Oral repair membrane with antibacterial properties, preparation method and application thereof

Abstract

The present invention provides an oral repair membrane with antibacterial properties, a preparation method and an application thereof, and belongs to the field of medical biomaterials. The oral repair membrane with antibacterial properties prepared by the present invention comprises an antibacterial nano-film layer, a first decellularized matrix layer and a second decellularized matrix layer; the first decellularized matrix layer and the second decellularized matrix layer have a loose surface and a dense surface; the nano-film layer is located on the dense surface of the first decellularized matrix layer; the loose surface of the first decellularized matrix layer is located on the dense surface of the second decellularized matrix layer. The process for preparing the oral repair membrane with antibacterial properties of the present invention is simple and rapid, and the prepared oral repair membrane is easy to use, has good tissue adhesion, hydrophilicity, low immunogenicity and degradation performance. When in use, the second decellularized matrix layer of the membrane faces the defective tissue, and repair is achieved as the membrane degrades. The oral repair membrane has broad application prospects in the field of oral medicine.

Description

Technical Field

[0001] The invention relates to the field of medical biomaterials, and in particular to an oral repair membrane with antibacterial properties, a preparation method and applications thereof. Background Art

[0002] Decellularized matrix is ​​the most widely used xenobiotic biomaterial in clinical practice. Its three-dimensional network structure is closest to the ideal natural scaffold and is widely used in the field of tissue regeneration and repair. It retains the natural extracellular matrix (ECM) structure and components, has a complete vascular system, and this multifunctional structure can induce cell differentiation and maturation. Due to the eutrophication of the decellularized matrix membrane, cells and bacteria will adhere and proliferate, and it has the disadvantages of weak active antibacterial and barrier properties. For example, the natural extracellular matrix biofilm disclosed in Chinese patent CN 106880872A and its preparation method and application do not consider the impact of bacterial infection on treatment; Chinese patent CN 219398361U discloses an oral repair membrane and related kit for guiding tissue regeneration, which uses the bladder basement membrane as a dense layer to play a barrier role, but does not consider that the bladder basement membrane is still easily attached by organisms and the membrane is damaged during surgery, thereby losing its barrier function.

[0003] Due to internal or external reasons, different degrees of infection may occur in the oral cavity at different stages of treatment, such as infection caused by membrane exposure during periodontitis treatment and oral repair membrane treatment. Periodontitis is an important cause that affects periodontal tissue regeneration during periodontal disease treatment. The presence of periodontal microbacteria, such as Porphyromonas gingivalis (Pg), can trigger excessive immune response, induce inflammation, lead to osteoclast activation and bone resorption. Microbial infection and the ensuing host immune response further affect the function of local periodontal ligament stem cells and enhance periodontitis. Periodontal regenerative surgeries, such as guided tissue regeneration and bone grafting, still have the risk of secondary infection.

[0004] Membrane exposure is one of the most commonly reported complications of guided tissue regeneration (GTR) and guided bone regeneration (GBR). The exposure of the barrier membrane after surgery will cause the microorganisms in the oral cavity to migrate and colonize to the exposed area. At this time, the membrane becomes a hotbed for bacteria, promoting bacterial attachment and proliferation, triggering acute inflammatory infiltration of the submembrane space, and causing the regeneration process to stop. Studies have shown that the proteases secreted by bacteria colonized on the barrier membrane will also accelerate the degradation of collagen barrier membranes (Bio-Gide collagen membrane and Haiao oral repair membrane), causing them to lose their barrier function prematurely, thereby reducing the treatment effect and increasing the risk of infection (a comparative study of the degradation of two collagen membranes exposed to the oral environment of Bai Peng. Chinese Journal of Oral Implantology, 2011, 16 (01): 56-57.). The above shows the necessity of preparing an oral repair membrane with active antibacterial properties and enhanced barrier properties.

[0005] Phase transition lysozyme (PTL) is a new amyloid protein assembly that is gentle, efficient and fast, and can form a transparent nanofilm with a tiny pore size. It has a long-lasting in vitro and in vivo broad-spectrum antibacterial effect on Gram-positive / negative and fungi, and is more effective than natural lysozyme (ACS Applied Materials & Interfaces, 2017.9(1): p.198-210.). In addition, the surface hydration of the nanofilm gives it anti-biofilm, hydrophobicity, blood compatibility and low cytotoxicity, and it can adhere to the surface of inorganic, organic, and microscopic active particles for a long time without affecting their normal functions. By using phase transition lysozyme technology, the membrane can provide active antibacterial effects.

