Multilayer artificial dura mater and preparation method thereof

Abstract

The invention relates to a multilayer artificial dura mater and a preparation method thereof. The multi-layer artificial dura mater comprises a modified allogeneic dura mater and a gel layer wrapping the allogeneic dura mater, the gel layer comprises a modified gel layer, a modified gel medium layer and a modified gel layer which are sequentially coated on the modified allogeneic dura mater; the modified gel layer is prepared from a modified gel solution; the modified gel solution is prepared from the following raw material components in parts by mass: 10 to 25 parts of polycaprolactone, 5 to 10 parts of carboxymethyl chitosan, 15 to 25 parts of recombinant humanized collagen, 20 to 40 parts of a modified gel medium, 1 to 3 parts of an antibacterial agent and 0.05 to 0.2 part of a photoinitiator. The medicine is more beneficial to treatment of large-area defects, leakage and adhesion are effectively prevented, and repair of the dura mater is promoted.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and in particular to a multi-layer artificial dura mater and a preparation method thereof. Background Art

[0002] The dura mater is a dense, double-layered membrane that wraps around the brain and spinal cord, nestled against the inside of the skull. Once incised or damaged due to trauma, tumors, or surgery, serious complications such as cerebrospinal fluid leakage, intracranial infection, and encephalocele can occur. Artificial dura mater is a key material for repairing dura mater defects in neurosurgery, and must meet four core requirements: preventing cerebrospinal fluid leakage, resisting adhesions, resisting infection, and promoting tissue regeneration.

[0003] The domestic market continues to grow. Traditional single-layer materials cannot simultaneously meet the requirements of preventing brain tissue adhesion on the inner surface and promoting fibroblast incorporation on the outer surface, resulting in unsatisfactory results in repairing dura mater defects. Summary of the Invention

[0004] To address the shortcomings of the existing technology, the present application aims to provide a multi-layer artificial dura mater and a preparation method. Specifically, the multi-layer artificial dura mater provided uses allogeneic dura mater as raw material and is coated with a gel layer. This effectively removes the immunogenicity of the allogeneic dura mater, reduces rejection, and helps reduce inflammatory reactions, making it more suitable for repairing patients with large defects.

[0005] The technical solutions include the following:

[0006] A multilayer artificial dura mater comprises a modified allogeneic dura mater and a gel layer wrapping the allogeneic dura mater; the gel layer comprises a modified gel layer, a modified gel medium layer and a modified gel layer sequentially wrapped on the modified allogeneic dura mater.

[0007] In one embodiment, the modified gel layer is prepared using a modified gel solution; the raw materials of the modified gel solution include the following components, by mass: 10-25 parts of polycaprolactone, 5-10 parts of carboxymethyl chitosan, 15-25 parts of recombinant humanized collagen and 20-40 parts of modified gel medium, 1-3 parts of antibacterial agent and 0.05-0.2 parts of photoinitiator.

[0008] After the modified gel solution components of the present application are combined, the elastic modulus, hardness and repair effect of the multi-layer artificial dura mater of the present application are better than those of other component combinations (other component combinations, for example, replace carboxymethyl chitosan with ordinary chitosan, or replace any one component).

[0009] In one embodiment, the raw materials of the modified gel solution include the following components, calculated by mass: 15-25 parts of polycaprolactone, 7-10 parts of carboxymethyl chitosan, 20-25 parts of recombinant humanized collagen and 25-40 parts of modified gel medium, 2-3 parts of antibacterial agent and 0.1-0.2 parts of photoinitiator.

[0010] In one embodiment, in the modified gel solution, the mass fraction of polycaprolactone is 10 parts, 15 parts, 20 parts, 25 parts, or a range between any two of the above values.

[0011] In one embodiment, in the modified gel solution, the mass fraction of carboxymethyl chitosan is 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or a range between any two of the above values.

[0012] In one embodiment, in the modified gel solution, the mass fraction of the recombinant humanized collagen is 15 parts, 18 parts, 20 parts, 22 parts, 25 parts, or a range between any two of the above values.

[0013] In one embodiment, in the modified gel solution, the weight fraction of the modified gel medium is 20 parts, 25 parts, 30 parts, 35 parts, 40 parts, or a range between any two of the above values.

[0014] In one embodiment, in the modified gel solution, the mass fraction of the antibacterial agent is 1 part, 2 parts, 3 parts, or a range between any two of the above values.

