的应用。
进一步地，所述组织修复包括骨缺损修复和牙周缺损修复。
本发明的有益效果为：
(1)本发明生物活性多级结构引导组织再生膜具有双层结构、不同孔隙率、不同亲疏水性等性质，一面为有机高分子组成的致密光滑层，一面为有机高分子材料、亲水性天然高分子材料和含镁微纳颗粒复合材料组成的疏松多孔层，该多级结构引导组织再生膜兼具良好力学性质、促细胞粘附增殖和促成骨成血管活性。双层结构克服了单一材料力学性能方面的缺点，具有适宜的力学强度和弹性模量，更有利于发挥诱导组织再生过程中发挥支撑和屏障作用。由复合材料组成的疏松多孔层具有更好的细胞粘附性，更有利于细胞的粘附增殖，并且生物活性镁离子的缓释作用能更有效促进细胞的成骨分化以及新生血管生成，因此能更有效协同促进新组织的生成。
本发明致密光滑层中选用有机高分子材料，以便于溶液浇铸法制备致密光滑层。
(2)本发明生物活性多级结构引导组织再生膜的制备采用溶液浇铸法和热致相分离法结合的工艺技术，使双层膜结构的界面紧密结合，使用中不易分裂断层和塌陷皱缩。同时，该方法相比于静电纺丝等方法具有更广泛的材料适应性，能适用于不同的高分子材料种类以及复合不同尺寸大小的微纳颗粒。
附图说明
图1.双层多级结构引导组织再生膜设计示意图。
图2.双层多级结构引导组织再生膜制备示意图。
图3.不同双层膜的表面图和切面图。
图4.不同双层膜的纵切面SEM图。
图5.不同膜材料的力学性能测试：A:弹性模量；B:断裂伸长率；C:最大应力。
图6.不同膜材料表面体外细胞粘附实验。
图7.不同膜材料体外促成骨活性研究：A:体外矿化钙结节染色；B:矿化定量测试结果。
图8.不同膜材料体外促血管新生活性研究。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
本发明提供一种兼具良好力学性质、促细胞粘附增殖和促成骨成血管活性的生物活性多级结构引导组织再生膜。所述引导组织再生膜具有多级结构，包括双层结构、不同孔隙率、不同亲疏水性等性质。本发明生物活性多级结构引导组织再生膜包括致密光滑层和疏松多孔层，所述致密光滑层由有机高分子材料制成，所述疏松多孔层由有机高分子材料、亲水性天然高分子材料和含镁微纳颗粒制成，所述有机高分子材料和亲水性天然高分子材料为生物可降解聚合物。
在具体实施方案中，所述有机高分子选自医用级聚乳酸(PLA)、聚羟基乙酸(PGA)、聚乳酸-羟基乙酸共聚物(PLGA)、聚己内酯(PCL)、聚氨酯、聚β-羟基丁酸(PHB)、PLA改性材料、PCL改性材料和PLGA改性材料中的至少一种。
在具体实施方案中，所述亲水性天然高分子选自医用级胶原、明胶、透明质酸、羧甲基纤维素、壳聚糖、丝素蛋白、海藻酸钠中的至少一种。
在具体实施方案中，所述含镁微纳颗粒选自镁金属颗粒、镁钙颗粒、镁锌颗粒、硅化镁颗粒、硼化镁颗粒、氢化镁颗粒、氧化镁颗粒的至少一种，粒径为10nm～100μm的微纳颗粒。
在具体实施方案中，疏松多孔层中，所述含镁微纳颗粒的质量百分比为1％～20％，有机高分子材料和亲水性天然高分子材料的质量比为(10-19)：(1-10)。
在一个优选实施方案中，所述含镁微纳颗粒粒径为1μm～100μm，更优选地为50～80μm。
在一个优选实施方案中，所述有机高分子选自聚乳酸-羟基乙酸共聚物(PLGA)，所述亲水性天然高分子选自明胶，所述含镁微纳颗粒选自镁金属颗粒。
在一个优选实施方案中，疏松多孔层中，有机高分子材料和亲水性天然高分子材料的质量比为10：1。
在一个优选实施方案中，疏松多孔层中，所述含镁微纳颗粒的质量百分比为1％～5％。
基于以上复合材料利用溶液浇铸法结合热致相分离法制备多级结构引导组织再生膜。双层结构通过逐层成型工艺制备，且层与层可紧密融合。根据热致相分离方法中后续除去稀释剂的方法又可分为萃取法和冻干法。多级结构膜的设计示意图见附图1，其制备步骤见附图2。
通过溶液浇铸法技术制备致密光滑层的方法：
