Preparation method, product and application of a bifunctional layer composite biofilm

Abstract

The present invention relates to a preparation method, product and application of a bifunctional layer composite biofilm. The preparation method includes: degreasing the membrane tissue; mixing the degreased membrane tissue with an eluent to remove cells, and freeze-drying to obtain a loose layer; mixing collagen, chitosan and acetic acid, and air-drying to form a film to obtain a dense layer; wetting the loose layer, attaching the dense layer to the loose layer, and freeze-drying; or wetting the dense layer, attaching the loose layer to the dense layer, and freeze-drying to obtain the biofilm. The biofilm prepared by the present invention has good mechanical properties and thermal stability, and its effect is better than that of a single-layer biofilm. The addition of chitosan can further improve the effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofilms, and relates to a preparation method, product and application of a bifunctional layer composite biofilm. Background Art

[0002] Guided bone tissue regeneration membranes are currently an innovative technology mainly used in periodontal treatment and oral implant fields. The main purpose is that due to the different healing speeds of different tissues in the damaged area, usually the growth speeds of fibroblasts and epithelial tissues are relatively fast, which will inhibit the growth of bone tissue. An implantable membrane material is placed between the soft tissue and the bone defect area to establish a biological barrier, so as to create a relatively closed bone regeneration environment without affecting the natural growth of the wound. It is a biocompatible material.

[0003] Currently, the most commonly used biofilms on the market are divided into absorbable biofilms and non-absorbable biofilms. Non-absorbable membranes are mainly metal membranes and polymer membranes. These membrane materials have excellent mechanical strength and can play a good shielding and supporting role. However, there are also huge defects. First of all, non-absorbable membranes usually have the disadvantages of high hardness and poor comfort, and have a poor fit with soft tissues. During activities, they will stimulate the wound and cause inflammatory reactions. Secondly, since the membrane cannot be absorbed and degraded in the body, a second operation is required to remove the implanted membrane from the damaged area, which not only increases the risk of infection but also prolongs the treatment and rehabilitation time.

[0004] Collagen, as one of the most widely used materials in biomedical applications, has low immunogenicity, good coordination with host cells, biodegradability, high strength and toughness. Collagen is a structural protein with biological functions, accounting for 1 / 3 of the total human protein, and is the main component of connective tissues or organs such as skin, cartilage, ligaments, and tendons. It can effectively promote cell division, proliferation and differentiation, induce platelet adhesion, promote platelet aggregation, and play an adhesive, compressive and filling role on the wound. Absorbable membranes are mainly collagen membranes, which have excellent biocompatibility and biodegradability in vivo, have a high matching degree with soft tissues and bone tissues in damaged areas of the human body, and can effectively cover the damaged area and enable bone tissue to grow in a shielding environment. At the same time, collagen-based absorbable biofilms do not require a second operation, reducing the risk of wound exposure and infection. In the application of biofilms, mechanical properties are also a factor that cannot be ignored. The quality of mechanical properties is directly related to the performance of biofilms in practical applications, especially in the medical field, such as skin repair and tissue engineering, the mechanical properties of biofilms are crucial for their functions and durability.

[0005] Therefore, providing a biodegradable biofilm with good mechanical properties is an urgent problem to be solved in this field. Summary of the Invention

[0006] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a preparation method, product and application of a dual-functional layer composite biofilm.

[0007] To achieve the purpose of this invention, the following technical solutions are adopted:

[0008] In the first aspect, the present invention provides a preparation method of a dual-functional layer composite biofilm, and the preparation method includes:

[0009] (1) Degreasing the membrane tissue;

[0010] (2) Mixing the degreased membrane tissue with an eluent to remove cells, and freeze-drying to obtain a loose layer;

[0011] Mixing collagen, chitosan and acetic acid, and air-drying to form a film to obtain a dense layer;

[0012] (3) Wetting the loose layer, attaching the dense layer to the loose layer, and freeze-drying;

[0013] Or, wetting the dense layer, attaching the loose layer to the dense layer, and freeze-drying to obtain the product.

[0014] The double-layer composite biofilm material prepared by the present invention has a dual-functional layer including a loose layer and a dense layer. The loose layer is composed of porcine acellular dermal matrix, and the dense layer is composed of porcine type I collagen and chitosan. Compared with traditional collagen biofilms, it has excellent tensile strength and thermal stability, and is suitable for application fields such as oral membranes and wound dressings.