[0015] In one embodiment, in the modified gel solution, the mass fraction of the photoinitiator is 0.05 parts, 0.1 parts, 0.15 parts, 0.2 parts, or a range between any two of the above values.

[0016] In one embodiment, the raw materials of the modified gel solution further include 250-300 parts of water.

[0017] In one embodiment, the polycaprolactone is a low molecular weight polycaprolactone, for example, a molecular weight of 1000-3000 Da. The modified gel solution composition can promote the use of low molecular weight polycaprolactone to form a dense barrier layer, significantly improve mechanical strength, and block cerebrospinal fluid leakage.

[0018] In one embodiment, the antimicrobial agent comprises chitosan quaternary ammonium salt.

[0019] In one embodiment, the photoinitiator includes ethyl 2,4,6-trimethylbenzoylphenylphosphonate.

[0020] In one embodiment, the raw material for preparing the modified gel medium layer consists of modified gel medium.

[0021] In one embodiment, the raw materials for preparing the modified gel medium include the following components, calculated by mass: 80-100 parts of gel medium, 13-15 parts of sodium alginate, 8-10 parts of hydroxyapatite nanoparticles and 20-25 parts of polyethylene glycol diacrylate.

[0022] The combination of components of the modified gel medium in the present application makes the elastic modulus and hardness of the multi-layer artificial dura mater of the present application, as well as the repair effect better than other forms of component combinations (other forms of component combinations, such as replacing hydroxyapatite nanoparticles with ordinary calcium phosphate powder, or replacing any one component).

[0023] In one embodiment, the raw materials for preparing the modified gel medium include the following components, calculated by mass: 85-95 parts of gel medium, 14-15 parts of sodium alginate, 8-9 parts of hydroxyapatite nanoparticles and 22-24 parts of polyethylene glycol diacrylate.

[0024] In one embodiment, in the modified gel medium, the mass fraction of the gel medium is 80 parts, 85 parts, 90 parts, 95 parts, 100 parts, or a range between any two of the above values.

[0025] In one embodiment, in the modified gel medium, the mass fraction of sodium alginate is 13 parts, 14 parts, 15 parts, or a range between any two of the above values.

[0026] In one embodiment, in the modified gel medium, the mass fraction of hydroxyapatite nanoparticles is 8 parts, 9 parts, 10 parts, or a range between any two of the above values.

[0027] In one embodiment, in the modified gel medium, the mass fraction of polyethylene glycol diacrylate is 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, or a range between any two of the above values.

[0028] In one embodiment, the gel medium is selected from one of tragacanth gum, gelatin, starch, cellulose derivatives or carbopol.

[0029] In one embodiment, a modified allogeneic dura mater is prepared using allogeneic dura mater and a modified chelating solution. The modified allogeneic dura mater serves as the core scaffold of the multilayer structure, providing a biomimetic structure to guide tissue regeneration and acting as a "sandwich layer" carrier to achieve compartmentalized functionalization (internal adhesion prevention and external repair promotion).

[0030] In one embodiment, the allogeneic dura mater is selected from the full-thickness dura mater of the bilateral temporoparietal regions with a thickness of 0.3-0.5 mm.

[0031] In one embodiment, the modified chelating solution comprises 0.1-0.3% by mass of edetate disodium, polyethylene glycol octylphenyl ether, and polycaprolactone in a mass ratio of (3-5):(2-3):1. The modified chelating solution is used to pretreat allogeneic dura mater, removing immunogenicity and enhancing biocompatibility through chemical modification, thereby addressing the core obstacle of allogeneic transplantation (rejection).

[0032] A method for preparing the multilayer artificial dura mater comprises the following steps:

[0033] S1. Soaking the modified allogeneic dura mater in the modified gel solution at 2-10°C for 10-12 hours, and then soaking it in the modified gel medium at 2-10°C for 10-12 hours to obtain a double-layer modified allogeneic dura mater;

[0034] S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again, place at 2-10°C for 10-12 hours, and trim the excess gel to a thickness of 2-5 mm;

[0035] S3. The dura mater trimmed in step S2 is subjected to vacuum freeze drying and sterilization to prepare the multi-layer artificial dura mater.

[0036] In one embodiment, the sterilization is performed by cobalt-60 irradiation.

[0037] In one embodiment, the preparation of the modified gel solution comprises the following steps:

[0038] S21. Dissolve 10-25 parts by mass of polycaprolactone, 5-10 parts of carboxymethyl chitosan, 15-25 parts of recombinant humanized collagen, and 20-40 parts of modified gel medium in 250-300 parts of hot water at 80-100°C, and stir at a constant temperature to obtain a preliminary mixed solution. Optionally, the constant temperature stirring time is 25-30 minutes.

