Artificial biomembrane patch and its preparation method and application

Abstract

The present application relates to an artificial biomembrane patch and a preparation method and application thereof. The artificial biomembrane patch comprises a repair layer and an anti-adhesion layer arranged on one side of the repair layer. The anti-adhesion layer has a porous structure. The anti-adhesion layer comprises a first polysaccharide polymer with an average cross-linking degree of 50% to 90% and a second polysaccharide polymer with an average cross-linking degree of 10% to 30%. The mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1 to 4:1, which effectively improves the anti-adhesion effect and biosafety of the artificial biomembrane patch.

Description

Technical Field

[0001] The present application relates to the technical field of biomaterials, and in particular to an artificial biomembrane patch and a preparation method and application thereof. Background Art

[0002] The brain is surrounded by three protective meningeal layers: the pia mater, the arachnoid mater, and the dura mater. The dura mater is the outermost connective tissue layer that surrounds and protects the brain and spinal cord. It is composed of fibroblasts and a large amount of extracellular collagen. The dura mater consists of two layers: the periosteum / endosteum layer and the meningeal layer. Among them, the meningeal layer is a relatively dense fibrous membrane structure that passes through the foramen magnum and is connected to the dura mater of the spinal cord. It has the functions of wrapping and protecting the brain and spinal cord, regulating the production and migration of neural progenitor cells, regulating the survival and proliferation of radial glial cells, preventing cerebrospinal fluid leakage and brain tissue adhesion, preventing superficial brain infection, and maintaining intracranial pressure. The outer layer of the dura mater is the periosteum or endosteal layer, which is usually adhered to the skull. Its porous surface is conducive to the regeneration of autologous dura mater and bone. The dura mater extends outward from the cranial nerves, migrates and combines with the nerve capsule, and extends downward to the spinal dura mater around the foramen magnum. The dura mater and spinal dura mater are important functional membrane tissues between the brain and skull, and between the spine and spinal cord, respectively, and have the same biological characteristics and functions.

[0003] Some factors such as trauma, tumor invasion, surgical resection and congenital malformations may damage the dura mater, leading to a variety of complications, including cerebrospinal fluid leakage, infection, meningitis and epilepsy. Since the dura mater tissue is non-regenerative, an artificial dura mater substitute is needed after neurosurgery to prevent adhesion. Therefore, there is an urgent need to find an artificial dura mater patch that can further improve the anti-adhesion effect. Summary of the invention

[0004] The present application provides an artificial biomembrane patch and a preparation method and application thereof, aiming to improve the anti-adhesion effect and biosafety of the artificial biomembrane patch.

[0005] In a first aspect of the present application, an artificial biomembrane patch is provided, comprising a repair layer and an anti-adhesion layer arranged on one side of the repair layer, the anti-adhesion layer having a porous structure, the anti-adhesion layer comprising a first polysaccharide polymer with an average cross-linking degree of 50% to 90% and a second polysaccharide polymer with an average cross-linking degree of 10% to 30%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1 to 4:1.

[0006] In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 2:1 to 4:1.

[0007] In some embodiments, the first polysaccharide polymer and the second polysaccharide polymer each independently comprise a biodegradable polysaccharide polymer.

[0008] In some embodiments, the biodegradable polysaccharide polymer includes at least one of chitosan, chitosan derivatives, cellulose, and cellulose derivatives.

[0009] In some embodiments, the material of the repair layer includes extracellular matrix.

[0010] In some embodiments, the average cross-linking degree of the first polysaccharide polymer is 60% to 90%.

[0011] In some embodiments, the average cross-linking degree of the second polysaccharide polymer is 15% to 30%.

[0012] In some embodiments, at least a portion of the first polysaccharide polymer and / or at least a portion of the second polysaccharide polymer in the anti-adhesion layer is connected to the repair layer through at least one of electrostatic interaction and secondary bond.

[0013] In some embodiments, the porosity of the anti-adhesion layer is 50% to 90%.

[0014] In some embodiments, the mass of the anti-adhesion layer per unit area on the repair layer is 1 mg / cm 2 ~6mg / cm 2 , optional 2mg / cm 2 ~4mg / cm 2 .

[0015] The second aspect of the present application provides a method for preparing an artificial biomembrane patch, comprising the following steps:

[0016] A solution containing a first polysaccharide polymer and a solution containing a second polysaccharide polymer are mixed and homogenized to obtain a film-forming solution; the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the film-forming solution is 1:1-4:1, the average cross-linking degree of the first polysaccharide polymer is 50%-90%, and the average cross-linking degree of the second polysaccharide polymer is 10%-30%;

[0017] The film-forming liquid is disposed on one side of the material of the repair layer to obtain an intermediate;

[0018] The solvent in the intermediate is removed to form a repair layer and an anti-adhesion layer with a porous structure to obtain the artificial biofilm patch.

[0019] In some embodiments, a first cross-linking monomer is mixed with a first solvent and subjected to a first emulsification treatment to obtain a first solution;

[0020] Mixing the first solution with a first cross-linking agent to perform a cross-linking reaction to obtain a first gel;

[0021] The first gel is subjected to ion replacement in a buffer solution and in water in sequence to obtain the solution containing the first polysaccharide polymer.

[0022] In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the membrane-forming solution is 2:1 to 4:1.

[0023] In some embodiments, the mass percentage of the first cross-linking monomer in the first solution is 2% to 4%.

[0024] In some embodiments, the first cross-linking monomer includes a polysaccharide polymer A having a viscosity of 20 mPa·s to 60 mPa·s.

[0025] In some embodiments, the mass ratio of the first cross-linking agent to the first solution is 1:1000-3:1000, and can be optionally 1:1000-2:1000.

[0026] In some embodiments, the mass ratio of the first gel to the buffer is 1:10 to 1:40.

[0027] In some embodiments, the mass ratio of the first gel to water is 1:10 to 1:40.

[0028] In some embodiments, the first cross-linking agent includes at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0029] In some embodiments, the buffer comprises at least one of a sodium phosphate buffer and a potassium phosphate buffer;

[0030] In some embodiments, the first solvent comprises water.

