Antibacterial oral repair composite film and preparation method thereof

Abstract

The present invention relates to the technical field of oral repair materials, and in particular to an antibacterial oral repair composite membrane and a preparation method thereof. The oral repair composite membrane is composed of an upper antibacterial membrane, a middle polycaprolactone fiber membrane, and a lower bone repair membrane; the raw materials for preparing the upper antibacterial membrane include the following components in parts by weight: 3-4 parts of cinnamon essential oil, 0.5-1 parts of xanthan gum, 0.5-1 parts of seed meal protein, 0.1-0.15 parts of tannic acid, 5-7 parts of silk fibroin, 1-1.5 parts of polylactic acid, 0.6-1 parts of chitosan, 1-1.5 parts of polyvinyl alcohol, and 1.5-2.5 parts of glycerol; the raw materials for preparing the lower bone repair membrane include the following components in parts by weight: 2-3 parts of nanohydroxyapatite, 3-5 parts of citric acid, 6-9 parts of lactic acid, 3-4 parts of tyrosine, 1-2 parts of glycolic acid, 2-3 parts of casein, 0.2-0.3 parts of hyperoside, and 1-2 parts of gelatin.

Description

Technical Field

[0001] The present invention relates to the technical field of oral restoration materials, and in particular to an antibacterial oral restoration composite film and a preparation method thereof. Background Art

[0002] In daily life, defects or loss of hard and soft tissues in the maxillofacial region can occur due to disease, trauma, and physiological degeneration. This can affect the morphological integrity of oral tissues and organs, and consequently, their normal function and aesthetic appearance. Oral prosthetic membranes create a biological barrier between oral soft tissue and bone defects, providing a favorable repair space for bone regeneration. They selectively block the entry of rapidly migrating fibroblasts and epithelial cells into the bone defect area, while promoting or even inducing bone regeneration and oral wound healing. Current oral prosthetic membranes suffer from the following issues: insufficient antibacterial properties. The oral cavity is a microbially rich environment, making it easy for bacteria to adhere and proliferate on the membrane surface, leading to infection and compromising repair effectiveness. They also suffer from poor bone integration. While oral prosthetic membranes made solely of materials such as polycaprolactone offer biocompatibility, good mechanical properties, and biodegradability, their ability to promote osteogenesis in bone defects is limited, making them inadequate for clinical needs. Furthermore, due to insufficient mechanical strength, some oral prosthetic membranes may become too soft and easily deform during use, failing to provide adequate support for bone regeneration and compromising repair effectiveness. With increasing attention to oral health, demand for oral prosthetic membranes is growing. Patients are increasingly demanding the performance of oral restoration membranes. They not only expect them to effectively repair oral tissue defects, but also to possess excellent antibacterial properties to reduce the risk of infection and enhance the restoration effect. Therefore, developing an oral restoration composite membrane with excellent antibacterial properties and a good restoration effect has important clinical significance and market value. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the present invention provides an antibacterial oral repair composite membrane and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] The invention discloses an antibacterial oral repair composite membrane, which consists of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane.

[0006] Furthermore, the middle-layer polycaprolactone fiber membrane is prepared using polycaprolactone as the main raw material through an electrospinning process. The preparation method includes the following steps: dissolving 8-12 parts by weight of polycaprolactone in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersing at 400-500W for 10-15 min to obtain a spinning solution with a concentration of 10wt%, and spraying the spinning solution onto a receiving roller with a rotation speed of 100-150 r / min through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm to form a middle-layer polycaprolactone fiber membrane.

[0007] Furthermore, the raw materials for preparing the upper antibacterial film include the following components in parts by weight: 3-4 parts of cinnamon essential oil, 0.5-1 part of xanthan gum, 0.5-1 part of seed meal protein, 0.1-0.15 part of tannic acid, 5-7 parts of silk fibroin, 1-1.5 parts of polylactic acid, 0.6-1 part of chitosan, 1-1.5 parts of polyvinyl alcohol, and 1.5-2.5 parts of glycerol.

[0008] Furthermore, the method for preparing the upper antibacterial film comprises the following steps:

[0009] A1: Sea buckthorn seed meal was dried and crushed through an 80-100 mesh sieve, added to ultrapure water, shaken at 45°C and 300 rpm for 1 hour, and centrifuged at 6000 r / min for 20 minutes. 1 mol / L sodium chloride was added to the precipitate, shaken at 30°C and 300 rpm for 40 minutes, and centrifuged at 6000 r / min for 20 minutes. 70-80 vol% ethanol was added to the precipitate, shaken at 40°C and 300 rpm for 1.5 hours, and centrifuged at 6000 r / min for 20 minutes. The supernatant was evaporated and concentrated, dialyzed in deionized water for 24 hours, and freeze-dried to obtain seed meal protein.

