Bacteriostatic oral rehabilitation composite film and preparation method thereof

Abstract

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

Description

Technical Field

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

[0002] In daily life, the soft and hard tissues of the human maxillofacial region may be defective or missing due to diseases, trauma, physiological degeneration and other factors, which will affect the integrity of the morphology of oral tissues and organs, and further affect their normal functions or the aesthetics of the oral and maxillofacial region. Oral repair membranes establish a biological barrier between oral soft tissues and bone defects, provide a good repair space for bone regeneration, selectively block fibroblasts and epithelial cells with faster migration speed from entering the bone defect area, and promote or even induce bone regeneration and oral wound healing. The current oral repair membranes have the following problems: insufficient antibacterial properties. The oral cavity is an environment rich in microorganisms. Bacteria are easy to adhere and proliferate on the surface of the repair membrane, leading to infection and affecting the repair effect; poor bone integration ability. Although the oral repair membranes prepared with materials such as polycaprolactone have biocompatibility, good mechanical properties and biodegradability, they have limited ability to promote osteogenesis at bone defects and are difficult to meet clinical needs; insufficient mechanical strength. Some oral repair membranes may have problems such as too soft texture and easy deformation during use, which cannot provide sufficient support for bone regeneration and affect the repair effect. As people pay more and more attention to oral health, the market demand for oral repair is growing. Patients have higher and higher requirements for the performance of oral repair membranes. They not only hope that the repair membrane can effectively repair oral tissue defects, but also hope that it has good antibacterial properties, reduces the risk of infection, and improves the repair effect. Therefore, the development of an oral repair composite membrane with good antibacterial properties and good repair 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 film and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions: The invention discloses an antibacterial oral repair composite membrane, which is composed of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane.

[0005] Furthermore, the middle-layer polycaprolactone fiber membrane is prepared using polycaprolactone as the main raw material through an electrospinning process, and the preparation method comprises 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 using an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm to spray the spinning solution onto a receiving roller with a rotation speed of 100-150 r / min to form a middle-layer polycaprolactone fiber membrane.

[0006] 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.

[0007] Furthermore, the method for preparing the upper antibacterial film comprises the following steps: A1: Take seabuckthorn seed meal, dry and grind it through a 80-100 mesh sieve, add it to ultrapure water, shake it at 45℃ and 300 rpm for 1 hour, centrifuge it at 6000 r / min for 20 minutes, add 1 mol / L sodium chloride to the precipitate, shake it at 30℃ and 300 rpm for 40 minutes, centrifuge it at 6000 r / min for 20 minutes, add 70-80 vol% ethanol to the precipitate, shake it at 40℃ and 300 rpm for 1.5 hours, centrifuge it at 6000 r / min for 20 minutes, evaporate and concentrate the supernatant, dialyze it in deionized water for 24 hours, and freeze-dry it to obtain seed meal protein; A2: adding the seed meal protein obtained in step A1 to an 80 vol% ethanol solution, stirring at 400-500 r / min for 10-15 min to obtain a seed meal protein solution, adding xanthan gum to deionized water, stirring at 700-800 r / min for 20-30 min to obtain a xanthan gum solution; 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, 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 10000-12000 r / min for 4-6 minutes to obtain an antibacterial emulsion; A4: Place the middle layer 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.

[0008] Furthermore, in step A2, the mass concentration of the seed meal protein in the ethanol solution is 10 mg / mL.

[0009] Furthermore, in step A2, the mass concentration of the xanthan gum in deionized water is 10 mg / mL.

[0010] Furthermore, the raw materials for preparing the lower 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.

[0011] Furthermore, the method for preparing the lower bone repair membrane comprises the following steps: B1: Disperse 40 nm nanohydroxyapatite in DMF, stir at 100-120℃ for 30 min, add citric acid, stir at 200-300 r / min 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; B2: Under nitrogen protection, add lactic acid, tyrosine, glycolic acid, modified hydroxyapatite and stannous chloride into toluene, reflux at 160℃ for 2-3 h, stir continuously at 200-300 rpm during the reaction, remove toluene by rotary evaporation, and heat for 10 -3 MPa, 160-180℃ for 4-5 h, dissolved with dichloromethane, slowly added with ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried under vacuum at 50-60℃ to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add casein to a pH 6.8 0.01 M PBS solution, stir magnetically overnight, adjust the pH to 9 with 1 mol / L NaOH, add hyperoside to deionized water and mix well, adjust the pH to 9, mix the two solutions, stir at 100-150 rpm for 24 h, dialyze with deionized water for 48 h, change the water every 6 h, and vacuum freeze-dry the dialysate to obtain modified casein; 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 polycaprolactone fiber membrane by electrospinning to form the lower bone repair membrane.