[0031] In some embodiments, the method for preparing the solution containing the second polysaccharide polymer comprises the following steps:

[0032] Mixing a second cross-linking monomer with a second solvent and subjecting the mixture to a second emulsification treatment to obtain a second solution;

[0033] The second solution is mixed with a second cross-linking agent to perform a cross-linking reaction to obtain the solution containing the second polysaccharide polymer.

[0034] In some embodiments, the mass percentage of the second cross-linking monomer in the second solution is 2% to 4%.

[0035] In some embodiments, the second cross-linking monomer includes a polysaccharide polymer B having a viscosity of 20 mPa·s to 60 mPa·s.

[0036] In some embodiments, the mass ratio of the second cross-linking agent to the second solution is 0.1:1000 to 0.4:1000, and can be optionally 0.1:1000 to 0.2:1000.

[0037] In some embodiments, the second cross-linking agent includes at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0038] In some embodiments, the second solvent comprises water.

[0039] The third aspect of the present application provides the use of the artificial biomembrane patch described in the first aspect of the present application or the artificial biomembrane patch prepared by the preparation method described in the second aspect of the present application in the preparation of dura mater or spinal dura mater repair products.

[0040] Compared with traditional technologies, the above-mentioned artificial biofilm patch has at least the following advantages:

[0041] By controlling the average cross-linking degree of the first polysaccharide polymer, the average cross-linking degree of the second polysaccharide polymer, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer of the above-mentioned artificial biomembrane patch within a reasonable range, the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer can be improved, scar hyperplasia can be reduced, and thus the anti-adhesion effect and biosafety of the artificial biomembrane patch can be effectively improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the sample and the testing machine fixture when testing the interlayer bonding strength in Test Example 1 of the present application.

[0043] Figure 2 The cell images and morphological test results in the cytotoxicity test of Experimental Example 2 of this application; Figure 2 Figures (a) to (c) in the figure are the test results of blank samples, negative samples and positive samples respectively. Figure 2 Figures (d) to (g) are the test results of Example 1, Example 3, Example 5, and Example 7, respectively. Figure 2 Figures (h) to (j) are the test results of Comparative Example 2, Comparative Example 4, and Comparative Example 6, respectively.

[0044] Figure 3 The anti-leakage test results of Test Example 3 of this application; Figure 3Figures (a) to (h) are the test results of Example 1, Example 3, Example 5, Example 7, Example 8, Example 9, Example 10, and Example 11, respectively. Figure 3 Figures (i) to (k) are the test results of Comparative Example 2, Comparative Example 4, and Comparative Example 6, respectively.

[0045] Figure 4 This is a morphological diagram of fibroblasts in Experimental Example 4 of the present application after different incubation times; Figure 4 Figures (a-1) to (a-5) are morphological diagrams of fibroblasts inoculated on the artificial biofilm patch prepared in Example 1 and incubated for 1 day, 2 days, 3 days, 5 days, and 7 days, respectively. Figure 4 Figures (b-1) to (b-5) are the morphological images of fibroblasts inoculated on commercially available artificial biofilm patches and incubated for 1d, 2d, 3d, 5d, and 7d, respectively.

[0046] Figure 5 This is a three-dimensional structural diagram of the artificial biomembrane patch prepared in Example 1 of the present application.

[0047] Description of reference numerals:

[0048] 1 Upper clamp; 2 Lower clamp; 3 Peeled portion of the specimen; 4 Unpeeled portion of the specimen. DETAILED DESCRIPTION

[0049] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0050] In this application, "first aspect", "second aspect", "third aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0051] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0052] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0053] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0054] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as those commonly understood by technicians in the technical field of this application. The terms used in this application and in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used in this application includes any and all combinations of one or more related listed items. The term "multiple" in this application means at least two, such as two, three, etc., unless otherwise clearly and specifically limited.

[0055] One embodiment of the present application provides an artificial biomembrane patch, comprising a repair layer and an anti-adhesion layer arranged on one side of the repair layer, the anti-adhesion layer having a porous structure, comprising a first polysaccharide polymer with an average cross-linking degree of 50% to 90% and a second polysaccharide polymer with an average cross-linking degree of 10% to 30%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1 to 4:1.

[0056] Postoperative adhesion refers to various abnormal tissue proliferations formed between organs and adjacent organs or tissues after surgical trauma. As adhesions grow, tissues will present different phenotypes, from a thin layer of fibrous membrane to a mixture of loose or dense fibrous tissue, nerves and blood vessels, and even scar tissue. Postoperative adhesions occur widely in various soft tissues such as the peritoneum, pericardium, uterus, tendons, and dura mater, often leading to chronic pain, dysfunction of adjacent organs, and some acute complications, which seriously reduce the quality of life of patients and even endanger their lives. Adhesions in which blood clots deposited within 1d-3d after surgery are gradually replaced by granulation tissue are called "membranous adhesions." The proliferation of fibroblasts / myofibroblasts and the formation of vascularized tissue 3d-14d after surgery are called "vascular adhesions." Therefore, reducing bleeding in the early stage, thereby reducing the formation of hematomas, and inhibiting the proliferation of fibroblasts can be used as an effective way to reduce adhesions.

[0057] The above-mentioned artificial biomembrane patch can provide mechanical support and early anti-adhesion barrier for brain tissue or spinal tissue. After being implanted into the wound site, the porous structure in the anti-adhesion layer of the artificial biomembrane patch interacts with platelets, thereby adsorbing platelets, accelerating the coagulation process, forming a gel isolation barrier layer, preventing early fibroblast penetration and adhesion, and avoiding early adhesion. Subsequently, a large number of fibroblasts and new capillary networks enter the repair layer to form new collagen tissue, gradually replacing the collagen tissue in the implanted artificial biomembrane patch. Controlling the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer, the average cross-linking degree of the second polysaccharide polymer, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer within the above range is conducive to improving the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer, reducing scar hyperplasia, thereby effectively improving the anti-adhesion effect and biosafety of the artificial biomembrane patch. It can be understood that the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer includes, but is not limited to, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, and 4:1.