[0039] S22. Add 1-3 parts of an antimicrobial agent (e.g., chitosan quaternary ammonium salt) to the preliminary mixed solution and disperse the solution by ultrasonication to obtain a secondary mixed solution. Optionally, the ultrasonic frequency is 40 kHz to 50 kHz; and the ultrasonic dispersion time is 10-15 minutes.

[0040] S23. Cool the mixed solution from the second step to 40-45°C, add 0.05-0.2 parts of a photoinitiator (e.g., ethyl 2,4,6-trimethylbenzoylphenylphosphonate), and stir in the dark. After cooling to room temperature, a modified gel solution is obtained. Optionally, the stirring time in the dark is 20-30 minutes; optionally, the stirring speed in the dark is 300-400 rpm.

[0041] In one embodiment, the rotation speed of the light-shielding stirring in step S23 is 300-400 r / min.

[0042] In one embodiment, the preparation of the modified gel medium comprises the following steps:

[0043] S211. According to parts by mass, 80-100 parts of the gel medium and 13-15 parts of sodium alginate were added to 250-300 parts of deionized water and stirred at a speed of 350-400r / min in a water bath at a constant temperature of 55 ℃ -60 ℃ for 20-30min to obtain a transparent viscous liquid;

[0044] S212. At 20-40° C., 8-10 parts of hydroxyapatite nanoparticles and 20-25 parts of polyethylene glycol diacrylate were added to a transparent viscous solution and ultrasonically dispersed at a frequency of 40 kHz to 50 kHz for 15-20 minutes to obtain a preliminary modified solution;

[0045] S213. Irradiate the preliminary modified solution with 365 nm ultraviolet light for 5-10 minutes to obtain a modified gel medium.

[0046] In one embodiment, the intensity of the ultraviolet light in step S213 is 10 mW / cm 2 .

[0047] In one embodiment, the preparation of the modified allogeneic dura mater comprises the following steps:

[0048] S11. Immersing the allogeneic dura mater in a modified chelating solution at 10-37° C. for 2-4 hours, controlling the pH to 6-10, to obtain a preliminarily modified allogeneic dura mater;

[0049] S12. Freeze-dry the preliminarily modified allogeneic dura mater at -35 to -20°C for 10 to 12 hours to obtain modified allogeneic dura mater.

[0050] Compared with traditional technologies, this application has the following beneficial effects:

[0051] 1. This application uses allogeneic dura mater as raw material. The gel layer obtained by modified gel solution and modified gel medium can help reduce inflammatory response, effectively prevent leakage and adhesion, and is particularly suitable for patients repairing large-area defects. It is simple to operate, low-cost, and less painful for patients.

[0052] 2. This application combines polycaprolactone, carboxymethyl chitosan, recombinant humanized collagen, and a modified gel medium. The polycaprolactone forms a dense barrier layer, significantly improving mechanical strength and blocking cerebrospinal fluid leakage. The recombinant humanized collagen directly participates in extracellular matrix reconstruction, accelerating endothelialization, increasing repair speed, and reducing the risk of immune rejection. Furthermore, the carboxymethyl chitosan effectively enhances anti-adhesion and antibacterial properties, promoting cell adhesion. The modified gel medium can make the elastic modulus and hardness of the multilayer artificial dura mater closer to those of normal dura mater, ensuring a complete and smooth repair area.

[0053] 3. This application combines four components, namely polycaprolactone, carboxymethyl chitosan, recombinant humanized collagen and modified gel medium, with an allogeneic dura mater scaffold, and then utilizes the structural characteristics of the modified gel layer, modified gel medium layer and modified gel layer. It is found that it has a significant synergistic effect, is more conducive to the treatment of large-area defects, effectively prevents leakage and adhesion, and promotes the repair of the dura mater. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0055] Figure 1 This is a process flow chart for preparing the multi-layer artificial dura mater of this application;

[0056] Figure 2 This is a process flow chart for preparing the modified gel solution of this application;

[0057] Figure 3 This is a flow chart of the preparation process of the modified gel medium of this application. DETAILED DESCRIPTION

[0058] To make the above-mentioned objects, features, and advantages of the present application more clearly understood, the specific embodiments of the present application are described in detail below. The following description sets forth many specific details to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without violating the scope of the present application. Therefore, the present application is not limited to the specific embodiments disclosed below.

