Double-layer oral repair membrane material and its preparation method

Abstract

The present invention discloses a double-layer oral repair membrane material and a preparation method thereof. The preparation method comprises the following steps: 1) Dispersing poly(lactic acid) microspheres containing osteogenic active materials in an aqueous solution of methacrylated protein to obtain a first mixture; spreading the first mixture, performing ultrasonic vibration, and drying to obtain a protein membrane; 2) Ultrasonically treating nano-scale osteogenic active material powder and nano-piezoelectric inorganic substances in an aqueous solution of a surface modifier to obtain surface-modified nano-powders; mixing the surface-modified nano-powders, poly(L-lactic acid), and a second solvent, and performing ultrasonic vibration to obtain an electrospinning solution; 3) Forming a fibrous membrane on the protein membrane by electrospinning the electrospinning solution, then washing and freeze-drying to obtain the double-layer oral repair membrane material. The double-layer oral repair membrane material obtained by the present invention has good hydrophilicity, piezoelectric properties, and softness.

Description

Technical Field

[0001] The present invention relates to a bilayer oral repair membrane material and a preparation method thereof. Background Art

[0002] In the fields of orthopedics and stomatology, for the treatment of bone defects caused by congenital malformations, trauma, tumors, inflammation, etc., it is usually necessary to adopt means to promote bone tissue healing, repair and regeneration. In the early 1980s, the concept of Guided Tissue Regeneration (GTR) was first proposed internationally. Based on the inference of the GTR concept, the tissue isolation membrane-guided bone regeneration technology is defined as the Guided Bone Regeneration (GBR) technology. Its core lies in establishing and maintaining a locally closed space on the surface of the bone defect by applying a tissue barrier membrane material (also called GBR membrane), so as to block the ingrowth of fibrous connective tissue, thereby avoiding the competitive inhibition phenomenon between different cells, enabling bone cells and bone tissue to fully mobilize their own healing abilities, and completing the process of high-quality bone regeneration and bone remodeling and reconstruction in the closed space without external adverse interference. Therefore, the GBR membrane is the key to the success of the GBR technology, and its own performance will directly affect the bone regeneration effect.

[0003] In current clinical operations of oral repair, this technology mainly uses natural bone powder or artificial bone powder as filling and is supplemented with GBR membrane coverage. GBR membrane materials are mainly divided into absorbable and non-absorbable types. Non-absorbable GBR membrane materials are mainly polytetrafluoroethylene (e-PTFE) and titanium metal, which have good mechanical strength and provide a stable bone regeneration and repair space. However, due to their non-absorbability, they need to be removed by a second operation after implantation, which will increase the pain of patients and the surgical cost. Absorbable GBR membrane materials are represented by collagen membranes, which can be completely degraded and absorbed in the body, so the harm caused by the second operation can be avoided. However, the collagen membrane has weak mechanical properties and a fast degradation rate in the body. The premature absorption of the membrane leads to the loss of its mechanical strength and the inability to support and maintain the bone forming space, and it is easy to cause premature ingrowth of fibrous tissue, thus interfering with bone regeneration in the surgical area. Although the GBR membranes prepared from absorbable synthetic materials (including polylactic acid, polycaprolactone, polyglycolic acid, etc.) have a slower degradation rate, they are still limited in clinical use due to their piezoelectric activity, membrane adhesion, and adhesiveness being lower than those of collagen membranes.

[0004] CN107029293A discloses a pericardial collagen membrane for guided bone regeneration, its preparation method and uses. This pericardial membrane is crosslinked after being treated by a unique decellularization technique and compounded with chitosan. The preparation method of the pericardial collagen membrane is as follows: removing large pieces of fat from porcine pericardium, then degreasing, soaking in alkali, removing alkali, enzymatic hydrolysis, compounding, crosslinking, and disinfecting. The pericardial membrane in this patent document has good biocompatibility and can extend the degradation time, but glutaraldehyde needs to be used as the crosslinking agent, and the introduction of chemical crosslinking agents poses a risk of potential toxic and side effects to the human body.

[0005] CN114732962A discloses a degradable antibacterial guided bone regeneration membrane, its preparation method and applications. The preparation method includes the following steps: (1) adding hydroxyapatite, a pore-forming agent, and ZIF-8 nanoparticles loaded with Cu 2+ into a polyester solution, and mixing evenly to obtain a casting solution; (2) casting the casting solution into a film, soaking it in deionized water and then taking it out and drying to obtain a degradable antibacterial guided bone regeneration membrane. The guided bone regeneration membrane obtained in this patent document focuses on antibacterial properties and does not involve hydrophilicity and piezoelectric activity.

[0006] CN115814170A discloses a preparation method and applications of a cross-scale bionic composite repair body for guided periodontal tissue regeneration. The preparation method includes: first, blending a polymer solution and a nano-reinforcement solution to prepare a blended sol, and constructing a bionic-structured composite film through assembly; then depositing a polyethylene glycol / polymer mixed layer on one side of the film, and performing sufficient chemical modification after drying; removing polyethylene glycol with hot water to obtain a rough surface, and spraying an inert polymer solution on this rough surface, and obtaining a heterogeneous bionic-structured material after mineralization treatment. The hydrophilicity of the repair body obtained in this patent document still needs to be improved, and it does not involve piezoelectric activity.