[0010] A2: Add the seed meal protein obtained in step A1 to an 80 vol% ethanol solution, and stir at 400-500 r / min for 10-15 min to obtain a seed meal protein solution. Add xanthan gum to deionized water, and stir at 700-800 r / min for 20-30 min to obtain a xanthan gum solution.

[0011] A3: Add the xanthan gum solution obtained in step A2 to the seed meal protein solution, stir at 70°C and 600-700 rpm for 3-4 hours, add tannic acid, and continue stirring for 1 hour to obtain a seed meal protein / xanthan gum / tannic acid complex solution, add cinnamon essential oil dropwise, and homogenize at 10,000-12,000 rpm for 4-6 minutes to obtain an antibacterial emulsion;

[0012] A4: Place the middle polycaprolactone fiber membrane at the bottom of the mold, mix 15-20wt% silk fibroin solution with chitosan and polylactic acid, stir at 300-400 rpm for 1-2 h, mix with the antibacterial emulsion obtained in step A3, add polyvinyl alcohol and glycerol, stir evenly, pour into the mold, and vacuum dry at 30-40°C to obtain the upper antibacterial membrane.

[0013] Furthermore, in step A2, the mass concentration of the seed meal protein in the ethanol solution is 10 mg / mL.

[0014] Furthermore, in step A2, the mass concentration of the xanthan gum in deionized water is 10 mg / mL.

[0015] Furthermore, the raw materials for preparing the lower bone repair membrane include the following components in parts by weight: 2-3 parts of nanohydroxyapatite, 3-5 parts of citric acid, 6-9 parts of lactic acid, 3-4 parts of tyrosine, 1-2 parts of glycolic acid, 2-3 parts of casein, 0.2-0.3 parts of hyperoside, and 1-2 parts of gelatin.

[0016] Furthermore, the preparation method of the lower bone repair membrane comprises the following steps:

[0017] B1: Disperse 40 nm nanohydroxyapatite in DMF, stir at 100-120°C for 30 min, add citric acid, stir at 200-300 rpm for 2-3 h, cool to room temperature, centrifuge at 8000 rpm for 10-15 min, wash the precipitate with deionized water, and vacuum dry to obtain modified hydroxyapatite;

[0018] B2: Under nitrogen protection, lactic acid, tyrosine, glycolic acid, modified hydroxyapatite and stannous chloride were added to toluene and refluxed at 160°C for 2-3 h. During the reaction, stirring was continued at 200-300 rpm. Toluene was removed by rotary evaporation. -3 MPa, 160-180 ° C for 4-5 hours, dissolved in dichloromethane, slowly added to ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried in vacuo at 50-60 ° C to obtain a lactic acid-tyrosine-hydroxyapatite copolymer;

[0019] B3: Add casein to a pH 6.8 0.01 M PBS solution and stir magnetically overnight. Adjust the pH to 9 with 1 mol / L NaOH. Add hyperoside to deionized water and mix thoroughly. Adjust the pH to 9. Mix the two solutions and stir at 100-150 rpm for 24 h. Dialyze with deionized water for 48 h, changing the water every 6 h. Vacuum freeze-dry the dialyzate to obtain modified casein.

[0020] B4: Add lactic acid-tyrosine-hydroxyapatite copolymer, modified casein and gelatin to hexafluoroisopropanol, ultrasonicate at 300-400W for 30 min to obtain 10-15wt% electrospinning solution, which is then sprayed onto the other side of the middle layer of polycaprolactone fiber membrane by electrospinning to form the lower layer bone repair membrane.

[0021] Furthermore, in step B1, the mass concentration of the nano-hydroxyapatite in DMF is 0.1-0.15 g / mL.

[0022] Furthermore, in step B2, the amount of stannous chloride used is 0.6-0.8 wt % of the lactic acid.

[0023] Furthermore, in step B3, the mass concentration of the casein in PBS is 10 mg / mL.

[0024] Furthermore, in step B3, the mass concentration of the hyperoside in deionized water is 1-1.5 g / L.

[0025] Furthermore, in step B4, during the electrospinning treatment, the flow rate is 2-3 mL / h, the speed of the receiving device is 100-150 rpm, the voltage is 15-20 kV, and the receiving distance is 15 cm.