[0012] Furthermore, in step B1, the mass concentration of the nano-hydroxyapatite in DMF is 0.1-0.15 g / mL.

[0013] Furthermore, in step B2, the amount of stannous chloride used is 0.6-0.8wt% of the lactic acid.

[0014] Furthermore, in step B3, the mass concentration of casein in PBS is 10 mg / mL.

[0015] Furthermore, in step B3, the mass concentration of hyperoside in deionized water is 1-1.5 g / L.

[0016] 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.

[0017] Furthermore, the present invention also provides a method for preparing the antibacterial oral repair composite film, comprising the following steps: placing the prepared upper antibacterial film, 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 20-30 MPa for 4-6 min to obtain the antibacterial oral repair composite film.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an antibacterial oral repair composite membrane, which adopts a three-layer structure design of an upper antibacterial membrane, a middle polycaprolactone fiber membrane and a lower bone repair membrane, giving the composite membrane multiple functions. The middle polycaprolactone fiber membrane has good biocompatibility and can provide good mechanical support, the upper antibacterial membrane can effectively inhibit bacterial growth and prevent infection, and the lower bone repair membrane can promote the repair of bone tissue, so that the composite membrane plays a comprehensive role in oral repair. 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. Seabuckthorn 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 help maintain a healthy oral environment. Biocompatible materials such as silk fibroin, polylactic acid and chitosan are used as the base material, which can be better compatible with surrounding tissues in the oral environment, is conducive to cell adhesion, proliferation and differentiation, reduces stimulation and immune response to oral tissues, and promotes 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 closely to the oral tissues and exert a continuous antibacterial and repair effect. Nano-hydroxyapatite is a bioactive material similar to the composition of human bone tissue, which can induce the growth and differentiation of bone cells and promote the repair and regeneration of bone tissue. After modification with citric acid, carboxyl and hydroxyl groups are connected on the surface of nano-hydroxyapatite, and the modified nano-hydroxyapatite is polymerized with polymer monomers to polymerize hydroxyapatite into polymers, which improves its dispersibility in composite materials and compatibility with other components, further enhancing the bone repair effect. Casein is modified by alkali treatment and combined with hyperoside through covalent bonds, which can regulate the activity and function of cells, enhance oral repair ability, enhance cross-linking degree, improve mechanical properties, and improve antibacterial effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The bone repair effects of the repair membranes described in Example 1 and Comparative Examples 1-4 of the present invention; Figure 2 The antibacterial effect of the repair film described in Example 1 and Comparative Examples 1-4 of the present invention; Figure 3 The mechanical properties of the repair films described in Examples 1-3 and Comparative Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention are purchased through commercial channels.

[0022] 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.

[0023] The middle-layer polycaprolactone fiber membrane is prepared by an electrospinning process with polycaprolactone as the main raw material. The preparation method includes the following steps: dissolving 12 g of polycaprolactone in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersing at 500W for 10 min to obtain a spinning solution with a concentration of 10wt%, and passing through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm, and spraying the spinning solution onto a receiving roller with a rotation speed of 150 r / min to form a middle-layer polycaprolactone fiber membrane.

[0024] 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.

