Preparation method of double-functional-layer composite biological membrane as well as product and application of double-functional-layer composite biological membrane

Abstract

The invention relates to a preparation method of a double-functional-layer composite biological membrane as well as a product and application of the double-functional-layer composite biological membrane. The preparation method comprises the following steps: performing degreasing treatment on membrane tissues; mixing the degreased membrane tissue with an eluent to remove cells, and performing freeze drying to obtain a loose layer; mixing collagen, chitosan and acetic acid, and air-drying to form a film, so as to obtain a compact layer; the loose layer is wetted, the compact layer is attached to the loose layer, and freeze drying is conducted; or wetting the compact layer, attaching the loose layer to the compact layer, and freeze-drying to obtain the composite material. The prepared biological membrane has good mechanical properties and thermal stability, the effect of the biological membrane is better than that of a single-layer biological membrane, and the effect can be further improved by adding chitosan.

Description

Technical Field

[0001] The invention belongs to the technical field of biofilms, and relates to a preparation method of a double-functional layer composite biofilm, a product and an application thereof. Background Art

[0002] Guided bone tissue regeneration membrane is an innovative technology currently used in periodontal treatment and oral implantation. Its main purpose is to establish a biological shield between the soft tissue and bone defect area, so as to create a relatively closed bone regeneration environment, while at the same time being a biocompatible material that does not affect the natural growth of the wound, because different tissues in the damaged area heal at different speeds. Normally, the faster growth of fibroblasts and epithelial tissue will inhibit the growth of bone tissue.

[0003] At present, the most commonly used biomembranes on the market are divided into absorbable biomembranes and non-absorbable biomembranes. Non-absorbable membranes are mainly metal membranes and polymer membranes. This type of membrane material has superior mechanical strength and can play a good shielding and supporting role. But at the same time, there are huge defects. First of all, non-absorbable membranes are usually accompanied by the disadvantages of high hardness and poor comfort. They have poor fit with soft tissues and can irritate wounds during activities, causing inflammatory reactions. Secondly, because the membrane cannot be absorbed and degraded in the body, a secondary operation is required to remove the implanted membrane from the damaged area, which not only increases the risk of infection but also prolongs the time of treatment and recovery.

[0004] Collagen is one of the most widely used materials in biomedical materials. It 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 protein in the human body. It is the main component of connective tissues or organs such as skin, cartilage, ligaments, tendons, etc. It can effectively promote cell division, proliferation and differentiation, induce platelet adhesion, promote platelet aggregation, and produce adhesion, compression and filling effects on wounds. The absorbable membrane is mainly a collagen membrane, which has excellent biocompatibility and in vivo degradability. It has a high degree of matching with the soft tissue and bone tissue in the damaged area of ​​the human body, and can effectively cover the damaged area to allow bone tissue to grow in a shielding environment. At the same time, collagen absorbable biofilms do not require secondary surgery, reducing the risk of wound exposure 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 to their function and durability.

[0005] Therefore, providing a degradable biofilm with good mechanical properties is a problem to be solved urgently in the art. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing a double-functional layer composite biofilm and its product and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a dual-functional layer composite biofilm, the preparation method comprising:

[0009] (1) Degreasing the membrane tissue;

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

[0011] Collagen, chitosan and acetic acid are mixed and air-dried into 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] Alternatively, the dense layer is moistened, the loose layer is attached to the dense layer, and freeze-dried to obtain the product.

[0014] The double-layer composite biomembrane material prepared by the present invention has a double functional layer including a loose layer and a dense layer, wherein the loose layer is composed of a porcine decellularized dermal matrix, and the dense layer is composed of porcine type I collagen and chitosan. Compared with the traditional collagen biomembrane, 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 membrane tissue to eluent is 1 g:(30-100) mL, wherein the specific point values ​​in (30-100) can be selected from 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 described one by one here.

[0016] Preferably, the membrane tissue comprises porcine peritoneum, porcine pericardium or porcine lower intestinal membrane.