[0006] In summary, there is an urgent need to prepare an oral repair membrane that has good mechanical properties and excellent antibacterial properties while ensuring the mechanical properties of the oral repair membrane. Summary of the invention

[0007] The purpose of the present invention is to provide an oral repair membrane with antibacterial properties, a preparation method and an application thereof.

[0008] The present invention provides an oral repair film with antibacterial properties, which comprises an antibacterial nano film layer, a first decellularized matrix layer and a second decellularized matrix layer;

[0009] The first decellularized matrix layer and the second decellularized matrix layer have a loose surface and a dense surface;

[0010] The nano-thin film layer is located on the dense surface of the first decellularized matrix layer;

[0011] The loose surface of the first decellularized matrix layer is located on the dense surface of the second decellularized matrix layer; the first decellularized matrix layer and the second decellularized matrix layer are prepared from animal tissues.

[0012] Furthermore, the first decellularized matrix layer includes 1 to 3 layers of decellularized matrix, each layer of the decellularized matrix has a loose surface facing downward and a dense surface facing upward, and each layer of the decellularized matrix is ​​staggered and stacked; the length of the first decellularized matrix layer is 20 to 60 mm, the width is 20 to 60 mm, and the thickness is 0.1 to 0.4 mm, and each layer of the decellularized matrix is ​​staggered and stacked; the contact surface of the first decellularized matrix layer and the second decellularized matrix layer is staggered and stacked.

[0013] The second decellularized matrix layer includes 2 to 5 layers of decellularized matrix, each layer of decellularized matrix has a loose surface facing downward and a dense surface facing upward; the second decellularized matrix layer is 3 to 8 mm shorter, 3 to 8 mm narrower, and 0.1 to 0.8 mm thicker than the first decellularized matrix layer; the second decellularized matrix layer is located in the center of the first decellularized matrix layer; the second decellularized matrix and the first decellularized matrix both have a loose surface facing downward and a dense surface facing upward.

[0014] Furthermore, the animal tissue is porcine small intestine, porcine pericardium, porcine skin, bovine skin or bovine pericardium; preferably, it is porcine small intestine.

[0015] Furthermore, the antibacterial nano film layer is a mixture of lysozyme and tri(2-carboxyethyl)phosphine, and the mixture is applied to the dense surface of the first decellularized matrix to form a thin film, and the coating amount is 50 to 200 μL / cm 2 , preferably 100 μL / cm 2 .

[0016] Furthermore, the porosity of the loose surface is 35% to 90%, and the pore size is greater than 35 μm; the porosity of the dense surface is 5% to 25%, and the pore size is less than 10 μm.

[0017] The present invention also provides a method for preparing the above-mentioned oral repair film with antibacterial properties, comprising the following steps:

[0018] (1) Decellularized matrix is ​​prepared using animal tissue;

[0019] (2) cutting the decellularized matrix to obtain a first decellularized matrix layer membrane sheet and a second decellularized matrix layer membrane sheet;

[0020] (3) preparing nanofilm reaction solution;

[0021] (4) covering the first decellularized matrix layer with the second decellularized matrix layer, and then coating the nanofilm reaction solution on the dense surface of the first decellularized matrix, standing, and freeze-drying to obtain;

[0022] The second decellularized matrix layer is located in the center of the first decellularized matrix layer;

[0023] The nano film reaction solution is a mixture of lysozyme and tri(2-carboxyethyl)phosphine;

[0024] 50~200μL / cm 2 The nano-thin film reaction solution is coated on the dense surface of the first decellularized matrix.

[0025] Furthermore, the step of obtaining the decellularized matrix comprises:

[0026] (1) Cleaning: Thaw the animal tissue and rinse it 8 to 10 times with purified water;

[0027] (2) Virus removal: Soak the biological tissue in step (1) in a 1-4 mol / L NaOH solution, shake for 15-20 min, and repeat 5-10 times;

[0028] (3) Degreasing: The material treated in step (2) is placed in a mixed solution of peracetic acid and ethanol and shaken for 15 to 20 minutes, and repeated 5 to 10 times;

[0029] (4) deproteinization: the material treated in step (3) is placed in a 2% to 8% sodium dodecyl sulfate solution for shaking and washing;

[0030] (5) Cleaning: Use purified water to ultrasonically clean the matrix for 5 to 10 times to obtain the decellularized matrix.

