Dry biofilm and its preparation method and application

Abstract

The present invention discloses a dry biofilm and a preparation method and application thereof, wherein the preparation of the dry biofilm comprises the following steps: placing the fresh biofilm in a biofilm protection solution and soaking it for 1 to 24 hours, drying it, and then sterilizing it by irradiation; wherein the biofilm protection solution is prepared from the following components: chondroitin sulfate, stabilizer, sodium hyaluronate, sulfur-containing compounds, and HC-HA / PTX3 complex. The dry biofilm prepared by the present invention can effectively preserve the active factors and collagen contained in the biofilm, and at the same time can avoid the biofilm material from being damaged and denatured during drying and irradiation inactivation of viruses or sterilization, maintain good flexibility and mechanical properties, and increase thickness, which is more conducive to clinical operation; the dry biofilm of the present invention is easy to store and transport, convenient for clinical surgical operations, and can be widely used in biopharmaceuticals and medical fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and more specifically, the present invention relates to a dry biofilm and a preparation method and application thereof. Background Art

[0002] In placental mammals, the amnion (about 0.02-0.05 mm thick) originates from the embryo and is a transparent, nerve-free, blood-free and lymphatic-free, low-antigenic and tough tissue composed of an epithelial cell layer, a basement membrane, a matrix layer, a fibroblast layer and a spongy layer. The umbilical cord connects the developing fetus to the placenta and is made of the amnion (UCAM) and Wharton's jelly.

[0003] Fresh amniotic membrane (including amniotic membrane from the umbilical cord) contains a variety of collagens, laminin, fibronectin, glycosaminoglycans, hyaluronic acid, etc. These active substances can promote the adhesion and migration of epithelial cells, induce epithelial differentiation, and prevent epithelial apoptosis; amniotic membrane cells and matrix also contain a variety of growth factors, such as nerve growth factor (NGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), transforming growth factor-α (TGF-α), transforming growth factor-β1 (TGF-β1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF); in addition, amniotic membrane cells and matrix also contain angiogenesis-inhibiting and anti-inflammatory proteins and some natural protease inhibitors, which can promote epithelial growth, inhibit inflammatory reactions, and inhibit excessive proliferation of fibrous tissue and neovascularization. Amniotic membrane plays an important role in promoting epithelial repair, healing and differentiation, inhibiting inflammation, anti-angiogenesis, inhibiting fibrosis and scarring, and preventing symblepharon.

[0004] Fresh and refrigerated amniotic membranes can retain various natural collagen and active factor components to the greatest extent, so they have better clinical effects. However, their main disadvantages are that they are difficult to store for a long time and have harsh storage conditions. They usually need to be stored at low temperatures (below -20°C), and their transportation requires cold chain transportation, which has high transportation costs. For example, the amniotic membrane product disclosed in U.S. Patents US006152142A and US006326019B1 is a Caesarean section placenta that is cleaned and then bluntly separated to remove the chorion, and the epithelium is spread on nitrocellulose filter paper, and then stored in DMEM:glycerol at a ratio of 1:1 and frozen in a -80°C refrigerator. The prepared amniotic membrane has not been decellularized, dried or sterilized, so the integrity of the amniotic membrane components and structure is retained to the greatest extent; but there are problems: ① The storage cost is too high and it is inconvenient to transport; ② It is frozen at low temperature during storage and needs to be thawed at room temperature when used. Both processes will cause the destruction of the collagen structure of the amniotic membrane and make it fragile; ③ Nitrocellulose filter paper is used as the substrate in the preparation, and the amniotic membrane needs to be torn off during use, making the already fragile amniotic membrane more likely to tear or curl, which brings many inconveniences to clinical operations; ④ The amniotic membrane has not been treated for virus inactivation, which has the risk of virus transmission.

[0005] In recent years, there are some freeze-dried and decellularized amniotic membrane products at home and abroad. The freeze-dried amniotic membrane can reach the ideal water content (generally 1-10%), but during the freezing, freezing, thawing, drying and storage process, there are many factors that induce protein denaturation (such as the selection of cryoprotectants and lyoprotectants, the selection of freezing rate, etc.), making the freeze-drying process difficult to control, time-consuming, and costly, which can easily cause protein denaturation and brittle amniotic membrane, making the amniotic membrane easy to tear during surgery, requiring repeated replacement, and prolonging the operation time. Decellularization itself is a "destructive" treatment method. While removing the cells in the amniotic membrane material, it also removes a large number of active factors present in the cells, leaving only the "scaffold" of the amniotic membrane, which is mainly composed of collagen.

[0006] Therefore, how to make the amniotic membrane material retain the various effective ingredients in the fresh amniotic membrane to the greatest extent, maintain its mechanical properties, and at the same time facilitate storage, transportation and clinical operation has become an urgent problem to be solved in the clinical application of biological amniotic membrane. Summary of the invention

[0007] Based on this, the purpose of the present invention is to provide a dry biomembrane and a preparation method and application thereof, wherein the dry biomembrane well retains various effective ingredients in fresh amniotic membrane, maintains mechanical properties, and is conducive to storage, transportation and clinical operation.

