CN117959502A - Oral cavity repairing film with antibacterial performance, preparation method and application thereof - Google Patents
Oral cavity repairing film with antibacterial performance, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an oral cavity repairing film with antibacterial performance, a preparation method and application thereof, and belongs to the field of medical biological materials. The oral cavity repairing film with antibacterial performance comprises an antibacterial nanometer film layer, a first acellular matrix layer and a second acellular matrix layer; the first acellular matrix layer and the second acellular matrix layer have a loose face and a dense face; the nanometer film layer is positioned on the compact surface of the first acellular matrix layer; the loose face of the first acellular matrix layer is located on the dense face of the second acellular matrix layer. The preparation method of the oral cavity repairing film with antibacterial property is simple and quick in process, and the prepared oral cavity repairing film is convenient to use and has good tissue adhesiveness, hydrophilicity, low immunogenicity and degradability. When in use, the second acellular matrix layer of the membrane faces the defective tissue, and the repair is realized along with the degradation of the membrane. The oral cavity repairing film has wide application prospect in the field of stomatology.
Description
Technical Field
The invention relates to the field of medical biological materials, in particular to an oral cavity repairing film with antibacterial performance, a preparation method and application thereof.
Background
The acellular matrix is the dissimilar biological material which is most widely applied in clinic at present, and the three-dimensional network structure of the acellular matrix is closest to an ideal natural bracket, so that the acellular matrix is widely applied to the field of tissue regeneration and repair. It retains the natural extracellular matrix (ECM) structure and components, has a complete vasculature, and this multifunctional structure can induce cell differentiation and maturation. Due to eutrophication of the decellularized matrix membrane, cells and bacteria can adhere and proliferate, and the disadvantages of weak active antibacterial property and barrier property exist. The natural extracellular matrix biological membrane as disclosed in Chinese patent CN 106880872A, and the preparation method and the application thereof do not consider the influence of bacterial infection on treatment; the oral cavity repairing film and the related kit for guiding tissue regeneration disclosed in Chinese patent CN 219398361U use the bladder basement membrane as a compact layer to play a barrier role, and the fact that the bladder basement membrane is still easy to be biological climbed and damaged in operation is not considered, so that the barrier role is lost.
Infection of varying degrees occurs in the mouth for internal or external reasons at different stages of treatment, such as infection by exposure to the membranes in periodontitis treatment and in oral restorative membrane treatment. Periodontitis is a major cause of affecting periodontal tissue regeneration in periodontal disease treatment, and the presence of periodontal microorganisms such as Porphyromonas gingivalis (Pg) can trigger excessive immune responses, induce inflammation, and lead to osteoclast activation and bone resorption. Microbial infection and the consequent host immune response in turn affect the function of local periodontal ligament stem cells, enhancing periodontitis. Periodontal regenerative surgery such as guided tissue regeneration, bone grafting, etc. still present the risk of secondary infections.
Membrane exposure is one of the most frequently reported complications of Guided Tissue Regeneration (GTR) and Guided Bone Regeneration (GBR). Exposure of the postoperative barrier membrane can cause microorganisms in the oral cavity to migrate and colonize the exposed area, at this time the membrane becomes a warm bed of bacteria, promoting bacterial attachment and proliferation, causing acute inflammatory infiltration of the submembranous space, resulting in cessation of the regeneration process. There are studies showing that proteases secreted by bacteria colonizing the barrier membrane also accelerate degradation of the collagen-like barrier membrane (Bio-Gide collagen membrane and haao oral repair membrane) and prematurely lose the barrier effect, thereby reducing the therapeutic effect and increasing the risk of infection (Bai Peng control study of degradation of both collagen membranes exposed to the oral environment. Journal of Chinese oral plantation, 2011,16 (01): 56-57.). The foregoing demonstrates the need for preparing a mouth repair film with active antibacterial and enhanced barrier properties.