[0031] Furthermore, the preparation method of the nano film reaction solution is: using 10mM Tris-HCl and NaOH solution with pH=12-13 to dissolve lysozyme and tri(2-carboxyethyl)phosphine hydrochloride to obtain a lysozyme and tri(2-carboxyethyl)phosphine solution with pH=7.0-7.4; then mixing and stirring for 2-5min to obtain the nano film reaction solution, wherein the concentration ratio of lysozyme to tri(2-carboxyethyl)phosphine is (0.5-2)g / L:20mol / L;

[0032] Furthermore, the concentration ratio of lysozyme to tris(2-carboxyethyl)phosphine is 1 g / L:20 mol / L.

[0033] The present invention also provides an application of the oral repair membrane with antibacterial properties prepared by the above method in the field of oral medicine.

[0034] The present invention provides an oral repair membrane with antibacterial properties, a preparation method and its application. The oral repair membrane prepared by the present invention has the following beneficial effects: the three-dimensional network structure of the acellular matrix used in the repair membrane is closest to the ideal natural scaffold, which is conducive to cell growth and attachment; the collagen albumin, growth factor and glycosaminoglycan contained in the repair membrane promote the growth of new blood vessels and tissue repair of defective parts; the oral repair membrane has excellent barrier protection performance: the nano film layer formed by lysozyme and tri(2-carboxyethyl)phosphine, its hydrophobicity and smooth surface are not conducive to cell attachment, and the smaller pore size can prevent external cells from entering the defective part and proliferating; at the same time, the nano film layer is formed by lysozyme and tri(2-carboxyethyl)phosphine through phase transition lysozyme reaction, while achieving the barrier, the active antibacterial effect of the oral repair membrane is improved, and the surface bacteria can be prevented from growing during the treatment of membrane exposure. And the membrane structure of the oral repair membrane of the present invention, which is large outside and small inside, can prevent food residues from entering the defective part and causing bacteria to grow during the treatment process; the membrane surface where bacteria are difficult to attach can prevent bacterial secretions from prematurely degrading the repair membrane, and prolong the barrier effect of the oral repair membrane. The present invention adopts staggered superposition film laying, so that each surface of the product has excellent mechanical properties and is conducive to suturing.

[0035] The preparation process of the invention is simple and rapid, and the prepared oral repair membrane is easy to use and has good tissue adhesion, hydrophilicity, low immunogenicity and degradation performance. When used, the second acellular matrix layer of the membrane is facing the defective tissue, and repair is achieved as the membrane degrades. The oral repair membrane has broad application prospects in the field of oral medicine.

[0036] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0037] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a picture of the freeze-dried membrane of the decellularized matrix.

[0039] Figure 2 The structure diagram of the oral repair membrane of the present invention is shown in FIG. 1, wherein 1 is a nano film layer, 2 is a first decellularized matrix layer, and 3 is a second decellularized matrix layer.

[0040] Figure 3 The scanning electron microscope image of the oral repair membrane of the present invention includes a) the loose surface of the decellularized matrix, b) the dense surface of the decellularized matrix, and c) the nano-thin film layer.

[0041] Figure 4 This is a screening experiment diagram of the content ratio of lysozyme and tri(2-carboxyethyl)phosphine. Among them, 11 groups of samples are lysozyme reduced by 10% from left to right until 0%, with a total of 11 groups of samples. DETAILED DESCRIPTION

[0042] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0043] Example 1: Prepared oral repair membrane with antibacterial properties

[0044] 1. Experimental Methods

[0045] 1. Preparation of decellularized matrix

[0046] 1. Cleaning: Thaw 4 g of porcine small intestine animal tissue and rinse 10 times with purified water.

[0047] 2. Virus removal: Soak the biological tissue in step (1) in 1M NaOH solution, shake for 20 minutes, and repeat 5 times.

[0048] 3. Degreasing: Place the material treated in step (2) in a mixed solution of peracetic acid and ethanol and shake for 15 minutes, repeating 5 times.

[0049] 4. Deproteinization: Place the material treated in step (3) in a 4% sodium dodecyl sulfate solution and shake and wash.