[0015] Preferably, the ratio of the membrane tissue to the eluent is 1 g:(30 - 100) mL, and the specific point values in (30 - 100) can be selected as 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc. Other specific point values within the above numerical range can be selected and will not be elaborated here one by one.

[0016] Preferably, the membrane tissue includes porcine peritoneum, porcine pericardium or porcine small intestine submucosa.

[0017] Preferably, the degreasing treatment includes scraping the fat of the membrane tissue and then washing it with a detergent.

[0018] Preferably, the detergent includes water, sodium chloride aqueous solution or PBS buffer solution.

[0019] Preferably, the eluent includes 2 - 5% of sodium percarbonate, 3 - 7% of sodium bicarbonate, 0.5 - 2% of Triton-X100 and 86 - 95% of water by mass percentage.

[0020] The mass percentage of sodium percarbonate can be selected as 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.; the mass percentage of sodium bicarbonate can be selected as 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, etc.; the mass percentage of Triton-X100 can be selected as 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, etc.; the mass percentage of water can be selected as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0021] Preferably, the temperature for cell removal is 35 - 40 °C, the time is 20 - 40 min, and the number of times is 5 - 8 times.

[0022] The temperature can be selected as 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, etc.; the time can be selected as 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc.; the number of times can be selected as 5 times, 6 times, 7 times, 8 times, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0023] Preferably, the mass ratio of chitosan, collagen, and acetic acid is (2 - 6):(2 - 6):(50 - 100).

[0024] Among them, the specific point values in (2 - 6) can be selected as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.; the specific point values in (50 - 100) can be selected as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc. Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0025] Preferably, the collagen is type I collagen.

[0026] Preferably, the preparation method of the collagen includes: mixing the defatted membrane tissue with an enzyme solution for enzymatic hydrolysis, centrifuging the enzymatically hydrolyzed solution, mixing the supernatant with a protein denaturing solution for precipitation, and mixing the precipitate with a salting-out solution to obtain it.

[0027] Preferably, the enzyme includes pepsin.

[0028] Preferably, the enzyme solution also includes acetic acid.

[0029] Preferably, the molar concentration of the enzyme in the enzyme solution is 0.5 - 1.5 mol / L, such as 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, 1.1 mol / L, 1.2 mol / L, 1.3 mol / L, 1.4 mol / L, 1.5 mol / L, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0030] Preferably, the temperature of the enzymatic hydrolysis is 5 - 10 °C and the time is 5 - 8 h.

[0031] The temperature can be selected as 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, etc., and the time can be selected as 5 h, 5.5 h, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0032] Preferably, the centrifugation speed is 5000 - 8000 rpm and the time is 5 - 10 min.

[0033] The speed can be selected as 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, etc., and the time can be selected as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0034] Preferably, the protein denaturation solution includes an aqueous sodium hydroxide solution.

[0035] Preferably, the salting - out solution includes a disodium hydrogen phosphate solution.

[0036] Preferably, the molar concentration of sodium hydroxide in the aqueous sodium hydroxide solution is 0.1 - 1 mol / L, such as 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.6 mol / L, 0.7 mol / L, 0.8 mol / L, 0.9 mol / L, 1 mol / L, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0037] Preferably, the volume ratio of the supernatant to the protein denaturation solution is 1:(1 - 10), and the specific point values in (1 - 10) can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. Other specific point values within the above numerical range can be selected and will not be elaborated one by one here.

[0038] Preferably, the ratio of precipitate to disodium hydrogen phosphate solution is 1 g:(10-100) mL, wherein the specific point values ​​in (10-100) can be selected from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0039] Preferably, the molar concentration of disodium hydrogen phosphate in the disodium hydrogen phosphate solution is 0.01-0.05 mol / L, for example, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, etc. Other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0040] Preferably, the disodium hydrogen phosphate solution also includes peracetic acid.

[0041] In a second aspect, the present invention provides a double-functional-layer composite biofilm prepared according to the method for preparing the double-functional-layer composite biofilm according to the first aspect.

[0042] In a third aspect, the present invention provides a use of the double-functional layer composite biofilm according to the second aspect in the preparation of an oral membrane or a wound dressing.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The double-layer biological composite membrane of the present invention comprises a loose layer prepared by a decellularized biological tissue membrane, which promotes the growth of bone tissue; and a dense layer prepared by extracting type I collagen from the biological tissue membrane, which isolates the growth of soft tissue and inhibits the growth of bone tissue.

[0045] 2. The present invention introduces chitosan into the dense layer, which can play an antibacterial and hemostatic role, and chitosan can also cross-link with type I collagen in situ to induce collagen molecules to self-polymerize, thereby improving the mechanical properties and thermal stability of the collagen membrane.