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0060] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0061] In this application, exemplary descriptions such as "in one of the implementation methods (or examples)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0062] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0063] In this document, when referring to a range, if the unit is followed only by the right endpoint, it means that the units of the left and right endpoints are the same. For example, 0.3-0.5M means that the units of the left endpoint "0.3" and the right endpoint "0.5" are both M (Mol / L).

[0064] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0065] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0066] The following are the requirements, models, brands or available sources of some of the raw materials / materials used in the examples, as shown in Table 1:

[0067] Table 1

[0068]

[0069]

[0070] Example 1

[0071] A multi-layer artificial dura mater:

[0072] Before preparing the multi-layer artificial dura mater, the modified gel medium, modified gel solution and modified allogeneic dura mater are prepared:

[0073] See Figure 3 , the preparation of the modified gel medium comprises the following steps:

[0074] S211. 80 g of tragacanth gum and 13 g of sodium alginate were added to 250 mL of deionized water and stirred at 350 r / min in a water bath at 60 ° C for 20 min to obtain a transparent viscous solution;

[0075] S212. At 20°C, 8 g of hydroxyapatite nanoparticles and 20 g of polyethylene glycol diacrylate were added to a transparent viscous solution and ultrasonically dispersed at a frequency of 40 kHz for 15 min to obtain a preliminary modified solution.

[0076] S213. The preliminary modified solution was irradiated with 365 nm ultraviolet light (light intensity: 10 mW / cm 2 ) irradiated for 5 min to obtain a modified gel medium;

[0077] See Figure 2 , the preparation of the modified gel solution comprises the following steps:

[0078] S21. Dissolve 10 g of polycaprolactone, 5 g of carboxymethyl chitosan, 15 g of recombinant humanized collagen, and 20 g of the modified gel medium in 250 mL of 80°C hot water and stir at constant temperature for 25 min to obtain a preliminary mixture;

[0079] S22. 1 g of chitosan quaternary ammonium salt was added to the preliminary mixture and ultrasonically dispersed at a frequency of 40 kHz for 10 min to obtain a secondary mixed solution;

[0080] S23. The mixture was cooled to 40 ° C in the second step, 0.05 g of ethyl 2,4,6-trimethylbenzoylphenylphosphonate was added, and the mixture was stirred at 300 r / min for 20 min in the dark, and then cooled to room temperature to obtain a modified gel solution;

[0081] The preparation of modified allogeneic dura mater includes the following steps:

[0082] S11. Immerse the allogeneic dura mater in a modified chelating solution (composed of 0.1% disodium edetate, polyethylene glycol octylphenyl ether, and polycaprolactone in a mass ratio of 3:2:1) at 10°C for 2 h, controlling the pH to 6, to obtain a preliminarily modified allogeneic dura mater.

[0083] S12. Freeze-dry the preliminarily modified allogeneic dura mater at -35°C for 10 h to obtain modified allogeneic dura mater.

[0084] See Figure 1 The steps for preparing the multi-layer artificial dura mater include:

[0085] S1. Soak the modified allogeneic dura mater in a modified gel solution at 2°C for 10 hours, then soak it in a modified gel medium at 2°C for 10 hours to obtain a double-layer modified allogeneic dura mater.

[0086] S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again and incubate at 2°C for 10 hours. Trim the excess gel to a thickness of 2 mm.

[0087] S3. The dura mater trimmed in step S2 is freeze-dried by vacuum and then sterilized by cobalt-60 irradiation to obtain a multi-layer artificial dura mater.

[0088] Example 2

[0089] A multi-layer artificial dura mater:

[0090] Before preparing the multi-layer artificial dura mater, the modified gel medium, modified gel solution and modified allogeneic dura mater are prepared:

[0091] The preparation of the modified gel medium comprises the following steps:

[0092] S211. 100 g of gelatin and 15 g of sodium alginate were added to 300 mL of deionized water and stirred at 400 rpm in a 60°C water bath for 30 min to obtain a transparent viscous solution.

[0093] S212. At 40°C, 10 g of hydroxyapatite nanoparticles and 25 g of polyethylene glycol diacrylate were added to a transparent viscous solution and ultrasonically dispersed at a frequency of 40 kHz for 20 min to obtain a preliminary modified solution.