[0050] 5. Cleaning: Use purified water to ultrasonically clean 10 times to obtain Figure 1 The decellularized matrix shown in the figure has a thickness of 0.1-0.2 mm and fibers ( Figure 1 ).

[0051] 6. Cut the decellularized matrix obtained in step 5 to obtain a decellularized matrix sheet with a length of 40 mm and a width of 30 mm. Alternately stack two layers of decellularized matrix sheets according to the fiber direction to obtain a first decellularized matrix layer.

[0052] 7. Cut the decellularized matrix obtained in step 5 to obtain a decellularized matrix sheet with a length of 35 mm and a width of 25 mm. Stack three layers of decellularized matrix sheets alternately along the fiber direction to obtain a second decellularized matrix layer.

[0053] (ii) preparing nanofilm reaction solution;

[0054] The nano film reaction liquid is a neutral mixed solution of lysozyme and tri(2-carboxyethyl)phosphine. The preparation steps of the nano film reaction liquid are as follows: using 10mM Tris-HCl and NaOH solution with pH=12-13 to dissolve lysozyme and tri(2-carboxyethyl)phosphine hydrochloride to obtain a lysozyme and tri(2-carboxyethyl)phosphine solution with pH=7.0-7.4; then mixing and stirring for 2-5 minutes to obtain the nano film reaction liquid, wherein the content ratio of lysozyme to tri(2-carboxyethyl)phosphine is 1g / L:20mol / L.

[0055] (III) Laying up and coating

[0056] The two sides of the decellularized matrix were scanned by scanning electron microscopy. The scanning electron microscopy images of the decellularized matrix are shown in Figure 3 As shown, the decellularized matrix membrane has a loose surface on one side and a dense surface on the other side; the porosity of the loose surface is 35% to 90%, and the pore size is >35 μm, and the porosity of the dense surface is 5% to 25%, and the pore size is <10 μm.

[0057] The second decellularized matrix layer membrane was placed on the freeze-drying plate, and then the first decellularized matrix layer membrane was staggered and overlapped; the second decellularized matrix layer was located in the center of the first decellularized matrix layer; the second decellularized matrix and the first decellularized matrix were both facing the loose side down and the dense side up, and the contact surface of the first decellularized matrix layer and the second decellularized matrix layer were both staggered and overlapped; then the concentration of 100 μL / cm 2 The nano-film reaction solution is coated on the dense surface of the first decellularized matrix, left to stand for 2 to 5 minutes, and finally freeze-dried and packaged to obtain an oral repair membrane.

[0058] Comparative Example 1 Preparation of oral repair membrane without nanofilm reaction solution

[0059] Based on the technical solution of Example 1, the nano film reaction liquid is not coated, and then vacuum freeze-dried and packaged to obtain an oral repair membrane.

[0060] The following experimental examples demonstrate the beneficial effects of the oral repair membrane with antibacterial properties prepared by the present invention.

[0061] Experimental Example 1. Screening experiment on the ratio of lysozyme and tri(2-carboxyethyl)phosphine in nanofilm reaction solution. 1. Experimental method

[0062] According to the technical scheme of Example 1, lysozyme and tris(2-carboxyethyl)phosphine hydrochloride were used to prepare lysozyme and tris(2-carboxyethyl)phosphine with a pH of 7.0 to 7.4, and then 1 mL of a mixed solution was prepared, wherein the lysozyme and tris(2-carboxyethyl)phosphine were 5 mg and 20 mM, respectively. The mixed solution was defined as 100% mixed solution when the lysozyme was 5 mg. Under the condition of quantitative 20 mM tris(2-carboxyethyl)phosphine, the lysozyme was reduced by 10% to 0%, and a total of 11 groups of samples were prepared. After the preparation was completed, the sample was allowed to stand at room temperature for 2 min, then mixed and photographed.

[0063] 2. Experimental Results

[0064] like Figure 4 As shown in the results, the higher the lysozyme content, the more turbid the mixed solution is, and the suspension can be seen macroscopically. Figure 3 (c), and with the use of a lower lysozyme content for the reaction to prepare the nano-film layer, the nano-film reaction solution can form a smooth, flawless coating layer on the decellularized matrix membrane. Therefore, in order to prevent the reaction speed from being too fast, which makes the film-forming speed difficult to control and affects the smoothness of the membrane surface, the content ratio of lysozyme and tri(2-carboxyethyl)phosphine used in the present invention is (0.5-2) g / L: 20 mol / L.