[0007] CN118267538A discloses an oral repair membrane and its preparation method. The oral repair membrane includes: a matrix, which is formed by overlapping and winding fiber filaments with diameters ranging from 10 nm to 100 μm and has a porous structure; and active particles, which are loaded on the surface and / or inside of the matrix, and the active particles are derived from piezoelectric materials (polyhydroxyalkanoates) and / or hydrophilic polymer materials. Although the softness of the oral repair membrane in this patent document is relatively small, its hydrophilicity and piezoelectric activity still need to be further improved.

[0008] CN117919520A discloses a nano-composite film for guided bone tissue regeneration, its preparation method and application. The composite film includes a polymer (one or more of polyvinyl alcohol, sodium alginate, chitosan and polylactic acid), a bioactive ceramic material (one or more of hydroxyapatite, β-tricalcium phosphate, bioactive glass and zirconia toughened alumina ceramic), and two-dimensional nano-materials (one or more of MXene, graphene oxide, reduced graphene oxide and MoS 2 (one or more of 2)); hydrogen bonds are formed between the polymer, the bioactive ceramic material and the two-dimensional nano-materials. The composite film of this patent document has good toughness, but its hydrophilicity still needs to be further improved, and piezoelectric activity is not involved. Summary of the Invention

[0009] In view of this, an object of the present invention is to provide a preparation method of a bilayer oral repair film material. The bilayer oral repair film material obtained by this preparation method has good hydrophilicity, piezoelectric properties, and good softness on the basis of being absorbable and degradable. Another object of the present invention is to provide a bilayer oral repair film material prepared by the above-mentioned preparation method.

[0010] The present invention adopts the following technical solutions to achieve the above objects.

[0011] On the one hand, the present invention provides a preparation method of a bilayer oral repair film material, including the following steps:

[0012] 1) Dispersing poly lactic acid microspheres containing osteogenic active materials in an aqueous solution of methacrylated protein to obtain a first mixture; spreading the first mixture and performing ultrasonic vibration to obtain a polymerized protein film; drying the polymerized protein film to obtain a protein film;

[0013] 2) Ultrasonically treating nano-scale osteogenic active material powder and nano-piezoelectric inorganic substances in an aqueous solution of a surface modifier to obtain surface-modified nano-powders; mixing the surface-modified nano-powders, poly-L-lactic acid and a second solvent, and performing ultrasonic vibration to obtain an electrospinning solution; wherein, the nano-piezoelectric inorganic substances are selected from nano-barium titanate or nano-zinc oxide; the aqueous solution of the surface modifier is selected from one of an aqueous solution of polyethylene glycol, an aqueous solution of polyglycolic acid and an aqueous solution of sodium citrate;

[0014] 3) Forming a fibrous film on the protein film by electrospinning the electrospinning solution, then washing and freeze-drying to obtain a bilayer oral repair film material;

[0015] Among them, steps 1) and 2) are not in a sequential order.

[0016] According to the preparation method of the present invention, preferably, in step 1), the nano-level osteogenic active material powder, polylactic acid, and pore-forming agent are mixed and heated to form a melt; the melt is sprayed into a first solvent to form microspheres, and polylactic acid microspheres containing the osteogenic active material are obtained;

[0017] Among them, the nano-level osteogenic active material powder is selected from one or more of nano-level bioactive glass, nano-level hydroxyapatite, and nano-level β-tricalcium phosphate;

[0018] Among them, the polylactic acid is selected from one or more of poly-L-lactic acid and poly-D-lactic acid, and must contain poly-L-lactic acid;

[0019] Among them, the pore-forming agent is selected from one or more of polyethylene glycol, polyglycolic acid, tributyl citrate, and polyhydroxybutyric acid;

[0020] Among them, the mass ratio of the nano-level osteogenic active material powder, polylactic acid, and pore-forming agent is 5-15:80-120:5-25.

[0021] According to the preparation method of the present invention, preferably:

[0022] The first solvent is selected from one or more of water, ethanol, and dimethyl sulfoxide;

[0023] The polylactic acid is a mixture of poly-D-lactic acid and poly-L-lactic acid; the mass ratio of the nano-level osteogenic active material powder, poly-D-lactic acid, poly-L-lactic acid, and pore-forming agent is 5-15:40-60:40-60:5-25.

[0024] According to the preparation method of the present invention, preferably, the methacrylated protein aqueous solution is a solution obtained by mixing methacrylated protein and water at a mass ratio of 1:3-1:10; among them, the methacrylated protein is selected from one of methacrylated type I collagen, methacrylated type II collagen, methacrylated silk fibroin, methacrylated recombinant collagen, and methacrylated gelatin.