[0008] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0009] The first aspect of the present invention provides a method for preparing a dry biofilm, comprising the following steps: placing a fresh biofilm in a biofilm protection solution and soaking it for 1 to 24 hours, drying it, and then sterilizing it by irradiation; wherein the biofilm protection solution is prepared from components having the following concentrations: 12 to 22 g / L chondroitin sulfate, 100 to 150 mg / L stabilizer, 5 to 40 g / L sodium hyaluronate, 8000 to 15000 ng / L sulfur-containing compounds, and 0.1 to 5 g / L HC-HA / PTX3 complex.

[0010] The second aspect of the present invention provides a dry biofilm prepared by the above preparation method.

[0011] The third aspect of the present invention provides the use of the above-mentioned dry biofilm in the preparation of materials for inhibiting infection and inflammatory response, reducing scar formation and protecting exposed nerve roots in ophthalmic surgery, skin defect repair, tendon repair and adhesion prevention, and spinal surgery.

[0012] The present invention has the following beneficial effects:

[0013] In the preparation method of the dry biofilm of the present invention, the fresh biofilm is immersed in a special biofilm protection liquid (a certain concentration of sulfur-containing compounds and HC-HA / PTX3 complex is added, and chondroitin sulfate, stabilizer and sodium hyaluronate act together), and then dried and irradiated. Under this scheme, the prepared dry biofilm can still effectively preserve the active factors and collagen contained in the fresh biofilm (the active factor loss rate is low, and the active factor preservation rate is above 60% after being sealed and stored at room temperature for 3 years), which can avoid the biofilm material from being damaged and denatured during drying and irradiation to inactivate viruses or sterilization, maintain good flexibility and mechanical properties, and the thickness of the dry biofilm is increased, thereby improving the maximum tensile strength, which is more convenient for operation in clinical surgery.

[0014] The dry biofilm prepared by the present invention is easy to store and transport, and overcomes the defects of wet biofilm that it is difficult to preserve for a long time and has harsh preservation conditions. It can be widely used in the fields of biopharmaceuticals and medicine to increase the gap between the protected tissue and the surrounding tissue, promote and regulate postoperative tissue regeneration, reduce the formation of scar tissue, increase anti-adhesion, and thus reduce postoperative complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the growth factor content of the biofilm prepared by different preparation methods in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0016] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0017] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0018] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0019] In some embodiments of the present invention, a method for preparing a dry biofilm is disclosed, comprising the following steps: placing a fresh biofilm in a biofilm protection solution and soaking it for 1 to 24 hours, drying it, and then sterilizing it by irradiation; wherein the biofilm protection solution is prepared from components having the following concentrations: 12 to 22 g / L chondroitin sulfate, 100 to 150 mg / L stabilizer, 5 to 40 g / L sodium hyaluronate, 8000 to 15000 ng / L sulfur-containing compounds, and 0.1 to 5 g / L HC-HA / PTX3 complex.

[0020] In some embodiments, the biofilm protection solution is prepared from components including the following concentrations: 14-16 g / L chondroitin sulfate, 100-120 mg / L stabilizer, 15-25 g / L sodium hyaluronate, 8000-12000 ng / L sulfur-containing compounds, and 0.8-1.2 g / L HC-HA / PTX3 complex. Among them, the HC-HA / PTX3 complex can be isolated from fetal tissues such as placenta, amniotic membrane and umbilical cord (in the present invention, the HC-HA / PTX3 complex is prepared by the method in reference application No. 202210284199). In the present invention, it is used for the first time as one of the main components for protecting biofilm growth factors and collagen in the preparation process of dry biofilm. It can effectively protect the growth factors (EGF, bFGF, HGF, TGF-β1, NGF) and collagen of the biofilm, reduce the loss of growth factors and collagen of the biofilm, and reduce the loss rate.

[0021] In some embodiments, the sulfur-containing compound is at least one of cysteine, cysteamine, 2-aminoethylthiosulfate, and thiourea. The sulfur-containing compound can promptly remove the free radicals generated by the biofilm sample stored in the preservation solution during irradiation, avoid the damage of the free radicals to the biofilm, and enable the biofilm to maintain the flexibility and excellent tensile strength of the fresh biofilm; at the same time, the sulfur-containing compound and the HC-HA / PTX3 complex play a synergistic role, so that the loss rate of growth factors (EGF, bFGF, HGF, TGF-β1, NGF) and collagen in the biofilm during the drying process is reduced, and the content of each growth factor and collagen in the biofilm after 6 months of storage is close to that of the fresh biofilm.

[0022] In some embodiments, the stabilizer is at least one of ascorbic acid, vitamin E, vitamin C, carotene, and SOD, preferably vitamin C.