Phase Transition Lysozyme (PTL) is a novel, mild, efficient and rapid amyloid-like protein assembly that can form transparent nanofilms with microscopic pore sizes. It has long-lasting in vitro and in vivo broad-spectrum antibacterial effect on gram positive/negative and fungi, and stronger killing effect than natural lysozyme (ACS APPLIED MATERIALS & Interfaces,2017.9 (1): p.198-210.). In addition, the surface hydration of the nano film makes the nano film have the performance of anti-biological film, hydrophobicity, blood compatibility, low cytotoxicity and the like, and can be permanently adhered to the surfaces of inorganic, organic and microscopic living particles without affecting the normal functions of the nano film. The active antibacterial effect of the membrane can be provided by using the phase-transition lysozyme technology.
In summary, there is a need to prepare an oral restoration film, which has good mechanical properties and excellent antibacterial properties on the premise of ensuring the mechanical properties of the oral restoration film.
Disclosure of Invention
The invention aims to provide an oral cavity repairing film with antibacterial performance, a preparation method and application thereof.
The invention provides an oral cavity repairing film with antibacterial performance, which comprises an antibacterial nanometer film layer, a first acellular matrix layer and a second acellular matrix layer;
the first acellular matrix layer and the second acellular matrix layer have a loose face and a dense face;
the nanometer film layer is positioned on the compact surface of the first acellular matrix layer;
the loose surface of the first acellular matrix layer is positioned on the compact surface of the second acellular matrix layer; the first acellular matrix layer and the second acellular matrix layer are prepared from animal tissue.
Further, the first acellular matrix layer comprises 1-3 layers of acellular matrixes, each layer of acellular matrix is loose and downward, compact and upward, and each layer of acellular matrix is overlapped in a staggered way; the length of the first acellular matrix layer is 20-60 mm, the width is 20-60 mm, the thickness is 0.1-0.4 mm, and acellular matrixes of each layer are overlapped in a staggered mode; the contact surfaces of the first acellular matrix layer and the second acellular matrix layer are overlapped in a staggered way.
The second acellular matrix layer comprises 2-5 layers of acellular matrixes, wherein each layer of acellular matrix is loose and downward and compact and upward; the second acellular matrix layer is 3-8 mm shorter and 3-8 mm narrower than the first acellular matrix layer, and the thickness of the second acellular matrix layer is 0.1-0.8 mm; the second acellular matrix layer is positioned in the center of the first acellular matrix layer; the second acellular matrix and the first acellular matrix are both loose face down and dense face up.
Further, the animal tissue is small intestine, pericardium, skin, cow hide or pericardium; preferably porcine small intestine.
Further, the antibacterial nano film layer is a film formed by mixing lysozyme and tri (2-carboxyethyl) phosphine and coating the mixed solution on the compact surface of the first acellular matrix, wherein the coating amount is 50-200 mu L/cm 2, preferably 100 mu L/cm 2.
Further, the porosity of the loose surface is 35% -90%, and the pore diameter is more than 35 mu m; the porosity of the compact surface is 5-25%, and the pore diameter is less than 10 mu m.
The invention also provides a preparation method of the oral cavity repairing film with antibacterial property, which comprises the following steps:
(1) Preparing a decellularized matrix by using animal tissues;
(2) Cutting the acellular matrix to obtain a first acellular matrix layer patch and a second acellular matrix layer patch;
(3) Preparing a nano film reaction solution;
(4) Covering the second acellular matrix layer with the first acellular matrix layer, coating the compact surface of the first acellular matrix with the nano film reaction solution, standing, and freeze-drying to obtain the nano film;
wherein the second acellular matrix layer is centered within the first acellular matrix layer;
the nano film reaction liquid is a mixed liquid of lysozyme and tri (2-carboxyethyl) phosphine;
And coating the nano film reaction liquid on the compact surface of the first acellular matrix at the concentration of 50-200 mu L/cm 2.
Further, the step of obtaining the acellular matrix comprises:
(1) Cleaning: thawing animal tissues and flushing the animal tissues with purified water for 8 to 10 times;
(2) Virus removal: soaking the biological tissue in the step (1) in a NaOH solution with the concentration of 1-4 mol/L, oscillating for 15-20 min, and repeating for 5-10 times;
(3) Degreasing: placing the material treated in the step (2) into a mixed solution of peroxyacetic acid and ethanol, and carrying out oscillation treatment for 15-20 min, and repeating for 5-10 times;
(4) Deproteinization: placing the treatment material in the step (3) in 2% -8% of sodium dodecyl sulfate solution for vibration cleaning;
(5) Cleaning: ultrasonically cleaning the cell substrate for 5 to 10 times by using purified water to obtain the acellular matrix.