[0025] The method for preparing the upper antibacterial film comprises the following steps: A1: Take seabuckthorn seed meal, dry and grind it through a 100-mesh sieve, add it into ultrapure water, shake it at 45℃ and 300 rpm for 1 h, centrifuge it at 6000 r / min for 20 min, add 1 mol / L sodium chloride to the precipitate, shake it at 30℃ and 300 rpm for 40 min, centrifuge it at 6000 r / min for 20 min, add 80 vol% ethanol to the precipitate, shake it at 40℃ and 300 rpm for 1.5 h, centrifuge it at 6000 r / min for 20 min, evaporate and concentrate the supernatant, dialyze it in deionized water for 24 h, and freeze-dry it to obtain seed meal protein; A2: Add 1 g of the seed meal protein obtained in step A1 to 100 mL of 80 vol% ethanol solution, stir at 500 r / min for 10 min to obtain a seed meal protein solution; add 1 g of xanthan gum to 100 mL of deionized water, stir at 800 r / min for 20 min to obtain a xanthan gum solution; A3: Add the xanthan gum solution obtained in step A2 to the seed meal protein solution, stir at 70°C and 700 rpm for 3 h, add 0.15 g of tannic acid, continue stirring for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution, add 4 g of cinnamon essential oil dropwise, and homogenize at 12000 r / min for 4 min to obtain an antibacterial emulsion; A4: Place the middle layer 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 dry it in a vacuum at 40 °C to obtain the upper antibacterial film.

[0026] 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.

[0027] The method for preparing the lower bone repair membrane comprises the following steps: 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, stirred at 300 r / min for 2 h, cooled to room temperature, centrifuged at 8000 rpm for 10 min, the precipitate was washed with deionized water, and vacuum dried to obtain modified hydroxyapatite; 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, and toluene was removed by rotary evaporation. - 3 MPa, 180℃ for 4 h, dissolved with dichloromethane, slowly added into ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried under vacuum at 60℃ to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add 3 g of casein to 300 mL of pH 6.8 0.01 M PBS solution, 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, mix well, adjust the pH to 9, mix the two solutions, stir at 150 rpm for 24 h, dialyze with deionized water for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain modified casein; B4: Add lactic acid-tyrosine-hydroxyapatite copolymer, modified casein and 2 g of gelatin into hexafluoroisopropanol, and ultrasonicate at 400W for 30 min to obtain a 15wt% electrospinning solution, which is 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 speed of the receiving device was 150 rpm, the voltage was 20 kV, and the receiving distance was 15 cm.

[0028] 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.

[0029] 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.

[0030] The middle-layer polycaprolactone fiber membrane is prepared by an electrospinning process with polycaprolactone as the main raw material. 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%, and 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.

[0031] 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.

[0032] The method for preparing the upper antibacterial film comprises the following steps: A1: Take seabuckthorn seed meal, dry and grind it through a 80-mesh sieve, add it into ultrapure water, shake it at 45℃ and 300 rpm for 1 h, centrifuge it at 6000 r / min for 20 min, add 1 mol / L sodium chloride to the precipitate, shake it at 30℃ and 300 rpm for 40 min, centrifuge it at 6000 r / min for 20 min, add 70 vol% ethanol to the precipitate, shake it at 40℃ and 300 rpm for 1.5 h, centrifuge it at 6000 r / min for 20 min, evaporate and concentrate the supernatant, dialyze it in deionized water for 24 h, and freeze-dry it to obtain seed meal protein; A2: Add 0.5 g of the seed meal protein obtained in step A1 to 50 mL of 80 vol% ethanol solution, stir at 400 r / min for 15 min to obtain a seed meal protein solution, take 0.5 g of xanthan gum and add it to 50 mL of deionized water, stir at 700 r / min for 30 min to obtain a xanthan gum solution; A3: Add the xanthan gum solution obtained in step A2 to the seed meal protein solution, stir at 70°C and 600 rpm for 4 h, add 0.1 g of tannic acid, continue stirring for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution, add 3 g of cinnamon essential oil dropwise, and homogenize at 10,000 r / min for 6 min to obtain an antibacterial emulsion; A4: Place the middle layer 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 dry it in a vacuum at 30°C to obtain the upper antibacterial film.

[0033] 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.