[0023] In some of the embodiments, the biofilm protective solution is prepared from components having the following concentrations: 14-16 g / L chondroitin sulfate, 100-120 mg / L vitamin C, 15-25 g / L sodium hyaluronate, 8000-12000 ng / L cysteine, and 0.8-1.2 g / L HC-HA / PTX3 complex.

[0024] In some embodiments, the pH of the biofilm protection solution is 7.2-7.4, and the osmotic pressure is 350-380 mOsm / L.

[0025] In some embodiments, the biological membrane can be amnion or umbilical cord-derived tissue (particularly Wharton's Jelly) of any animal (preferably mammal), preferably amnion.

[0026] In some embodiments, the drying process is drying, freeze drying, natural air drying or vacuum drying.

[0027] In some embodiments, the drying temperature is 35-50°C, and the drying time is 30-120 minutes. Preferably, the drying temperature is 42-48°C, and the drying time is 40-50 minutes; more preferably, the drying temperature is 45°C, and the drying time is 45 minutes, and the dried biofilm obtained by drying will not be over-dried.

[0028] In some embodiments, the freeze-drying temperature is -50°C to -80°C, preferably -50°C to -60°C. The soaked biofilm tissue is placed in a freeze dryer so that it is suspended in the freeze dryer for freeze drying. Before freeze drying, one or more biofilm tissues can be optionally placed on a drying device, and the drying device can be arranged with grooves in a grid arrangement. The grid on a single drying device can have the same uniform size, or can include multiple sizes designed for different surgical applications. Of course, any size and shape can be used for a drying device. The drying device can also have raised ridges or blades without grooves that limit the product space. A pad membrane such as a nitrocellulose filter membrane, a PE membrane, a silica gel membrane, etc. can also be placed on the drying device.

[0029] In some embodiments, the natural air drying comprises the steps of placing the soaked biofilm on a substrate and drying it naturally, wherein the substrate includes but is not limited to a plate, a bowl, a sieve or a frame.

[0030] In some embodiments, the irradiation is gamma ray irradiation or high energy electron beam irradiation, and the radiation dose of the irradiation is 20 to 45 kGy.

[0031] In some embodiments, after the drying step, the step of performing a composite thickening treatment on the biofilm is also included, wherein the biofilm is immersed in a medical grade polymer polysaccharide solution with a mass concentration of 1 to 5% for 1 to 3 minutes to thicken the biofilm film to 0.2 to 0.5 mm. Preferably, the medical grade polymer polysaccharide is medical grade hyaluronic acid with a molecular weight of 1 to 1.5 million or high viscosity medical grade sodium carboxymethyl cellulose. Increasing the thickness of the biofilm can protect the biological activity of the biofilm to a certain extent.

[0032] In some of the embodiments, after the drying step, the step of superimposing a polymer material film on the surface of the biofilm is also included, wherein a polymer material solution (such as collagen solution, poly(lactic-co-glycolic acid), PLGA) is spread on the biofilm and then dried to obtain a biofilm with a double-layer structure.

[0033] In some embodiments, after the drying step, the step of laminating the two biofilm layers together in contact with each other using an adhesive to form a biofilm laminated product is further included. The adhesive can be any biological adhesive known to those skilled in the art, preferably a biocompatible adhesive, including but not limited to natural adhesives such as fibronectin, fibrin or synthetic adhesives.

[0034] In some embodiments, after the drying step, the step of cross-linking the two biofilms to form a biofilm laminate is also included. The cross-linking agent is any cross-linking agent known to those skilled in the art, and the cross-linking method is any cross-linking method known to those skilled in the art, including but not limited to chemical cross-linking, peptide cross-linking, UV cross-linking, radiation cross-linking, fibronectin cross-linking, fibrinogen cross-linking, and hydrogel cross-linking.

[0035] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Example 1 A method for preparing a dry biological amniotic membrane

[0037] This embodiment provides a method for preparing a dry biological amniotic membrane, comprising the following steps:

[0038] (1) Take the fetal membranes of healthy cesarean section and rinse them clean with sterile tissue buffer or physiological saline in a sterile environment;

[0039] (2) Use a blunt instrument to separate the amnion and chorion of the fetal membrane, discard the chorion, and retain the amnion;

[0040] (3) Remove the connective tissue and spongy layer of the amniotic membrane;

[0041] (4) Treat with sterile tissue buffer and antibiotic balance solution;

[0042] (5) placing the treated amniotic membrane in a container containing a biofilm protection solution for immersion for 12 hours; wherein the biofilm protection solution is prepared from the following components: 15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 10000 ng (10000 ng / L) cysteine, 1 g (1 g / L) HC-HA / PTX3 complex, 1000 mL water for injection, pH 7.2-7.4, osmotic pressure 350-380 mOsm / L;

[0043] (6) Place the soaked amniotic membrane with the membrane matrix facing downward on the glass, and then place the drying device with the biofilm in an incubator preheated to 45° C. for about 30 minutes;

[0044] (7) Cut the dried amniotic membrane into appropriate sizes, package and seal;

[0045] (8) Irradiate with γ-rays at a dose of 25 kGy for 24 hours and store at room temperature.