[0026] Furthermore, the present invention also provides a method for preparing the antibacterial oral repair composite membrane, comprising the following steps: placing the prepared upper antibacterial membrane, middle polycaprolactone fiber membrane and lower bone repair membrane under vacuum drying at 25°C for 24 hours, and hot pressing at 40°C and 20-30 MPa for 4-6 minutes to obtain the antibacterial oral repair composite membrane.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides an antibacterial composite membrane for oral restoration. The composite membrane utilizes a three-layer structure consisting of an upper antibacterial membrane, a middle polycaprolactone fiber membrane, and a lower bone repair membrane, endowing the composite membrane with multiple functions. The middle polycaprolactone fiber membrane exhibits excellent biocompatibility and provides excellent mechanical support. The upper antibacterial membrane effectively inhibits bacterial growth and prevents infection. The lower bone repair membrane promotes bone tissue repair, resulting in a comprehensive composite membrane for oral restoration. The upper antibacterial film contains cinnamon essential oil, tannic acid, chitosan and other antibacterial ingredients, which work synergistically to enhance the antibacterial effect and better prevent oral infections. Sea buckthorn seed meal protein is extracted to form a complex with xanthan gum and tannic acid, and cinnamon essential oil with antibacterial effect is added to form an emulsion loaded with cinnamon essential oil, which can inhibit the growth of oral pathogens. The synergistic effect of multiple ingredients makes the composite film have excellent antibacterial properties and helps maintain a healthy oral environment. Biocompatible materials such as silk fibroin, polylactic acid and chitosan are used as the base material. It can be well compatible with surrounding tissues in the oral environment, which is conducive to cell adhesion, proliferation and differentiation, reducing irritation and immune response to oral tissues, and promoting the repair and regeneration of oral tissues. Ingredients such as polyvinyl alcohol help to form a uniform and stable membrane structure, so that the film can fit tightly to the oral tissues and exert a sustained antibacterial and repair effect. Nanohydroxyapatite is a bioactive material with similar composition to human bone tissue, capable of inducing the growth and differentiation of bone cells and promoting the repair and regeneration of bone tissue. After modification with citric acid, carboxyl and hydroxyl groups are attached to the surface of the nanohydroxyapatite. The modified nanohydroxyapatite is then polymerized with monomers, incorporating the hydroxyapatite into the polymer. This improves its dispersibility in the composite material and its compatibility with other components, further enhancing bone repair. Casein is modified using an alkali treatment method and covalently bound to hyperoside, which can regulate cell activity and function, enhance oral repair capabilities, increase cross-linking, improve mechanical properties, and enhance antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The bone repair effects of the repair membranes described in Example 1 and Comparative Examples 1-4 of the present invention;

[0031] Figure 2 The antibacterial effect of the repair film described in Example 1 and Comparative Examples 1-4 of the present invention;

[0032] Figure 3The mechanical properties of the repair films described in Examples 1-3 and Comparative Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific examples, but the present invention is not limited to the following examples. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0034] Example 1: An antibacterial oral repair composite membrane, consisting of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane.

[0035] The middle-layer polycaprolactone fiber membrane is prepared with polycaprolactone as the main raw material by electrospinning process. The preparation method includes the following steps: 12 g of polycaprolactone is dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersed at 500W for 10 min to obtain a spinning solution with a concentration of 10wt%. The spinning solution is sprayed onto a receiving roller with a rotation speed of 150 r / min through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm to form a middle-layer polycaprolactone fiber membrane.

[0036] The raw materials for preparing the upper antibacterial film include the following components in parts by weight: 4 parts of cinnamon essential oil, 1 part of xanthan gum, 1 part of seed meal protein, 0.15 parts of tannic acid, 7 parts of silk fibroin, 1.5 parts of polylactic acid, 1 part of chitosan, 1.5 parts of polyvinyl alcohol, and 2.5 parts of glycerol.

[0037] The method for preparing the upper antibacterial film comprises the following steps:

[0038] A1: Sea buckthorn seed meal was dried and ground, passed through a 100-mesh sieve, added to ultrapure water, shaken at 45°C and 300 rpm for 1 hour, and centrifuged at 6000 rpm for 20 minutes. 1 mol / L sodium chloride was added to the precipitate, shaken at 30°C and 300 rpm for 40 minutes, and centrifuged at 6000 rpm for 20 minutes. 80 vol% ethanol was added to the precipitate, shaken at 40°C and 300 rpm for 1.5 hours, and centrifuged at 6000 rpm for 20 minutes. The supernatant was evaporated and concentrated, dialyzed in deionized water for 24 hours, and freeze-dried to obtain seed meal protein.

[0039] A2: Add 1 g of the seed meal protein obtained in step A1 to 100 mL of 80 vol% ethanol solution and stir at 500 rpm for 10 min to obtain a seed meal protein solution. Add 1 g of xanthan gum to 100 mL of deionized water and stir at 800 rpm for 20 min to obtain a xanthan gum solution.

[0040] A3: The xanthan gum solution obtained in step A2 was added to the seed meal protein solution, stirred at 70°C and 700 rpm for 3 h, 0.15 g of tannic acid was added, and stirring was continued for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution. 4 g of cinnamon essential oil was added dropwise, and homogenization was carried out at 12,000 rpm for 4 min to obtain an antibacterial emulsion.