[0025] According to the preparation method of the present invention, preferably, in the first mixture, the concentration of the polylactic acid microspheres containing the osteogenic active material is 1-10 wt%.

[0026] According to the preparation method of the present invention, preferably, in step 2), the nano-scale osteogenic active material powder and the nano-piezoelectric inorganic substance are ultrasonically treated in an aqueous solution of a surface modifier, followed by solid-liquid separation and drying to obtain the surface-modified nano-powder; wherein, the nano-scale osteogenic active material powder is selected from one or more of nano-scale bioactive glass, nano-scale hydroxyapatite, and nano-scale β-tricalcium phosphate; the mass ratio of the nano-scale osteogenic active material powder to the nano-piezoelectric inorganic substance is 1 to 1.05:1.

[0027] According to the preparation method of the present invention, preferably, in step 2), the mass-volume concentration of the aqueous solution of the surface modifier is 2 to 8% (w / v); the mass ratio of the surface-modified nano-powder to poly-L-lactic acid is 1:10 to 1:100.

[0028] According to the preparation method of the present invention, preferably, the second solvent is selected from one of hexafluoroisopropanol, N,N-dimethylformamide, trifluoroethanol, tetrahydrofuran, and dichloromethane.

[0029] According to the preparation method of the present invention, preferably, the parameters of the electrospinning include: the rate of the micro-injection pump is 5 to 10 mL / h, the high-voltage is 18 to 30 kV, and the distance between the receiver and the needle of the injection pump is 20 to 25 cm.

[0030] On the other hand, the present invention also provides a bilayer oral repair membrane material, which is prepared according to the preparation method described above.

[0031] The bilayer oral repair membrane material prepared by the present invention is based on absorbable degradation, and also has good hydrophilicity, piezoelectric properties, and softness; among them, good hydrophilicity is beneficial to soft tissue adhesion and reduces the risk of gum atrophy / membrane exposure; good piezoelectric properties are beneficial to promoting osteogenesis, achieving high-quality bone regeneration and bone remodeling, and improving osteogenic efficiency; good softness is beneficial to the attachment of the overall membrane. Description of the Drawings

[0032] Figure 1 SEM image of the protein membrane of the first layer in Example 1.

[0033] Figure 2 SEM image of the fiber membrane of the second layer in Example 1. Detailed Description of the Invention

[0034] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0035] A preparation method of a double-layer oral repair membrane material of the present invention includes the following steps: 1) a step of preparing a protein membrane; 2) a step of preparing an electrospinning solution; 3) a step of forming the double-layer oral repair membrane material. Among them, steps 1) and 2) have no sequential order and can be carried out simultaneously. The following is a detailed description.

[0036] <Step of preparing the protein membrane>

[0037] In the present invention, polylactic acid microspheres containing osteogenic active materials are dispersed in a methacrylated protein aqueous solution to obtain a first mixture; the first mixture is spread out and subjected to ultrasonic vibration to obtain a polymerized protein membrane; the polymerized protein membrane is dried to obtain a protein membrane. This is beneficial to improving the hydrophilicity and piezoelectric properties of the obtained double-layer oral repair membrane material, and it can be absorbed and degraded, and the degradation time is longer than that of a simple collagen membrane.

[0038] In the present invention, the polylactic acid microspheres containing osteogenic active materials are prepared by the following steps: mixing nano-scale osteogenic active material powder, polylactic acid, and a pore-forming agent, and heating to form a melt; spraying the melt into a first solvent to form microspheres, obtaining polylactic acid microspheres containing osteogenic active materials. This is beneficial to forming uniform microspheres.

[0039] In this step, the mass ratio of the nano-scale osteogenic active material powder, polylactic acid, and the pore-forming agent is 5-15:80-120:5-25, preferably 5-12:80-110:8-20, and more preferably 5-10:80-100:10-15. This is beneficial to taking into account hydrophilicity and piezoelectric properties.

[0040] In this step, the nano-scale osteogenic active material powder is selected from one or more of nano-scale bioactive glass, nano-scale hydroxyapatite, and nano-scale β-tricalcium phosphate, preferably nano-scale bioactive glass. The particle size range is 20-150 nm. Bioactive glass is a silicate-based material, and its main components include SiO 2 、Na 2 O、CaO、P 2 O 5 . The source of bioactive glass is not particularly limited and can be commercially purchased.

[0041] In this step, the polylactic acid is selected from one or more of poly-L-lactic acid (PLLA) and poly-D-lactic acid (PDLA), and must contain poly-L-lactic acid. In certain specific embodiments, the polylactic acid is a mixture of poly-D-lactic acid and poly-L-lactic acid, and the mass ratio of poly-D-lactic acid to poly-L-lactic acid can be 40-60:40-60, preferably 40-55:40-55, more preferably 40-50:40-50, and the amount of poly-L-lactic acid is greater than or equal to the amount of poly-D-lactic acid. The molecular weight of the polylactic acid is 50,000-200,000 g / mol (which can be denoted as 50,000-200,000 Da). The optical purity of poly-D-lactic acid and poly-L-lactic acid can be greater than 95%.

