Preparation method and application of collagen matrix membrane

Abstract

The invention discloses a collagen matrix membrane preparation method and application thereof.The collagen matrix membrane which is excellent in mechanical property and long in degradation period is prepared by taking animal casings as raw materials and sequentially including the steps of membrane laying, gradient freeze-drying and the like, sufficient bone regeneration space and time can be better provided in the early stage of periodontal disease healing, and the collagen matrix membrane can be used for treating periodontal diseases. The clinical application is better met; moreover, the collagen matrix membrane prepared by the invention is loaded with a plurality of growth fiber factors beneficial to the growth of periodontium, so that the regeneration and repair of the periodontium are facilitated, and the treatment effect is improved.

Description

Technical Field

[0001] The invention relates to the technical field of biomembrane preparation, and in particular to a preparation method of a collagen matrix membrane and application thereof. Background Art

[0002] GTR membrane, or Guided Tissue Regeneration Membrane, is a medical technology used to regenerate periodontal structures. GTR membrane regenerates periodontal structures by using a barrier membrane (periodontal regeneration membrane). Its core function is to isolate the epithelium and connective tissue, providing time and space for osteoblasts to form new alveolar bone, cementum and periodontal membrane. This technology is widely used in the clinical treatment of periodontal disease, especially in the treatment of gingival recession. GTR membrane is a key factor in the successful implementation of GTR surgery and plays an irreplaceable role in the regeneration of periodontal tissues.

[0003] GTR membranes are divided into non-absorbable membranes and bio-absorbable membranes; they can provide space for periodontal tissue regeneration. Non-absorbable membranes have been gradually eliminated due to their difficulty in degradation; collagen membranes are common bio-absorbable membranes, but the current mechanical properties of collagen membranes are poor and they degrade quickly in the body, affecting the treatment effect of periodontal disease. Summary of the invention

[0004] Therefore, based on the above background, the present invention provides a method for preparing a collagen matrix membrane and its application, which not only has excellent mechanical properties but also has a longer degradation cycle, and can better meet clinical applications.

[0005] The technical solution of the present invention is:

[0006] A method for preparing a collagen matrix membrane comprises the following steps:

[0007] 1) washing the casings of mammals and soaking them in water;

[0008] 2) soaking the casings from step 1) in a virus removal solution, and then washing with purified water;

[0009] 3) soaking the casing treated in step 2) in a degreasing solution for multiple times, and then washing it with purified water;

[0010] 4) soaking the casing treated in step 3) in a sodium dodecyl sulfate solution, and then washing it with purified water;

[0011] 5) The casings processed in step 4) are spread out and laid flat, stacked in multiple layers, pressed into films multiple times, and then subjected to gradient freeze drying in a freeze dryer.

[0012] Preferably, the mammal in step 1) is selected from pigs, cattle and sheep.

[0013] Preferably, the devirion solution in step 2) is selected from at least one of sodium hydroxide solution, sodium hypochlorite solution, formic acid, and hydrogen peroxide solution.

[0014] Preferably, the concentration of the sodium dodecyl sulfate solution in step 4) is 0.1-0.3 wt %.

[0015] During the soaking in the sodium dodecyl sulfate solution in step 4), the weight ratio of casing to sodium dodecyl sulfate solution is 1:(5-6), the soaking temperature is a constant temperature of 20-26° C., and the soaking time is 3-9 hours.

[0016] Preferably, the degreasing liquid in step 3) is prepared from chloroform and methanol in a volume ratio of (2-4):1.

[0017] In step 3), the number of times of soaking in the degreasing liquid is 2-5 times, the duration of each soaking is 16-48 hours, and the soaking temperature is a constant temperature of 20-26°C.

[0018] Furthermore, in step 2), sodium hydroxide solution and hydrogen peroxide solution are used for step-by-step treatment for detoxification.

[0019] Specifically, the soaking treatment is firstly carried out with a sodium hydroxide solution, the concentration of which is 0.5-1.5 mol / L, and then the soaking treatment is carried out with a hydrogen peroxide solution, the concentration of which is 30 wt%;

[0020] When soaking in hydrogen peroxide solution, the weight ratio of casing to hydrogen peroxide solution is 1:(5-6), the soaking temperature is 20-26°C; the soaking time is 16-36h, and the water is changed every 45-60min.

[0021] Furthermore, in step 5), the casing treated in step 4) is first soaked in a growth factor solution for multiple times, then spread out and laid flat, and multiple layers are stacked and pressed into a film.