[0058] In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 2: 1 to 4: 1. Controlling the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer to be within the above range is conducive to further improving the binding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer, further reducing scar hyperplasia, and thus further improving the anti-adhesion effect of the artificial biomembrane patch.

[0059] In some embodiments, the first polysaccharide polymer and the second polysaccharide polymer each independently comprise a biodegradable polysaccharide polymer.

[0060] In some embodiments, the biodegradable polysaccharide polymer includes at least one of chitosan, chitosan derivatives, cellulose, and cellulose derivatives.

[0061] In some embodiments, the chitosan derivative includes at least one of carboxymethyl chitosan, alkylated chitosan, quaternized chitosan, and acylated chitosan.

[0062] In some embodiments, the cellulose derivative includes at least one of methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

[0063] In some embodiments, the material of the repair layer includes an extracellular matrix.

[0064] In some embodiments, the average cross-linking degree of the first polysaccharide polymer is 60% to 90%. Controlling the average cross-linking degree of the first polysaccharide polymer within the above range is conducive to further improving the bonding force between the first polysaccharide polymer in the anti-adhesion layer and the repair layer, further reducing scar hyperplasia, and thus further improving the anti-adhesion effect of the artificial biomembrane patch.

[0065] In some embodiments, the average cross-linking degree of the second polysaccharide polymer is 15% to 30%. Controlling the average cross-linking degree of the second polysaccharide polymer within the above range is conducive to further improving the bonding force between the second polysaccharide polymer in the anti-adhesion layer and the repair layer, further reducing scar hyperplasia, and thus further improving the anti-adhesion effect of the artificial biomembrane patch.

[0066] In some embodiments, at least part of the first polysaccharide polymer and / or at least part of the second polysaccharide polymer in the anti-adhesion layer is connected to the repair layer through at least one of electrostatic action and secondary bonds. Thus, it is beneficial to further enhance the binding force between the first polysaccharide polymer and / or the second polysaccharide polymer in the anti-adhesion layer and the repair layer, and further enhance the anti-adhesion effect of the artificial biofilm patch. The secondary bonds include but are not limited to hydrogen bonds and van der Waals forces.

[0067] In some embodiments, the porosity of the anti-adhesion layer is 50% to 90%.

[0068] In some embodiments, the mass of the anti-adhesion layer per unit area of ​​the repair layer is 1 mg / cm 2 ~6mg / cm 2 , optional 2mg / cm 2 ~4mg / cm 2 .

[0069] Another embodiment of the present application provides a method for preparing an artificial biomembrane patch, comprising the following steps:

[0070] A solution containing a first polysaccharide polymer and a solution containing a second polysaccharide polymer are mixed and homogenized to obtain a film-forming solution; the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the film-forming solution is 1:1-4:1, the average cross-linking degree of the first polysaccharide polymer is 50%-90%, and the average cross-linking degree of the second polysaccharide polymer is 10%-30%;

[0071] Disposing a film-forming liquid on one side of the material of the repair layer to obtain an intermediate;

[0072] The solvent in the intermediate is removed to form a repair layer and an anti-adhesion layer with a porous structure, thereby obtaining an artificial biofilm patch.

[0073] In the above preparation method, by controlling the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer, the average cross-linking degree of the second polysaccharide polymer, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer within the above range, it is beneficial to improve the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer, reduce scar hyperplasia, and thus effectively improve the anti-adhesion effect and biosafety of the artificial biomembrane patch.

[0074] In some embodiments, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the membrane-forming solution is 2: 1 to 4: 1. This is conducive to further improving the binding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer, further reducing scar hyperplasia, and thus further improving the anti-adhesion effect of the artificial biomembrane patch.

[0075] In some embodiments, the ratio of the mass of the membrane-forming liquid to the area of ​​the material of the repair layer contacting the membrane-forming liquid is 2 g:10 cm 2 ~3.2g:10cm 2 .

[0076] In some embodiments, the method for preparing a solution comprising a first polysaccharide polymer comprises the following steps:

[0077] Mixing a first cross-linking monomer with a first solvent and performing a first emulsification treatment to obtain a first solution;

[0078] Mixing the first solution with the first cross-linking agent to perform a cross-linking reaction to obtain a first gel;

[0079] The first gel is subjected to ion replacement in a buffer solution and in water in turn to obtain a solution containing the first polysaccharide polymer. Optionally, the process conditions of the cross-linking reaction include: the cross-linking time is more than 6 hours.

[0080] In the above embodiment, the first emulsification treatment can promote the first cross-linking monomer in the first solution to fully react with the first cross-linking agent, and the first gel is ion-exchanged by using buffer and water, which is beneficial to remove the residual first cross-linking agent in the solution containing the first polysaccharide polymer.

[0081] In some embodiments, the mass percentage of the first cross-linking monomer in the first solution is 2% to 4%. It is understood that the mass percentage includes, but is not limited to: 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%.

[0082] In some embodiments, the first cross-linking monomer includes a polysaccharide polymer A having a viscosity of 20 mPa·s to 60 mPa·s. It is understood that the above viscosity includes but is not limited to: 20 mPa·s, 30 mPa·s, 40 mPa·s, 50 mPa·s, and 60 mPa·s.

[0083] In some embodiments, the mass ratio of the first cross-linking agent to the first solution is 1:1000~3:1000, and can be optionally 1:1000~2:1000. This is conducive to further improving the binding force between the first polysaccharide polymer in the anti-adhesion layer and the repair layer, further reducing scar hyperplasia, and thus further improving the anti-adhesion effect of the artificial biofilm patch. It can be understood that the mass ratio of the first cross-linking agent to the first solution includes but is not limited to: 1:1000, 1.5:1000, 2:1000, 2.5:1000, 3:1000.

[0084] In some embodiments, the mass ratio of the first gel to the buffer is 1:10 to 1:40.

[0085] In some embodiments, the mass ratio of the first gel to water is 1:10 to 1:40.

[0086] In some embodiments, the first cross-linking agent includes at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0087] In some embodiments, the buffer comprises at least one of a sodium phosphate buffer and a potassium phosphate buffer.

[0088] In some embodiments, the first solvent includes water.