[0042] According to an embodiment of the present invention, the mass ratio of the nano-scale osteogenic active material powder, poly-D-lactic acid, poly-L-lactic acid and the pore-forming agent can be 5-15:40-60:40-60:5-25.

[0043] In the present invention, the pore-forming agent can be selected from one or more of polyethylene glycol, polyglycolic acid, tributyl citrate and polyhydroxybutyric acid, preferably polyethylene glycol, and the molecular weight of polyethylene glycol is greater than or equal to 800 and less than or equal to 4000. For example, it can be polyethylene glycol 800 (denoted as PEG 800 ), polyethylene glycol 1000 (denoted as PEG 1000 ). The first solvent is selected from one or more of water, ethanol and dimethyl sulfoxide, preferably water.

[0044] According to an embodiment of the present invention, the nano-scale osteogenic active material powder, polylactic acid and the pore-forming agent are mixed, and then heated and blended in a torque rheometer at 190-220 °C for 10-20 min to form a melt; the melt is sprayed into the first solvent through a microfluidic extrusion device to form microspheres, and stirring is continued for 30 min-1 h, and then washed repeatedly with water at 35-45 °C to remove the residual pore-forming agent, and after washing, it is dried in vacuum to obtain the polylactic acid microspheres containing the osteogenic active material.

[0045] In the present invention, the aperture of the nozzle used in the microfluidic extrusion device is 20-50 μm.

[0046] In the present invention, the methacrylated protein aqueous solution is a solution obtained by mixing methacrylated protein and water in a mass ratio of 1:3-1:10. Stirring can be carried out during mixing until completely dissolved. The mass ratio of methacrylated protein to water is preferably 1:6-1:10, more preferably 1:8-1:9.

[0047] The methacrylated protein is selected from one of methacrylated type I collagen, methacrylated type II collagen, methacrylated silk fibroin, methacrylated recombinant collagen and methacrylated gelatin, preferably methacrylated gelatin.

[0048] In the first mixture of the present invention, the concentration of the poly(lactic acid) microspheres containing the osteogenic active material is 1 to 10 wt%, preferably 3 to 9 wt%, and more preferably 5 to 8 wt%.

[0049] In the present invention, the first mixture can be spread on a mold by solution casting, then left standing for 15 to 30 min, and then subjected to ultrasonic vibration in an ultrasonic oscillator to obtain a polymerized protein film. The frequency of the ultrasonic vibration is 50 to 300 kHz, preferably 150 to 300 kHz, and more preferably 240 to 300 kHz. In this way, a large number of free radicals can be generated by the reaction of the porous surface of the microspheres with water and air under the action of ultrasonic vibration, thereby catalyzing the free radical polymerization of the carbon-carbon double bonds on the side chains of the methacrylated protein.

[0050] In the present invention, the polymerized protein film is placed in a vacuum oven and dried at 45 to 55 °C for 2 to 4 h to obtain a protein film. The thickness of the protein film can be 200 to 500 μm.

[0051] <Steps for preparing the electrospinning solution>

[0052] In the present invention, the nano-scale osteogenic active material powder and the nano-piezoelectric inorganic substance are ultrasonically treated in an aqueous solution of a surface modifier to obtain surface-modified nano-powders; the surface-modified nano-powders, poly(L-lactic acid), and a second solvent are mixed and ultrasonically vibrated to obtain an electrospinning solution. This is beneficial to obtaining a bilayer oral repair membrane material with good softness and good piezoelectric properties.

[0053] In the present invention, the nano-piezoelectric inorganic substance is selected from nano-barium titanate or nano-zinc oxide, preferably nano-barium titanate. The particle size of the nano-piezoelectric inorganic substance is 50 to 150 nm.

[0054] In the present invention, the aqueous solution of the surface modifier is selected from one of an aqueous solution of polyethylene glycol, an aqueous solution of polyglycolic acid, and an aqueous solution of sodium citrate, preferably an aqueous solution of polyethylene glycol. The molecular weight of the polyethylene glycol in the aqueous solution of polyethylene glycol is 1000 to 4000. For example, it can be polyethylene glycol 2000 (denoted as PEG 2000 ). The mass-volume concentration of the aqueous solution of the surface modifier is 2 to 8% (w / v), preferably 3 to 8% (w / v), and more preferably 4 to 8% (w / v).

[0055] In this step, the nano-scale osteogenic active material powder is selected from one or more of nano-scale bioactive glass, nano-scale hydroxyapatite, and nano-scale β-tricalcium phosphate, preferably nano-scale bioactive glass. The particle size range is 20 to 150 nm. Bioactive glass is a silicate-based material, and its main components include SiO 2 、Na 2O, CaO, P 2 O 5 There is no particular limitation on the source of the bioactive glass, and it can be commercially available. The mass ratio of the nano-level osteogenic active material powder to the nano-piezoelectric inorganic substance is 1 to 1.05:1.