[0022] Preferably, the concentrations of the growth factors in the growth factor solution are:

[0023] VEGF: 30-50μg / g, FGF: 40-70μg / g, TGF-β1: 15-28μg / g, BMP: 44-55μg / g.

[0024] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:(3-8), and the soaking times are 6-8 times;

[0025] In this step, the material is immersed in a constant temperature oscillator at a rotation speed of 300-500 rpm and a constant temperature of 30°C; the immersion time is 60-90 min / time, and the cycle is 6-8 times. Before each immersion in the growth factor solution, the material needs to be cleaned with PBS (pH 7.0).

[0026] Based on the same inventive concept, the present invention applies the collagen matrix membrane prepared by the above preparation method to the preparation of periodontal regeneration membrane.

[0027] The beneficial effects achieved by the technical solution of the present invention are:

[0028] The present invention uses animal casings as raw materials, such as pig casings, and undergoes multi-step processing to prepare a collagen matrix membrane with excellent mechanical properties and a longer degradation cycle, which can be used for periodontal treatment and can provide sufficient bone regeneration space and time in the early stage of periodontal disease healing, thus better meeting clinical application requirements.

[0029] Furthermore, the collagen matrix membrane prepared by the present invention is loaded with a variety of growth fiber factors that are beneficial to the growth of periodontal tissues, so as to be more conducive to the regeneration and repair of periodontal tissues and improve the therapeutic effect;

[0030] The invention uses animal casings which are cheap and widely available as raw materials, and has low preparation cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Attached Figure 1 This is a picture of the finished product of the collagen matrix membrane of Example 1.

[0032] Attached Figure 2 This is a SEM microscopic picture of the collagen matrix membrane of Example 1.

[0033] Attached Figure 3 The pictures are of the degradation experimental process of the collagen matrix membrane of the examples and comparative examples, wherein (a) is comparative example 1 and (b) is example 1. DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The technical solution of the present invention is a method for preparing a collagen matrix membrane, which comprises the following steps:

[0036] (1) washing the pig casings and soaking them in water;

[0037] In this step, frozen pig casings can be used. After thawing, they are cut into long sections of about 2-5 meters in length, cleaned and cut into long strips, washed with purified water until there is no foreign matter on the surface, and then soaked in water for multiple times, each time for 2-5 minutes, and repeated 2-3 times;

[0038] 2) soaking the casing treated in step 1) in a virus removal solution, and then washing it with purified water;

[0039] The virus removal solution in this step is selected from at least one of sodium hydroxide solution, sodium hypochlorite solution, formic acid, and hydrogen peroxide solution.

[0040] Preferably, the devirion solution is selected from sodium hydroxide solution and hydrogen peroxide solution. Specifically, the treatment is performed step by step using sodium hydroxide solution and hydrogen peroxide solution in sequence. Specifically, the casing of step 1) is first soaked in sodium hydroxide solution, and then washed with purified water after soaking; then the treated casing is soaked in hydrogen peroxide solution, and then washed with purified water after soaking;

[0041] The alkali solution used in this step is a sodium hydroxide solution with a concentration of 0.5-1.5 mol / L.

[0042] When soaking in alkali solution, the weight ratio of casing to alkali solution is 1:(5-6), the soaking temperature is 0-10°C, and the soaking time is 0.5-2h.

[0043] In this step, alkaline solution is used to decompose nucleic acids, dissolve cell membranes and cytoplasmic components, and achieve decellularization.

[0044] The concentration of the hydrogen peroxide solution was 30 wt %.

[0045] In this step, when the hydrogen peroxide solution is used for soaking, the weight ratio of casing to hydrogen peroxide solution is 1:(5-6), the soaking temperature is 20-26°C, the soaking time is 16-36h, and the water is changed every 45-60min.

[0046] The purpose of using hydrogen peroxide in this step is to utilize its strong oxidizing property to destroy the nucleic acid structure so as to inactivate viruses in animal-derived materials.

[0047] 3) Degreasing

[0048] Soak the casing treated in step 3) in degreasing liquid for multiple times, and then wash it with purified water after soaking;

[0049] The degreasing solution in this step is prepared by mixing chloroform and methanol in a volume ratio of (2-4):1.

[0050] The soaking times in this step are 2-5 times, each soaking time is 16-48 hours, and the soaking temperature is a constant temperature of 20-26°C.