将有机高分子溶于有机溶剂中，加热搅拌使其充分溶解形成透明溶液，制备质量分数为10％～30％的有机高分子溶液；在具体实施方案中，所用有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环、四氢呋喃、二甲基甲酰胺的一种或混合溶剂。
将有机高分子溶液浇铸到洁净平整的玻璃模具内，放入真空干燥箱中抽真空2～5分钟，脱去其中的气泡，真空度可为0～100Pa，然后在室温或加热条件下继续干燥至有机溶剂完全挥发，得到致密光滑层，随即进行后续疏松多孔层的制备。
致密光滑层的厚度可为0.5mm～2mm，或根据实际需求制备。
通过萃取法热致相分离技术制备疏松多孔层的方法：
将有机高分子溶于有机溶剂中，加热搅拌使其充分溶解，制备质量分数为10％～30％的有机高分子溶液；在具体实施方案中，所用稀释剂有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环、四氢呋喃的一种或混合溶剂。
将亲水性天然高分子溶于有机溶剂六氟异丙醇或三氟乙醇，搅拌使其充分溶解，根据不同高分子溶解度制备相应溶液。
将有机高分子溶液和亲水性天然高分子溶液混合，加入含镁微纳颗粒粉末，搅拌均匀，获得混合溶液。
将上述混合溶液加入到上述溶液浇铸法得到的致密光滑层表面，铺展均匀，放置3～10分钟使致密光滑层的界面材料微溶于溶液中，将样品快速冷冻(-20℃～-80℃)，冷冻1～2小 时。然后加入萃取剂，萃取剂选自甲醇、乙醇、正己烷、乙醚、乙酸乙酯、丙酮等对于上述高分子材料的非良性溶剂中的一种或混合溶液。将加入萃取剂的样品放入4℃冰箱，12小时后换液继续萃取，2～4次确保全部稀释剂有机溶剂被萃取出，最后，真空干燥得到双层多级结构膜。
通过冻干法热致相分离技术制备疏松多孔层的方法：
将有机高分子溶于有机溶剂中，加热搅拌使其充分溶解，制备质量分数为10％～30％的有机高分子溶液；在具体实施方案中，所用稀释剂有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环、四氢呋喃的一种或混合溶剂。
将亲水性天然高分子溶于有机溶剂六氟异丙醇或三氟乙醇，搅拌使其充分溶解，根据不同高分子溶解度制备相应溶液。
将有机高分子溶液和亲水性天然高分子溶液混合，加入含镁微纳颗粒粉末，搅拌均匀，获得混合溶液。
将上述混合溶液加入到上述溶液浇铸法得到的有致密光滑层表面，铺展均匀，放置3～10分钟使致密光滑层的界面材料微溶于溶液中，将样品快速冷冻(-20℃～-80℃或液氮)，冷冻后放入冻干机在-40～-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到双层多级结构膜。
疏松多孔层厚度可为1mm～5mm，或根据实际需求制备厚度。
实施例1
双层膜PPMG的制备：将PLGA高分子(3.0g)加入二氯甲烷(20mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。然后将10mL所制备的PLGA溶液浇铸到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，将模具放入真空干燥箱中抽真空2分钟，脱去其中的气泡。然后在室温条件下继续干燥至有机溶剂完全挥发，得到致密的有机高分子膜。将明胶(0.15g)加入六氟异丙醇(1mL)中，加热至50℃充分搅拌溶解至澄清透明溶液，然后将该溶液加入上述PLGA溶液(10mL)中，充分搅拌均匀，再加入镁金属颗粒(0.075g，粒径为50～80μm)，充分搅拌分散均匀。将混合溶液加入上述模具中已经干燥的致密PLGA膜表面，铺展均匀，形成厚度约2mm的溶液层。静置3分钟待溶液界面将干燥的有机层微微溶解，然后将模具放入液氮中快速冷冻。然后放入冷冻干燥机中，在-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到双层多级结构膜。
实施例2