[0065] Experimental Example 2: Mechanical properties and water absorption multiples of oral repair membranes with antibacterial properties 1. Experimental methods

[0066] Mechanical property test: The oral repair membranes prepared in Example 1 and Comparative Example 1 were tested using a tensile tester with a test size of 15×15 mm. The tensile strength and elongation at break of the membrane were recorded during the test. The elongation at break was calculated using the formula: (elongation at break of the membrane / original length of the membrane)*100%.

[0067] Water absorption multiple: Allow a membrane of known weight to absorb water for 1 minute, then suspend it in the air to control the water for 1 minute and weigh it. Calculate the water absorption multiple by comparing the weight of the membrane before and after water absorption.

[0068] 2. Experimental Results

[0069] The experimental results are shown in Table 1.

[0070] Table 1 Mechanical properties and water absorption test

[0071] Group Example 1 Comparative Example 1 Tensile strength / N 88.7 86.5 Water absorption multiple 7.73 7.65

[0072] The tensile strength and elongation at break of the membrane were recorded by a tensile tester. The elongation at break was calculated by the formula: (elongation at break of the membrane / original length of the membrane)*100%. The elongation at break of the oral repair membrane of Example 1 was 23.55%.

[0073] Table 1 shows the results of testing the tensile strength and water absorption performance of Example 1 and Comparative Example 1. It can be seen that the tensile strength and water absorption performance of Example 1 are better than those of Comparative Example 1, indicating that Example 1 has excellent mechanical properties and hydrophilicity. Good mechanical properties can prevent the membrane from being damaged during suture pulling in clinical use, and good hydrophilicity allows the oral repair membrane to be used in a wet oral environment, enhances adhesion performance, and prevents falling off.

[0074] Experimental Example 2: Antibacterial performance test of oral repair membrane with antibacterial performance

[0075] 1. Antibacterial performance test of oral repair membrane

[0076] Take 1×10 6 100 μL of CFU / mL Staphylococcus aureus and Escherichia coli suspensions were respectively coated on LB agar medium. The oral repair membranes of Example 1 and Comparative Example 1 were cut into circles with a diameter of 10 mm, sterilized in a high-temperature steam sterilizer (220°C, 30 min), and placed in the center of the LB agar medium, and each group of experiments was repeated 3 times.

[0077] The obtained culture dish was placed upside down in a 37°C constant temperature incubator and cultured for 14 hours. The diameter of the inhibition zone was measured and the results are shown in the following table.

[0078] Table 2 Comparison of antibacterial properties

[0079]

[0080] It can be seen from Table 2 that the oral repair membrane prepared in Comparative Example 1 cannot play an active antibacterial role, while the oral repair membrane prepared in Experimental Example 1 can effectively fight against common oral bacteria, wherein the anti-Gram-positive bacteria and anti-Gram-negative bacteria capabilities are about twice that of Comparative Example 1.

[0081] 2. Test on antibacterial rate of oral repair membrane

[0082] The oral repair membranes of Example 1 and Comparative Example 1, which were sterilized in a high-temperature steam sterilizer (220°C, 30 min) and had a size of 15×15 mm, were mixed with 1×10 5 CFU / mL Staphylococcus aureus and Escherichia coli suspensions were mixed and incubated at 37°C. After 24 hours, the total number of bacteria in the suspension was detected using the LB agar plate dilution method, and each group was repeated 3 times. Staphylococcus aureus and Escherichia coli were treated in the same way as blank groups. The antibacterial rate was calculated as: (1-(total number of bacteria in the experimental group / total number of bacteria in the blank group))×100%, and the results are shown in the following table.

[0083] Table 3 Antibacterial rate

[0084] Group Example 1 Comparative Example 1 Anti-Staphylococcus aureus rate / % 98.6±0.008 35.1±0.016 Anti-E. coli rate % 99.0±0.017 37.8±0.002

[0085] It can be seen from Table 3 that the antibacterial rate of the oral repair membrane prepared in Experimental Example 1 is significantly higher than that in Comparative Example 1.