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

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

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

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

[0021] Preferably, the temperature for removing cells 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℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc. The time can be selected as 20 min, 22min, 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 range can be selected, so they will not be elaborated here.

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

[0024] 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., and 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 range can be selected, and they will not be listed here one by one.

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

[0026] Preferably, the method for preparing 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 denaturing solution for precipitation, and mixing the precipitate with a salting-out solution to obtain the collagen.

[0027] Preferably, the enzyme comprises 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, for example, 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, which will not be described one by one here.

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

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

[0032] Preferably, the centrifugal 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 also be selected, so they will not be elaborated here.

[0034] Preferably, the protein denaturing solution comprises an aqueous sodium hydroxide solution.

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

[0036] Preferably, the molar concentration of sodium hydroxide in the sodium hydroxide aqueous solution is 0.1-1 mol / L, for example 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, which will not be described one by one here.

[0037] Preferably, the volume ratio of the supernatant to the protein denaturing solution is 1:(1-10), wherein the specific point values ​​in (1-10) can be selected from 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 described 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] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

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

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

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

[0051] Type III collagen was purchased from MacLean 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, which comprises:

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

[0056] (2) Mix 3 g of cleaned porcine peritoneum with 50 mL of 1 mol / L pepsin acetic acid solution, stir slowly 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 white collagen 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). The ratio of the precipitate to the disodium hydrogen phosphate solution is 1 g:50 mL. Filter and rinse the filtered collagen white precipitate with purified water 4 times to obtain the final type I collagen.

[0057] Preparation Example 2

[0058] This preparation example provides a method for preparing collagen, which comprises:

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

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

[0061] Preparation Example 3

[0062] This preparation example provides a method for preparing collagen, which comprises:

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

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

[0065] Preparation Example 4

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

[0067] Preparation Example 5

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

[0069] Example 1

[0070] This embodiment provides a method for preparing a dual-functional layer composite biofilm, the preparation method comprising:

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

[0072] (2) 3 g of washed porcine peritoneum was mixed with 180 mL of an eluent, wherein the eluent included, by mass percentage, 3% sodium percarbonate, 1% Triton-X100, 4.5% sodium bicarbonate and the remainder water, and the mixture was shaken and washed 6 times on a shaker at 37°C, each wash time being 30 min. After wash, the mixture was rinsed 8 times with purified water, pre-frozen at -30°C for 1 hour, frozen at -15°C for 5 h, and kept warm at 0°C for 4 h to obtain a loose layer.

[0073] (3) chitosan, collagen prepared in Preparation Example 1, and acetic acid were mixed in a mass ratio of 3:3:80, and air-dried to form a film to obtain a dense layer;

[0074] (4) The dense layer is moistened in water and placed on a smooth flat plate. The loose layer is then attached to it and freeze-dried 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 embodiment provides a method for preparing a dual-functional layer composite biofilm, the preparation method comprising:

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

[0078] (2) 3 g of washed porcine peritoneum was mixed with 90 mL of an eluent, wherein the eluent included 2% sodium percarbonate, 2% Triton-X100, 7% sodium bicarbonate and the remainder water, and the mixture was shaken and washed 8 times at 35°C on a shaker, each time for 20 min. After washing, the mixture was rinsed 8 times with purified water, pre-frozen at -30°C for 1 hour, frozen at -15°C for 5 h, and kept warm at 0°C for 4 h to obtain a loose layer.

[0079] (3) chitosan, collagen prepared in Preparation Example 2, and acetic acid were mixed in a mass ratio of 1:1:50, and air-dried to form a film to obtain a dense layer;

[0080] (4) The dense layer is moistened in water and placed on a smooth flat plate. The loose layer is then attached to it and freeze-dried 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 embodiment provides a method for preparing a dual-functional layer composite biofilm, the preparation method comprising:

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

[0084] (2) 3 g of washed porcine peritoneum was mixed with 300 mL of an eluent, wherein the eluent included, by mass percentage, 5% sodium percarbonate, 0.5% Triton-X100, 3% sodium bicarbonate and the remainder water, and the mixture was shaken and washed 5 times at 40°C on a shaker, each time for 40 min. After washing, the mixture was rinsed 8 times with purified water, pre-frozen at -30°C for 1 hour, frozen at -15°C for 5 h, and kept warm at 0°C for 4 h to obtain a loose layer.