[0046] Example 2 A method for preparing a dry biological amniotic membrane

[0047] This embodiment provides a method for preparing a dry biological amniotic membrane, comprising the following steps:

[0048] Steps (1) to (5) are the same as in Example 1;

[0049] (6) Place the soaked amniotic membrane in a freeze dryer and freeze-dry at -60°C;

[0050] (7) cutting the freeze-dried amniotic membrane into appropriate sizes, packaging, and sealing;

[0051] (8) Irradiate with γ-rays at a dose of 40 kGy for 24 hours and store at room temperature.

[0052] Example 3 A method for preparing a dry biological amniotic membrane

[0053] The present embodiment provides a method for preparing a dry biological amniotic membrane, except that in step (5), the biological membrane protective solution is prepared from the following components: 22 g chondroitin sulfate, 150 mg vitamin C, 40 g sodium hyaluronate, 15000 ng (15000 ng / L) of cysteine, 5 g (5 g / L) of HC-HA / PTX3 complex, and 1000 mL of water for injection, and the other steps are the same as those in Example 1.

[0054] Example 4 A method for preparing a dry biological amniotic membrane

[0055] The present embodiment provides a method for preparing a dry biological amniotic membrane, except that in step (5), the biological membrane protective solution is prepared from the following components: 12 g chondroitin sulfate, 100 mg vitamin C, 5 g sodium hyaluronate, 8000 ng (8000 ng / L) of cysteine, 0.1 g (0.1 g / L) of HC-HA / PTX3 complex, and 1000 mL of water for injection, and the other steps are the same as those in Example 1.

[0056] Example 5 A method for preparing a dry biological amniotic membrane

[0057] The present embodiment provides a method for preparing a dry biological amniotic membrane, except that in step (5), the biological membrane protective solution is prepared from the following components: 15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 10000 ng (10000 ng / L) cysteine, 1 g (1 g / L) HC-HA / PTX3 complex, and 1000 mL water for injection, and the other steps are the same as those in Example 1.

[0058] Example 6 A method for preparing a dry biological amniotic membrane

[0059] The present embodiment provides a method for preparing a dry biological amniotic membrane, except that in step (5), the biological membrane protective solution is prepared from the following components: 12 g chondroitin sulfate, 100 mg vitamin C, 5 g sodium hyaluronate, 8000 ng (8000 ng / L) of cysteine, 0.1 g (0.1 g / L) of HC-HA / PTX3 complex, and 1000 mL of water for injection, and the other steps are the same as those in Example 1.

[0060] Comparative Example 1

[0061] The present comparative example provides a method for preparing a dry biological amniotic membrane. Except that in step (5), the biological membrane protective solution is prepared from the following components: 15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 300 g glycerol, and 1000 mL water for injection, the other steps are the same as those in Example 1.

[0062] Comparative Example 2

[0063] The present comparative example provides a method for preparing a dry biological amniotic membrane, except that in step (5), the biological membrane protective solution is prepared from the following components: 15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 20000 ng (20000 ng / L) cysteine, and 1000 mL water for injection, and the other steps are the same as those in Example 1.

[0064] Comparative Example 3

[0065] The present comparative example provides a method for preparing a dry biological amniotic membrane. Except that in step (5), the biological membrane protective solution is prepared from the following components: 15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 300 g glycerol, 2 g (2 g / L) of HC-HA / PTX3 complex, and 1000 mL of water for injection, the other steps are the same as those in Example 1.

[0066] Comparative Example 4

[0067] The present comparative example provides a method for preparing a dry biological amnion. Except that in step (5), the biological membrane protection liquid is 8% glycerol (a biological membrane preservative commonly used in the art), the other steps are the same as those of Example 1.

[0068] Comparative Example 5

[0069] The present comparative example provides a method for preparing a dry biological amniotic membrane. The amniotic membrane is directly dried using the drying method of Example 1 without using a biological membrane protection solution for immersion treatment. That is, based on Example 1, step (5) is omitted.

[0070] Comparative Example 6

[0071] This comparative example provides a method for preparing a wet biological amniotic membrane. The amniotic membrane treated according to the method of Example 1 is cut into appropriate sizes, placed in a biological membrane preservation solution (15 g chondroitin sulfate, 100 mg vitamin C, 20 g sodium hyaluronate, 300 g glycerol, 20 mL dimethyl sulfoxide, 10000 ng (10000 ng / L) cysteine, 1 g (1 g / L) HC-HA / PTX3 complex, 1000 mL water for injection, pH 7.2-7.4, osmotic pressure 350-380 mOsm / L), transferred to a -20°C insulation container, and then irradiated with γ rays with an irradiation dose of 25 kGy for 24 hours, and stored at -20°C.