双层膜PPM的制备：将PLGA高分子(3.0g)加入二氯甲烷(20mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。然后将10mL所制备的PLGA溶液浇铸到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，将模具放入真空干燥箱中抽真空2分钟，脱去其中的气泡。然后在室温条件下继续干燥至有机溶剂完全挥发，得到致密的有机高分子膜。向PLGA溶液(10mL)中加入镁金属颗粒(0.075g，粒径为50～80μm)，充分搅拌分散均匀。将混合溶液加入上述模具中已经干燥的致密PLGA膜表面，铺展均匀，形成厚度约2mm的溶液层。静置3分钟待溶液界面将干燥的有机层微微溶解，然后将模具放入液氮中快速冷冻。然后放入冷冻干燥机中，在-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到双层多级结构膜。
实施例3
双层膜PP的制备：将PLGA高分子(3.0g)加入二氯甲烷(20mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。然后将10mL所制备的PLGA溶液浇铸到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，将模具放入真空干燥箱中抽真空2分钟，脱去其中的气泡。然后在室温条件下继续干燥至有机溶剂完全挥发，得到致密的有机高分子膜。将PLGA溶液(10mL)中加入上述模具中已经干燥的致密PLGA膜表面，铺展均匀，形成厚度约2mm的溶液层。静置3分钟待溶液界面将干燥的有机层微微溶解，然后将模具放入液氮中快速冷冻。然后放入冷冻干燥机中，在-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到双层多级结构膜。
实施例4
致密膜P的制备：将PLGA高分子(3.0g)加入二氯甲烷(20mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。然后将10mL所制备的PLGA溶液浇铸到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，将模具放入真空干燥箱中抽真空2分钟，脱去其中的气泡。然后在室温条件下继续干燥至有机溶剂完全挥发，得到致密的有机高分子膜。
实施例5
多孔膜PM的制备：将PLGA高分子(1.5g)加入二氯甲烷(10mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。向其中加入镁金属颗粒(0.075g)，充分搅拌分散均匀。将混合溶液加入到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，将模具放入真空干燥箱中抽真空2分钟，脱去其中的气泡。然后在室温条件下继续干燥至有机溶剂 完全挥发，得到致密的有机高分子膜。
实施例6
多孔膜PMG的制备：将PLGA高分子(1.5g)加入二氯甲烷(10mL)中，充分搅拌溶解至澄清透明溶液，制备15w/v％的PLGA溶液。将明胶(0.15g)加入六氟异丙醇(1mL)中，加热至50℃充分搅拌溶解至澄清透明溶液，然后将该溶液加入上述PLGA溶液中，充分搅拌均匀，再加入镁金属颗粒(0.075g，粒径为50～80μm)，充分搅拌分散均匀。将混合溶液加入到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，然后将模具放入液氮中快速冷冻。然后放入冷冻干燥机中，在-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到多孔层PMG膜。
实施例7
明胶膜Gel的制备：将明胶(1.5g)加入六氟异丙醇(10mL)中，加热至50℃充分搅拌溶解至澄清透明溶液。将溶液加入到洁净平整的玻璃模具内，形成厚度约2mm的均匀溶液层，然后将模具放入液氮中快速冷冻。然后放入冷冻干燥机中，在-60℃下冻干48小时以上，确保所有溶剂被完全除去，得到明胶膜Gel。