[0089] In some embodiments, the method for preparing a solution comprising a second polysaccharide polymer comprises the following steps:

[0090] Mixing a second cross-linking monomer with a second solvent and subjecting the mixture to a second emulsification treatment to obtain a second solution;

[0091] The second solution is mixed with the second cross-linking agent to undergo a cross-linking reaction to obtain a solution containing a second polysaccharide polymer. It is understood that the process conditions of the second emulsification treatment may be the same as or different from the process conditions of the first emulsification treatment. The second cross-linking monomer reacts with the second cross-linking agent to obtain a second polysaccharide polymer.

[0092] In some embodiments, the mass percentage of the second cross-linking monomer in the second solution is 2% to 4%. It is understood that the mass percentage includes, but is not limited to: 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%.

[0093] In some embodiments, the second cross-linking monomer includes a polysaccharide polymer B having a viscosity of 20 mPa·s to 60 mPa·s. It is understood that the above viscosity includes but is not limited to: 20 mPa·s, 30 mPa·s, 40 mPa·s, 50 mPa·s, and 60 mPa·s.

[0094] In some embodiments, polysaccharide polymer A and polysaccharide polymer B each independently include at least one of chitosan, chitosan derivatives, cellulose and cellulose derivatives. Optionally, chitosan derivatives include at least one of carboxymethyl chitosan, alkylated chitosan, quaternized chitosan and acylated chitosan. Optionally, cellulose derivatives include at least one of methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose.

[0095] In some embodiments, the mass ratio of the second cross-linking agent to the second solution is 0.1:1000~0.4:1000, and can be optionally 0.1:1000~0.2:1000. Thus, it is beneficial to further enhance the binding force between the second polysaccharide polymer in the anti-adhesion layer and the repair layer, further reduce scar hyperplasia, and thus further enhance the anti-adhesion effect of the artificial biofilm patch. It is understood that the mass ratio of the second cross-linking agent to the second solution includes but is not limited to: 0.1:1000, 0.15:1000, 0.2:1000, 0.25:1000, 0.3:1000, 0.35:1000, 0.4:1000.

[0096] In some embodiments, the second cross-linking agent includes at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0097] In some embodiments, the second solvent includes water.

[0098] By adjusting the process conditions of the above-mentioned preparation method of the present application, the artificial biomembrane patch in the above-mentioned embodiment of the present application can be obtained.

[0099] Another embodiment of the present application provides the use of the artificial biomembrane patch mentioned above in the present application or the artificial biomembrane patch prepared by the above preparation method in the present application in preparing a repair product of the dura mater or spinal dura mater.

[0100] In order to further illustrate the present application, the technical scheme of the present application is described in detail below in conjunction with specific examples. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in the field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0101] Example 1

[0102] The artificial biofilm patch was prepared as follows:

[0103] (1) Preparation of extracellular matrix (material for repair layer)

[0104] (1.1) Remove excess fat and accessory tissue from the animal membrane tissue, and wash it repeatedly with purified water or physiological saline until the blood is removed; then extract it with an organic reagent at a ratio of 1:6 (w / v), shake and defat at room temperature, 1200 rpm, and then replace the organic reagent and continue to shake and defat for 16 hours; discard the organic reagent, add purified water at a ratio of 1:20 (w / v), shake and wash at room temperature, 1200 rpm, and shake and wash 10 times, 10 minutes / time, to obtain the defatted animal membrane tissue;

[0105] (1.2) Add alkaline solution to the defatted animal membrane tissue at a solid-liquid ratio of 1:10 (w / v), shake at room temperature, 120 rpm, for 60 min, wash, and repeat three times to obtain the extracellular matrix (material for the repair layer).

[0106] (2) Preparation of a solution containing a first polysaccharide polymer and a solution containing a second polysaccharide polymer

[0107] (2.1) Preparation of a solution containing a first polysaccharide polymer

[0108] (2.1.1) Weigh a certain amount of carboxymethyl chitosan (first cross-linking monomer) with a viscosity of 37.2 mPa·s, prepare a solution with a mass concentration of 2% with water for injection (first solvent), stir it with an emulsifier, place it in 2°C~8°C to swell for more than 15 hours, stir it again after the swelling end point, and stir it for about 30 minutes to obtain the first solution;

[0109] (2.1.2) adding a first crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) to the first solution, allowing the solution to stand for crosslinking for more than 6 hours to form a first gel, wherein the mass ratio of the first crosslinking agent to the first solution is 1:1000;

[0110] (2.1.3) preparing PBS buffer, with the mass ratio of the first gel to the PBS buffer being 1:20, placing the first gel in the PBS buffer and allowing it to stand to replace ions using the osmotic pressure principle, and the solution replacement lasts for 7 hours;

[0111] (2.1.4) Repeat step (2.1.3), this time using water for injection as the replacement fluid, with the mass ratio of water for injection to the first gel being 20:1, to obtain a solution containing the first polysaccharide polymer (a sol containing the first polysaccharide polymer).

[0112] (2.2) Preparation of a solution containing a second polysaccharide polymer

[0113] (2.2.1) Weigh a certain amount of carboxymethyl chitosan (second cross-linking monomer) with a viscosity of 37.2 mPa·s, prepare a solution with a mass concentration of 2% with water for injection (second solvent), stir it with an emulsifier, place it in 2°C~8°C to swell for more than 15 hours, stir it again after the swelling end point, and stir it for about 30 minutes to obtain a second solution;

[0114] (2.2.2) Add the second cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) into the second solution, the mass ratio of the second cross-linking agent to the second solution being 0.1:1000, and stir the mixture for 5 minutes using an emulsifier to obtain a solution containing a second polysaccharide polymer.

[0115] (2.2.3) Preparation of membrane-forming solution

[0116] The solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended in a mass ratio of 2:1, and homogenized and stirred using a vacuum emulsifier. The homogenization speed is set to 1500 rpm, the stirring speed is set to 45 rpm, the homogenization time is set to 5 min, and the stirring time is set to 8 min to obtain a film-forming solution.