[0072] Experimental Example 1 Effect of different biofilm preparation methods on the preservation of biofilm active factors

[0073] This test example detected the contents of various growth factors in fresh amniotic membrane, dry biological amniotic membrane prepared in Examples 1 to 2, Comparative Examples 1 to 5, and wet biological amniotic membrane prepared in Comparative Example 6.

[0074] 1. Experimental Grouping

[0075] Experimental group 1: dry biological amniotic membrane prepared in Example 1

[0076] Experimental Group 2: Dry biological amniotic membrane prepared in Example 2

[0077] Control group CK: fresh amniotic membrane treated in steps (1) to (4) in Example 1, with surface moisture absorbed.

[0078] Control group 1: dry biological amniotic membrane prepared in comparative example 1

[0079] Control group 2: dry biological amniotic membrane prepared in comparative example 2

[0080] Control group 3: dry biological amniotic membrane prepared in comparative example 3

[0081] Control group 4: dry biological amniotic membrane prepared in comparative example 4

[0082] Control group 5: dry biological amniotic membrane prepared in comparative example 5

[0083] Control group 6: wet-state preserved biological amniotic membrane prepared in comparative example 6 (surface moisture is absorbed)

[0084] 2. Experimental methods

[0085] Weigh a certain amount of experimental group samples and control group samples respectively, and ultrasonically crush them. Weigh 30 mg of amniotic membrane and add it to 1 ml of 1×PBS buffer. Oscillate on a shaker at room temperature for 24 hours, then centrifuge at 4°C, 12000 rpm for 30 minutes, and take the supernatant for quantitative detection (the content of HGF and other factors in fresh amniotic membrane is relatively high, so it can be diluted to a certain proportion for detection. Whether other materials should be diluted depends on the specific situation).

[0086] According to the instructions of each growth factor detection kit, the contents of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), β1 transforming growth factor (TGF-β1), and nerve growth factor (NGF) in the experimental group samples and the control group samples were detected respectively, and the contents of each growth factor in the CK (fresh amniotic membrane) of the control group were compared with those in the control group, so as to evaluate the preservation effect of active factors in the dry amniotic membrane prepared in each experimental group and the control group.

[0087] 3. Experimental results

[0088] The results are as follows Figure 1 shown.

[0089] from Figure 1 The results show that compared with the control group CK (fresh amniotic membrane), the growth factors in all sample groups showed a downward trend. However, compared with the control groups 1 to 5, the decline in growth factors in experimental groups 1 and 2 was significantly smaller, indicating that after being protected by the biofilm protection solution of the present invention, the dry amniotic membrane was prepared by drying or freeze-drying, and the active factor preservation rate was high; the growth factor preservation rate in the dry amniotic membrane of experimental groups 1 and 2 was basically the same as that in the wet bio-amniotic membrane of control group 6. It shows that after being protected by the biofilm protection solution of the present invention, the dry amniotic membrane was prepared by drying or freeze-drying, which can avoid the damage and denaturation of the biofilm material during the drying process and irradiation sterilization.

[0090] Compared with Example 1, the biofilm protective solution of Comparative Example 1 does not add cysteine ​​and HC-HA / PTX3 complex, and adds glycerol; the biofilm protective solution of Comparative Example 2 does not add HC-HA / PTX3 complex; the biofilm protective solution of Comparative Example 3 replaces cysteine ​​with glycerol; the content of each growth factor is significantly reduced compared with the dry amniotic membrane of Example 1, among which the effect of Comparative Example 2 is relatively good, which indicates that the addition of cysteine ​​in the biofilm protective solution plays a very important role in maintaining the growth factor content, and cysteine ​​and HC-HA / PTX3 complex can play a synergistic effect to significantly reduce the growth factor loss rate.

[0091] Compared with Example 1, in Comparative Example 4, 8% glycerol was used as the biofilm protection liquid, and the content of each growth factor in the prepared dry amniotic membrane dropped sharply, indicating that conventional biofilm preservatives are not suitable for the preparation of dry biofilms.

[0092] Compared with Example 1, in Comparative Example 5, no biofilm protection solution was used, and the content of each growth factor in the prepared dry amniotic membrane was only 40% of the content of each growth factor in the dry amniotic membrane of Example 1, proving that in order to ensure the content of growth factors when preparing dry amniotic membrane, biofilm protection solution must be added for protection. Experimental Example 2 Effect of different biofilm preparation methods on biofilm collagen protection

[0093] In order to verify the protective effect of the dry biofilm preparation method of the present invention on biofilm collagen, this experimental example used the parallel reaction monitoring (PRM) technology of mass spectrometry to detect the abundance of type I collagen, type III collagen, type IV collagen, type VI collagen, type VII collagen, type XII collagen, fibronectin and laminin in each experimental group of fresh amniotic membrane, dry bio-amniotic membrane prepared in Examples 1 and 2 of the present invention, dry bio-amniotic membrane prepared in Comparative Examples 1-5, and wet bio-amniotic membrane prepared in Comparative Example 6.