[0046] 3. The tensile strength and thermal stability of the composite membrane are improved. The acellular dermal matrix method can preserve the complete three-dimensional structure of the dermal tissue as much as possible, and the structure of collagen will not be destroyed. The introduction of chitosan into the dense layer can also cross-link with type I collagen in situ, inducing the self-polymerization of collagen molecules, thereby improving the mechanical properties and thermal stability of the collagen membrane. DETAILED DESCRIPTION

[0047] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solution of the present invention in conjunction with the preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0048] The sources of the active ingredients contained in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown, and the necessary auxiliary ingredients contained in other commercially available raw materials are not elaborated):

[0049] Pepsin was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] Triton-X100 was purchased from Macklin Reagent Co., Ltd.

[0051] Type III collagen was purchased from Macklin Reagent Co., Ltd.

[0052] Chitosan was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0053] Preparation Example 1

[0054] This preparation example provides a method for preparing collagen, and the preparation method includes:

[0055] (1) Select fresh pig peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer.

[0056] (2) Mix 3 g of the washed pig peritoneum with 50 mL of 1 mol / L pepsin acetic acid solution, slowly stir at 8 °C for 6 h, and centrifuge at 7000 rpm for 7 min. Mix the supernatant with 0.5 mol / L sodium hydroxide solution to obtain a collagen white precipitate. The volume ratio of the supernatant to the sodium hydroxide solution is 1:5. Salt out the precipitate with 0.02 mol / L disodium hydrogen phosphate solution (the solvent is peracetic acid), and the ratio of the precipitate to the disodium hydrogen phosphate solution is 1 g:50 mL. Filter, and wash the filtered collagen white precipitate 4 times with purified water to obtain the final type I collagen.

[0057] Preparation Example 2

[0058] This preparation example provides a method for preparing collagen, and the preparation method includes:

[0059] (1) Select fresh pig peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer.

[0060] (2) Mix 3 g of the washed porcine peritoneum with 50 mL of an acetic acid solution of 0.5 mol / L pepsin, slowly stir for 5 h at 10 °C, and centrifuge for 10 min under the centrifugation condition of 5000 rpm. Mix the supernatant with 1 mol / L sodium hydroxide solution to obtain a collagen white precipitate. The volume ratio of the supernatant to the sodium hydroxide solution is 1:1. Salt out the precipitate with 0.05 mol / L disodium hydrogen phosphate solution (the solvent is peracetic acid), and the ratio of the precipitate to the disodium hydrogen phosphate solution is 1 g:100 mL. Filter, and wash the filtered collagen white precipitate 4 times with purified water to obtain the final type I collagen.

[0061] Preparation Example 3

[0062] This preparation example provides a method for preparing collagen, and the preparation method includes:

[0063] (1) Select fresh porcine peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer solution.

[0064] (2) Mix 3 g of the washed porcine peritoneum with 50 mL of an acetic acid solution of 1.5 mol / L pepsin, slowly stir for 8 h at 5 °C, and centrifuge for 5 min under the centrifugation condition of 8000 rpm. Mix the supernatant with 0.1 mol / L sodium hydroxide solution to obtain a collagen white precipitate. The volume ratio of the supernatant to the sodium hydroxide solution is 1:10. Salt out the precipitate with 0.01 mol / L disodium hydrogen phosphate solution (the solvent is peracetic acid), and the ratio of the precipitate to the disodium hydrogen phosphate solution is 1 g:10 mL. Filter, and wash the filtered collagen white precipitate 4 times with purified water to obtain the final type I collagen.

[0065] Preparation Example 4

[0066] This preparation example provides a method for preparing collagen, which is only different from Preparation Example 1 in that in step (2), "acetic acid solution of 1 mol / L pepsin" is replaced with "acetic acid solution of 0.1 mol / L pepsin", and other operations remain unchanged.

[0067] Preparation Example 5

[0068] This preparation example provides a method for preparing collagen, which is only different from Preparation Example 1 in that in step (2), "acetic acid solution of 1 mol / L pepsin" is replaced with "acetic acid solution of 2 mol / L pepsin", and other operations remain unchanged.

[0069] Example 1

[0070] This example provides a method for preparing a bifunctional layer composite biomembrane, and the preparation method includes:

[0071] (1) Select fresh porcine peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer solution.