[0094] S213. The preliminary modified solution was irradiated with 365 nm ultraviolet light (light intensity: 10 mW / cm 2 ) irradiated for 10 min to obtain a modified gel medium;

[0095] The preparation of the modified gel solution comprises the following steps:

[0096] S21. Dissolve 25 g of polycaprolactone, 10 g of carboxymethyl chitosan, 25 g of recombinant humanized collagen, and 40 g of modified gel medium in 300 mL of 100°C hot water and stir at constant temperature for 30 min to obtain a preliminary mixture;

[0097] S22 was added to the preliminary mixture 3g chitosan quaternary ammonium salt, ultrasonic dispersion at a frequency of 40kHz for 15min to obtain a secondary mixed solution;

[0098] S23. The mixture was cooled to 45 ° C in the second step, 0.2 g of ethyl 2,4,6-trimethylbenzoylphenylphosphonate was added, and the mixture was stirred at 400 r / min for 30 min in the dark, and then cooled to room temperature to obtain a modified gel solution;

[0099] The preparation of modified allogeneic dura mater includes the following steps:

[0100] S11. Immerse the allogeneic dura mater in a modified chelating solution (composed of 0.3% disodium edetate, polyethylene glycol octylphenyl ether, and polycaprolactone, in a mass ratio of 5:3:1) at 37°C for 4 hours, with the pH controlled at 10, to obtain a preliminarily modified allogeneic dura mater.

[0101] S12. Freeze-dry the preliminarily modified allogeneic dura mater at -20°C for 12 h to obtain modified allogeneic dura mater.

[0102] The steps for preparing the multi-layer artificial dura mater include:

[0103] S1. Soak the modified allogeneic dura mater in a modified gel solution at 10°C for 12 hours, then soak it in a modified gel medium at 10°C for 12 hours to obtain a double-layer modified allogeneic dura mater.

[0104] S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again and incubate at 10°C for 12 hours. Trim the excess gel to a thickness of 5 mm.

[0105] S3. The dura mater trimmed in step S2 is freeze-dried by vacuum and then sterilized by cobalt-60 irradiation to obtain a multi-layer artificial dura mater.

[0106] Example 3

[0107] A multi-layer artificial dura mater:

[0108] Before preparing the multi-layer artificial dura mater, the modified gel medium, modified gel solution and modified allogeneic dura mater are prepared:

[0109] The preparation of the modified gel medium comprises the following steps:

[0110] S211. 85 g of starch and 14 g of sodium alginate were added to 270 mL of deionized water and stirred at 370 rpm in a 60°C water bath for 22 min to obtain a transparent viscous solution.

[0111] S212. At 25°C, 9 g of hydroxyapatite nanoparticles and 22 g of polyethylene glycol diacrylate were added to a transparent viscous solution and ultrasonically dispersed at a frequency of 40 kHz for 17 min to obtain a preliminary modified solution.

[0112] S213. The preliminary modified solution was irradiated with 365 nm ultraviolet light (light intensity: 10 mW / cm 2 ) irradiated for 7 min to obtain a modified gel medium;

[0113] The preparation of the modified gel solution comprises the following steps:

[0114] S21. Dissolve 15 g of polycaprolactone, 7 g of carboxymethyl chitosan, 20 g of recombinant humanized collagen, and 25 g of modified gel medium in 270 mL of 85°C hot water and stir at constant temperature for 27 min to obtain a preliminary mixture;

[0115] S22. 2 g of chitosan quaternary ammonium salt was added to the preliminary mixture and ultrasonically dispersed at a frequency of 40 kHz for 12 min to obtain a secondary mixed solution;

[0116] S23. The mixture was cooled to 42 ° C in the second step, 0.1 g of ethyl 2,4,6-trimethylbenzoylphenylphosphonate was added, and stirred at a speed of 320 r / min for 22 min in the dark, and then cooled to room temperature to obtain a modified gel solution;

[0117] The preparation of modified allogeneic dura mater includes the following steps:

[0118] S11. Immerse the allogeneic dura mater in a modified chelating solution (composed of 0.2% disodium edetate, polyethylene glycol octylphenyl ether, and polycaprolactone, in a mass ratio of 4:2.5:1) at 15°C for 3 hours, controlling the pH to 7, to obtain a preliminarily modified allogeneic dura mater.

[0119] S12. Freeze-dry the preliminarily modified allogeneic dura mater at -30°C for 11 h to obtain modified allogeneic dura mater.