[0041] A4: Place the middle polycaprolactone fiber membrane at the bottom of the mold, use 7 g of silk fibroin to prepare a 20 wt% silk fibroin solution, mix it with 1 g of chitosan and 1.5 g of polylactic acid, stir at 400 rpm for 1 h, mix it with the antibacterial emulsion obtained in step A3, add 1.5 g of polyvinyl alcohol and 2.5 g of glycerol, stir evenly, pour it into the mold, and vacuum dry it at 40°C to obtain the upper antibacterial membrane.

[0042] The raw materials for preparing the lower bone repair membrane include the following components in parts by weight: 3 parts of nano-hydroxyapatite, 5 parts of citric acid, 9 parts of lactic acid, 4 parts of tyrosine, 2 parts of glycolic acid, 3 parts of casein, 0.3 parts of hyperoside, and 2 parts of gelatin.

[0043] The preparation method of the lower bone repair membrane comprises the following steps:

[0044] B1: 3 g of 40 nm nanohydroxyapatite was dispersed in 20 mL of DMF, stirred at 120 °C for 30 min, 5 g of citric acid was added, and the mixture was stirred at 300 rpm for 2 h. The mixture was cooled to room temperature and centrifuged at 8000 rpm for 10 min. The precipitate was washed with deionized water and dried in vacuo to obtain modified hydroxyapatite.

[0045] B2: Under nitrogen protection, 9 g of lactic acid, 4 g of tyrosine, 2 g of glycolic acid, 72 mg of modified hydroxyapatite and stannous chloride were added to toluene and refluxed at 160 °C for 3 h. During the reaction, stirring was continued at 300 rpm. Toluene was removed by rotary evaporation. - 3 MPa, 180 ° C for 4 h, dissolved in dichloromethane, slowly added to ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried in vacuo at 60 ° C to obtain lactic acid-tyrosine-hydroxyapatite copolymer;

[0046] B3: Add 3 g of casein to 300 mL of a 0.01 M PBS solution at pH 6.8 and stir magnetically overnight. Adjust the pH to 9 with 1 mol / L NaOH. Add 0.3 g of hyperoside to 200 mL of deionized water and mix thoroughly. Adjust the pH to 9. Mix the two solutions and stir at 150 rpm for 24 h. Dialyze the solution against deionized water for 48 h, changing the water every 6 h. The dialyzate is vacuum freeze-dried to obtain modified casein.

[0047] B4: Lactic acid-tyrosine-hydroxyapatite copolymer, modified casein, and 2 g of gelatin were added to hexafluoroisopropanol and ultrasonicated at 400W for 30 min to obtain a 15wt% electrospinning solution, which was then sprayed onto the other side of the middle polycaprolactone fiber membrane by electrospinning to form a lower bone repair membrane. During the electrospinning process, the flow rate was 3 mL / h, the receiving device speed was 150 rpm, the voltage was 20 kV, and the receiving distance was 15 cm.

[0048] This embodiment also provides a method for preparing the antibacterial oral repair composite membrane, comprising the following steps: placing the prepared upper antibacterial membrane, middle polycaprolactone fiber membrane, and lower bone repair membrane under vacuum drying at 25°C for 24 h, and hot pressing at 40°C and 30 MPa for 4 min to obtain the antibacterial oral repair composite membrane.

[0049] Example 2: An antibacterial oral repair composite membrane, consisting of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane.

[0050] The middle-layer polycaprolactone fiber membrane is prepared with polycaprolactone as the main raw material by electrospinning process. The preparation method includes the following steps: 8 g of polycaprolactone is dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersed at 400W for 15 min to obtain a spinning solution with a concentration of 10wt%. The spinning solution is sprayed onto a receiving roller with a rotation speed of 100 r / min through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm to form a middle-layer polycaprolactone fiber membrane.

[0051] The raw materials for preparing the upper antibacterial film include the following components in parts by weight: 3 parts of cinnamon essential oil, 0.5 parts of xanthan gum, 0.5 parts of seed meal protein, 0.1 parts of tannic acid, 5 parts of silk fibroin, 1 part of polylactic acid, 0.6 parts of chitosan, 1 part of polyvinyl alcohol, and 1.5 parts of glycerol.