[0051] In this step, chloroform and methanol solution are used to effectively dissolve and remove the grease on the surface of the casing.

[0052] 4) deproteinization;

[0053] Soak the casing treated in step 2) in a sodium dodecyl sulfate solution, and then wash it with purified water after soaking;

[0054] The concentration of the sodium dodecyl sulfate solution used in this step is 0.1-0.3 wt %.

[0055] When soaking in sodium dodecyl sulfate solution, the weight ratio of casing to sodium dodecyl sulfate solution is 1:(5-6), the soaking temperature is a constant temperature of 20-26°C, and the soaking time is 3-9h.

[0056] In this step, sodium dodecyl sulfate is used to dissolve the cell membrane, mainly to separate DNA and protein.

[0057] 5) Soak the casing treated in step 4) in a growth factor solution, then spread it out and lay it flat, stack multiple layers (5-20 layers), press it into a film, and then use a freeze dryer for gradient freeze drying. The concentrations of the growth factors in the growth factor solution used in this step are:

[0058] VEGF: 30-50μg / g, FGF: 40-70μg / g, TGF-β1: 15-28μg / g, BMP: 44-55μg / g.

[0059] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:(3-8), and the soaking times are 6-8 times;

[0060] In this step, the growth factor solution is used for immersion in a constant temperature oscillator, the rotation speed is 300-500rpm, and the temperature is constant at 30°C; the immersion time is 60-90min / time, and the cycle is 6-8 times. Before each immersion in the growth factor solution, the raw material needs to be washed with PBS (pH7.0).

[0061] In this step, a hydraulic cutting machine can be used for pressing, with the following parameters: distance from the upper pressing plate to the workbench: 5.2 cm, pressing time: 1.8-2.5 s / time, pressing times: 3-4 times. Compared with single pressing (forming into sheets), multiple pressing in this step is less likely to cause delamination during the subsequent use of the finished product.

[0062] The specific operation steps of gradient freeze drying using a freeze dryer can be seen below:

[0063] a. Temperature (℃): -65; Vacuum (Pa): 0; Time (h): 1-4.5;

[0064] b. Temperature (℃): -55; Vacuum (Pa): 0; Time (h): 0.5-1;

[0065] c. Temperature (℃): -30; Vacuum (Pa): 0; Time (h): 0.5-1;

[0066] d. Temperature (℃): -30; Vacuum (Pa): 26-30; Time (h): 8-12;

[0067] e. Temperature (℃): -25; Vacuum (Pa): 26-30; Time (h): 8-12;

[0068] f. Temperature (℃): 0; Vacuum (Pa): 26-30; Time (h): 1-1.5;

[0069] g, temperature (℃): 5; vacuum (pa): 26-30; time (h): 1-1.5;

[0070] h, temperature (℃): 5; vacuum (pa): 26-30; time (h): 1-1.5;

[0071] i. Temperature (℃): 16; Vacuum (Pa): 26-30; Time (h): 1-1.5;

[0072] j. Temperature (℃): 16; Vacuum (Pa): 0; Time (h): 1-2.

[0073] Further, more specifically, the specific operating steps of gradient freeze-drying using a freeze dryer can be seen in Table 1.

[0074] Table 1: Gradient freeze-drying steps

[0075] step Temperature(℃) Vacuum(pa) Time (h) step Temperature(℃) Vacuum(pa) Time (h) 1 -65 0 2 6 0 28 1 2 -55 0 1 7 5 28 1 3 -30 0 1 8 5 28 1 4 -30 28 8 9 16 28 1 5 -25 28 8 10 16 0 2

[0076] See Table 1. The steps of gradient freeze-drying are as follows:

[0077] a. Temperature (℃): -65; Vacuum (Pa): 0; Time (h): 2;

[0078] b. Temperature (℃): -55; Vacuum (Pa): 0; Time (h): 1;

[0079] c. Temperature (℃): -30; Vacuum (Pa): 0; Time (h): 1;

[0080] d. Temperature (℃): -30; Vacuum (Pa): 28; Time (h): 8;

[0081] e. Temperature (℃): -25; Vacuum (Pa): 28; Time (h): 8;

[0082] f. Temperature (℃): 0; Vacuum (Pa): 28; Time (h): 1;

[0083] g, temperature (℃): 5; vacuum (pa): 28; time (h): 1;

[0084] h, temperature (℃): 5; vacuum (pa): 28; time (h): 1;

[0085] i. Temperature (℃): 16; Vacuum (Pa): 28; Time (h): 1;

[0086] j. Temperature (℃): 16; Vacuum (Pa): 0; Time (h): 2.