[0117] (3) Double-layer composite

[0118] (3.1) Lay a low-density polyethylene film flat and tightly against the bottom surface of the mold for use as a paving board, and lay the repair layer material obtained in step (1.2) with the smooth surface facing up on the low-density polyethylene film to completely cover the bottom surface of the mold;

[0119] (3.2) Add the film-forming liquid obtained in step (2.2.3) to the material of the repair layer in step (3.1), and spread the film-forming liquid on the material of the repair layer by gravity casting, and let it stand for more than 5 hours to form a double-layer intermediate; wherein the ratio of the mass of the film-forming liquid to the area of ​​the material of the repair layer contacting the film-forming liquid is 2.0 g:10 cm 2 ;

[0120] (3.3) subjecting the double-layer structure formed in step (3.2) to vacuum freeze-drying;

[0121] (3.4) The dried sample of step (3.3) is pressed and cut to form the required thickness and size requirements, and is packaged and terminally sterilized to obtain a final product, an artificial biofilm patch, which includes a repair layer and an anti-adhesion layer arranged on one side of the repair layer, the anti-adhesion layer is formed by the above-mentioned film-forming liquid, and the anti-adhesion layer has a porous structure, the porosity of the anti-adhesion layer is 77.03%, the average cross-linking degree of the first polysaccharide polymer (carboxymethyl chitosan) in the anti-adhesion layer is 58.14%, the average cross-linking degree of the second polysaccharide polymer (carboxymethyl chitosan) in the anti-adhesion layer is 11.25%, the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 2:1, and the mass of the anti-adhesion layer per unit area of ​​the repair layer is 2.6 mg / cm 2 .

[0122] Example 2

[0123] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first cross-linking agent to the first solution in step (2.1.2) is 2:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 71.24%, and the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer is 64.25%.

[0124] Example 3

[0125] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first cross-linking agent to the first solution in step (2.1.2) is 3:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 64.07%, and the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer is 81.95%.

[0126] Example 4

[0127] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second cross-linking agent to the second solution in step (2.2.2) is 0.3:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 73.25%, and the average cross-linking degree of the second polysaccharide polymer in the anti-adhesion layer is 17.65%.

[0128] Example 5

[0129] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second cross-linking agent to the second solution in step (2.2.2) is 0.4:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 70.35%, and the average cross-linking degree of the second polysaccharide polymer in the anti-adhesion layer is 23.11%.

[0130] Example 6

[0131] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the membrane-forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended in a mass ratio of 1:1, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 82.35%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1.

[0132] Example 7

[0133] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the membrane-forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended in a mass ratio of 4:1, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 68.35%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 4:1.

[0134] Example 8

[0135] The preparation method of the artificial biofilm patch is basically the same as that of Example 1, except that in step (3.2), the ratio of the mass of the membrane-forming liquid to the area of ​​the material of the repair layer contacting the membrane-forming liquid is 2.2 g:10 cm 2 In the artificial biomembrane patch obtained in step (3.4), the porosity of the anti-adhesion layer is 78.35%, and the mass of the anti-adhesion layer per unit area of ​​the repair layer is 2.8 mg / cm 2 .

[0136] Example 9

[0137] The preparation method of the artificial biofilm patch is basically the same as that of Example 1, except that in step (3.2), the ratio of the mass of the membrane-forming liquid to the area of ​​the material of the repair layer contacting the membrane-forming liquid is 2.6 g:10 cm 2 In the artificial biomembrane patch obtained in step (3.4), the porosity of the anti-adhesion layer is 79.51%, and the mass of the anti-adhesion layer per unit area of ​​the repair layer is 3.1 mg / cm 2 .

[0138] Example 10

[0139] The preparation method of the artificial biofilm patch is basically the same as that of Example 1, except that in step (3.2), the ratio of the mass of the film-forming liquid to the area of ​​the material of the repair layer contacting the film-forming liquid is 3 g:10 cm 2 In the artificial biomembrane patch obtained in step (3.4), the porosity of the anti-adhesion layer is 80.32%, and the mass of the anti-adhesion layer per unit area of ​​the repair layer is 3.3 mg / cm 2 .

[0140] Embodiment 11

[0141] The preparation method of the artificial biofilm patch is basically the same as that of Example 1, except that in step (3.2), the ratio of the mass of the membrane-forming liquid to the area of ​​the material of the repair layer contacting the membrane-forming liquid is 3.2 g:10 cm 2 In the artificial biomembrane patch obtained in step (3.4), the porosity of the anti-adhesion layer is 83.22%, and the mass of the anti-adhesion layer per unit area of ​​the repair layer is 3.6 mg / cm 2 .

[0142] Comparative Example 1

[0143] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first cross-linking agent to the first solution in step (2.1.2) is 0.5:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 89.35%, and the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer is 35.15%.

[0144] Comparative Example 2

[0145] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the first polysaccharide polymer, the mass ratio of the first cross-linking agent to the first solution in step (2.1.2) is 4:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 61.28%, and the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer is 92.25%.

[0146] Comparative Example 3

[0147] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second cross-linking agent to the second solution in step (2.2.2) is 0.05:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 83.26%, and the average cross-linking degree of the second polysaccharide polymer in the anti-adhesion layer is 4.23%.

[0148] Comparative Example 4

[0149] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the solution containing the second polysaccharide polymer, the mass ratio of the second cross-linking agent to the second solution in step (2.2.2) is 0.5:1000, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 73.41%, and the average cross-linking degree of the second polysaccharide polymer in the anti-adhesion layer is 32.15%.

[0150] Comparative Example 5

[0151] The preparation method of the artificial biomembrane patch is basically the same as that of Example 1, except that: in the preparation of the membrane-forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended in a mass ratio of 0.5:1, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 93.21%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 0.5:1.

[0152] Comparative Example 6

[0153] The preparation method of the artificial biomembrane patch is basically the same as that in Example 1, except that: in the preparation of the membrane-forming solution in step (2.2.3), the solution containing the first polysaccharide polymer and the solution containing the second polysaccharide polymer are blended in a mass ratio of 5:1, the porosity of the anti-adhesion layer of the artificial biomembrane patch obtained in step (3.4) is 58.14%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 5:1.