[0056] According to an embodiment of the present invention, the nano-level osteogenic active material powder and the nano-piezoelectric inorganic substance are placed in an aqueous solution of a surface modifier and ultrasonically treated, followed by solid-liquid separation and drying to obtain surface-modified nano-powders. The volume ratio of the sum of the masses of the nano-level osteogenic active material powder and the nano-piezoelectric inorganic substance to the aqueous solution of the surface modifier can be 1 g:8 to 12 mL, preferably 1 g:9 to 12 mL, for example, it can be 1 g:10 mL. In the present invention, the frequency of the ultrasonic treatment can be 30 to 80 kHz, preferably 45 to 80 kHz, more preferably 60 to 80 kHz. The temperature during the ultrasonic treatment can be 60 to 85 °C, preferably 65 to 85 °C, more preferably 75 to 80 °C. The time of the ultrasonic treatment can be 10 to 40 min, preferably 15 to 40 min, more preferably 20 to 30 min. After the ultrasonic treatment, solid-liquid separation is carried out. The solid-liquid separation can be filtration. After the solid-liquid separation, the solid is dried, and the drying temperature can be 55 to 85 °C.

[0057] In certain embodiments, the surface-modified nano-powders and poly(L-lactic acid) are mixed, and the mixture is added to a second solvent and ultrasonically vibrated for 1 to 3 h, and then continuously stirred in a closed container for 4 to 8 h to obtain an electrospinning solution. The frequency of the ultrasonic vibration can be 30 to 80 kHz, preferably 45 to 80 kHz, more preferably 60 to 80 kHz.

[0058] In the present invention, the mass ratio of the surface-modified nano-powders to poly(L-lactic acid) is 1:10 to 1:100, preferably 1:10 to 1:50, more preferably 1:10 to 1:25. The concentration of poly(L-lactic acid) in the electrospinning solution is 5 to 20 wt%, preferably 5 to 15 wt%, more preferably 5 to 10 wt%. In this step, the molecular weight of poly(L-lactic acid) can be 50,000 to 100,000 g / mol.

[0059] In the present invention, the second solvent is selected from one of hexafluoroisopropanol, N,N-dimethylformamide, trifluoroethanol, tetrahydrofuran, and dichloromethane, preferably hexafluoroisopropanol.

[0060] <Steps for forming the double-layer oral repair membrane material>

[0061] An electrospinning solution is used to form a fibrous membrane on the protein membrane by electrospinning, and then it is washed and freeze-dried to obtain the double-layer oral repair membrane material. This is beneficial for obtaining a repair membrane with a thickness within a specific range and for improving the hydrophilicity and piezoelectric properties of the obtained repair membrane material.

[0062] The parameters of the electrospinning include: the rate of the micro-injection pump is 5-10 mL / h, the high-voltage is 18-30 kV, and the distance between the receiver and the needle of the injection pump is 20-25 cm. The receiver can be a plate-shaped receiver.

[0063] In some specific embodiments, the protein film can be fixed on the plate-shaped receiver, and an electrospun solution is formed into a fibrous film on the protein film by electrospinning. After electrospinning, washing is carried out. The washing can be carried out with water for injection multiple times to remove the residual second solvent, and then vacuum freeze-drying is carried out at a temperature below -30 °C for 20-36 h.

[0064] The present invention also provides a bilayer oral repair membrane material, which is prepared according to the preparation method described above. The thickness of the bilayer oral repair membrane material is 400 μm-1 mm, preferably 500-900 μm, and more preferably 500-890 μm.

[0065] The first layer of the bilayer oral repair membrane material of the present invention is a cross-linked protein film containing poly(lactic acid) / osteogenic active material composite microspheres, which contacts the gingiva during use. The protein film has good hydrophilicity, which is beneficial to the adhesion and growth of tissue cells and reduces the risk of gingival atrophy or membrane exposure; the second layer is an irregular poly(lactic acid) fiber layer containing piezoelectric / osteogenic active material powder, which contacts the bone defect site filled with bone powder during use and has good piezoelectric effect, which is beneficial to promoting osteogenesis. Moreover, the bilayer oral repair membrane material of the present invention has good softness and good overall adhesion.

[0066] The test methods used in the following examples are described as follows:

[0067] SEM test: Test is carried out using ZEISS GeminiSEM 300.

[0068] The raw materials used in the following examples are described as follows:

[0069] Methacrylated gelatin, nano-scale bioactive glass powder, poly(L-lactic acid) (PLLA), poly(D-lactic acid) (PDLA) are from Chengdu Meiyi Boya Material Technology Co., Ltd. Nano-barium titanate is purchased from Nanjing Hongde Nano Materials Co., Ltd.