[0087] In this step, the compressed membrane is freeze-dried using the gradient freeze-drying parameters in the above table. Compared with constant temperature freeze-drying, the thickness of the finished product can be more uniform, and it is less likely to stratify during subsequent use. Gradient freeze-drying has less impact on the internal fiber structure of the product, and the product has stronger water absorption.

[0088] Compared with constant temperature drying, gradient freeze-drying is more efficient and shortens the production cycle.

[0089] The relevant properties of the collagen matrix membrane prepared by the present invention were tested, and the test results are shown below:

[0090] ① The collagen matrix membrane is loaded with a variety of growth factors, and the contents of the loaded growth factors are shown in Table 2 below:

[0091] Table 2:

[0092]

[0093]

[0094] ②The thickness of the collagen matrix membrane is 0.1-0.89mm.

[0095] The total protein content of collagen matrix membrane is 90%-98.5%;

[0096] The tensile strength (wet state) of the collagen matrix membrane is 17-35Mpa, and the tear force (wet state) is 5-8N;

[0097] The water absorption of the collagen matrix membrane is 3.5-5.0 g / g;

[0098] The in vitro degradation time of the collagen matrix membrane is 20-27 days.

[0099] The test basis for the above performance detection is as follows:

[0100] YY / T1794-2021 General technical requirements for oral collagen membranes In vitro degradation test method: Use 0.02 mg / mL collagenase solution to simulate the product use environment In vitro degradation reference standards: GB / T16886.9-2001 Biological evaluation of medical devices Part 9 Qualitative and quantitative framework for potential degradation products. GB / T16886.13-2001 Biological evaluation of medical devices Part 13: Qualitative and quantitative degradation products of polymer medical devices. YY / T0474-2004 In vitro degradation test of poly L-lactide resin and products for surgical implants.

[0101] The method of using the present invention is to soak the biofilm with physiological saline, cover the wound and then suture it, so that if stratification occurs during subsequent use, it will affect the healing process.

[0102] Embodiment 1: A method for preparing a collagen matrix membrane, comprising the following steps:

[0103] (1) washing the pig casings and soaking them in water;

[0104] In this step, frozen pig casings can be used. After thawing, they are cut into long sections of about 2-5 meters in length, cleaned and cut into long strips, washed with purified water until there is no foreign matter on the surface, and then soaked in water for multiple times, each time for 2 minutes, and repeated twice;

[0105] 2) soaking the casings from step 1) in a virus removal solution, and then washing with purified water;

[0106] In this step, the casings are treated in steps using a sodium hydroxide solution and a hydrogen peroxide solution in sequence. Specifically, the casings of step S1 are first soaked in a sodium hydroxide solution, and then washed with purified water after the soaking. Then, the treated casings are soaked in a hydrogen peroxide solution, and then washed with purified water after the soaking.

[0107] The alkali solution used in this step is a sodium hydroxide solution with a concentration of 0.8 mol / L.

[0108] When soaking in alkali solution, the weight ratio of casing to alkali solution is 1:6, the soaking temperature is 4°C, and the soaking time is 90 minutes.

[0109] In this step, alkaline solution is used to decompose nucleic acids, dissolve cell membranes and cytoplasmic components, and achieve decellularization.

[0110] The hydrogen peroxide solution in this step is a 30 wt % hydrogen peroxide solution.

[0111] When soaking in hydrogen peroxide solution, the weight ratio of casing to hydrogen peroxide solution is 1:5, the soaking temperature is 20°C; the soaking time is 28 hours, and the water is changed every 60 minutes.

[0112] 3) soaking the casing treated in step 2) in a degreasing solution for multiple times, and then washing it with purified water;

[0113] The degreasing solution in this step is prepared by using chloroform and methanol in a volume ratio of 4:1.

[0114] The soaking times in this step are 3 times, and the soaking time of each time is 16h, 24h, and 48h respectively, and the soaking temperature is a constant temperature of 20°C.

[0115] 4) soaking the casing treated in step 3) in a sodium dodecyl sulfate solution, and then washing it with purified water;

[0116] The concentration of the sodium dodecyl sulfate solution used in this step is 0.1 wt %.

[0117] When soaking in sodium dodecyl sulfate solution, the weight ratio of casing to sodium dodecyl sulfate solution is 1:5, the soaking temperature is a constant 20°C, and the soaking time is 8 hours.