[0154] Test Method

[0155] (1) Test on average cross-linking degree of the first polysaccharide polymer and the second polysaccharide polymer

[0156] (1.1) Reagent preparation:

[0157] Prepare an acetate buffer solution with a pH of 5.4 and a concentration of 2 mol / L: Measure 86 mL of 2 mol / L sodium acetate solution and add 14 mL of 2 mol / L acetic acid to mix. Check the corrected pH value with a pH meter.

[0158] Prepare ninhydrin colorimetric solution: weigh 85 mg of ninhydrin and 15 mg of reduced ninhydrin, and dissolve them in 10 mL of ethylene glycol methyl ether.

[0159] Preparation of reduced ninhydrin: Weigh 5g of ninhydrin and dissolve it in 125mL of boiling purified water to obtain a yellow solution. Dissolve 5g of vitamin C in 250mL of warm purified water, and add the vitamin C solution dropwise to the ninhydrin solution while stirring. Precipitation will continue to appear. Continue stirring for 15 minutes after the addition, then cool to 4°C in a refrigerator, filter the precipitate, collect the precipitate, wash it with cold water 3 times, dry it in a desiccator, and store it for later use.

[0160] Prepare 60% ethanol: Pipette 60 mL of anhydrous ethanol and add purified water to make up to 100 mL.

[0161] (1.2) Preparation of standard curve: Take a 2.5 mmol / L amino acid standard solution as the stock solution and prepare the standard curve according to Table 1 below.

[0162] Table 1

[0163]

[0164] (1.3) Sample solution preparation: Accurately weigh 300 mg of the sample before cross-linking and the sample after cross-linking into a 50 mL test tube, add water to dissolve and make up to volume.

[0165] (1.4) Color development: Pipette 1 mL of each standard solution and each sample solution into a test tube, add 1 mL of pH 5.4, 2 mol / L acetate buffer and 1 mL of ninhydrin colorimetric solution, mix well, heat in a 100℃ boiling water bath for 15 min, cool with tap water. After standing for 5 min, add 3 mL of 60% ethanol for dilution.

[0166] (1.5) Determination: UV spectrophotometer was used for determination at a wavelength of 570 nm. 570nm The value is the ordinate, and the concentration of the amino acid standard is the abscissa. The standard curve is drawn to calculate the amino acid concentration in the sample. The average cross-linking degree is calculated by the change in amino acid content before and after cross-linking of the carboxymethyl chitosan solution.

[0167]

[0168] Where: C1—amino acid concentration of sample before cross-linking (mmol / L);

[0169] C2—amino acid concentration of sample after cross-linking (mmol / L);

[0170] m1—sample weight before cross-linking (mg);

[0171] m2—weight of sample after cross-linking (mg).

[0172] When testing the average crosslinking degree of the first polysaccharide polymer, the sample before crosslinking refers to a solution containing the first crosslinking monomer before crosslinking, such as the first solution, and the sample after crosslinking refers to a solution containing the first polysaccharide polymer after crosslinking.

[0173] When testing the average crosslinking degree of the second polysaccharide polymer, the sample before crosslinking refers to a solution containing the second crosslinking monomer before crosslinking, such as the second solution, and the sample after crosslinking refers to a solution containing the second polysaccharide polymer after crosslinking.

[0174] (2) Porosity test of anti-adhesion layer

[0175] The porosity of the anti-adhesion layer was tested with reference to GB / T 33052-2016 “Determination of porosity of microporous functional films - Hexadecane absorption method”.

[0176] Take an artificial biofilm patch sample, about 12 cm in area 2 , measure the length a, width b and thickness c, accurately weigh the sample mass and record it as m1, immerse the sample in a container filled with anhydrous ethanol, take out the sample after 30 minutes, wipe off the anhydrous ethanol on the surface, quickly weigh the mass of the sample that is full of anhydrous ethanol, record it as m2, and the porosity P of the anti-adhesion layer is calculated as follows:

[0177]

[0178] Where: m1 is the dry weight of the sample, in g;

[0179] m2 is the mass of the sample after it is fully absorbed by anhydrous ethanol, in g;

[0180] a is the length of the sample, in mm;

[0181] b is the width of the sample, in mm;

[0182] c is the thickness of the sample, in mm;

[0183] ρ is the density of anhydrous ethanol, which is 0.79 g / cm 3 .

[0184] Test Example 1 Determination of interlayer bonding strength

[0185] Please refer to Figure 1, the artificial biofilm patch samples prepared in Examples 1-11 and Comparative Examples 1-6 were cut into strip samples with a width of 15.0±0.1mm and a length of 30mm, 3M double-sided tape was tightly adhered to the surface of the repair layer of the sample, one end of the sample was pre-peeled 10mm, and the peeled two sides were clamped on the upper clamp 1 and the lower clamp 2 of the test machine, respectively, so that the longitudinal axis of the peeled part 3 of the sample coincided with the center line of the upper clamp 1 and the lower clamp 2, and the tightness was appropriate. During the test, the unpeeled part 4 of the sample was T-shaped with the stretching direction, and a test speed of 300±50mm / min was selected. The peeling force during the peeling process of the sample was recorded, and the interlayer bonding strength was calculated according to the following formula: interlayer bonding strength (kN / m) = maximum peeling force (N) / sample width (mm), and the test results are shown in Table 2.

[0186] Table 2

[0187]

[0188] It can be seen from Table 2 that compared with comparative examples 1-5, the interlayer bonding strength between the repair layer and the anti-adhesion layer of the artificial biomembrane patch prepared in Examples 1-11 is higher, indicating that Examples 1-11 control the average cross-linking degree of the first polysaccharide polymer in the anti-adhesion layer, the average cross-linking degree of the second polysaccharide polymer, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer within a reasonable range, thereby improving the bonding force between the first polysaccharide polymer and the second polysaccharide polymer in the anti-adhesion layer and the repair layer. The improvement of the bonding force can reduce scar hyperplasia, thereby improving the anti-adhesion effect of the artificial biomembrane patch.

[0189] Test Example 2 Cytotoxicity Test

[0190] The artificial biomembrane patch samples prepared in Example 1, Example 3, Example 5, Example 7, Comparative Example 2, Comparative Example 4, and Comparative Example 6 were subjected to cytotoxicity test.