[0070] Preparation Example 1

[0071] The nano-scale bioactive glass powder, PDLA, PLLA and PEG 1000Weigh the materials according to a mass ratio of 10:40:40:10, then add them to a rheometer, heat up to 200 °C and blend for 20 min to obtain a uniform melt. Among them, the particle size of the nano-bioactive glass powder is 20 nm, and the molecular weights of PDLA and PLLA are both 100 kDa. Spray the melt into purified water (the first solvent) through a microfluidic extruder with a diameter of 10 μm, stir for 1 h, and then repeatedly rinse with purified water 5 times to remove the residual PEG. 1000 Then, dry it in a vacuum at 50 °C for 8 h to obtain poly(lactic acid) microspheres containing osteogenic active materials.

[0072] Example 1

[0073] 1) Disperse the poly(lactic acid) microspheres containing osteogenic active materials prepared in Preparation Example 1 in an aqueous solution of methacrylated protein (an aqueous solution formed by mixing methacrylated gelatin and water in a ratio of 1:8 and stirring at 37 °C until completely dissolved). The concentration of the poly(lactic acid) microspheres is 8 wt%, and stir magnetically at room temperature for 15 min until the microspheres are dispersed to obtain a first mixture; spread the first mixture on a mold by the solution casting method, let it stand at room temperature for 20 min, and then ultrasonically vibrate it in an ultrasonic oscillator for 15 min, with a vibration frequency of 240 kHz, to obtain a polymerized protein film; place the polymerized protein film in a vacuum drying oven and dry it at 50 °C for 3 h to obtain a protein film, which is the protein film of the first layer. Its thickness is 450 μm. The SEM results of the protein film of the first layer are shown in Figure 1 .

[0074] 2) Mix the nano-bioactive glass powder and nano-barium titanate evenly according to a mass ratio of 1:1, then place them in an aqueous solution of polyethylene glycol (8% (w / v), PEG 2000 )), ultrasonically treat them at 80 °C for 30 min, filter, wash the filter cake with purified water, and dry the filter cake to obtain surface-modified nano-powders. Mix the surface-modified nano-powders and PLLA at a mass ratio of 1:25, then add them to hexafluoroisopropanol (the second solvent) and mix, ultrasonically vibrate for 1 h, and then continue to stir in a closed container at room temperature for 6 h to form an electrospinning solution with a PLLA concentration of 10 wt%; among them, the particle size of nano-barium titanate is 50 nm, the particle size of the nano-bioactive glass powder is 20 nm, the molecular weight of PLLA is 70 kDa, and the optical purity is greater than 95%. Among them, the ratio of the sum of the masses of the nano-bioactive glass powder and nano-barium titanate to the volume of the aqueous solution of polyethylene glycol is 1 g:10 mL.

[0075] 3) Fix the protein film on a plate receiver, form a fibrous film on the protein film by electrospinning the electrospinning solution, then wash it 3 times with deionized water to remove the residual solvent, and freeze-dry it at -30 °C for 24 h to obtain a bilayer oral repair membrane material. The thickness is 890 μm. The parameters of electrospinning are as follows: the rate of the micro-injection pump is 8 mL / h, the high-voltage is 25 kV, and the distance between the receiver and the needle of the injection pump is 23 cm. The SEM results of the fibrous film of the second layer are shown in Figure 2 .

[0076] Preparation Example 2

[0077] Weigh nano-scale bioactive glass powder, PDLA, PLLA and PEG 1000 in a mass ratio of 5:40:40:15, then add them to a rheometer, heat up to 210 °C and blend for 15 min to obtain a uniform melt. Among them, the particle size of the nano-scale bioactive glass powder is 20 nm, and the molecular weights of PDLA and PLLA are both 100 kDa. Spray the melt into purified water (the first solvent) through a microfluidic extruder with a diameter of 10 μm, stir for 1 h, and then rinse repeatedly with purified water 5 times to remove the residual PEG 1000 , and then vacuum dry at 50 °C for 8 h to obtain poly(lactic acid) microspheres containing osteogenic active materials.

[0078] Example 2

[0079] 1) Disperse the poly(lactic acid) microspheres containing osteogenic active materials prepared in Preparation Example 2 in an aqueous solution of methacrylated protein (an aqueous solution formed by mixing methacrylated gelatin and water in a ratio of 1:9 and stirring at 37 °C until completely dissolved). The concentration of the poly(lactic acid) microspheres is 5 wt%, and stir magnetically at room temperature for 15 min until the microspheres are dispersed to obtain a first mixture; spread the first mixture on a mold by the solution casting method, let it stand at room temperature for 20 min, and then ultrasonically vibrate it in an ultrasonic oscillator for 15 min, with a vibration frequency of 240 kHz, to obtain a polymerized protein film; place the polymerized protein film in a vacuum drying oven and dry it at 50 °C for 3 h to obtain a protein film. Its thickness is 330 μm.