[0094] 1. Experimental Grouping

[0095] Experimental group 1: dry biological amniotic membrane prepared in Example 1

[0096] Experimental Group 2: Dry biological amniotic membrane prepared in Example 2

[0097] Control group CK: fresh amniotic membrane treated in steps (1) to (4) in Example 1, with surface moisture absorbed.

[0098] Control group 1: dry biological amniotic membrane prepared in comparative example 1

[0099] Control group 2: dry biological amniotic membrane prepared in comparative example 2

[0100] Control group 3: dry biological amniotic membrane prepared in comparative example 3

[0101] Control group 4: dry biological amniotic membrane prepared in comparative example 4

[0102] Control group 5: dry biological amniotic membrane prepared in comparative example 5

[0103] Control group 6: wet biological amniotic membrane prepared in comparative example 6 (surface moisture is absorbed)

[0104] 2. The PRM mass spectrometry identification method was used to quantitatively identify the target proteins in each sample. Based on the sample total protein loading concentration of 0.4 μg / μl and the internal standard peptide loading concentration of 0.004 μg / μl, (target protein abundance value*0.004 / internal standard peptide abundance value*0.4)*100% was used to calculate the proportion of each target protein in the total protein in each experimental group. The specific results are shown in Table 1.

[0105] Table 1 The proportion of target protein in total protein in each experimental group

[0106]

[0107] 3. Calculate the OR value, that is, the ratio of the proportion of each target protein in the total protein in each experimental group to the proportion of the target protein in the total protein in the sample 5 of the control group (the dry biological amniotic membrane prepared in comparative example 5 was not soaked in the biological membrane protection liquid, and the amniotic membrane was directly dried by the drying method of Example 1). The results are shown in Table 2.

[0108] The significance of OR value: OR value equal to 1 indicates that the treatment method of biological amniotic membrane during the drying process has no effect on the protection of collagen; OR value greater than 1 indicates that the treatment method of biological amniotic membrane during the drying process promotes the protection of collagen; OR value less than 1 indicates that the treatment method of biological amniotic membrane during the drying process inhibits the protection of collagen. The larger the OR value, the more effective the treatment method of biological amniotic membrane during the drying process is for the protection of collagen.

[0109] Table 2 OR values ​​of each experimental group (n=5)

[0110]

[0111]

[0112] 4. Results Analysis

[0113] From the results in Table 2, it can be seen that the OR values ​​of the samples in each group are greater than 1, indicating that the treatment methods of experimental group 1, experimental group 2, control group 1, control group 2, control group 3, and control group 4 have a certain protective effect on the target collagen during the drying process of the biological amniotic membrane. However, the OR values ​​in experimental group 1 and experimental group 2 are much greater than the OR values ​​in control group 1, control group 2, control group 3, and control group 4. Therefore, when preparing dry biomembranes, soaking treatment with the protective solution of the present invention before drying has a more significant protective effect on amniotic membrane collagen, and is equivalent to the protective effect of wet biomembranes on amniotic membrane collagen (p<0.05).

[0114] Experimental Example 3 Preservation effect of biofilm at different storage time

[0115] This example detects the preservation effects of the dry biological amniotic membrane prepared in Example 1 and the wet biological amniotic membrane prepared in Comparative Example 6 when stored at room temperature for 6 months and 3 years. The above test is divided into 4 groups, namely Group A (dry biological amniotic membrane stored at room temperature for 6 months), Group B (wet biological amniotic membrane stored at -20°C low temperature and wet state for 6 months), Group C (dry biological amniotic membrane stored at room temperature for 3 years) and Group D (wet biological amniotic membrane stored at -20°C low temperature and wet state for 1 year). Referring to the method of Test Example 1, the contents of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), β1 transforming growth factor (TGF-β1), and nerve growth factor (NGF) in Group A, Group B, Group C, and Group D are detected respectively, and the ratio of the content of each growth factor in the samples of Group A, Group B, Group C, and Group D to the content of each growth factor in the fresh amniotic membrane is calculated. The specific results are shown in Table 3.

[0116] Table 3 Effects of different storage time on the preservation of biofilm active factors

[0117]

[0118] As can be seen from Table 3, the wet biological amniotic membrane (Comparative Example 6) has an active factor preservation rate of more than 68% when stored in a low-temperature wet state at -20°C for 6 months; the active factor preservation rate is about 50% when stored in a low-temperature wet state at -20°C for 1 year; the dry biological amniotic membrane prepared by the present invention (Example 1) has an active factor preservation rate of more than 70% when stored in a sealed state at room temperature for 6 months; and the active factor preservation rate can still reach more than 60% when stored in a sealed state at room temperature for 3 years. Therefore, the dry biological amniotic membrane prepared by the present invention is more conducive to the long-term preservation of biological membrane materials and has a good preservation effect. The active factor preservation rate of the wet biological amniotic membrane stored at room temperature for 3 years is higher than the active factor preservation rate of the wet biological amniotic membrane stored at -20°C for 1 year.