[0118] 5) Soak the casing treated in step 4) in a growth factor solution, then spread it out and lay it flat, stack 10 layers, press it into a film multiple times using a hydraulic cutting machine, and then freeze dry it in a freeze dryer. The concentrations of the growth factors in the growth factor solution used in this step are:

[0119] VEGF: 30 μg / g, FGF: 55 μg / g, TGF-β1: 16 μg / g, BMP: 50 μg / g.

[0120] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:5, and the soaking times are 8 times;

[0121] In this step, the growth factor solution is used for immersion in a constant temperature oscillator, the rotation speed is 350 rpm, and the temperature is constant at 30°C; the immersion time is 75 min / time, and the cycle is 8 times. Before each immersion in the growth factor solution, the raw material needs to be cleaned with PBS (pH 7.0).

[0122] In this step, a hydraulic cutting machine can be used for pressing, parameters: distance from upper pressing plate to workbench: 5.2cm, pressing time: 1.8-1.9s / time, pressing times: 3 times.

[0123] The specific operation of gradient freeze-drying using a freeze dryer in this step is shown in Table 1 above.

[0124] The finished product of the collagen matrix membrane prepared in this example is shown in Figure 1, and its SEM internal structure is shown in Figure 2 .

[0125] Its performance is as follows:

[0126] The content of the growth factors loaded is shown in Table 3 below:

[0127] Table 3:

[0128] Growth factor type Content (average value) VEGF 4408.7 (pg / g) FGF 6573.4 (pg / g) TGF-β1 3458.9 (pg / g) BMP 9.89227 (μg / g)

[0129] The total protein content of collagen matrix membrane is 98.5%;

[0130] The tensile strength (wet state) of the collagen matrix membrane is 34.75 MPa, and the tear force (wet state) is 7.6 N;

[0131] Water absorption is 5.0g / g;

[0132] The in vitro degradation time is 27 days.

[0133] Embodiment 2: A method for preparing a collagen matrix membrane, comprising the following steps:

[0134] Take the pig casing, clean it, and soak it in water;

[0135] In this step, frozen pig casings can be used. After thawing, they are cut into long sections of about 2-5 meters in length, cleaned and cut into long strips, washed with purified water until there is no foreign matter on the surface, and then soaked in water for multiple times, each time for 2 minutes, and repeated twice;

[0136] 2) soaking the casing treated in step 1) in a virus removal solution, and then washing it with purified water;

[0137] This step uses sodium hydroxide solution and hydrogen peroxide solution for treatment in sequence. Specifically, the casings of step 1) are first soaked in sodium hydroxide solution, and then washed with purified water; then the treated casings are soaked in hydrogen peroxide solution, and then washed with purified water;

[0138] The alkali solution used in this step is a sodium hydroxide solution with a concentration of 0.5 mol / L.

[0139] When soaking in alkali solution, the weight ratio of casing to alkali solution is 1:6, the soaking temperature is 4°C, and the soaking time is 30 minutes.

[0140] In this step, alkaline solution is used to decompose nucleic acids, dissolve cell membranes and cytoplasmic components, and achieve decellularization.

[0141] The hydrogen peroxide solution in this step is a 30 wt % hydrogen peroxide solution.

[0142] When soaking in hydrogen peroxide solution, the weight ratio of casing to hydrogen peroxide solution is 1:5, the soaking temperature is 20°C; the soaking time is 28 hours, and the water is changed every 60 minutes.

[0143] 3) soaking the casing treated in step 2) in a degreasing solution for multiple times, and then washing it with purified water;

[0144] The degreasing solution in this step is prepared by using chloroform and methanol in a volume ratio of 4:1.

[0145] The soaking times in this step are 3 times, and the soaking time of each time is 16h, 24h, and 48h respectively, and the soaking temperature is a constant temperature of 20°C.

[0146] 4) soaking the casing treated in step 3) in a sodium dodecyl sulfate solution, and then washing it with purified water;

[0147] The concentration of the sodium dodecyl sulfate solution used in this step is 0.1 wt %.

[0148] When soaking in sodium dodecyl sulfate solution, the weight ratio of casing to sodium dodecyl sulfate solution is 1:5, the soaking temperature is constant at 20°C, and the soaking time is 3 hours.