[0052] The method for preparing the upper antibacterial film comprises the following steps:

[0053] A1: Sea buckthorn seed meal was dried and ground, passed through an 80-mesh sieve, added to ultrapure water, shaken at 45°C and 300 rpm for 1 hour, and centrifuged at 6000 rpm for 20 minutes. 1 mol / L sodium chloride was added to the precipitate, shaken at 30°C and 300 rpm for 40 minutes, and centrifuged at 6000 rpm for 20 minutes. 70 vol% ethanol was added to the precipitate, shaken at 40°C and 300 rpm for 1.5 hours, and centrifuged at 6000 rpm for 20 minutes. The supernatant was evaporated and concentrated, dialyzed in deionized water for 24 hours, and freeze-dried to obtain seed meal protein.

[0054] A2: Add 0.5 g of the seed meal protein obtained in step A1 to 50 mL of 80 vol% ethanol solution, and stir at 400 rpm for 15 min to obtain a seed meal protein solution. Add 0.5 g of xanthan gum to 50 mL of deionized water, and stir at 700 rpm for 30 min to obtain a xanthan gum solution.

[0055] A3: The xanthan gum solution obtained in step A2 was added to the seed meal protein solution, stirred at 70°C and 600 rpm for 4 h, 0.1 g of tannic acid was added, and stirring was continued for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution. 3 g of cinnamon essential oil was added dropwise, and homogenization was carried out at 10,000 rpm for 6 min to obtain an antibacterial emulsion.

[0056] A4: Place the middle polycaprolactone fiber membrane at the bottom of the mold, use 5 g of silk fibroin to prepare a 15 wt% silk fibroin solution, mix it with 0.6 g of chitosan and 1 g of polylactic acid, stir at 300 rpm for 2 h, mix it with the antibacterial emulsion obtained in step A3, add 1 g of polyvinyl alcohol and 1.5 g of glycerol, stir evenly, pour it into the mold, and vacuum dry it at 30°C to obtain the upper antibacterial membrane.

[0057] The raw materials for preparing the lower bone repair membrane include the following components in parts by weight: 2 parts of nano-hydroxyapatite, 3 parts of citric acid, 6 parts of lactic acid, 3 parts of tyrosine, 1 part of glycolic acid, 2 parts of casein, 0.2 parts of hyperoside, and 1 part of gelatin.

[0058] The preparation method of the lower bone repair membrane comprises the following steps:

[0059] B1: 2 g of 40 nm nanohydroxyapatite was dispersed in 20 mL of DMF, stirred at 100 °C for 30 min, 3 g of citric acid was added, and the mixture was stirred at 200 rpm for 3 h. The mixture was cooled to room temperature and centrifuged at 8000 rpm for 10 min. The precipitate was washed with deionized water and dried in vacuo to obtain modified hydroxyapatite.

[0060] B2: Under nitrogen protection, 6 g of lactic acid, 3 g of tyrosine, 1 g of glycolic acid, modified hydroxyapatite and 36 mg of stannous chloride were added to toluene and refluxed at 160 °C for 2 h. During the reaction, stirring was continued at 200 rpm. Toluene was removed by rotary evaporation. - 3 MPa, 160 ° C for 5 h, dissolved in dichloromethane, slowly added to ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried in vacuo at 50 ° C to obtain lactic acid-tyrosine-hydroxyapatite copolymer;

[0061] B3: Add 2 g of casein to 2000 mL of a 0.01 M PBS solution at pH 6.8 and stir magnetically overnight. Adjust the pH to 9 with 1 mol / L NaOH. Add 0.2 g of hyperoside to 200 mL of deionized water and mix thoroughly. Adjust the pH to 9. Mix the two solutions and stir at 100 rpm for 24 h. Dialyze against deionized water for 48 h, changing the water every 6 h. The dialyzate is vacuum freeze-dried to obtain modified casein.

[0062] B4: Lactic acid-tyrosine-hydroxyapatite copolymer, modified casein, and 1 g of gelatin were added to hexafluoroisopropanol and ultrasonicated at 300W for 30 min to obtain a 10wt% electrospinning solution, which was then sprayed onto the other side of the middle polycaprolactone fiber membrane by electrospinning to form a lower bone repair membrane. During the electrospinning process, the flow rate was 2 mL / h, the receiving device speed was 100 rpm, the voltage was 15 kV, and the receiving distance was 15 cm.

[0063] This embodiment also provides a method for preparing the antibacterial oral repair composite membrane, comprising the following steps: placing the prepared upper antibacterial membrane, middle polycaprolactone fiber membrane, and lower bone repair membrane under vacuum drying at 25°C for 24 h, and hot pressing at 40°C and 20 MPa for 6 min to obtain the antibacterial oral repair composite membrane.

[0064] Example 3: An antibacterial oral repair composite membrane, consisting of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane.

[0065] The middle-layer polycaprolactone fiber membrane is prepared with polycaprolactone as the main raw material by electrospinning process. The preparation method includes the following steps: 10 g of polycaprolactone is dissolved in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersed at 450W for 12 min to obtain a spinning solution with a concentration of 10wt%. The spinning solution is sprayed onto a receiving roller with a rotation speed of 120 r / min through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm to form a middle-layer polycaprolactone fiber membrane.