实施例8
膜材料的结构特征：根据医疗器械生物学评价的国家标准GB/T16886.18-2011及GB/T16886.19-2011，基于已经建立的方法，利用SEM表征支架表面、断面的物理结构形貌。
结果表明，纵切面TEM图显示双层膜PP、PPM和PPMG都具有明显的双层结构，其中一层为致密结构，一层为多孔结构。相比于PP和PPM，PPMG的多孔层中孔径更大更均匀，表面亲水高分子的加入更有利于形成疏松多孔结构。不同双层膜的表面照片及PPMG的侧面照片见附图3，纵切面SEM测试结果见附图4。
实施例9
膜材料的力学性能：根据医疗器械生物学评价的国家标准GB/T8813-2008(ISO 844:2004)的要求和标准，利用拉伸法测定膜材料的弹性模量、拉伸强度、断裂伸长率和最大应力等。
结果表明，致密膜P具有最大的断裂伸长率，而明胶膜Gel具有最大应力，表明致密膜P具有较好的拉伸性而强度不高，而明胶膜几乎没有拉伸性而脆性大。通过形成双层多级结构膜，在弹性模量、拉伸强度、断裂伸长率和最大应力等方面得到平衡，获得比较均衡的力学性能。测试结果见附图5。
实施例10
膜材料体细胞粘附的研究：将正常成骨细胞(MC 3T3-E1)接种在不同膜材料上，包括PP、PPM和PPMG三种不同膜材料，正常培养24小时，然后用钙黄绿素(Calcein)染色细胞，荧光显微镜观察膜材料表面细胞粘附情况。
结果表明，三种膜材料中PPMG膜表面细胞数量最多，PP膜表面细胞数量最少，说明单纯的疏水性有机高分子表面对细胞粘附性较差，而加入亲水性高分子(如明胶)则可提高膜表面对细胞的粘附能力，有利于细胞粘附增殖。测试结果见附图6。
实施例11
膜材料体外促成骨活性的研究：将正常成骨细胞(MC 3T3-E1)接种在不同膜材料上，包括PP、PPMG，正常培养3天后加入成骨诱导剂，细胞成骨诱导21天后，用10％中性福尔马林固定细胞，用1％茜素红染色30分钟后显微镜拍照观察矿化钙结节
结果表明，三种膜材料中PPMG膜表面成骨细胞矿化结节数量最多，PP膜表面成骨细胞矿化结节数量最少，说明单纯的疏水性有机高分子膜对成骨细胞的促成骨分化能力较弱，而PPMG膜不仅具有较好的细胞粘附性，而且可以释放镁离子，有效促进细胞成骨分化形成矿化钙结节。测试结果见附图7。
实施例12
膜材料体外促血管新生活性的研究：将人脐静脉细胞融合细胞(Eahy-926)接种于多孔材料后常规培养，培养12-16h后，相差显微镜观察细胞形态并拍照，用分析软件Image J系统计数每个孔5个随机选定的低倍视野下的分支以及管腔。
结果表明，三种膜材料中PPMG膜表面脐静脉细胞形成的官腔数量最多，PP膜表面脐静脉细胞形成官腔数量最少，说明单纯的疏水性有机高分子膜对脐静脉细胞的促血管新生能力较弱，而PPMG膜不仅具有较好的细胞粘附性，而且可以释放镁离子，有效促进脐静脉细胞的血管新生活性形成官腔。测试结果见附图8。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (13)

  1. 生物活性多级结构引导组织再生膜，其特征在于，所述生物活性多级结构引导组织再生膜包括致密光滑层和疏松多孔层，所述致密光滑层由有机高分子材料制成，所述疏松多孔层由有机高分子材料、亲水性天然高分子材料和含镁微纳颗粒制成，所述有机高分子材料和亲水性天然高分子材料为生物可降解聚合物。
  2. 根据权利要求1所述的生物活性多级结构引导组织再生膜，其特征在于，所述有机高分子材料选自医用级聚乳酸、聚羟基乙酸、聚乳酸-羟基乙酸共聚物、聚己内酯、聚氨酯、聚β-羟基丁酸、PLA改性材料、PCL改性材料和PLGA改性材料中的至少一种；
    所述亲水性天然高分子材料选自医用级胶原、明胶、透明质酸、羧甲基纤维素、壳聚糖、丝素蛋白和海藻酸钠中的至少一种；
    所述含镁微纳颗粒选自镁金属颗粒、镁钙颗粒、镁锌颗粒、硅化镁颗粒、硼化镁颗粒、氢化镁颗粒和氧化镁颗粒的至少一种；