[0149] 5) Soak the casing treated in step 4) in a growth factor solution, then spread it out and lay it flat, stack 10 layers, press it into a film multiple times using a hydraulic cutting machine, and then freeze dry it in a freeze dryer. The concentrations of the growth factors in the growth factor solution used in this step are:

[0150] VEGF: 30 μg / g, FGF: 40 μg / g, TGF-β1: 15 μg / g, BMP: 44 μg / g.

[0151] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:5, and the soaking times are 8 times;

[0152] In this step, the growth factor solution is used for immersion in a constant temperature oscillator, the rotation speed is 350 rpm, and the temperature is constant at 30°C; the immersion time is 75 min / time, and the cycle is 8 times. Before each immersion in the growth factor solution, the raw material needs to be cleaned with PBS (pH 7.0).

[0153] In this step, a hydraulic cutting machine can be used for pressing, parameters: distance from upper pressing plate to workbench: 5.2cm, pressing time: 1.8-1.9s / time, pressing times: 3 times.

[0154] The specific operation of gradient freeze-drying using a freeze dryer in this step is shown in Table 1 above.

[0155] Its performance is as follows:

[0156] The content of the growth factors loaded is shown in Table 4 below:

[0157] Table 4:

[0158] Growth factor type Content (average value) VEGF 4058.1(pg / g) FGF 5990.2 (pg / g) TGF-β1 3234.4 (pg / g) BMP 9.12085μg / g)

[0159] The total protein content of the collagen matrix membrane was 92.1%;

[0160] The tensile strength (wet state) of the collagen matrix membrane is 20.11 MPa, and the tear force (wet state) is 5.9 N;

[0161] Water absorption is 4.1g / g;

[0162] The in vitro degradation time is 25 days.

[0163] Embodiment 3: A method for preparing a collagen matrix membrane, comprising the following steps:

[0164] (1) washing the pig casings and soaking them in water;

[0165] In this step, frozen pig casings can be used. After thawing, they are cut into long sections of about 2-5 meters in length, cleaned and cut into long strips, washed with purified water until there is no foreign matter on the surface, and then soaked in water for multiple times, each time for 2 minutes, and repeated twice;

[0166] 2) soaking the casing treated in step 1) in a virus removal solution, and then washing it with purified water;

[0167] In this step, the casings are treated in steps using a sodium hydroxide solution and a hydrogen peroxide solution in sequence. Specifically, the casings of step 1) are first soaked in a sodium hydroxide solution, and then washed with purified water after the soaking. Then, the treated casings are soaked in a hydrogen peroxide solution, and then washed with purified water after the soaking.

[0168] The alkali solution used in this step is a sodium hydroxide solution with a concentration of 1.5 mol / L.

[0169] When soaking in alkali solution, the weight ratio of casing to alkali solution is 1:6, the soaking temperature is 4°C, and the soaking time is 120 minutes.

[0170] In this step, alkaline solution is used to decompose nucleic acids, dissolve cell membranes and cytoplasmic components, and achieve decellularization.

[0171] The hydrogen peroxide solution in this step is a 30 wt % hydrogen peroxide solution.

[0172] When soaking in hydrogen peroxide solution, the weight ratio of casing to hydrogen peroxide solution is 1:5, the soaking temperature is 20°C; the soaking time is 36 hours, and the water is changed every 60 minutes.

[0173] 3) soaking the casing treated in step 2) in a degreasing solution for multiple times, and then washing it with purified water;

[0174] The degreasing solution in this step is prepared by using chloroform and methanol in a volume ratio of 4:1.

[0175] The soaking times in this step are 3 times, and the soaking time of each time is 16h, 24h, and 48h respectively, and the soaking temperature is a constant temperature of 20°C.

[0176] 4) soaking the casing treated in step 3) in a sodium dodecyl sulfate solution, and then washing it with purified water;

[0177] The concentration of the sodium dodecyl sulfate solution used in this step is 0.3 wt %.

[0178] When soaking in sodium dodecyl sulfate solution, the weight ratio of casing to sodium dodecyl sulfate solution is 1:5, the soaking temperature is constant at 20°C, and the soaking time is 9 hours.

[0179] 5) Soak the casing treated in step 4) in a growth factor solution, then spread it out and lay it flat, stack 10 layers, press it into a film multiple times using a hydraulic cutting machine, and then freeze dry it in a freeze dryer. The concentrations of the growth factors in the growth factor solution used in this step are:

[0180] VEGF: 50 μg / g, FGF: 70 μg / g, TGF-β1: 28 μg / g, BMP: 55 μg / g.