[0120] The steps for preparing the multi-layer artificial dura mater include:

[0121] S1. Soak the modified allogeneic dura mater in a modified gel solution at 5°C for 11 hours, then soak it in a modified gel medium at 5°C for 11 hours to obtain a double-layer modified allogeneic dura mater.

[0122] S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again and incubate at 5°C for 11 hours. Trim the excess gel to a thickness of 3 mm.

[0123] S3. The dura mater trimmed in step S2 is freeze-dried by vacuum and then sterilized by cobalt-60 irradiation to obtain a multi-layer artificial dura mater.

[0124] Example 4

[0125] A multi-layer artificial dura mater:

[0126] Before preparing the multi-layer artificial dura mater, the modified gel medium, modified gel solution and modified allogeneic dura mater are prepared:

[0127] The preparation of the modified gel medium comprises the following steps:

[0128] S211. 95 g of carboxyvinyl polyol and 14 g of sodium alginate were added to 280 mL of deionized water and stirred at 380 r / min in a water bath at 60 ° C for 27 min to obtain a transparent viscous solution;

[0129] S212. 9 g of hydroxyapatite nanoparticles and 24 g of polyethylene glycol diacrylate were added to the transparent viscous solution at 35°C and ultrasonically dispersed at a frequency of 40 kHz for 18 min to obtain a preliminary modified solution.

[0130] S213. The preliminary modified solution was irradiated with 365 nm ultraviolet light (light intensity: 10 mW / cm 2 ) irradiated for 8 min to obtain a modified gel medium;

[0131] The preparation of the modified gel solution comprises the following steps:

[0132] S21. Dissolve 20 g of polycaprolactone, 8 g of carboxymethyl chitosan, 22 g of recombinant humanized collagen, and 35 g of modified gel medium in 280 mL of 94°C hot water and stir at constant temperature for 28 min to obtain a preliminary mixture;

[0133] S22. 2 g of chitosan quaternary ammonium salt was added to the preliminary mixture and ultrasonically dispersed at a frequency of 40 kHz for 14 min to obtain a secondary mixed solution;

[0134] S23. The mixture was cooled to 44 ° C in the second step, 0.14g of ethyl 2,4,6-trimethylbenzoylphenylphosphonate was added, and the mixture was stirred at 380r / min for 28min in the dark, and then cooled to room temperature to obtain a modified gel solution;

[0135] The preparation of modified allogeneic dura mater includes the following steps:

[0136] S11. Immerse the allogeneic dura mater in a modified chelating solution (composed of 0.25% disodium edetate, polyethylene glycol octylphenyl ether, and polycaprolactone, in a mass ratio of 4.5:2.7:1) at 32°C for 3 h, controlling the pH to 8, to obtain a preliminarily modified allogeneic dura mater.

[0137] S12. Freeze-dry the preliminarily modified allogeneic dura mater at -25°C for 11.5 hours to obtain modified allogeneic dura mater.

[0138] The steps for preparing the multi-layer artificial dura mater include:

[0139] S1. Soak the modified allogeneic dura mater in a modified gel solution at 28°C for 11.5 hours, then soak it in a modified gel medium at 8°C for 11.5 hours to obtain a double-layer modified allogeneic dura mater.

[0140] S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again and incubate at 8°C for 11.5 hours. Trim the excess gel to a thickness of 4 mm.

[0141] S3. The dura mater trimmed in step S2 is freeze-dried by vacuum and then sterilized by cobalt-60 irradiation to obtain a multi-layer artificial dura mater.

[0142] Comparative Example 1

[0143] Comparative Example 1 differs from Example 1 in that the addition of modified allogeneic dura mater is omitted in Comparative Example 1, and the remaining steps are exactly the same as those in Example 1.

[0144] Comparative Example 2

[0145] Comparative Example 2 differs from Example 1 in that the addition of the modified gel solution is omitted in Comparative Example 2, and the remaining steps in Comparative Example 2 are exactly the same as those in Example 1.

[0146] Comparative Example 3

[0147] Comparative Example 3 differs from Example 1 in that, in Comparative Example 3, the modified gel medium in the modified gel solution is replaced with chitosan (CAS: 9012-76-4), and the remaining steps in Comparative Example 3 are exactly the same as those in Example 1.