[0119] Test Example 4 Thickness Change and Mechanical Properties Test of Dry Biofilm

[0120] In order to verify the mechanical properties of the dry biomembrane provided by the present invention, this test example measured the thickness of the bio-amniotic membrane of the control group CK, experimental group 1, experimental group 2, control group 1, control group 2, control group 3, control group 4, and control group 5 in a fresh state (i.e., the fresh amniotic membrane treated by steps (1) to (4)) and the thickness after drying (wherein the control group CK was not dried). The maximum breaking force of the bio-amniotic membrane samples prepared by the control group CK, experimental group 1, experimental group 2, control group 1, control group 2, control group 3, control group 4, and control group 5 was tested by an electronic universal testing machine (model: RGM-6002T), and the tensile strength of each sample was calculated (tensile strength = maximum breaking force (N) / [sample thickness (mm)*width (mm)]. The results are shown in Table 4.

[0121] Table 4 Thickness, maximum breaking force and tensile strength test of each group of samples (n=5)

[0122]

[0123] From the results in Table 4, it can be seen that, except for control group 4 and control group 5, the thickness of the biological amniotic membrane samples in each group (after drying) is thicker than that in the fresh state, and the thickening range of experimental group 1 and experimental group 2 is larger than that of other groups. Compared with other groups, the maximum breaking force of the biological amniotic membrane samples in experimental group 1 and experimental group 2 is significantly improved, and the maximum breaking force is smaller than that of the control group CK. After calculation, the tensile strength of the biological amniotic membrane samples in experimental group 1 and experimental group 2 is improved compared with that of the control group CK, while the tensile strength of the biological amniotic membrane samples in control groups 1 to 5 is very different from that of the control group CK, especially the control group 5, which has the worst effect.

[0124] The results of this test example show that compared with the conventional drying method (control group 5) and the biological amniotic membrane prepared by soaking in the protective solution and then drying in comparative examples 1 to 4, the dry biological membrane prepared by soaking in the protective solution provided by the present invention and then drying has a thicker thickness, a greater maximum breaking force, and a higher tensile strength, so its mechanical properties are better, the flexibility is better, and it is more suitable for clinical operation. This may be because during the drying process of the biological membrane, the sulfur-containing compounds in the preservation solution can promptly remove the free radicals generated during irradiation, avoid the damage of the free radicals to the biological membrane, and work together with HC-HA / PTX3 and other components to effectively protect the collagen, so that the prepared dry biological amniotic membrane has good flexibility and mechanical properties.

[0125] Experimental Example 5 Application of dry amniotic membrane in spinal surgery

[0126] 60 adult New Zealand rabbits weighing 2.0-3.0 kg were selected, regardless of gender. According to the different epidural coverings at the vertebral lamina defect, the 60 rabbits were randomly divided into a dry amniotic membrane group, a polylactic acid membrane group and a blank control group, with 20 rabbits in each group.

[0127] Under aseptic operation, the posterior midline incision was about 20 mm long, and the L5 spinous process was cut off layer by layer. The L5 lamina was bitten off with a micro-lamina rongeur to create a 10 mm × 5 mm dura mater exposed area, and the epidural fat was removed. After thorough hemostasis, the animals were randomly grouped before surgery, and the dry biological amniotic membrane prepared by the present invention (sample prepared in Example 2) and the polylactic acid membrane were respectively covered on the epidural membrane, and the non-covered group was used as a blank control group. All animals were caged and raised under the same conditions after surgery, and no drugs were taken.

[0128] 1. Specimen collection and processing

[0129] Five animals were sacrificed in each group at 2, 4, 8, and 12 weeks after surgery. After the animals were sacrificed, the L5 segment of the spine, including the sacrospinal muscle and vertebral attachments, was completely removed, fixed with formalin solution, and then embedded in paraffin and sliced ​​with a thickness of 5 μm. The tissue sections were stained with HE, Masson, and picric acid-sirius red.

[0130] 2. Optical microscope observation

[0131] The distribution of epidural scars and their relationship with the dura mater and nerve roots were observed under a common optical microscope.

[0132] 3. Computer image analysis (Axioplan2 image, USA)

[0133] The cross-sectional area of ​​the irregular scar was calculated, and its ratio to the spinal canal area was used to determine the scar index.

[0134] 4. Statistical analysis

[0135] Analysis of variance and q test were used.