[0181] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:5, and the soaking times are 8 times;

[0182] In this step, the growth factor solution is used for immersion in a constant temperature oscillator, the rotation speed is 350 rpm, and the temperature is constant at 30°C; the immersion time is 75 min / time, and the cycle is 8 times. Before each immersion in the growth factor solution, the raw material needs to be cleaned with PBS (pH 7.0).

[0183] In this step, a hydraulic cutting machine can be used for pressing, parameters: distance from upper pressing plate to workbench: 5.2cm, pressing time: 1.8-1.9s / time, pressing times: 3 times.

[0184] The specific operation of gradient freeze-drying using a freeze dryer in this step is shown in Table 1 above.

[0185] Its performance is as follows:

[0186] The content of the growth factors loaded is shown in Table 5 below:

[0187] Table 5:

[0188] Growth factor type Content (average value) VEGF 4372.7 (pg / g) FGF 6289.8 (pg / g) TGF-β1 3349.4 (pg / g) BMP 9.61399μg / g)

[0189] The total protein content of the collagen matrix membrane was 94.7%;

[0190] The tensile strength (wet state) of the collagen matrix membrane is 29.62 MPa, and the tear force (wet state) is 6.3 N;

[0191] Water absorption is 4.3g / g;

[0192] The in vitro degradation time is 21 days.

[0193] Comparative Example 1: A method for preparing a collagen matrix membrane, comprising the following steps:

[0194] (1) washing the pig casings and soaking them in water;

[0195] In this step, frozen pig casings can be used. After thawing, they are cut into long sections of about 2-5 meters in length, cleaned and cut into long strips, washed with purified water until there is no foreign matter on the surface, and then soaked in water for multiple times, each time for 2 minutes, and repeated twice;

[0196] 2) soaking the casing treated in step 1) in a virus removal solution, and then washing it with purified water;

[0197] The devirion solution is a sodium hydroxide solution and a hydrogen peroxide solution. Specifically, the sodium hydroxide solution and the hydrogen peroxide solution are used in sequence for treatment in steps. Specifically, the casing of step 1) is first soaked in the sodium hydroxide solution, and then washed with purified water after soaking; then the treated casing is soaked in the hydrogen peroxide solution, and then washed with purified water after soaking;

[0198] The alkali solution used in this step is a sodium hydroxide solution with a concentration of 0.8 mol / L.

[0199] When soaking in alkali solution, the weight ratio of casing to alkali solution is 1:6, the soaking temperature is 4°C, and the soaking time is 90 minutes.

[0200] In this step, alkaline solution is used to decompose nucleic acids, dissolve cell membranes and cytoplasmic components, and achieve decellularization.

[0201] The hydrogen peroxide solution in this step is a 30 wt % hydrogen peroxide solution.

[0202] When soaking in hydrogen peroxide solution, the weight ratio of casing to hydrogen peroxide solution is 1:5, the soaking temperature is 20°C; the soaking time is 28 hours, and the water is changed every 60 minutes.

[0203] 3) Degreasing

[0204] Soak the casing treated in step 2) in degreasing liquid for multiple times, and then wash it with purified water after soaking;

[0205] The degreasing solution in this step is prepared by using chloroform and methanol in a volume ratio of 4:1.

[0206] The soaking times in this step are 3 times, and the soaking time of each time is 16h, 24h, and 48h respectively, and the soaking temperature is a constant temperature of 20°C.

[0207] 4) deproteinization;

[0208] Soak the casing treated in step 3) in a sodium dodecyl sulfate solution, and then wash it with purified water after soaking;

[0209] The concentration of the sodium dodecyl sulfate solution used in this step is 0.1 wt %.

[0210] When soaking in sodium dodecyl sulfate solution, the weight ratio of casing to sodium dodecyl sulfate solution is 1:5, the soaking temperature is a constant 20°C, and the soaking time is 8 hours.

[0211] 5) The casing treated in step 4) is soaked in a growth factor solution, then spread out and laid flat, 10 layers are stacked, and then pressed into a film multiple times using a hydraulic cutting machine, and then freeze-dried at a constant temperature.

[0212] The concentrations of the growth factors in the growth factor solutions used in this step are:

[0213] VEGF: 30 μg / g, FGF: 55 μg / g, TGF-β1: 16 μg / g, BMP: 50 μg / g.