Further, the preparation method of the nano film reaction liquid comprises the following steps: dissolving lysozyme and Tris (2-carboxyethyl) phosphine hydrochloride by using 10mM Tris-HCl and NaOH solution with pH=12-13, and preparing lysozyme and Tris (2-carboxyethyl) phosphine solution with pH=7.0-7.4; and then mixing and stirring for 2-5 min to obtain the nano film reaction liquid, wherein the concentration ratio of lysozyme to tri (2-carboxyethyl) phosphine is (0.5-2) g/L:20mol/L;
further, the concentration ratio of lysozyme and tris (2-carboxyethyl) phosphine was 1g/L:20mol/L.
The invention also provides application of the oral cavity repairing film with antibacterial property prepared by the method in the field of stomatology.
The invention provides an oral cavity repairing film with antibacterial performance, a preparation method and application thereof. The oral cavity repairing film prepared by the invention has the following beneficial effects: the three-dimensional network structure of the acellular matrix used by the repair membrane is closest to an ideal natural bracket, and is beneficial to cell ingrowth and climbing; the collagen, the growth factors and the glycosaminoglycan contained in the collagen promote the growth of new blood vessels and the tissue repair of defect parts; the oral repair film has excellent barrier protection performance: the hydrophobicity and smooth surface of the nano film layer formed by lysozyme and tri (2-carboxyethyl) phosphine are not beneficial to cell adhesion, and the smaller aperture can prevent external cells from entering the defect part to proliferate; meanwhile, lysozyme and tri (2-carboxyethyl) phosphine form a nano film layer through phase transition lysozyme reaction, so that the active antibacterial effect of the oral cavity repairing film is improved while a barrier is realized, and surface bacteria breeding in the treatment of exposing the film can be avoided. The membrane structure with large outside and small inside of the oral cavity repairing membrane can prevent food residues from entering the defect part to cause bacterial growth in the treatment process; the membrane surface to which bacteria are difficult to adhere can avoid the premature degradation of the repair membrane caused by bacterial secretion, and the barrier effect of the oral repair membrane is prolonged. The invention uses staggered superposition film laying, so that the mechanical properties of each surface of the product are excellent, and the stitching is facilitated.
The preparation process is simple and quick, and the prepared oral cavity repairing film is convenient to use and has good tissue adhesiveness, hydrophilicity, low immunogenicity and degradability. When in use, the second acellular matrix layer of the membrane faces the defective tissue, and the repair is realized along with the degradation of the membrane. The oral cavity repairing film has wide application prospect in the field of stomatology.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a diagram of a decellularized matrix lyophilized pellet.
Fig. 2 is a schematic structural view of the oral restoration film of the present invention. Wherein 1 is a nano film layer, 2 is a first acellular matrix layer, and 3 is a second acellular matrix layer.
Fig. 3 is a scanning electron microscope image of the oral restoration film of the present invention. Wherein a) the acellular matrix loose side, b) the acellular matrix dense side; c) A nano-film layer.
FIG. 4 is a graph showing a screening experiment of lysozyme and tris (2-carboxyethyl) phosphine content ratio. Wherein, the number of the 11 groups of samples is reduced by 10 percent from left to right for lysozyme to 0 percent, and the total number of the 11 groups of samples is 11.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Example 1 dental restoration film with antibacterial Properties prepared
1. Experimental method
(One) preparation of acellular matrix
1. Cleaning: 4g of porcine small intestine animal tissue was thawed and rinsed 10 times with purified water.
2. Virus removal: soaking the biological tissue in the step (1) in a 1M NaOH solution, vibrating for 20min, and repeating for 5 times.
3. Degreasing: and (3) placing the material treated in the step (2) in a mixed solution of peroxyacetic acid and ethanol, and carrying out shaking treatment for 15min, and repeating for 5 times.
4. Deproteinization: and (3) placing the treated material in the step (3) in a 4% sodium dodecyl sulfate solution for vibration cleaning.