[0034] The method for preparing the lower bone repair membrane comprises the following steps: 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, stirred at 200 r / min for 3 h, cooled to room temperature, centrifuged at 8000 rpm for 10 min, the precipitate was washed with deionized water, and vacuum dried to obtain modified hydroxyapatite; 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, and toluene was removed by rotary evaporation. - 3 MPa, 160℃ for 5 h, dissolved with dichloromethane, slowly added with ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried under vacuum at 50℃ to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add 2 g of casein to 2000 mL of PBS solution (pH 6.8, 0.01 M), 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, mix well, adjust the pH to 9, mix the two solutions, stir at 100 rpm for 24 h, dialyze with deionized water for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain modified casein; B4: Add lactic acid-tyrosine-hydroxyapatite copolymer, modified casein and gelatin 1 g into hexafluoroisopropanol, and ultrasonicate at 300W for 30 min to obtain a 10wt% electrospinning solution, which is 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 speed of the receiving device was 100 rpm, the voltage was 15 kV, and the receiving distance was 15 cm.

[0035] 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.

[0036] 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.

[0037] The middle-layer polycaprolactone fiber membrane is prepared by an electrospinning process with polycaprolactone as the main raw material. The preparation method includes the following steps: dissolving 10 g of polycaprolactone in a mixed solvent of dichloromethane and N,N-dimethylformamide (v:v=4:1), ultrasonically dispersing at 450W for 12 min to obtain a spinning solution with a concentration of 10wt%, and passing through an electrospinning device with a voltage of 15 kV, a flow rate of 2 mL / h, and a receiving distance of 15 cm, and spraying the spinning solution onto a receiving roller with a rotation speed of 120 r / min to form a middle-layer polycaprolactone fiber membrane.

[0038] 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.

[0039] The method for preparing the upper antibacterial film comprises the following steps: A1: Take seabuckthorn seed meal, dry and grind it through a 90-mesh sieve, add it into ultrapure water, shake it at 45℃ and 300 rpm for 1 h, centrifuge it at 6000 r / min for 20 min, add 1 mol / L sodium chloride to the precipitate, shake it at 30℃ and 300 rpm for 40 min, centrifuge it at 6000 r / min for 20 min, add 75 vol% ethanol to the precipitate, shake it at 40℃ and 300 rpm for 1.5 h, centrifuge it at 6000 r / min for 20 min, evaporate and concentrate the supernatant, dialyze it in deionized water for 24 h, and freeze-dry it to obtain seed meal protein; A2: Add 0.8 g of the seed meal protein obtained in step A1 to 80 mL of 80 vol% ethanol solution, stir at 450 r / min for 12 min to obtain a seed meal protein solution, take 0.8 g of xanthan gum and add it to 80 mL of deionized water, stir at 750 r / min for 25 min to obtain a xanthan gum solution; A3: Add the xanthan gum solution obtained in step A2 to the seed meal protein solution, stir at 70°C and 650 rpm for 3.5 h, add 0.12 g of tannic acid, continue stirring for 1 h to obtain a seed meal protein / xanthan gum / tannic acid complex solution, add 3.5 g of cinnamon essential oil dropwise, and homogenize at 11000 r / min for 5 min to obtain an antibacterial emulsion; A4: Place the middle layer 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 film.

[0040] 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.

[0041] The method for preparing the lower bone repair membrane comprises the following steps: 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, stirred at 250 r / min for 2.5 h, cooled to room temperature, centrifuged at 8000 rpm for 12 min, the precipitate was washed with deionized water, and vacuum dried to obtain modified hydroxyapatite; 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, and toluene was removed by rotary evaporation. -3 MPa, 170℃ for 4.5 h, dissolved with dichloromethane, slowly added into ethanol for precipitation, filtered, the filter cake was washed with ethanol and deionized water, and dried under vacuum at 55℃ to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add 2.5 g of casein to 250 mL of PBS solution (pH 6.8, 0.01 M), 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 well, adjust the pH to 9, mix the two solutions, stir at 120 rpm for 24 h, dialyze with deionized water for 48 h, change the water every 6 h, and freeze-dry the dialysate to obtain modified casein; B4: Add lactic acid-tyrosine-hydroxyapatite copolymer, modified casein and 1.5 g of gelatin into hexafluoroisopropanol, and ultrasonicate at 350W for 30 min to obtain a 12wt% electrospinning solution, which is 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 speed of the receiving device was 120 rpm, the voltage was 18 kV, and the receiving distance was 15 cm.

[0042] 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.