[0214] When the growth factor solution is used for soaking, the weight ratio of casing to growth factor solution is 1:5, and the soaking times are 8 times;

[0215] In this step, the growth factor solution is used for immersion in a constant temperature oscillator, the rotation speed is 350 rpm, and the temperature is constant at 30°C; the immersion time is 75 min / time, and the cycle is 8 times. Before each immersion in the growth factor solution, the raw material needs to be cleaned with PBS (pH 7.0).

[0216] In this step, a hydraulic cutting machine can be used for pressing, parameters: distance from upper pressing plate to workbench: 5.2cm, pressing time: 1.8-1.9s / time, pressing times: 3 times.

[0217] The conditions of constant temperature freeze drying in this comparative example are as follows:

[0218] Temperature (℃): -30℃; Vacuum (pa): 28; Time (h): 72.

[0219] Its performance is as follows:

[0220] The content of the growth factors loaded is shown in Table 6 below:

[0221] Table 6:

[0222] Growth factor type Content (average value) VEGF 3057.3 (pg / g) FGF 4610.1(pg / g) TGF-β1 2450.2 (pg / g) BMP 8.13281μg / g)

[0223] The total protein content of the collagen matrix membrane was 86.7%;

[0224] The tensile strength (wet state) of the collagen matrix membrane is 9.83 MPa, and the tear force (wet state) is 2.9 N;

[0225] Water absorption is 2.1g / g;

[0226] The in vitro degradation time is 16 days.

[0227] Figure 3 The degradation experimental process of the collagen matrix membrane prepared in Comparative Example 1 and Example 1 is shown in the figure.

[0228] Example 1:

[0229] During the experiment, there was an obvious stratification phenomenon on the 10th day, but there was no such phenomenon in Example 1.

[0230] Comparative Example 2: Existing Products

[0231] Oral absorbable biofilm (Landu)

[0232] Water absorption (average value): 3.47g / g

[0233] Tear force (average): 3.94N

[0234] Tensile strength (average value): 14.95MPa

[0235] Degradation time: 20d.

[0236] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a collagen matrix membrane, characterized in that: It includes the following steps: 1) washing the casings of mammals and soaking them in water; 2) soaking the casings from step 1) in a virus removal solution, and then washing with purified water; 3) soaking the casing treated in step 2) in a degreasing solution for multiple times, and then washing it with purified water; 4) soaking the casing treated in step 3) in a sodium dodecyl sulfate solution, and then washing it with purified water; 5) The casings processed in step 4) are spread out and laid flat, stacked in multiple layers, pressed into films multiple times, and then subjected to gradient freeze drying in a freeze dryer.

2. The method for preparing a collagen matrix membrane according to claim 1, characterized in that: The mammal in step 1) is selected from pigs, cattle and sheep.

3. The method for preparing a collagen matrix membrane according to claim 1, characterized in that: The virus removal solution in step 2) At least one selected from sodium hydroxide solution, sodium hypochlorite solution, formic acid, and hydrogen peroxide solution.

4. The method for preparing a collagen matrix membrane according to claim 1, characterized in that: The concentration of the sodium dodecyl sulfate solution in step 4) is 0.1-0.3 wt %.

5. The method for preparing a collagen matrix membrane according to claim 1, characterized in that: The degreasing liquid in step 3) is prepared by mixing chloroform and methanol in a volume ratio of (2-4):

1.

6. The method for preparing a collagen matrix membrane according to claim 5, characterized in that: In step 3), the degreasing liquid is used for soaking: 2-5 times, each soaking time is 16-48 hours, and the soaking temperature is constant at 20-26°C.

7. The method for preparing a collagen matrix membrane according to claim 1, characterized in that: In step 2), sodium hydroxide solution and hydrogen peroxide solution are used for step-by-step treatment to carry out detoxification.

8. The method for preparing a collagen matrix membrane according to any one of claims 1 to 7, characterized in that: In step 6), the casing treated in step 5) is first soaked in a growth factor solution for multiple times, then spread out and laid flat, and after multiple layers are stacked, pressed into a film.

9. The method for preparing a collagen matrix membrane according to claim 8, characterized in that: The concentrations of the growth factors in the growth factor solutions are: VEGF: 30-50μg / g, FGF: 40-70μg / g, TGF-β1: 15-28μg / g, BMP: 44-55μg / g.

10. Use of the collagen matrix membrane according to any one of claims 1 to 9 in the preparation of periodontal regeneration membrane.

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