[0080] 2) Mix nano-scale bioactive glass powder and nano-barium titanate with a mass ratio of 1:1 evenly, and then place them in an aqueous solution of polyethylene glycol (8% (w / v), PEG 2000) In it, ultrasonic treatment is carried out at 80 °C for 40 min, then filtered, the filter cake is washed with purified water, and the filter cake is dried to obtain surface-modified nano powder. The surface-modified nano powder is mixed with PLLA at a mass ratio of 1:10, and then added to hexafluoroisopropanol (the second solvent) for mixing, ultrasonic vibration is carried out for 1 h, and then stirring is continued for 6 h at room temperature in a closed container to form an electrospinning solution with a PLLA concentration of 5 wt%; among them, the particle size of barium titanate nano is 50 nm, the particle size of nano bioactive glass powder is 20 nm, the molecular weight of PLLA is 70 kDa, and the optical purity is greater than 95%. Among them, the ratio of the sum of the masses of nano bioactive glass powder and barium titanate nano to the volume of polyethylene glycol aqueous solution is 1 g:10 mL.

[0081] 3) Fix the protein film on a plate receiver, form a fibrous film on the protein film by electrospinning the electrospinning solution, then wash it 3 times with water for injection to remove the residual solvent, and freeze-dry it at -30 °C for 24 h to obtain a double-layer oral repair film material. The thickness is 820 μm. The parameters of electrospinning are: the rate of the micro-injection pump is 8 mL / h, the high-voltage is 25 kV, and the distance between the receiver and the needle of the injection pump is 23 cm.

[0082] Example 3

[0083] Carry out according to the steps of Example 1. The thickness of the obtained protein film is 210 μm.

[0084] The thickness of the obtained double-layer oral repair film material is 830 μm.

[0085] Example 4

[0086] Carry out according to the steps of Example 1. The thickness of the obtained protein film is 210 μm.

[0087] The thickness of the obtained double-layer oral repair film material is 500 μm.

[0088] Comparative Example 1

[0089] The difference from Example 1 is only that step 2) is different. In this comparative example, barium titanate nano and nano bioactive glass powder are not added, and PLLA is used to form an electrospinning solution. The thickness of the obtained double-layer film is 860 μm.

[0090] Comparative Example 2

[0091] The difference from Example 1 is only that step 1) is not included. In this comparative example, only a fibrous film is formed, and the protein film is not involved.

[0092] Experimental Example

[0093] 1. Dielectric constant test

[0094] In the present invention, a quasi-static d33 tester is used to directly measure the piezoelectric constants of the film materials of Examples 1 to 4 and the comparative examples. None of the film materials are polarized. Five points are taken for each sample for measurement and the average value is taken. The results are shown in Table 1.

[0095] Table 1

[0096] Serial Number Piezoelectric Constant d33 (pC / N) Example 1 2.25±0.31 Example 2 2.17±0.26 Example 3 2.23±0.18 Example 4 2.18±0.23 Comparative Example 1 0.91±0.05 Comparative Example 2 1.31±0.07 Pure PLLA Electrospun Membrane 0.75±0.09

[0097] As can be seen from Table 1, the double-layer repair film materials obtained in the present invention have relatively high piezoelectric constants, reaching more than 2.1 pC / N, and are significantly improved without polarization. This is beneficial to promoting osteogenesis and improving the osteogenesis efficiency.

[0098] 2. Hydrophilicity test

[0099] In the present invention, a contact angle tester is used to measure the surface contact angles of the double-layer repair films (protein films on the gingiva side) obtained in Examples 1 to 4 and pure PLLA electrospun films prepared by an electrospinning process at room temperature. Three samples are tested for each group of samples, and three positions are tested for each sample. The average value is calculated. The test results are shown in Table 2 below.

[0100] Table 2

[0101] Serial Number Contact Angle Example 1 43.18°±2.95° Example 2 45.26°±2.78° Example 3 43.95°±2.57° Example 4 44.09°±2.36° Pure PLLA Electrospun Membrane 91.53°±3.71°

[0102] As can be seen from Table 2, the protein film on the gingiva side of the double-layer repair film material of the present invention has good hydrophilicity and shows good adhesion to the surface of endothelial cells. Therefore, the inventor believes that the repair film prepared by the present invention is beneficial to the adhesion of endothelial cells, thereby reducing the risk of gingival recession / membrane exposure.

[0103] 3. Softness test

[0104] In the present invention, the softness of Examples 1 to 4 is measured according to the method of GB / T 8942-2002. The softness value is the maximum vector sum of the bending resistance of the film material and the frictional force at the gap, expressed in millinewtons (mN). The smaller the softness value, the softer the film material and the better the corresponding adhesiveness. When the softness of the film material is below 1000 mN, it has good adhesiveness. The results are shown in Table 3.

[0105] Table 3

[0106] Serial Number Softness (mN) Example 1 753 Example 2 740 Example 3 894 Example 4 761

[0107] As shown in Table 3, the double-layer repair membrane materials of the present invention have good softness. However, when the fiber membrane of the second layer accounts for a higher proportion, its softness becomes worse, which may be because the fiber membrane of the second layer has a higher proportion of polylactic acid, which has a certain brittleness and affects the softness of the finished product.

[0108] In addition, the average pore size of the fiber membrane of comparative example 2 was measured to be 1.2 μm, and the porosity was 93%. The present invention controls the electrospinning parameters to obtain a fiber membrane with irregular pores and a sufficiently small average pore size (1.2 μm) to achieve a better physical barrier effect and maintain the stability of the osteogenic space.