[0148] Performance testing:

[0149] After the multi-layer artificial dura mater obtained in Examples 1-4 and Comparative Examples 1-3 was implanted in rabbits for 24 weeks, samples were taken for biomechanical property testing. The rabbits were processed as follows: routine dura mater skin preparation, disinfection, and draping were performed, and the full-thickness dura mater of the bilateral temporal and parietal regions was removed. A dura mater defect with a diameter of 4 mm was created using a trephine drill. The entire experiment was divided into seven groups, and seven products were implanted in the defects respectively. After 24 weeks, samples were taken for biomechanical property testing. The biomechanical properties mainly include elastic modulus and hardness tests. The results obtained are shown in Table 2 below:

[0150] Table 2

[0151] Elastic modulus (Mpa) Hardness (Kpa) Example 1 1.51 3.18 Example 2 1.52 3.23 Example 3s 1.46 3.17 Example 4 1.48 3.20 Comparative Example 1 1.13 2.67 Comparative Example 2 1.15 2.65 Comparative Example 3 1.12 2.71

[0152] As shown in Table 2 above, the elastic modulus of the examples is generally higher than that of the comparative examples, indicating that the synergistic effect of the various raw materials in the modified gel solution and the modified allogeneic dura mater in the present application effectively improves the elastic modulus of the new tissue.

[0153] Minipigs aged 4-6 months were used for dural defect repair. After anesthesia, skin preparation, and disinfection, full-thickness dural defects with a diameter of 9 mm were created bilaterally in the temporoparietal region. Four groups were divided into two groups, each using the multilayer artificial dura mater obtained in Example 1 and Comparative Examples 1-3. Samples were collected and observed 6, 12, and 24 weeks after surgery.

[0154] Through experiments, it can be obtained that the modified gel solution (Comparative Example 1) alone is basically not filled, and incomplete repair is also observed in the independent comparative example 2 experimental group, the surface coverage is uneven and the repair tissue is thinner than that of the experimental group of Example 1. In the experimental group of Example 1, transparent meningeal cells can be seen in the repair area, and there is good integration between the repair tissue and the normal meninges. The comparative example 3 experimental group has a worse repair effect than the simple comparative example 2 experimental group, the repair tissue is thinner, and the unrepaired area is larger, indicating that the modified gel solution prepared by chitosan combined with polycaprolactone, carboxymethyl chitosan, and recombinant humanized collagen is less effective in repairing large-area meningeal defects.

[0155] The above experiments show that the multi-layer artificial dura mater of the present application is not only biocompatible and has no immune rejection reaction, but also can help reduce inflammatory reactions and promote the repair and reconstruction of the meningeal matrix; the generated meninges are transparent, with good repair effects, and the elastic modulus and hardness are closer to those of normal meninges, and the repair area is complete and smooth.

[0156] The invention is good and can be applied to patients whose meningeal defects are too large (for example, a lesion with a diameter of 9 mm. Compared with the existing technology that can only repair a defect with a diameter of 4 mm, the product provided by this application has a repair area four times larger) or patients who lack normal meningeal tissue around the injury; the production process is simple, the material is easy to obtain, and the cost is low. The use of this composite material only requires one operation, the operation is simple, and it can greatly reduce the pain suffered by the patient.

[0157] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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.

[0158] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims, and the specification may be used to interpret the content of the claims.

Claims

1. A multi-layer artificial dura mater, characterized in that: The multi-layer artificial dura mater comprises a modified allogeneic dura mater and a gel layer wrapping the allogeneic dura mater; The gel layer comprises a modified gel layer, a modified gel medium layer and a modified gel layer sequentially wrapped on the modified allogeneic dura mater; The modified gel layer is prepared by using a modified gel solution; The raw materials of the modified gel solution include the following components in parts by mass: 10-25 parts of polycaprolactone, 5-10 parts of carboxymethyl chitosan, 15-25 parts of recombinant humanized collagen and 20-40 parts of modified gel medium, 1-3 parts of antibacterial agent and 0.05-0.2 parts of photoinitiator.

2. The multi-layer artificial dura mater according to claim 1, characterized in that: The raw materials of the modified gel solution include the following components in parts by mass: 10-15 parts of polycaprolactone, 5-7 parts of carboxymethyl chitosan, 15-20 parts of recombinant humanized collagen and 20-25 parts of modified gel medium, 1-2 parts of antibacterial agent and 0.05-0.1 parts of photoinitiator; Optionally, the raw materials of the modified gel solution include the following components in parts by mass: 15-25 parts of polycaprolactone, 7-10 parts of carboxymethyl chitosan, 20-25 parts of recombinant humanized collagen and 25-40 parts of modified gel medium, 2-3 parts of antibacterial agent and 0.1-0.2 parts of photoinitiator.