[0072] (2) Mix 3 g of the washed porcine peritoneum with 180 mL of eluent. The eluent comprises 3% sodium percarbonate, 1% Triton-X100, 4.5% sodium bicarbonate and the balance water by mass percentage. Shake and wash it 6 times on a shaker at 37 °C, with each desorption time being 30 min. After desorption, rinse it 8 times with purified water, pre-freeze it at -30 °C for 1 hour, freeze it at -15 °C for 5 h, and keep it at 0 °C for 4 h to obtain the loose layer;

[0073] (3) Mix chitosan, the collagen prepared in Preparation Example 1 and acetic acid in a mass ratio of 3:3:80, and air-dry it into a film to obtain the dense layer;

[0074] (4) Wet the dense layer in water and place it on a smooth flat plate, then attach the loose layer thereto, and freeze-dry it into a film. The freeze-drying time is 12 h, the temperature is -30 °C, and the pressure is 1 mbar.

[0075] Example 2

[0076] This example provides a method for preparing a dual-functional layer composite biological membrane, and the preparation method includes:

[0077] (1) Select fresh porcine peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer solution.

[0078] (2) Mix 3 g of the washed porcine peritoneum with 90 mL of eluent. The eluent comprises 2% sodium percarbonate, 2% Triton-X100, 7% sodium bicarbonate and the balance water by mass percentage. Shake and wash it 8 times on a shaker at 35 °C, with each desorption time being 20 min. After desorption, rinse it 8 times with purified water, pre-freeze it at -30 °C for 1 hour, freeze it at -15 °C for 5 h, and keep it at 0 °C for 4 h to obtain the loose layer;

[0079] (3) Mix chitosan, the collagen prepared in Preparation Example 2 and acetic acid in a mass ratio of 1:1:50, and air-dry it into a film to obtain the dense layer;

[0080] (4) Wet the dense layer in water and place it on a smooth flat plate, then attach the loose layer thereto, and freeze-dry it into a film. The freeze-drying time is 12 h, the temperature is -30 °C, and the pressure is 1 mbar.

[0081] Example 3

[0082] This example provides a method for preparing a dual-functional layer composite biological membrane, and the preparation method includes:

[0083] (1)Select fresh porcine peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer solution.

[0084] (2)Mix 3 g of the washed porcine peritoneum with 300 mL of eluent. The eluent includes 5% sodium percarbonate, 0.5% Triton-X100, 3% sodium bicarbonate and the balance of water by mass percentage. Shake and wash it 5 times at 40 °C on a shaker, with each washing time being 40 min. After washing, rinse it 8 times with purified water, pre-freeze it at -30 °C for 1 hour, freeze it at -15 °C for 5 h, and keep it at 0 °C for 4 h to obtain the loose layer;

[0085] (3)Mix chitosan, the collagen prepared in Preparation Example 3 and acetic acid in a mass ratio of 3:3:25, and air-dry it into a film to obtain the dense layer;

[0086] (4)Wet the dense layer in water and place it on a smooth flat plate, then attach the loose layer thereto, and freeze-dry it into a film. The freeze-drying time is 12 h, the temperature is -30 °C, and the pressure is 1 mbar.

[0087] Example 4

[0088] This example provides a method for preparing a dual-functional layer composite biomembrane. The difference from Example 1 is only that in step (2), the eluent does not contain sodium percarbonate, and the reduced mass is proportionally distributed to the masses of sodium bicarbonate and Triton-X100, and other operations remain unchanged.

[0089] Example 5

[0090] This example provides a method for preparing a dual-functional layer composite biomembrane. The difference from Example 1 is only that in step (2), the eluent does not contain sodium bicarbonate, and the reduced mass is proportionally distributed to the masses of sodium percarbonate and Triton-X100, and other operations remain unchanged.

[0091] Example 6

[0092] This example provides a method for preparing a dual-functional layer composite biomembrane. The difference from Example 1 is only that in step (2), the eluent does not contain Triton-X100, and the reduced mass is proportionally distributed to the masses of sodium percarbonate and sodium bicarbonate, and other operations remain unchanged.

[0093] Example 7

[0094] This example provides a method for preparing a dual-functional layer composite biofilm. The difference from Example 1 is only that in step (3), "chitosan, the collagen prepared in Preparation Example 4, and acetic acid are mixed in a mass ratio of 3:3:80, and air-dried to form a film, thus obtaining the dense layer", and other operations remain unchanged.