[0085] (3) chitosan, collagen prepared in Preparation Example 3 and acetic acid were mixed in a mass ratio of 3:3:25, and air-dried to form a film to obtain a dense layer;

[0086] (4) The dense layer is moistened in water and placed on a smooth flat plate. The loose layer is then attached to it and freeze-dried 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 embodiment provides a method for preparing a dual-functional layer composite biofilm, which differs from Embodiment 1 only in that the eluent in step (2) does not contain sodium percarbonate, and its reduced mass is proportionally distributed to the masses of sodium bicarbonate and Triton-X100, and other operations remain unchanged.

[0089] Example 5

[0090] This embodiment provides a method for preparing a dual-functional layer composite biofilm, which differs from Embodiment 1 only in that the eluent in step (2) does not contain sodium bicarbonate, and its reduced mass is proportionally distributed to the mass of sodium percarbonate and Triton-X100, and other operations remain unchanged.

[0091] Example 6

[0092] This embodiment provides a method for preparing a dual-functional layer composite biofilm, which differs from Embodiment 1 only in that the eluent in step (2) does not contain Triton-X100, and its reduced mass is proportionally distributed to the mass of sodium percarbonate and sodium bicarbonate, and other operations remain unchanged.

[0093] Example 7

[0094] This embodiment provides a method for preparing a dual-functional layer composite biofilm, which differs from Embodiment 1 only in that step (3) is "mixing chitosan, collagen prepared in Preparation Example 4 and acetic acid in a mass ratio of 3:3:80, and air-drying to form a film to obtain a dense layer", and other operations remain unchanged.

[0095] Example 8

[0096] This embodiment provides a method for preparing a dual-functional layer composite biofilm, which differs from Embodiment 1 only in that step (3) is "mixing chitosan, collagen prepared in Preparation Example 5 and acetic acid in a mass ratio of 3:3:80, and air-drying to form a film to obtain a 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 with PBS buffer for 8 times.

[0100] (2) Mix 3 g of washed porcine peritoneum with 180 mL of eluent, which includes, by mass percentage, 3% sodium percarbonate, 1% Triton-X100, 4.5% sodium bicarbonate and the remainder water. Wash the mixture six times on a shaker at 37°C, with each wash lasting 30 min. Rinse the mixture eight times with purified water, prefreeze at -30°C for 1 hour, freeze at -15°C for 5 h, and keep warm at 0°C for 4 h to obtain a loose layer.

[0101] Comparative Example 2

[0102] This comparative example provides a collagen film, and the preparation method thereof is as follows: chitosan, the collagen prepared in Preparation Example 1, and acetic acid are mixed in a mass ratio of 3:3:80, and air-dried to form a film.

[0103] Comparative Example 3

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

[0105] Comparative Example 4

[0106] This comparative example provides a method for preparing a double-functional layer composite biofilm, which differs from Example 1 only in that step (2) is "rinsing 3 g of the washed porcine peritoneum with purified water for 8 times, pre-freezing at -30°C for 1 hour, freezing at -15°C for 5 hours, and keeping warm at 0°C for 4 hours, to obtain", and other operations remain unchanged.

[0107] Comparative Example 5

[0108] This comparative example provides a method for preparing a dual-functional layer composite biofilm, which differs from Example 1 only in that step (3) is "mixing collagen and acetic acid in a mass ratio of 3:40, air-drying to form a film, and then immersing the collagen film in a chitosan acetic acid solution (the mass ratio of chitosan to acetic acid is 3:40), air-drying, and obtaining", and other operations remain unchanged.