[0043] The difference between Comparative Example 1 and Example 1 is that no seed meal protein is added; The difference between Comparative Example 2 and Example 1 is that no tannic acid is added; The difference between Comparative Example 3 and Example 1 is that hydroxyapatite is not added; The only difference between Comparative Example 4 and Example 1 is that casein is used instead of modified casein.

[0044] 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, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. After sodium pentobarbital anesthesia, the rats' mandibular hair was shaved, and an incision of about 1.5 cm long was made along the lower edge of the rat's 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 the operation, and the bone volume reconstruction rate was calculated. The bone volume reconstruction rate = [(bone loss volume on the first day - bone loss volume at each time point) / bone loss volume on the first day] × 100%. The results are as follows Figure 1 shown.

[0045] Figure 1 The results showed that at the 8th week, 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 tissues and prevent the invasion of connective tissue.

[0046] Experimental Example 2: Each group took 100 μL of 1.5×10 8 CFU / mL of Porphyromonas gingivalis liquid was evenly spread on the blood agar plate, and the composite membranes prepared in Example 1 and Comparative Examples 1-4 were cut into composite membrane 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 the 3rd and 21st days. The results are as follows: Figure 2 shown.

[0047] Figure 2 The results showed that the diameter of the antibacterial 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, the cross-linking degree decreased, and 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 antibacterial zone diameter decrease rate of Example 1 group was the lowest, and the stability was better.

[0048] Experimental Example 3: The tensile strength of the antibacterial oral repair membranes 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.

[0049] Figure 3 The results showed that the tensile strength of Examples 1-3 was better than that of Comparative Examples 1-4, wherein Comparative Example 1 did not add seed meal protein, the stability of the antibacterial emulsion formed decreased, and the mechanical properties of the repair composite membrane prepared decreased; Comparative Example 2 did not add tannic acid, the degree of cross-linking decreased, and the mechanical properties decreased; Comparative Example 3 did not add hydroxyapatite, and the mechanical properties decreased; Comparative Example 4 did not modify casein, the degree of cross-linking decreased, and the mechanical properties decreased.

[0050] 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. Under the concept 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 film, characterized in that: It is composed 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 an 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 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 method for preparing the upper antibacterial film comprises the following steps: A1: Take seabuckthorn seed meal, dry it, grind it, sieve it, add it into ultrapure water, shake it, centrifuge it, add sodium chloride to precipitate it, shake it, centrifuge it, add ethanol to precipitate it, shake it, centrifuge it, evaporate and concentrate the supernatant, dialyze it, and freeze-dry it to obtain seed meal protein; A2: adding the seed meal protein obtained in step A1 to the ethanol solution, stirring to obtain a seed meal protein solution, taking xanthan gum and adding it to deionized water, stirring to obtain a xanthan gum solution; A3: adding the xanthan gum solution obtained in step A2 to the seed meal protein solution, stirring, adding tannic acid, continuing stirring to obtain a seed meal protein / xanthan gum / tannic acid complex solution, adding cinnamon essential oil dropwise, homogenizing, and obtaining an antibacterial emulsion; A4: placing the middle layer polycaprolactone fiber membrane at the bottom of the mold, mixing 15-20wt% silk fibroin solution with chitosan and polylactic acid, stirring, mixing with the antibacterial emulsion obtained in step A3, adding polyvinyl alcohol and glycerol, stirring evenly, pouring into the mold, and vacuum drying to obtain the upper antibacterial membrane; The raw materials for preparing the lower 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 method for preparing the lower bone repair membrane comprises the following steps: B1: Disperse nano-hydroxyapatite 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 toluene was removed by rotary evaporation. -3 MPa, 160-180°C, dissolve with dichloromethane, slowly add ethanol to precipitate, filter, wash the filter cake with ethanol and deionized water, and vacuum dry to obtain lactic acid-tyrosine-hydroxyapatite copolymer; B3: Add casein to PBS solution, stir magnetically overnight, adjust pH to 9 with NaOH, add hyperoside to deionized water and mix well, adjust 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 to spray it to the other side of the middle layer polycaprolactone fiber membrane to form the lower bone repair membrane.

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

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

6. A method for preparing an 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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