[0086] 3. Testing of antibacterial adhesion properties of oral repair membranes

[0087] The membranes that had been tested for antibacterial rate of oral repair membranes were taken out, and 0.5 mL of sterile water was used to wash the floating bacteria on the membranes. Then, 1 mL of sterile water was used to soak the membranes, and the bacteria on the membranes were shaken off. The total number of bacteria was detected using the LB agar plate dilution method, and each group was repeated 3 times. The results are shown in Table 4.

[0088] Table 4 Comparison of antibacterial adhesion performance

[0089]

[0090] The experimental results show that the oral restoration membrane with a nano-film layer can effectively kill bacteria, and due to the presence of the nano-film layer, when its surface is exposed to a bacterial environment, it can prevent bacterial adhesion, which can effectively prevent bacterial infection after the membrane is exposed.

[0091] 4. Anti-Porphyromonas gingivalis performance of oral repair membrane

[0092] The sterilized oral repair membranes of Example 1 and Comparative Example 1 with a size of 15×15 mm were mixed with 1×10 5 CFU / mL Porphyromonas gingivalis suspension was mixed and placed in 37℃ for anaerobically incubation. After 24h, the bacterial suspension was taken out and the OD was measured. 600 The absorbance was used to evaluate the antibacterial performance of each group, and each group was repeated 3 times. Pure Porphyromonas gingivalis was treated in the same way as the blank group. The antibacterial rate was calculated as follows: (1-(OD 600 / Blank group OD 600 ))×100%, the results are shown in Table 5.

[0093] Table 5 Comparison of anti-Porphyromonas gingivalis performance

[0094] Group Example 1 Comparative Example 1 Antibacterial rate 98.1％ 31.6％

[0095] It can be seen from Table 5 that the antibacterial oral repair film prepared in Example 1 can effectively inhibit Porphyromonas gingivalis that can cause periodontitis, and the inhibition rate reaches 98.1%, which is much higher than that of Comparative Example 1.

[0096] The present invention provides an oral repair membrane with antibacterial properties, a preparation method and its application. The oral repair membrane prepared by the present invention has the following beneficial effects: the three-dimensional network structure of the acellular matrix used in the repair membrane is closest to the ideal natural scaffold, which is conducive to cell growth and attachment; the collagen albumin, growth factor and glycosaminoglycan contained in the repair membrane promote the growth of new blood vessels and tissue repair of defective parts; the oral repair membrane has excellent barrier protection performance: the nano film layer formed by lysozyme and tri(2-carboxyethyl)phosphine, its hydrophobicity and smooth surface are not conducive to cell attachment, and the smaller pore size can prevent external cells from entering the defective part and proliferating; at the same time, the nano film layer is formed by lysozyme and tri(2-carboxyethyl)phosphine through phase transition lysozyme reaction, while achieving the barrier, the active antibacterial effect of the oral repair membrane is improved, and the surface bacteria can be prevented from growing during the treatment of membrane exposure. And the membrane structure of the oral repair membrane of the present invention, which is large outside and small inside, can prevent food residues from entering the defective part and causing bacteria to grow during the treatment process; the membrane surface where bacteria are difficult to attach can prevent bacterial secretions from prematurely degrading the repair membrane, and prolong the barrier effect of the oral repair membrane. The present invention adopts staggered superposition film laying, so that each surface of the product has excellent mechanical properties and is conducive to suturing.

[0097] The preparation process of the invention is simple and rapid, and the prepared oral repair membrane is easy to use and has good tissue adhesion, hydrophilicity, low immunogenicity and degradation performance. When used, the second acellular matrix layer of the membrane is facing the defective tissue, and repair is achieved as the membrane degrades. The oral repair membrane has broad application prospects in the field of oral medicine.