[0109] Test Example 1

[0110] Mechanical properties evaluation

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

[0112] Five samples were tested for each group of composite membrane materials, and the average value was taken. The experiment was repeated three times to obtain the elongation at break (%) and the maximum tensile force at break (N).

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

[0114] Where Ts is the tensile strength, F is the maximum tensile force (N) on which the material is subjected when it breaks; S is the area of ​​the fractured section of the material (mm 2 ).

[0115] Table 1

[0116]

[0117] The experimental results are shown in Table 1. The composite membrane formed by the decellularized matrix and collagen I ensures the tensile strength of the biofilm while making the material have good toughness. The addition of chitosan has a positive effect on the mechanical properties of the material, because chitosan will cross-link with collagen to increase the mechanical strength of the biofilm; the degree of decellularization of the porcine peritoneum will also affect the elongation at break. When the degree of treatment is low, the close arrangement of the biofilm structure will cause the elongation at break to decrease. When the degree of treatment is high, the mechanical strength of the biofilm will be destroyed; the composition of the decellularized solution will affect the above effects. Sodium percarbonate, sodium bicarbonate, and Triton-X100 have a certain synergistic effect. The lack of elution solution will cause incomplete elution and poor decellularization effect. Similarly, low pepsin concentration will also cause the same effect, and the product toughness and elongation at break will decrease. At the same time, the lack of acellular matrix layer, the mechanical properties of the single collagen layer are poor, because the cross-linking 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 double-functional layer composite biofilm was tested by a shrinkage temperature instrument. Take a 50 mm × 3 mm material (the sample thickness is less than 3 mm), fully immerse the sample in water at a temperature of 20 ± 2 ° C, increase the water temperature, control the heating rate to 2 ° C / min, and stir it continuously to make it flow evenly until the sample begins to shrink, and immediately record the temperature.

[0121] Table 2

[0122]

[0123] The composite membrane formed by the decellularized matrix and collagen I has good thermal stability and a high shrinkage temperature. Insufficient de-washing of the biofilm using a de-washing agent and insufficient decellularization of pepsin have little effect on the shrinkage temperature, but excessive addition of pepsin will destroy the basic structure of the collagen fibers, causing a decrease in the thermal stability of the biofilm. At the same time, the lack of a decellularized matrix layer (Comparative Example 2) leads to a significant decrease in the thermal stability of the thermally stable biofilm, and the shrinkage temperature of the separate collagen membrane layer is low.

[0124] Test Example 3

[0125] Antibacterial performance test results

[0126] The antibacterial property of the double-functional layer composite biofilm was tested using the standard plate count method.

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

[0128] Table 3

[0129]

[0130] The addition of chitosan will enhance the antibacterial ability of the biofilm and reduce the bacterial colony accordingly. Pepsin and decellularization solution have a positive effect on the antibacterial ability of the biofilm. The higher the pepsin concentration and the more thorough the decellularization, the lower the number of bacterial colonies. No chitosan added, different chitosan addition methods and no decellularization will all lead to 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-mentioned embodiments to illustrate the preparation method of a dual-functional layer composite biofilm of the present invention, its product and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0132] The preferred embodiments of the present invention are 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0133] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

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.

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 eluent comprises, by mass percentage, 2-5% sodium percarbonate, 3-7% sodium bicarbonate, 0.5-2% Triton-X100 and 86-95% water.

4. The method for preparing a double-functional layer composite biofilm according to claim 1, characterized in that: The temperature for removing cells is 35-40°C, the time is 20-40 min, and the number of times is 5-8 times.

5. The method for preparing a double-functional layer composite biofilm according to claim 1, characterized in that: The mass ratio of chitosan, collagen and acetic acid is (2-6):(2-6):(50-100).

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

7. The method for preparing a dual-functional layer composite biofilm according to claim 1, characterized in that: The preparation method of 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.

8. The method for preparing a double-functional layer composite biofilm according to claim 7, characterized in that: Such enzymes include pepsin.

9. 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 8.

10. Use of the double-functional layer composite biofilm according to claim 9 in preparing oral membranes or wound dressings.

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