[0095] Example 8

[0096] This example provides a method for preparing a dual-functional layer composite biofilm. The difference from Example 1 is only that in step (3), "chitosan, the collagen prepared in Preparation Example 5, and acetic acid are mixed in a mass ratio of 3:3:80, and air-dried to form a film, thus obtaining the dense layer", and other operations remain unchanged.

[0097] Comparative Example 1

[0098] This comparative example provides a matrix membrane, and its preparation method is as follows:

[0099] (1) Select fresh porcine peritoneum, scrape off the fat on the membrane tissue, and wash it repeatedly 8 times with PBS buffer solution.

[0100] (2) Mix 3 g of the washed porcine peritoneum with 180 mL of eluent. The eluent includes 3% of sodium percarbonate, 1% of Triton-X100, 4.5% of sodium bicarbonate, and the balance of water by mass percentage. Shake and wash it 6 times on a shaker at 37 °C, with each desorption time being 30 min. After desorption, rinse it 8 times with purified water, pre-freeze it at -30 °C for 1 hour, freeze it at -15 °C for 5 h, and keep it at 0 °C for 4 h, thus obtaining the loose layer.

[0101] Comparative Example 2

[0102] This comparative example provides a collagen membrane, and its preparation method is: Mix chitosan, the collagen prepared in Preparation Example 1, and acetic acid in a mass ratio of 3:3:80, and air-dry to form a film, thus obtaining it.

[0103] Comparative Example 3

[0104] This comparative example provides a method for preparing a dual-functional layer composite biofilm. The difference from Example 1 is only that in step (3), "collagen and acetic acid are mixed in a mass ratio of 6:80, and air-dried to form a film, thus obtaining it", and other operations remain unchanged.

[0105] Comparative Example 4

[0106] This comparative example provides a method for preparing a dual-functional layer composite biofilm. The difference from Example 1 is only that in step (2), "3 g of the washed porcine peritoneum is rinsed 8 times with purified water, pre-frozen at -30 °C for 1 hour, frozen at -15 °C for 5 h, and kept at 0 °C for 4 h, thus obtaining it", and other operations remain unchanged.

[0107] Comparative Example 5

[0108] This comparative example provides a method for preparing a dual-functional layer composite biofilm. The difference from Example 1 is only that in step (3), "collagen and acetic acid are mixed at a mass ratio of 3:40, and after air-drying to form a film, the collagen film is immersed in a chitosan acetic acid solution (the mass ratio of chitosan to acetic acid is 3:40), and then air-dried to obtain it", and other operations remain unchanged.

[0109] Test Example 1

[0110] Mechanical property evaluation

[0111] Test method: The tensile properties of the dual-functional layer composite biofilm are tested using a universal testing machine. According to the method specified for Type 2 specimens in GB / T528-2009, the test is carried out at a tensile rate of 1 mm / min. The shape of the composite film is a long strip with a size of 50×10 mm, and its thickness is measured using a thickness tester. The two ends of the material sample are fixed with the clips of a texture analyzer.

[0112] Five samples of each composite film material are tested, and the average value is taken. The experiment is repeated three times to obtain the elongation at break (%) and the maximum tensile force (N) at break.

[0113] Ts (MPa) = F (N) / S (mm 2 )

[0114] where Ts is the tensile strength, F is the maximum tensile force (N) that the material receives at break; S is the area of the cross-section of the material at break (mm 2 )

[0115] Table 1

[0116]

[0117] The experimental results are shown in Table 1. The composite membrane formed by the acellular matrix and type I collagen has good toughness while ensuring the tensile strength of the biological membrane. The addition of chitosan has a positive effect on the mechanical properties of the material because chitosan will crosslink with collagen to increase the mechanical strength of the biological membrane. The degree of decellularization of porcine peritoneum also affects the elongation at break. When the treatment degree is low, the tight arrangement of the biological membrane structure will cause the elongation at break to decrease. When the treatment degree is high, the mechanical strength of the biological membrane will be damaged. The composition of the decellularization solution will affect the above effects. Sodium percarbonate, sodium bicarbonate, and Triton-X100 have certain synergistic effects. Incomplete elution due to the lack of eluent will result in poor decellularization effect. Similarly, low pepsin concentration will also cause the same effect, resulting in a decrease in product toughness and elongation at break. At the same time, the lack of the acellular matrix layer makes the mechanical properties of the single-layer structure of the collagen layer poor because the crosslinking method of chitosan and collagen will also affect the tensile strength and elongation at break.