3. The multi-layer artificial dura mater according to claim 1 or 2, characterized in that: The raw materials for preparing the modified gel medium layer consist of modified gel medium; Optionally, the raw materials for preparing the modified gel medium include the following components in parts by mass: 80-100 parts of gel medium, 13-15 parts of sodium alginate, 8-10 parts of hydroxyapatite nanoparticles and 20-25 parts of polyethylene glycol diacrylate; Optionally, the raw materials for preparing the modified gel medium include the following components in parts by mass: 85-95 parts of gel medium, 14-15 parts of sodium alginate, 8-9 parts of hydroxyapatite nanoparticles and 22-24 parts of polyethylene glycol diacrylate.

4. The multi-layer artificial dura mater according to claim 3, characterized in that: The gel medium is selected from one of tragacanth gum, gelatin, starch, cellulose derivatives or carbopol.

5. The multi-layer artificial dura mater according to any one of claims 1, 2 and 4, characterized in that: The antibacterial agent includes chitosan quaternary ammonium salt, and the photoinitiator includes 2,4,6-trimethylbenzoylphenyl phosphonic acid ethyl ester.

6. The multi-layer artificial dura mater according to any one of claims 1, 2 and 4, characterized in that: The modified allogeneic dura mater is prepared by using allogeneic dura mater and modified chelating solution; Optionally, the allogeneic dura mater is selected from the full-thickness dura mater of bilateral temporoparietal regions, with a thickness of 0.3-0.5 mm; Optionally, the modified chelating liquid is composed of 0.1-0.3% by mass concentration of disodium edetate, polyethylene glycol octylphenyl ether, and polycaprolactone, and the mass ratio thereof is (3-5): (2-3):

1.

7. A method for preparing the multilayer artificial dura mater according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1. Soaking the modified allogeneic dura mater in the modified gel solution at 2-10°C for 10-12 hours, and then soaking it in the modified gel medium at 2-10°C for 10-12 hours to obtain a double-layer modified allogeneic dura mater; S2. Soak the double-layer modified allogeneic dura mater in the modified gel solution again, place at 2-10°C for 10-12 hours, and trim the excess gel to a thickness of 2-5 mm; S3. The dura mater trimmed in step S2 is subjected to vacuum freeze drying and sterilization to prepare the multi-layer artificial dura mater.

8. The method according to claim 7, characterized in that The preparation of the modified gel solution comprises the following steps: S21. The polycaprolactone, carboxymethyl chitosan, recombinant humanized collagen and modified gel medium are dissolved in 250-300 parts of 80-100 ° C hot water and stirred at a constant temperature to obtain a preliminary mixture; optionally, the constant temperature stirring time is 25-30min; S22. The antibacterial agent is added to the preliminary mixture, and ultrasonically dispersed to obtain a secondary mixed solution; optionally, the ultrasonic frequency is 40kHz-50kHz; optionally, the ultrasonic dispersion time is 10-15min; S23. The mixed solution of the second step is cooled to 40-45 ° C, the photoinitiator is added, stirred in the dark, and cooled to room temperature to obtain a modified gel solution; optionally, the stirring time in the dark is 20-30min; optionally, the stirring speed in the dark is 300-400r / min.

9. The method according to claim 7 or 8, characterized in that The preparation of the modified gel medium comprises the following steps: S211. The gel medium and the sodium alginate are added to 250-300 parts of deionized water and stirred at a speed of 350-400 r / min in a water bath at a constant temperature of 55 ℃ -60 ℃ for 20-30 min to obtain a transparent viscous liquid; S212. Adding the hydroxyapatite nanoparticles and the polyethylene glycol diacrylate to a transparent viscous liquid at 20-40° C. and ultrasonically dispersing the mixture at a frequency of 40 kHz to 50 kHz for 15-20 min to obtain a preliminary modified solution. S213. The preliminary modified solution was irradiated with 365 nm ultraviolet light for 5-10 min to obtain a modified gel medium; optionally, the ultraviolet light intensity was 10 mW / cm 2 .

10. The method according to claim 9, characterized in that The preparation of the modified allogeneic dura mater comprises the following steps: S11. Immersing the allogeneic dura mater in the modified chelating solution at 10-37° C. for 2-4 h, controlling the pH to 6-10, to obtain a preliminarily modified allogeneic dura mater; S12. Freeze-dry the preliminarily modified allogeneic dura mater at -35 to -20°C for 10 to 12 hours to obtain modified allogeneic dura mater.

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