[0191] Cell preparation: Rat fibroblasts were revived in MEM medium containing 10% FBS (fetal bovine serum) and cultured at 37°C, 5% CO2, and saturated humidity. The cells were passaged 2-3 times until the cells reached the logarithmic growth phase. The cells were digested with trypsin, collected, and the cell concentration was adjusted to 1×10 5 The following experiments were performed with 10 cells / mL.

[0192] Sample preparation: Take the sample for processing, according to the extraction ratio: 6cm 2 / mL, extraction medium: 0.5mL of MEM culture medium, extraction conditions: 37°C, 125rpm shaking extraction for 72h.

[0193] Preparation of negative samples: Place MEM medium containing 10% FBS into a 50 mL sterile centrifuge tube and shake and extract at 37°C for 72 hours.

[0194] Positive sample preparation: Prepare MEM complete medium containing 10% DMSO (dimethyl sulfoxide).

[0195] Blank sample preparation: MEM medium containing 10% FBS.

[0196] Experimental method: Add the cell suspension to a 96-well plate, 100 μL per well, for a total of 1×10 4 cells / well, cultured at 37℃, 5% CO2 for 24h. After the culture, discard the culture medium in the plate, add sample extract (four dose groups of 100%, 50%, 25% and 12.5%), negative control, blank control and positive control, 6 wells in each group, and place in a 37℃, 5% CO2 saturated humidity incubator. After 24 hours of culture, take photos and record under the microscope, discard the culture medium in the plate, add 50μL (1mg / mL) of MTT (thiazolyl blue) dye to each well, and culture in a 37℃, 5% CO2 saturated humidity incubator for 2h. After the culture, discard the liquid in the culture plate, add 100μL of isopropanol to each well, shake and mix in the dark for 30min, and place in the microplate reader for absorbance detection with 570nm as the detection wavelength and 650nm as the reference wavelength.

[0197] Analysis of experimental results: The relative proliferation rate of cells was calculated according to the following formula based on the mean absorbance of each group.

[0198] Relative value increase rate (%) = 100*ODe / ODb

[0199] Where: ODe—average optical density of 100% extract of test sample;

[0200] ODb—average blank optical density;

[0201] The raw experimental data were processed and the toxicity of the materials was evaluated according to Tables 3 and 4.

[0202] Table 3 Cytotoxicity grading criteria

[0203]

[0204] Table 4 Cytotoxicity grading criteria

[0205]

[0206] Table 5 Relative growth rate and toxicity level

[0207]

[0208] From Table 3~Table 5 and Figure 2 It can be seen that Comparative Examples 2, 4, and 6 exhibited obvious cytotoxic reactions, which was caused by the high average crosslinking degree of the first polysaccharide polymer, the average crosslinking degree of the second polysaccharide polymer, or the mass proportion of the first polysaccharide polymer in the anti-adhesion layer in the artificial biomembrane patches prepared in Comparative Examples 2, 4, and 6. Examples 1, 3, 5, and 7 did not exhibit cytotoxic reactions; and Examples 2, 4, 6, and 8-11 did not exhibit cytotoxic reactions, indicating that the artificial biomembrane patches prepared in Examples 1-11 have biological safety.

[0209] Test Example 3 Leakage Test

[0210] The artificial biomembrane patch samples prepared in Example 1, Example 3, Example 5, Example 7, Example 8, Example 9, Example 10, Comparative Example 2, Comparative Example 4, Comparative Example 6, and Comparative Example 8 were subjected to anti-leakage test.

[0211] Place the sample in the attached drawing of the specification of Chinese patent CN219552202U Figure 1 The sample was fixed in the anti-leakage detection device shown in the figure. For easy observation, the saline in the cylindrical funnel was dyed with methylene blue solution. By pressing the inflatable part, a pressure of 40 mmHg was applied to the liquid storage bag to press the sample and the corresponding pressure value was recorded by the pressure gauge. The test results are shown in Figure 3 shown.

[0212] Figure 3 It is shown that under a pressure of 40 mmHg, Comparative Examples 2, 4 and 6 all exhibit different degrees of methylene blue solution leakage, while the artificial biomembrane patches prepared in Examples 1, 3, 5, 7, 8, 9, 10 and 11 are able to prevent leakage.

[0213] Test Example 4 Fibroblast proliferation test

[0214] The artificial biomembrane patch samples prepared in Example 1 and samples of similar products on the market (pure extracellular matrix material) were taken for fibroblast proliferation test, wherein the similar products on the market only have a repair layer but no anti-adhesion layer.

[0215] Cell culture: The cells were inoculated in a culture medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% CO2. Cells in the logarithmic growth phase were digested and passaged with 0.25% trypsin. Cells from the 3rd to 5th generations were taken for further testing.

[0216] Cell proliferation: The trimmed samples were placed in a 24-well plate, and well-growing P3 cells were taken and 10 5Cells were inoculated on the sample surface (3cm*4cm), and three parallel samples were set up. After incubation for 1d, 2d, 3d, 5d, and 7d, 20µL of MTT solution (5mg / mL) was added to each well, and the well was placed in an incubator for incubation for 3h~4h. After taking out, the liquid in the well plate was gently aspirated with a pipette, and 150µL of DMSO was added to each well. The absorbance was measured at 570nm using an enzyme reader within 15 minutes. The test results are as follows: Figure 4 shown.

[0217] Depend on Figure 4 It can be seen that the artificial biomembrane patch prepared in Example 1 inhibits the proliferation of fibroblasts after implantation, can act as a mechanical barrier, and reduce the effect of surgical adhesion, which further illustrates that the artificial biomembrane patch prepared in the present application has a relatively excellent anti-adhesion effect.

[0218] Test Example 5 Microstructure Analysis

[0219] Micro-CT was used to analyze the three-dimensional pore structure of the artificial biofilm patch sample prepared in Example 1. The sample was placed in a detection instrument, and the detection parameters were set as follows: voltage 60 KV, current 80 μA, integration time 700 s, number of projections 1440, resolution 2 μm, and the entire area of ​​the sample was selected and the porosity was analyzed.