5. Cleaning: the resulting decellularized matrix was ultrasonically washed 10 times with purified water to give a decellularized matrix sheet with a thickness of 0.1-0.2mm as shown in FIG. 1, and fibers on the decellularized matrix sheet were visualized (FIG. 1).
6. Cutting the acellular matrix obtained in the step 5 to obtain acellular matrix membranes with the length of 40mm and the width of 30mm, and overlapping 2 layers of acellular matrix membranes in a staggered manner according to the fiber direction to obtain a first acellular matrix layer.
7. Cutting the acellular matrix obtained in the step 5 to obtain acellular matrix membranes with the length of 35mm and the width of 25mm, and overlapping 3 acellular matrix membranes in a staggered manner according to the fiber direction to obtain a second acellular matrix layer.
Secondly, preparing a nano film reaction solution;
The nano-film reaction liquid is a neutral mixed liquid of lysozyme and tri (2-carboxyethyl) phosphine, and the preparation steps of the nano-film reaction liquid are as follows: dissolving lysozyme and Tris (2-carboxyethyl) phosphine hydrochloride by using 10mM Tris-HCl and NaOH solution with pH=12-13, and preparing lysozyme and Tris (2-carboxyethyl) phosphine solution with pH=7.0-7.4; and then mixing and stirring for 2-5 min to obtain the nano film reaction liquid, wherein the content ratio of lysozyme to tri (2-carboxyethyl) phosphine is 1g/L:20mol/L.
(III) lay-up and coating
Scanning the two sides of the acellular matrix by a scanning electron microscope, wherein a scanning electron microscope image of the acellular matrix is shown as figure 3, and the acellular matrix membrane is loose on one side and compact on the other side; wherein the porosity of the loose surface is 35-90%, the pore diameter is more than 35 mu m, the porosity of the compact surface is 5-25%, and the pore diameter is less than 10 mu m.
Placing the second acellular matrix layer membrane sheet on a freeze-drying disc, and then overlapping and covering the first acellular matrix layer membrane sheet in a staggered manner; the second acellular matrix layer is positioned in the center of the first acellular matrix layer; the second acellular matrix and the first acellular matrix are loose and downward and compact and upward, and the contact surfaces of the first acellular matrix layer and the second acellular matrix layer are staggered and overlapped; then coating nano film reaction liquid on the compact surface of the first acellular matrix according to 100 mu L/cm 2, standing for 2-5 min, and finally vacuum freeze-drying and packaging to obtain the oral cavity repairing film.
Comparative example 1 preparation of oral repair film without nano film reaction liquid
Based on the technical scheme of the embodiment 1, the nano film reaction liquid is not coated, and then the oral cavity restoration film is obtained through vacuum freeze-drying and packaging.
The beneficial effects of the oral cavity repairing film with antibacterial property prepared by the invention are proved by experimental examples.
Experimental example 1, screening experiment I and experiment method for lysozyme and tris (2-carboxyethyl) phosphine content ratio in nano-film reaction solution
Lysozyme and tris (2-carboxyethyl) phosphine hydrochloride were prepared using the protocol of example 1 to give lysozyme and tris (2-carboxyethyl) phosphine having ph=7.0 to 7.4, followed by 1mL of a mixed solution, wherein lysozyme and tris (2-carboxyethyl) phosphine were sequentially 5mg:20mM. When the lysozyme content in the mixed solution was defined as 5mg, the mixed solution was 100%, and under the quantitative condition of 20mM tris (2-carboxyethyl) phosphine, the lysozyme content was reduced by 10% to 0% in sequence, and 11 groups of samples were prepared in total. After the preparation is completed, the sample is kept stand at room temperature for 2min, and then evenly mixed and photographed.
2. Experimental results
As shown in FIG. 4, the results showed that the higher the lysozyme content, the more turbid the mixed solution, and the macroscopic suspension was visible. As shown in fig. 3 (c), the nano-film reaction solution can form a smooth and flawless coating layer on the decellularized matrix membrane with lower lysozyme content for the reaction preparation of the nano-film layer. Therefore, in order to prevent the film forming speed from influencing the film surface smoothness due to the excessively high reaction speed, the content ratio of lysozyme to tri (2-carboxyethyl) phosphine used in the invention is (0.5-2) g/L:20mol/L.