[0066] The raw materials for preparing the upper antibacterial film include the following components in parts by weight: 3.5 parts of cinnamon essential oil, 0.8 parts of xanthan gum, 0.8 parts of seed meal protein, 0.12 parts of tannic acid, 6 parts of silk fibroin, 1.2 parts of polylactic acid, 0.8 parts of chitosan, 1.2 parts of polyvinyl alcohol, and 2 parts of glycerol.

[0067] The method for preparing the upper antibacterial film comprises the following steps:

[0068] A1: Sea buckthorn seed meal was dried and ground, passed through a 90-mesh sieve, added to ultrapure water, shaken at 45°C and 300 rpm for 1 hour, and centrifuged at 6000 rpm for 20 minutes. 1 mol / L sodium chloride was added to the precipitate, shaken at 30°C and 300 rpm for 40 minutes, and centrifuged at 6000 rpm for 20 minutes. 75 vol% ethanol was added to the precipitate, shaken at 40°C and 300 rpm for 1.5 hours, and centrifuged at 6000 rpm for 20 minutes. The supernatant was evaporated and concentrated, dialyzed in deionized water for 24 hours, and freeze-dried to obtain the seed meal protein.

[0069] A2: Add 0.8 g of the seed meal protein obtained in step A1 to 80 mL of an 80 vol% ethanol solution, and stir at 450 rpm for 12 min to obtain a seed meal protein solution. Add 0.8 g of xanthan gum to 80 mL of deionized water, and stir at 750 rpm for 25 min to obtain a xanthan gum solution.

[0070] A3: The xanthan gum solution obtained in step A2 was added to the seed meal protein solution, stirred at 70°C and 650 rpm for 3.5 h, 0.12 g of tannic acid was added, and stirring was continued for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution. 3.5 g of cinnamon essential oil was added dropwise, and homogenization was carried out at 11,000 rpm for 5 min to obtain an antibacterial emulsion.

[0071] A4: Place the middle polycaprolactone fiber membrane at the bottom of the mold, use 6 g of silk fibroin to prepare an 18 wt% silk fibroin solution, mix it with 0.8 g of chitosan and 1.2 g of polylactic acid, stir at 350 rpm for 1.5 h, mix it with the antibacterial emulsion obtained in step A3, add 1.2 g of polyvinyl alcohol and 2 g of glycerol, stir evenly, pour it into the mold, and vacuum dry it at 35°C to obtain the upper antibacterial membrane.

[0072] The raw materials for preparing the lower bone repair membrane include the following components in parts by weight: 2.5 parts of nano-hydroxyapatite, 4 parts of citric acid, 8 parts of lactic acid, 3.5 parts of tyrosine, 1.5 parts of glycolic acid, 2.5 parts of casein, 0.25 parts of hyperoside, and 1.5 parts of gelatin.

[0073] The preparation method of the lower bone repair membrane comprises the following steps:

[0074] B1: 2.5 g of 40 nm nanohydroxyapatite was dispersed in 20 mL of DMF, stirred at 110 °C for 30 min, 4 g of citric acid was added, and the mixture was stirred at 250 rpm for 2.5 h. The mixture was cooled to room temperature and centrifuged at 8000 rpm for 12 min. The precipitate was washed with deionized water and dried in vacuo to obtain modified hydroxyapatite.

[0075] B2: Under nitrogen protection, 8 g of lactic acid, 3.5 g of tyrosine, 1.5 g of glycolic acid, modified hydroxyapatite and 56 mg of stannous chloride were added to toluene and refluxed at 160 °C for 2.5 h. During the reaction, stirring was continued at 250 rpm. Toluene was removed by rotary evaporation. -3 MPa, 170 ° C for 4.5 h, dissolved in dichloromethane, slowly added to ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried in vacuo at 55 ° C to obtain lactic acid-tyrosine-hydroxyapatite copolymer;

[0076] B3: Add 2.5 g of casein to 250 mL of a 0.01 M PBS solution at pH 6.8, stir magnetically overnight, adjust the pH to 9 with 1 mol / L NaOH, add 0.25 g of hyperoside to 200 mL of deionized water, mix thoroughly, and adjust the pH to 9. Mix the two solutions and stir at 120 rpm for 24 h. Dialyze against deionized water for 48 h, changing the water every 6 h. The dialyzate is vacuum freeze-dried to obtain modified casein.