Claims

1. Application of an oral repair membrane with antibacterial properties in the preparation of medical devices in the field of oral medicine, characterized in that: The oral repair film with antibacterial properties comprises an antibacterial nano film layer, a first decellularized matrix layer and a second decellularized matrix layer; The first decellularized matrix layer and the second decellularized matrix layer have a loose surface and a dense surface; the porosity of the loose surface is 35% to 90%, and the pore size is greater than 35 μm; the porosity of the dense surface is 5% to 25%, and the pore size is less than 10 μm; The nano-thin film layer is located on the dense surface of the first decellularized matrix layer; The loose surface of the first decellularized matrix layer is located on the dense surface of the second decellularized matrix layer; the first decellularized matrix layer and the second decellularized matrix layer are prepared from animal tissues; the animal tissues are porcine small intestine, porcine pericardium, porcine skin, bovine skin or bovine pericardium; The nano-film layer is a thin film formed by coating the nano-film reaction solution on the dense surface of the first decellularized matrix, and the coating amount is 50 to 200 μL / cm 2 The preparation method of the nano film reaction solution is as follows: using 10mM Tris-HCl and NaOH solution with a pH of 12 to 13 to dissolve lysozyme and tri(2-carboxyethyl)phosphine hydrochloride to obtain a lysozyme and tri(2-carboxyethyl)phosphine solution with a pH of 7.0 to 7.4; then mixing and stirring for 2 to 5 minutes to obtain the nano film reaction solution, wherein the content ratio of lysozyme to tri(2-carboxyethyl)phosphine is (0.5 to 2) g / L: 20 mol / L; The first decellularized matrix layer includes 1 to 3 layers of decellularized matrix, each layer of the decellularized matrix has a loose surface facing downward and a dense surface facing upward, and each layer of the decellularized matrix is ​​stacked alternately; the length of the first decellularized matrix layer is 20 to 60 mm, the width is 20 to 60 mm, and the thickness is 0.1 to 0.4 mm, and each layer of the decellularized matrix is ​​stacked alternately; the contact surface of the first decellularized matrix layer and the second decellularized matrix layer is stacked alternately; The second decellularized matrix layer includes 2 to 5 layers of decellularized matrix, each layer of decellularized matrix has a loose surface facing downward and a dense surface facing upward; the second decellularized matrix layer is 3 to 8 mm shorter, 3 to 8 mm narrower, and 0.1 to 0.8 mm thicker than the first decellularized matrix layer; the second decellularized matrix layer is located in the center of the first decellularized matrix layer; the second decellularized matrix and the first decellularized matrix both have a loose surface facing downward and a dense surface facing upward.

2. The use according to claim 1, characterized in that: The animal tissue is porcine small intestine.

3. The use according to claim 1, characterized in that: The coating volume is 100 μL / cm 2 .

4. The use according to claim 1, characterized in that: The method for preparing the oral repair film with antibacterial properties comprises the following steps: (1) Decellularized matrix is ​​prepared using animal tissue; (2) cutting the decellularized matrix to obtain a first decellularized matrix layer membrane sheet and a second decellularized matrix layer membrane sheet; (3) Preparation of nanofilm reaction solution; (4) Covering the first decellularized matrix layer with the second decellularized matrix layer, and then coating the nano film reaction solution on the dense surface of the first decellularized matrix, allowing it to stand, and freeze-drying to obtain; The second decellularized matrix layer is located in the center of the first decellularized matrix layer; The preparation method of the nano film reaction solution is as follows: using 10mM Tris-HCl and NaOH solution with a pH of 12 to 13 to dissolve lysozyme and tri(2-carboxyethyl)phosphine hydrochloride to prepare a lysozyme and tri(2-carboxyethyl)phosphine solution with a pH of 7.0 to 7.4; then mixing and stirring for 2 to 5 minutes to obtain the nano film reaction solution, wherein the content ratio of lysozyme to tri(2-carboxyethyl)phosphine is (0.5 to 2) g / L: 20 mol / L; 50~200μL / cm 2 The nano-thin film reaction solution is coated on the dense surface of the first decellularized matrix.

5. The use according to claim 4, characterized in that: The step of obtaining the decellularized matrix comprises: (1) Cleaning: Thaw the animal tissue and rinse it 8 to 10 times with purified water; (2) Virus removal: Soak the biological tissue in step (1) in a 1-4 mol / L NaOH solution, shake for 15-20 min, and repeat 5-10 times; (3) Degreasing: Place the material treated in step (2) in a mixed solution of peracetic acid and ethanol and shake for 15 to 20 minutes, repeating 5 to 10 times; (4) Deproteinization: The material treated in step (3) is placed in a 2% to 8% sodium dodecyl sulfate solution for shaking and washing; (5) Cleaning: Use purified water to perform ultrasonic cleaning for 5 to 10 times to obtain a decellularized matrix.

6. The use according to claim 4, characterized in that: The content ratio of lysozyme to tri(2-carboxyethyl)phosphine is 1 g / L:20 mol / L.

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