Experimental example 2 mechanical properties, water absorption multiple detection one, experiment method of oral cavity repairing film with antibacterial property
Mechanical property test: the oral repair films prepared in example 1 and comparative example 1 were tested using a tensile tester with a test specification of 15×15mm. Recording tensile strength and elongation at break of the membrane in the test process, and calculating an elongation at break calculation formula: (elongation at break of the membrane/original length of the membrane) ×100%.
Water absorption multiple: the membrane with known weight is absorbed for 1min, then suspended for controlling water for 1min and weighed, and the water absorption multiple is calculated according to the weight ratio of the membrane before and after water absorption.
2. Experimental results
The experimental results are shown in table 1.
TABLE 1 detection of mechanical Properties and Water absorption times
| Group of | Example 1 | Comparative example 1 |
| Tensile Strength/N | 88.7 | 86.5 |
| Water absorption multiple | 7.73 | 7.65 |
And recording tensile strength and elongation at break of the membrane by a tensile tester, wherein the calculation formula of the elongation at break is as follows: 100% of elongation at break of the film/original length of the film, 23.55% of elongation at break of the oral repair film of example 1.
Table 1 shows the results of testing the tensile strength and water absorption properties of example 1 and comparative example 1, and it can be seen that the tensile strength and water absorption properties of example 1 are superior to those of comparative example 1, indicating that example 1 has excellent mechanical properties and hydrophilicity. The membrane can be prevented from being damaged in the suture and pulling process of clinical use due to good mechanical property, and the oral cavity repairing membrane can be used in an oral cavity wet environment due to good hydrophilicity, so that the attaching performance is enhanced, and the membrane is prevented from falling off.
Experimental example 2 antibacterial property detection of oral prosthetic film having antibacterial property
1. Oral cavity repair film antibacterial performance test
100. Mu.L of 1X 10 6 CFU/mL staphylococcus aureus and escherichia coli suspension were each plated on LB agar medium. The mouth repair films of example 1 and comparative example 1 were cut into circles with a diameter of 10mm, sterilized by a high-temperature steam sterilizer (220 ℃ C., 30 min), and then placed in the center of LB agar medium, respectively, and each group of experiments was repeated 3 times.
The resulting dish was placed upside down in a incubator at 37℃for 14 hours, and the diameter of the inhibition zone was measured, and the results are shown in the following table.
Table 2 comparison of antibacterial properties
As can be seen from table 2, the oral repair film prepared in comparative example 1 does not have a good active antibacterial effect, and the oral repair film prepared in experimental example 1 can effectively combat common oral bacteria. Wherein, the capacity of resisting gram positive bacteria and gram negative bacteria is about 2 times of that of comparative example 1.
2. Oral repair film antibacterial rate test
The oral repair membranes of example 1 and comparative example 1 sterilized by high temperature steam sterilization cabinet (220 ℃,30 min) with a gauge of 15 x 15mm were mixed with 1x 10 5 CFU/mL of staphylococcus aureus and escherichia coli suspension, respectively, and incubated at 37 ℃. After 24h the total number of bacteria in the suspension was checked using LB agar plate dilution, 3 replicates per group. The same treatment was performed on staphylococcus aureus and escherichia coli, respectively, as a blank group. The calculation formula of the bacteriostasis rate is as follows: (1- (total number of bacteria in experimental group/total number of bacteria in blank group)). Times.100%, the results are shown in the following table.
TABLE 3 antibacterial Rate
| Group of | Example 1 | Comparative example 1 |
| Anti-staphylococcus aureus rate/% | 98.6±0.008 | 35.1±0.016 |
| Anti-colibacillus rate% | 99.0±0.017 | 37.8±0.002 |
As can be seen from table 3, the oral repair film prepared in experimental example 1 has a significantly higher antibacterial rate than comparative example 1.