[0077] B4: Lactic acid-tyrosine-hydroxyapatite copolymer, modified casein, and 1.5 g of gelatin were added to hexafluoroisopropanol and ultrasonicated at 350W for 30 min to obtain a 12 wt% electrospinning solution, which was then sprayed onto the other side of the middle polycaprolactone fiber membrane by electrospinning to form a lower bone repair membrane. During the electrospinning process, the flow rate was 2.5 mL / h, the receiving device speed was 120 rpm, the voltage was 18 kV, and the receiving distance was 15 cm.

[0078] This embodiment also provides a method for preparing the antibacterial oral repair composite membrane, comprising the following steps: placing the prepared upper antibacterial membrane, middle polycaprolactone fiber membrane, and lower bone repair membrane under vacuum drying at 25°C for 24 h, and hot pressing at 40°C and 25 MPa for 5 min to obtain the antibacterial oral repair composite membrane.

[0079] The only difference between Comparative Example 1 and Example 1 is that no seed meal protein is added;

[0080] The only difference between Comparative Example 2 and Example 1 is that no tannic acid is added;

[0081] The only difference between Comparative Example 3 and Example 1 is that no hydroxyapatite is added;

[0082] The only difference between Comparative Example 4 and Example 1 is that casein is used instead of modified casein.

[0083] Experimental Example 1: 30 male SD rats weighing (200±20) g were selected and randomly divided into 5 groups, with 6 rats in each group, namely Example 1 Group, Comparative Example 1 Group, Comparative Example 2 Group, Comparative Example 3 Group, and Comparative Example 4 Group. After anesthesia with sodium pentobarbital, the rats' mandibular hair was shaved, and an incision of about 1.5 cm long was made along the lower edge of the rats' mandible to expose the lingual bone plate of the mandibular angle. A perforated bone defect with a diameter of 3 mm was prepared at the intersection of 5 mm from the lower edge of the mandible and 5 mm from the posterior side of the mandibular angle. The composite membranes of Example 1 and Comparative Examples 1-4 were attached to the bone defects respectively. The bone defect volume was measured 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks after surgery, and the bone volume reconstruction rate was calculated. The bone volume reconstruction rate = [(bone damage volume on the first day - bone damage volume at each time point) / bone damage volume on the first day] × 100%. The results are as follows Figure 1 shown.

[0084] Figure 1 The results showed that at week 8, the bone volume reconstruction rates of Example 1 and Comparative Examples 1-4 were 93.7%, 85.23%, 89.11%, 75.01% and 80.17%, respectively. The induced bone reconstruction rate of the composite membrane of Example 1 was higher than that of Comparative Examples 1-4. The oral repair composite membrane of the present invention can effectively promote oral bone repair, maintain the space required for bone repair for a long time, reduce the pressure of surrounding soft tissue and prevent the invasion of connective tissue.

[0085] Experimental Example 2: Each group took 100 μL of 1.5×10 8 The bacterial solution of Porphyromonas gingivalis with a concentration of CFU / mL was evenly spread on a blood agar plate. The composite films prepared in Example 1 and Comparative Examples 1-4 were cut into composite film discs with a diameter of 6 mm and placed in the center of the blood agar plate respectively. The diameters of the inhibition zones were measured after 3 days and 21 days. The results are as follows: Figure 2 shown.

[0086] Figure 2 The results showed that the diameter of the inhibition zone of Example 1 was larger than that of Comparative Examples 1-4. Comparative Example 1 did not add seed meal protein, and the antibacterial activity decreased. Comparative Example 2 did not add tannic acid with antioxidant and antibacterial effects, and the antibacterial activity decreased. Comparative Example 3 did not add hydroxyapatite with antibacterial effects, and the antibacterial activity decreased. Comparative Example 4 did not modify casein, and the cross-linking degree decreased. Moreover, no hyperoside with antibacterial effect was introduced, and the antibacterial activity decreased. All groups of composite membranes maintained a certain antibacterial activity after 21 days, among which the inhibition zone diameter decrease rate of Example 1 group was the lowest, and the stability was better.

[0087] Experimental Example 3: The tensile strength of the antibacterial oral restoration films prepared in Examples 1-3 and Comparative Examples 1-4 was tested using a universal mechanical testing machine according to the national standard "Determination of tensile properties of plastics Part 3: Test conditions for films and sheets" GB / T 1040.3-2006. The test distance was 30 mm, the tensile rate was 100 mm / min, the test environment temperature was 23°C, and the humidity was 45%-55%. The results are as follows: Figure 3 shown.

[0088] Figure 3 The results showed that the tensile strength of Examples 1-3 was better than that of Comparative Examples 1-4. In Comparative Example 1, no seed meal protein was added, the stability of the antibacterial emulsion formed decreased, and the mechanical properties of the repair composite membrane prepared decreased. In Comparative Example 2, no tannic acid was added, the degree of cross-linking decreased, and the mechanical properties decreased. In Comparative Example 3, no hydroxyapatite was added, and the mechanical properties decreased. In Comparative Example 4, casein was not modified, the degree of cross-linking decreased, and the mechanical properties decreased.