[0118] Test Example 2

[0119] Thermal stability evaluation

[0120] The thermal stability of the dual-functional layer composite biological membrane was examined by a shrinkage temperature instrument. Take a material with a size of 50 mm×3 mm (the sample thickness is less than 3 mm). The sample was first fully immersed in water at a temperature of 20±2°C, and then the water temperature was increased at a heating rate of 2°C / min while constantly stirring to make it flow evenly until the sample began to shrink, and the temperature was immediately recorded.

[0121] Table 2

[0122]

[0123] The composite membrane formed by the acellular matrix and type I collagen has good thermal stability and a high shrinkage temperature. Insufficient washing of the biological membrane with the detergent and insufficient decellularization with pepsin have little effect on the shrinkage temperature. However, excessive addition of pepsin will damage the basic structure of collagen fibers, resulting in a decrease in the thermal stability of the biological membrane. At the same time, the lack of the acellular matrix layer (Comparative Example 2) leads to a significant decrease in the thermal stability of the thermally stable biological membrane, and the shrinkage temperature of the single collagen membrane layer is low.

[0124] Test Example 3

[0125] Antibacterial performance experimental results

[0126] The antibacterial property of the dual-functional layer composite biological membrane was detected, and the standard plate count method was used to test the antibacterial ability of the biological membrane.

[0127] The composite biofilms obtained from the examples and comparative examples were cut into 10 mm × 10 mm square pieces and placed in a methicillin-resistant Staphylococcus aureus (MRSA) solution. Then, they were centrifuged for 5 minutes under centrifugation conditions of 3000 rpm. The precipitate was washed three times with an equal amount of PBS, and the PBS washing solutions were combined. 100 μL was taken and spread on an LB agar plate, and cultured at a temperature of 37°C for 24 h. The obtained bacterial colonies were counted to compare the differences in antibacterial ability between different groups.

[0128] Table 3

[0129]

[0130] The addition of chitosan will enhance the antibacterial ability of the biofilm, and the number of bacterial colonies will decrease accordingly. Pepsin and the desorption solution have a beneficial effect on the antibacterial ability of the biofilm. The higher the pepsin concentration and the more thorough the desorption, the lower the number of bacterial colonies. The absence of chitosan, differences in chitosan addition methods, and non-decellularization will all cause a decrease in antibacterial ability and an increase in the number of bacterial colonies.

[0131] The applicant declares that the present invention uses the above examples to illustrate a preparation method, its products and applications of a bifunctional layer composite biofilm of the present invention. However, the present invention is not limited to the above examples, that is, it does not mean that the present invention must rely on the above examples to be implemented. Those skilled in the art should understand that any improvement to the present invention, equivalent substitution of each raw material of the products of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0132] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0133] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A method for preparing a double-functional layer composite biofilm, characterized in that: The preparation method comprises: (1) Degreasing the membrane tissue; (2) Mixing the defatted membrane tissue with the eluent to remove cells, freeze-drying, and obtaining a loose layer; Collagen, chitosan and acetic acid are mixed and air-dried into a film to obtain a dense layer; (3) Wetting the loose layer, attaching the dense layer to the loose layer, and freeze-drying; Alternatively, the dense layer is moistened, the loose layer is attached to the dense layer, and freeze-dried to obtain the product; The eluent comprises, by mass percentage, 2-5% sodium percarbonate, 3-7% sodium bicarbonate, 0.5-2% Triton-X100 and 86-95% water; The temperature for removing cells is 35-40°C, the time for a single removal is 20-40 min, and the number of times is 5-8 times; The mass ratio of chitosan, collagen and acetic acid is (2-6):(2-6):(50-100).

2. The method for preparing a dual-functional layer composite biofilm according to claim 1, characterized in that: The ratio of membrane tissue to eluent is 1 g: (30-100) mL.

3. The method for preparing a double-functional layer composite biofilm according to claim 1, characterized in that: The collagen is type I collagen.

4. The method for preparing a double-functional layer composite biofilm according to claim 1, characterized in that: The preparation method of the collagen comprises: mixing the defatted membrane tissue with an enzyme solution for enzymatic hydrolysis, centrifuging the solution after enzymatic hydrolysis, mixing the supernatant with a protein denaturation solution for precipitation, and mixing the precipitation with a salting-out solution to obtain the collagen.

5. The method for preparing a double-functional layer composite biofilm according to claim 4, characterized in that: Such enzymes include pepsin.

6. A double-functional-layer composite biofilm prepared according to the method for preparing a double-functional-layer composite biofilm according to any one of claims 1 to 5.

7. Use of the double-functional layer composite biofilm according to claim 6 in preparing oral membranes or wound dressings.

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