[0136] 5. Results

[0137] Observation under a common microscope showed that 2 weeks after surgery, a large amount of granulation tissue filled the defect outside the dura mater in the blank group, extending from the dorsal side to the lateral side of the spinal canal, and some reached the nerve root. Granulation tissue and fibroblast proliferation were inactive in the polylactic acid membrane group and the dry biological amniotic membrane group. The matrix and basement membrane of the amniotic membrane sheet were swollen and loose, with a few fibroblasts and inflammatory cells infiltrating. 4 weeks after surgery, the blank group had more collagen fibers, some fibroblasts transformed into fibrocytes, and the capillaries decreased. The polylactic acid membrane group and the dry biological amniotic membrane group had fewer fibroblasts, scarce collagen fibers, and an epidural space. The amniotic membrane was fused with the fibrous tissue behind it, with a fuzzy structure and no adhesion with the dura mater; the collagen density was low, and there was no obvious inflammatory reaction. At 8 weeks, the granulation tissue was fibrotic, the collagen in the blank group was dense, the dura mater adhered to the scar, and bone tissue was formed at the defect of the original lamina, and there was a scar between it and the dura mater. The polylactic acid membrane group had low collagen density and no obvious inflammatory reaction; there was little epidural scar and no adhesion to the dura mater; the polylactic acid membrane degraded into small red-stained, structureless fragments. At 12 weeks after surgery, a large number of collagen fibers were observed in the blank group, which were tightly and disorderly arranged, and the cell components basically disappeared. The dura mater and scar were densely adhered and connected to the new vertebral lamina. At 8 and 12 weeks after surgery in the dry amniotic membrane group, the scar area was reduced, the cell components were scarce, there was no adhesion on the dura mater surface under the new bone plate, some specimens had epidural fat regeneration, and the amniotic membrane was replaced by collagen.

[0138] The microcomputer image processing system calculated the ratio of the scar area invading the spinal canal to the area of ​​the entire spinal canal. The three slices were measured and the average was taken to calculate the relative scar index in the spinal canal. Within 2 weeks, the repair of the lamina defect was mainly granulation tissue formation, with less collagen fibers, and the scar index difference was not significant (p>0.05). At 4 weeks, the collagen fibers increased and the scar was initially formed. The dry biological amniotic membrane group had the smallest value, showing the dry biological amniotic membrane's adhesion prevention effect (p<0.01). At 8 weeks, the polylactic acid membrane gradually expanded into a gel, exerting its adhesion prevention properties, but it was still worse than the dry biological amniotic membrane group (p<0.01). At 12 weeks, the scar index of the dry biological amniotic membrane group and the polylactic acid membrane group was smaller than that of the blank group (p<0.01), but the difference between the two groups was significant (p<0.05) (see Table 5).

[0139] Table 5 Comparison of relative scar index of dry amniotic membrane at different times (n=5; %)

[0140] Grouping 2 weeks 4 weeks 8 weeks 12 weeks Blank group 3.87±0.53 8.69±0.67 15.07±0.73 12.07±0.87 Polylactic acid film group 3.30±0.68 7.74±0.59** 11.03±0.86** 7.98±0.91** Dry amniotic membrane group 3.11±0.45 <![CDATA[5.18±0.40** △△ ]]> <![CDATA[8.00±0.59** △△ ]]> <![CDATA[6.98±0.63** △ ]]>

[0141] q test: compared with the blank group **p<0.01; compared with polylactic acid film △p<0.05, △△p<0.01

[0142] From the results in Table 5, it can be seen that the dry biological amniotic membrane provided by the present invention can protect the exposed nerve roots during spinal surgery by inhibiting infection and inflammatory response, reducing the release of inflammatory mediators, reducing local fibroblast infiltration, reducing scar formation, and preventing adhesion.

[0143] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a dry biofilm, characterized in that: The following steps are involved: The fresh biofilm is placed in a biofilm protection solution and soaked for 1 to 24 hours, dried, and then irradiated for sterilization; wherein the biofilm protection solution is prepared from components with the following concentrations: 15 g / L chondroitin sulfate, 100 mg / L vitamin C, 20 g / L sodium hyaluronate, 10000 ng / L cysteine, and 1.0 g / L HC-HA / PTX3 complex.

2. The method for preparing a dry biofilm according to claim 1, characterized in that: The drying process is drying, freeze drying, natural air drying or vacuum drying; And / or, the drying temperature is 35-50° C., and the drying time is 30-120 min; And / or, the freeze-drying temperature is -50°C to -80°C.

3. The method for preparing a dry biofilm according to claim 2, characterized in that: The drying temperature is 42-48°C, and the drying time is 40-50 min; and / or the freeze-drying temperature is -50°C to -60°C.

4. The method for preparing a dry biofilm according to claim 1, characterized in that: The irradiation is gamma ray irradiation or high energy electron beam irradiation, and the radiation dose of the irradiation is 20 to 45 kGy.

5. The method for preparing a dry biofilm according to claim 1, characterized in that: The biological membrane is amniotic membrane or umbilical cord derived tissue.

6. The dry biofilm prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the dry biofilm according to claim 6 in preparing materials for inhibiting infection and inflammatory response, reducing scar formation and protecting exposed nerve roots in ophthalmic surgery, skin defect repair, tendon repair and adhesion prevention, and spinal surgery.

Images