[0220] Depend on Figure 5 It can be seen that the three-dimensional structure of the artificial biomembrane patch prepared in Example 1 presents a clear layered structure. The upper layer is the anti-adhesion layer, which is loose and porous, and the lower layer is the repair layer containing the extracellular matrix, which is arranged relatively densely. The transition layer between the upper and lower layers is formed by electrostatic interaction and secondary bonds between the first polysaccharide polymer and / or the second polysaccharide polymer in the anti-adhesion layer and the extracellular matrix in the repair layer.

[0221] 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.

[0222] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. An artificial biomembrane patch, characterized in that: The invention comprises a repair layer and an anti-adhesion layer arranged on one side of the repair layer, wherein the anti-adhesion layer has a porous structure, and the anti-adhesion layer contains a first polysaccharide polymer with an average cross-linking degree of 50% to 90% and a second polysaccharide polymer with an average cross-linking degree of 10% to 30%, and the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 1:1 to 4:1; The material of the repair layer is extracellular matrix.

2. The artificial biomembrane patch according to claim 1, characterized in that: The mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the anti-adhesion layer is 2:1 to 4:

1.

3. The artificial biomembrane patch according to claim 2, characterized in that: The first polysaccharide polymer and the second polysaccharide polymer each independently comprise a biodegradable polysaccharide polymer.

4. The artificial biomembrane patch according to claim 3, characterized in that: The biodegradable polysaccharide polymer includes at least one of chitosan, chitosan derivatives, cellulose and cellulose derivatives.

5. The artificial biomembrane patch according to any one of claims 1 to 4, characterized in that: The artificial biofilm patch has at least one of the following characteristics (1) to (2): (1) The average cross-linking degree of the first polysaccharide polymer is 60% to 90%; (2) The average cross-linking degree of the second polysaccharide polymer is 15% to 30%.

6. The artificial biomembrane patch according to any one of claims 1 to 4, characterized in that: At least a portion of the first polysaccharide polymer and / or at least a portion of the second polysaccharide polymer in the anti-adhesion layer is connected to the repair layer through at least one of electrostatic action and secondary bond.

7. The artificial biomembrane patch according to any one of claims 1 to 4, characterized in that: The porosity of the anti-adhesion layer is 50% to 90%.

8. The artificial biomembrane patch according to any one of claims 1 to 4, characterized in that: The mass of the anti-adhesion layer per unit area on the repair layer is 1 mg / cm 2 ~6mg / cm 2 .

9. The artificial biomembrane patch according to any one of claims 1 to 4, characterized in that: The mass of the anti-adhesion layer per unit area on the repair layer is 2 mg / cm 2 ~4mg / cm 2 .

10. A method for preparing an artificial biomembrane patch, characterized in that: The steps include: A solution containing a first polysaccharide polymer and a solution containing a second polysaccharide polymer are mixed and homogenized to obtain a film-forming solution; the mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the film-forming solution is 1:1 to 4:1, the average cross-linking degree of the first polysaccharide polymer is 50% to 90%, and the average cross-linking degree of the second polysaccharide polymer is 10% to 30%; The film-forming liquid is disposed on one side of the material of the repair layer to obtain an intermediate; removing the solvent in the intermediate to form a repair layer and an anti-adhesion layer with a porous structure to obtain the artificial biomembrane patch; Wherein, the material of the repair layer is extracellular matrix.

11. The preparation method according to claim 10, characterized in that: The method for preparing the solution containing the first polysaccharide polymer comprises the following steps: Mixing a first cross-linking monomer with a first solvent and performing a first emulsification treatment to obtain a first solution; Mixing the first solution with a first cross-linking agent to perform a cross-linking reaction to obtain a first gel; The first gel is subjected to ion replacement in a buffer solution and in water in sequence to obtain the solution containing the first polysaccharide polymer.

12. The preparation method according to claim 11, characterized in that: The mass ratio of the first polysaccharide polymer to the second polysaccharide polymer in the membrane-forming solution is 2:1 to 4:

1.

13. The preparation method according to claim 11, characterized in that: The preparation method satisfies at least one of the following conditions (1) to (8): (1) The mass percentage of the first cross-linking monomer in the first solution is 2% to 4%; (2) the first cross-linking monomer includes a polysaccharide polymer A having a viscosity of 20 mPa·s to 60 mPa·s; (3) The mass ratio of the first cross-linking agent to the first solution is 1:1000 to 3:1000; (4) The mass ratio of the first gel to the buffer is 1:10 to 1:40; (5) The mass ratio of the first gel to water is 1:10 to 1:40; (6) the first cross-linking agent includes at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (7) The buffer comprises at least one of a sodium phosphate buffer and a potassium phosphate buffer; (8) The first solvent includes water.

14. The preparation method according to claim 13, characterized in that: The mass ratio of the first cross-linking agent to the first solution is 1:1000 to 2:1000.

15. The preparation method according to any one of claims 10 to 14, characterized in that: The method for preparing the solution containing the second polysaccharide polymer comprises the following steps: Mixing a second cross-linking monomer with a second solvent and subjecting the mixture to a second emulsification treatment to obtain a second solution; The second solution is mixed with a second cross-linking agent to perform a cross-linking reaction to obtain the solution containing the second polysaccharide polymer.

16. The preparation method according to claim 15, characterized in that: The preparation method satisfies at least one of the following conditions (1) to (5): (1) The mass percentage of the second cross-linking monomer in the second solution is 2% to 4%; (2) the second cross-linking monomer includes a polysaccharide polymer B having a viscosity of 20 mPa·s to 60 mPa·s; (3) The mass ratio of the second cross-linking agent to the second solution is 0.1:1000 to 0.4:1000; (4) the second cross-linking agent comprises at least one of 1,4-butanediol diglycidyl ether and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (5) The second solvent includes water.

17. The preparation method according to claim 16, characterized in that: The mass ratio of the second cross-linking agent to the second solution is 0.1:1000 to 0.2:1000.

18. Use of the artificial biomembrane patch according to any one of claims 1 to 9 or the artificial biomembrane patch prepared by the preparation method according to any one of claims 10 to 17 in preparing a repair product of dura mater or spinal dura mater.

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