[0109] In summary, the piezoelectric constant of the double-layer oral repair membrane material prepared by the present invention can be improved without polarization; in addition, it has good hydrophilicity and low softness. The double-layer oral repair membrane material prepared by the present invention has good piezoelectric properties, which is beneficial to promote osteogenesis and achieve high-quality bone regeneration and bone remodeling and reconstruction; it has good hydrophilicity and good softness, which is beneficial to soft tissue adhesion, reduces the risk of gingival atrophy / membrane exposure, and is beneficial to the attachment of the overall membrane.

[0110] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A method for preparing a double-layer oral repair membrane material, characterized in that: The steps include: 1) mixing nano-scale osteogenic active material powder, polylactic acid and a pore-forming agent, and heating to form a melt; spraying the melt into a first solvent to form microspheres, thereby obtaining polylactic acid microspheres containing osteogenic active material; dispersing the polylactic acid microspheres containing osteogenic active material in a methacryloyl protein aqueous solution to obtain a first mixture; spreading the first mixture, and ultrasonically vibrating to obtain a polymerized protein film; and drying the polymerized protein film to obtain a protein film; 2) placing nano-scale osteogenic active material powder and nano-piezoelectric inorganic substance in a surface modifier aqueous solution and subjecting it to ultrasonic treatment to obtain surface-modified nano-powder; mixing the surface-modified nano-powder, poly-L-lactic acid and a second solvent, subjecting them to ultrasonic vibration to obtain an electrospinning solution; wherein the nano-piezoelectric inorganic substance is selected from nano-barium titanate or nano-zinc oxide; and the surface modifier aqueous solution is selected from one of a polyethylene glycol aqueous solution, a polyglycolic acid aqueous solution and a sodium citrate aqueous solution; 3) electrospinning the electrospinning solution to form a fiber membrane on the protein membrane, followed by washing and freeze-drying to obtain a double-layer oral repair membrane material; Among them, step 1) and step 2) are in no particular order; Wherein, the nano-scale osteogenic active material powder is selected from one or more of nano-scale bioactive glass, nano-scale hydroxyapatite and nano-scale β-tricalcium phosphate; the polylactic acid is selected from one or more of poly-L-lactic acid and poly-D-lactic acid, and must contain poly-L-lactic acid; the pore-forming agent is selected from one or more of polyethylene glycol, polyglycolic acid, tributyl citrate and polyhydroxybutyric acid; The mass ratio of nano-scale osteogenic active material powder, polylactic acid and pore-forming agent is 5-15:80-120:5-25; The methacrylylated protein aqueous solution is a solution obtained by mixing methacrylylated protein and water in a mass ratio of 1:3 to 1:10, and the methacrylylated protein is selected from one of methacrylylated type I collagen, methacrylylated type II collagen, methacrylylated silk fibroin, methacrylylated recombinant collagen and methacrylylated gelatin.

2. The preparation method according to claim 1, characterized in that: The first solvent is selected from one or more of water, ethanol and dimethyl sulfoxide; The polylactic acid is a mixture of poly (dextrorotatory) lactic acid and poly (l-lactic acid); the mass ratio of the nano-scale osteogenic active material powder, poly (dextrorotatory) lactic acid, poly (l-lactic acid) and pore-forming agent is 5-15:40-60:40-60:5-25.

3. The preparation method according to claim 1, characterized in that: In the first mixture, the concentration of the polylactic acid microspheres containing the osteogenic active material is 1-10 wt %.

4. The preparation method according to claim 1, characterized in that: In step 2), the nano-scale osteogenic active material powder and the nano-piezoelectric inorganic substance are placed in an aqueous solution of a surface modifier for ultrasonic treatment, solid-liquid separation, and drying to obtain a surface-modified nano-powder; wherein the nano-scale osteogenic active material powder is selected from one or more of nano-scale bioactive glass, nano-scale hydroxyapatite and nano-scale β-tricalcium phosphate; the mass ratio of the nano-scale osteogenic active material powder to the nano-piezoelectric inorganic substance is 1 to 1.05:

1.

5. The preparation method according to claim 4, characterized in that: In step 2), the mass volume concentration of the surface modifier aqueous solution is 2 to 8% (w / v); the mass ratio of the surface modified nanopowder to the poly-L-lactic acid is 1:10 to 1:

100.

6. The preparation method according to claim 1, characterized in that: The second solvent is selected from one of hexafluoroisopropanol, N,N-dimethylformamide, trifluoroethanol, tetrahydrofuran and dichloromethane.

7. The preparation method according to claim 1, characterized in that: The parameters of the electrospinning include: the rate of the microinjection pump is 5-10 mL / h, the high voltage is 18-30 kV, and the distance between the receiver and the injection pump needle is 20-25 cm.

8. A double-layer oral repair membrane material, characterized in that: It is prepared according to the preparation method according to any one of claims 1 to 7.

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