3. Antibacterial adhesion performance detection of oral repair film
The membrane subjected to the oral restoration membrane bacteriostasis rate test is taken out, and planktonic bacteria on the membrane are rinsed with 0.5mL of sterile water. The membrane was then immersed in 1mL of sterile water and bacteria on the membrane were shaken off, and the total number of bacteria was measured using LB agar plate dilution, 3 replicates per group, and the results are shown in table 4.
Table 4 comparison of antibacterial adhesion properties
Experimental results show that the oral cavity repairing film with the nano film layer can effectively kill bacteria, and can prevent bacteria from adhering when the surface of the oral cavity repairing film is exposed in a bacterial environment due to the existence of the nano film layer, so that bacterial infection after the film is exposed can be effectively prevented.
4. Anti-Porphyromonas gingivalis performance of oral repair film
The sterilized, 15X 15mm size oral repair membranes of example 1 and comparative example 1 were mixed with 1X 10 5 CFU/mL Porphyromonas gingivalis suspension, respectively, and placed in anaerobic incubation at 37 ℃. After 24 hours, the bacterial suspensions OD 600 absorbance was taken out and assayed to evaluate the antibacterial properties of each group, each group being repeated 3 times. The same treatment was performed on pure Porphyromonas gingivalis as a blank group. The calculation formula of the bacteriostasis rate is as follows: (1- (experimental group OD 600/blank group OD 600)). Times.100%, the results are shown in Table 5.
TABLE 5 comparison of Porphyromonas gingivalis resistance
| Group of | Example 1 | Comparative example 1 |
| Antibacterial rate | 98.1％ | 31.6％ |
As can be seen from Table 5, the antibacterial oral restoration film prepared in example 1 can effectively inhibit Porphyromonas gingivalis which can cause periodontitis, and the antibacterial rate is 98.1% which is far higher than that of comparative example 1.
The invention provides an oral cavity repairing film with antibacterial performance, a preparation method and application thereof. The oral cavity repairing film prepared by the invention has the following beneficial effects: the three-dimensional network structure of the acellular matrix used by the repair membrane is closest to an ideal natural bracket, and is beneficial to cell ingrowth and climbing; the collagen, the growth factors and the glycosaminoglycan contained in the collagen promote the growth of new blood vessels and the tissue repair of defect parts; the oral repair film has excellent barrier protection performance: the hydrophobicity and smooth surface of the nano film layer formed by lysozyme and tri (2-carboxyethyl) phosphine are not beneficial to cell adhesion, and the smaller aperture can prevent external cells from entering the defect part to proliferate; meanwhile, lysozyme and tri (2-carboxyethyl) phosphine form a nano film layer through phase transition lysozyme reaction, so that the active antibacterial effect of the oral cavity repairing film is improved while a barrier is realized, and surface bacteria breeding in the treatment of exposing the film can be avoided. The membrane structure with large outside and small inside of the oral cavity repairing membrane can prevent food residues from entering the defect part to cause bacterial growth in the treatment process; the membrane surface to which bacteria are difficult to adhere can avoid the premature degradation of the repair membrane caused by bacterial secretion, and the barrier effect of the oral repair membrane is prolonged. The invention uses staggered superposition film laying, so that the mechanical properties of each surface of the product are excellent, and the stitching is facilitated.
The preparation process is simple and quick, and the prepared oral cavity repairing film is convenient to use and has good tissue adhesiveness, hydrophilicity, low immunogenicity and degradability. When in use, the second acellular matrix layer of the membrane faces the defective tissue, and the repair is realized along with the degradation of the membrane. The oral cavity repairing film has wide application prospect in the field of stomatology.
Claims (10)
1. An oral repair film having antibacterial properties, characterized in that: the antibacterial nano-film comprises an antibacterial nano-film layer, a first acellular matrix layer and a second acellular matrix layer;
the first acellular matrix layer and the second acellular matrix layer have a loose face and a dense face;
the nanometer film layer is positioned on the compact surface of the first acellular matrix layer;
the loose surface of the first acellular matrix layer is positioned on the compact surface of the second acellular matrix layer; the first acellular matrix layer and the second acellular matrix layer are prepared from animal tissue.