    优选地，所述含镁微纳颗粒的粒径为10nm～100μm；
    优选地，所述含镁微纳颗粒的粒径为1μm～100μm；
    优选地，所述含镁微纳颗粒的粒径为为50～80μm。
  3. 根据权利要求1所述的生物活性多级结构引导组织再生膜，其特征在于，所述含镁微纳颗粒占疏松多孔层的质量百分比为1％～20％；
    所述疏松多孔层中有机高分子材料和亲水性天然高分子材料的质量比为(10-19)：(1-10)。
  4. 根据权利要求1所述的生物活性多级结构引导组织再生膜，其特征在于，所述致密光滑层的厚度为0.5mm～2mm，所述疏松多孔层的厚度为1mm～5mm。
  5. 权利要求1-4任一项所述生物活性多级结构引导组织再生膜的制备方法，其特征在于，包括如下步骤：
    (1)采用溶液浇铸法制备致密光滑层；
    (2)在所述致密光滑层上，采用热致相分离技术制备疏松多孔层。
  6. 根据权利要求5所述的制备方法，其特征在于，所述溶液浇铸法制备致密光滑层的步骤包括：将有机高分子材料溶于有机溶剂中，获得有机高分子溶液；将有机高分子溶液浇铸到洁净平整的模具内，放入真空干燥箱中抽真空脱去其中的气泡，然后在室温或加热条件下干燥至有机溶剂完全挥发，得到致密光滑层。
  7. 根据权利要求6所述的制备方法，其特征在于，所述有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环、四氢呋喃和二甲基甲酰胺的一种或混合溶剂；
    所述有机高分子溶液的质量百分比浓度为10％～30％；
    所述抽真空的时间为2～5分钟，抽真空至真空度为0～100Pa。
  8. 根据权利要求5所述的制备方法，其特征在于，所述热致相分离技术制备疏松多孔层的步骤包括：
    将有机高分子材料溶于有机溶剂中，获得有机高分子溶液；
    将亲水性天然高分子材料溶于有机溶剂中，获得亲水性天然高分子溶液；
    将有机高分子溶液和亲水性天然高分子溶液混合，向其中加入含镁微纳颗粒粉末，搅拌均匀，获得混合溶液；
    将上述混合溶液加入到致密光滑层表面，静置一定时间使界面融合，快速冷冻进行相分离，然后通过萃取法或冻干法除去有机溶剂，即在所述致密光滑层上形成疏松多孔层。
  9. 根据权利要求8所述的制备方法，其特征在于，所述萃取法除去有机溶剂的步骤为：相分离后加入萃取剂，将加入萃取剂的样品置于4℃，12小时后更换萃取剂继续萃取，多次萃取确保有机溶剂被萃取出，最后真空干燥；
    所述萃取剂为所述有机高分子材料和亲水性天然高分子材料的非良性溶剂；
    优选地，所述萃取剂选自甲醇、乙醇、正己烷、乙醚、乙酸乙酯和丙酮中的一种或混合溶液；
    所述冻干法除去有机溶剂的步骤为：相分离后将样品放入冻干机在-40～-60℃下冻干48小时以上，确保所有溶剂被完全除去。
  10. 根据权利要求8所述的制备方法，其特征在于，所述有机高分子材料所用有机溶剂为二氯甲烷、氯仿、碳酸二甲酯、碳酸二乙酯、1,4-二氧六环和四氢呋喃的一种或混合溶剂；
    所述亲水性天然高分子材料所用有机溶剂为六氟异丙醇或三氟乙醇；
    优选地，所述有机高分子溶液的质量百分比浓度为10％～30％。
  11. 根据权利要求8所述的制备方法，其特征在于，所述静置的时间为3～10分钟；
    所述快速冷冻的温度为-20℃～-80℃，时间为1～2小时。
  12. 权利要求1-4任一项所述生物活性多级结构引导组织再生膜在制备组织修复用制品中的应用。
  13. 根据权利要求13所述的应用，其特征在于，所述组织修复包括骨缺损修复和牙周缺损修复。
PCT/CN2022/124150 2022-10-09 2022-10-09 生物活性多级结构引导组织再生膜及其制备方法 WO2024077418A1 (zh)

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