[0089] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

Claims

1. An antibacterial oral repair composite membrane, characterized in that: It consists of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane; The middle layer polycaprolactone fiber membrane is prepared by electrospinning process using polycaprolactone as the main raw material; The raw materials for preparing the upper antibacterial film include the following components in parts by weight: 3-4 parts of cinnamon essential oil, 0.5-1 part of xanthan gum, 0.5-1 part of seed meal protein, 0.1-0.15 part of tannic acid, 5-7 parts of silk fibroin, 1-1.5 parts of polylactic acid, 0.6-1 part of chitosan, 1-1.5 parts of polyvinyl alcohol, and 1.5-2.5 parts of glycerol; The method for preparing the upper antibacterial film comprises the following steps: A1: Dry and crush seabuckthorn seed meal, sieve it, add it to ultrapure water, shake it, centrifuge it, add 1 mol / L sodium chloride to the precipitate, shake it, centrifuge it, add ethanol to the precipitate, shake it, centrifuge it, evaporate and concentrate the supernatant, dialyze it, and freeze-dry it to obtain the seed meal protein. A2: Add the seed meal protein obtained in step A1 to the ethanol solution and stir to obtain a seed meal protein solution; add xanthan gum to deionized water and stir to obtain a xanthan gum solution; A3: Add the xanthan gum solution obtained in step A2 to the seed meal protein solution, stir, add tannic acid, continue stirring to obtain a seed meal protein / xanthan gum / tannic acid complex solution, add cinnamon essential oil dropwise, and homogenize to obtain an antibacterial emulsion; A4: Place the middle polycaprolactone fiber membrane at the bottom of the mold, mix 15-20 wt% silk fibroin solution with chitosan and polylactic acid, stir, mix with the antibacterial emulsion obtained in step A3, add polyvinyl alcohol and glycerol, stir evenly, pour into the mold, and vacuum dry to obtain the upper antibacterial membrane; The raw materials for preparing the lower layer bone repair membrane include the following components in parts by weight: 2-3 parts of nano-hydroxyapatite, 3-5 parts of citric acid, 6-9 parts of lactic acid, 3-4 parts of tyrosine, 1-2 parts of glycolic acid, 2-3 parts of casein, 0.2-0.3 parts of hyperoside, and 1-2 parts of gelatin; The preparation method of the lower bone repair membrane comprises the following steps: B1: Disperse nanohydroxyapatite in DMF, stir, add citric acid, stir to react, cool, centrifuge, wash the precipitate with deionized water, and vacuum dry to obtain modified hydroxyapatite; B2: Under nitrogen protection, lactic acid, tyrosine, glycolic acid, modified hydroxyapatite and stannous chloride were added to toluene, refluxed, and the toluene was removed by rotary evaporation. -3 MPa, 160-180 ° C, dissolved in dichloromethane, slowly added to ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and vacuum dried to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add casein to PBS solution, stir magnetically overnight, adjust the pH to 9 with NaOH, add hyperoside to deionized water and mix well, adjust the pH to 9, mix the two solutions, stir, dialyze, and freeze-dry to obtain modified casein; B4: Add lactic acid-tyrosine-hydroxyapatite copolymer, modified casein and gelatin to hexafluoroisopropanol, ultrasonicate to obtain 10-15wt% electrospinning solution, and use electrospinning method to spray it to the other side of the middle layer polycaprolactone fiber membrane to form the lower layer bone repair membrane.

2. The antibacterial oral repair composite membrane according to claim 1, characterized in that In step A2, the mass concentration of the seed meal protein in the ethanol solution is 10 mg / mL; the mass concentration of xanthan gum in deionized water is 10 mg / mL.

3. The antibacterial oral repair composite membrane according to claim 2, characterized in that In step B1, the mass concentration of the nano-hydroxyapatite in DMF is 0.1-0.15 g / mL.

4. The antibacterial oral repair composite membrane according to claim 3, characterized in that In step B2, the amount of stannous chloride used is 0.6-0.8 wt % of the lactic acid.

5. The antibacterial oral repair composite membrane according to claim 4, characterized in that In step B3, the mass concentration of casein in PBS solution is 10 mg / mL; the mass concentration of hyperoside in deionized water is 1-1.5 g / L.

6. A method for preparing the antibacterial oral repair composite film according to any one of claims 1 to 5, characterized in that: The following steps are involved: The prepared upper antibacterial film, middle polycaprolactone fiber film and lower bone repair film are vacuum dried and hot pressed to obtain an antibacterial oral repair composite film.

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