2. The oral repair film with antibacterial properties according to claim 1, characterized in that: the first acellular matrix layer comprises 1-3 layers of acellular matrixes, each layer of acellular matrix is loose and downward, compact and upward, and each layer of acellular matrix is overlapped in a staggered way; the length of the first acellular matrix layer is 20-60 mm, the width is 20-60 mm, the thickness is 0.1-0.4 mm, and acellular matrixes of each layer are overlapped in a staggered mode; the contact surfaces of the first acellular matrix layer and the second acellular matrix layer are overlapped in a staggered way.
The second acellular matrix layer comprises 2-5 layers of acellular matrixes, wherein each layer of acellular matrix is loose and downward and compact and upward; the second acellular matrix layer is 3-8 mm shorter and 3-8 mm narrower than the first acellular matrix layer, and the thickness of the second acellular matrix layer is 0.1-0.8 mm; the second acellular matrix layer is positioned in the center of the first acellular matrix layer; the second acellular matrix and the first acellular matrix are both loose face down and dense face up.
3. The oral repair film with antibacterial properties according to claim 2, characterized in that: the animal tissue is small intestine, pericardium, skin, cow hide or pericardium; preferably porcine small intestine.
4. The oral repair film with antibacterial properties according to claim 3, characterized in that: the antibacterial nanometer film layer is a film formed by mixing lysozyme and tri (2-carboxyethyl) phosphine and coating the mixed solution on the compact surface of the first acellular matrix, wherein the coating amount is 50-200 mu L/cm 2, preferably 100 mu L/cm 2.
5. The oral repair film with antibacterial properties according to any one of claims 1-4, characterized in that: the porosity of the loose surface is 35% -90%, and the pore diameter is more than 35 mu m; the porosity of the compact surface is 5-25%, and the pore diameter is less than 10 mu m.
6. A method for preparing an oral restoration film having antibacterial properties according to any one of claims 1 to 5, characterized in that: the method comprises the following steps:
(1) Preparing a decellularized matrix by using animal tissues;
(2) Cutting the acellular matrix to obtain a first acellular matrix layer patch and a second acellular matrix layer patch;
(3) Preparing a nano film reaction solution;
(4) Covering the second acellular matrix layer with the first acellular matrix layer, coating the compact surface of the first acellular matrix with the nano film reaction solution, standing, and freeze-drying to obtain the nano film;
wherein the second acellular matrix layer is centered within the first acellular matrix layer;
the nano film reaction liquid is a mixed liquid of lysozyme and tri (2-carboxyethyl) phosphine;
And coating the nano film reaction liquid on the compact surface of the first acellular matrix at the concentration of 50-200 mu L/cm 2.
7. The method according to claim 6, wherein: the step of obtaining a decellularized matrix comprises:
(1) Cleaning: thawing animal tissues and flushing the animal tissues with purified water for 8 to 10 times;
(2) Virus removal: soaking the biological tissue in the step (1) in a NaOH solution with the concentration of 1-4 mol/L, oscillating for 15-20 min, and repeating for 5-10 times;
(3) Degreasing: placing the material treated in the step (2) into a mixed solution of peroxyacetic acid and ethanol, and carrying out oscillation treatment for 15-20 min, and repeating for 5-10 times;
(4) Deproteinization: placing the treatment material in the step (3) in 2% -8% of sodium dodecyl sulfate solution for vibration cleaning;
(5) Cleaning: ultrasonically cleaning the cell substrate for 5 to 10 times by using purified water to obtain the acellular matrix.
8. The method according to claim 7, wherein: the preparation method of the nano film reaction liquid comprises the following steps: dissolving lysozyme and Tris (2-carboxyethyl) phosphine hydrochloride by using 10mM Tris-HCl and NaOH solution with pH=12-13, and preparing lysozyme and Tris (2-carboxyethyl) phosphine solution with pH=7.0-7.4; and then mixing and stirring for 2-5 min to obtain the nano film reaction liquid, wherein the concentration ratio of lysozyme to tri (2-carboxyethyl) phosphine is (0.5-2) g/L:20mol/L.
9. The method according to claim 8, wherein: the concentration ratio of lysozyme and tris (2-carboxyethyl) phosphine is 1g/L:20mol/L.
10. Use of an oral repair film with antibacterial properties prepared according to the method of any one of claims 1 to 9 in the field of stomatology.
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