Artificial dura mater and preparation method thereof

Abstract

The present invention provides an artificial dura mater and a preparation method thereof, and relates to the technical field of medical repair materials. The artificial dura mater is mainly composed of a degradable repair membrane layer and an anti-seepage protective layer composited on the degradable repair membrane layer, wherein: the degradable repair membrane layer is composited by a plurality of fiber-oriented sheets, and the orientation direction of the oriented fibers in the fiber-oriented sheets is consistent. The oriented fibers with the same orientation direction are conducive to the attachment of cells, have high biocompatibility, and have good repair and regeneration effects; at the same time, the patch composited by fiber-oriented sheets with different orientation directions in the present application has better mechanical strength. In addition, the anti-seepage protective layer can play a good anti-seepage role, and can also prevent fibroblasts, muscles, and vascular tissue cells from growing into the artificial dura mater to cause adhesion. Therefore, the artificial dura mater of the present application has the advantages of high biocompatibility, good mechanical strength, and excellent anti-seepage and adhesion performance.

Description

Technical Field

[0001] The invention relates to the technical field of medical repair materials, in particular to an artificial dura mater and a preparation method thereof. Background Art

[0002] The dura mater is a thick and tough double-layer membrane. The outer layer is called the periosteum, which is the periosteum on the inner surface of the skull and is only loosely attached to the skull cap. The inner layer is called the meningeal layer, which is thicker and tougher than the outer layer and is connected to the dura mater at the foramen magnum. The main function of the dura mater is to protect the brain.

[0003] During neurosurgery craniotomy, the dura mater must be cut open. Dura mater bleeding, electrocoagulation hemostasis, tumor invasion of dura mater resection, traumatic dura mater injury, etc. can all lead to dura mater defects. At the same time, the integrity of the dura mater is very important in neurosurgery. Dura mater repair materials play an important role in restoring the integrity of the dura mater, protecting brain tissue, and preventing complications such as cerebrospinal fluid leakage, intracranial infection, brain herniation, and epilepsy.

[0004] At present, there are dozens of dura mater repair materials on the market at home and abroad, which can be roughly divided into four categories: autologous fascia, allogeneic materials, xenogeneic materials, natural and artificial synthetic materials.

[0005] In current clinical applications, autologous fascia reconstruction of the dura mater is still the standard for clinical application, but the incidence of early postoperative cerebrospinal fluid leakage and subcutaneous effusion is still high, which can easily lead to complications such as adhesions, and may cause non-healing or dysfunction of the donor site. Allogeneic and xenogeneic materials cannot completely avoid immune rejection, and residual antigens can cause varying degrees of inflammatory reactions, resulting in fibrosis and brain adhesions. It is easy to produce scars and irritation to brain tissue. The source of allogeneic materials is limited, making it difficult to form commercial production. At the same time, due to the xenogeneic or xenogeneic source, the risk of virus transmission cannot be completely avoided. For example, the source of artificial meninges derived from bovine materials has been greatly reduced after the outbreak of mad cow disease in Europe, and its safety has also been greatly questioned. Natural and synthetic materials include non-absorbable materials and absorbable materials. Non-absorbable materials such as polytetrafluoroethylene and polyurethane have good tissue compatibility, but as permanent foreign bodies, rejection reactions are difficult to avoid, and they are prone to aseptic inflammatory reactions and stimulate granulation tissue formation. Existing absorbable materials such as polydioxanone and polyglycolic acid have good biocompatibility and mechanical properties, but they are prone to adhesion, cerebrospinal fluid leakage, and have poor flexibility and conformability, which affects the repair effect.

[0006] Therefore, it is necessary and urgent to research and develop an artificial dura mater repair material with better biocompatibility, mechanical strength, softness, and anti-penetration and anti-adhesion properties.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The first object of the present invention is to provide an artificial dura mater, which has the advantages of high biocompatibility, good mechanical strength and excellent anti-penetration and adhesion performance compared with the existing artificial dura mater through the mutual cooperation of a degradable repair membrane layer and an anti-seepage protective layer.

[0009] The second object of the present invention is to provide a method for preparing an artificial dura mater.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0011] The present invention provides an artificial dura mater, which comprises a degradable repair membrane layer and an anti-seepage protective layer composited on the degradable repair membrane layer;

[0012] The degradable repair film layer is prepared by compounding multiple fiber orientation sheets; the fiber orientation sheet is formed into oriented fibers by electrostatic spinning, and the orientation directions of the oriented fibers in the fiber orientation sheet are consistent;

[0013] The orientation directions of the fibers in each fiber orientation sheet in the degradable repair film layer are different.

[0014] Furthermore, the oriented fibers are obtained by electrostatic spinning of degradable polyurethane or polylactic acid;

[0015] Preferably, the weight average molecular weight Mw of the degradable polyurethane is 18W to 22W;

[0016] Preferably, the weight average molecular weight Mw of the polylactic acid is 18W to 28W;

[0017] Preferably, the diameter of the oriented fibers in the fiber oriented sheet is 1 nm to 20 μm, and the porosity is 10 to 40%.

[0018] Furthermore, the anti-seepage protective layer is prepared from degradable polyurethane by a casting method;

[0019] The anti-seepage protective layer is a degradable polyurethane film layer without a pore structure.

[0020] Furthermore, the total thickness of the artificial dura mater is 0.1 to 0.3 mm, wherein:

[0021] The thickness of the degradable repair film layer is 0.1-0.29 mm, and the thickness of the anti-seepage protection layer is 0.02-0.1 mm.

[0022] Furthermore, the tensile strength of the artificial dura mater is 9-45 N / cm; the elongation at break is 90-750%; the suture strength is 4-25 N, and the softness is 130.55 mN-376.09 mN.

[0023] Furthermore, the degradable repair film layer is prepared by compounding two layers of fiber oriented sheets;

[0024] Preferably, the two-layer fiber-oriented sheet comprises a first fiber-oriented sheet and a second fiber-oriented sheet, and the first fiber-oriented sheet and the second fiber-oriented sheet have different orientation directions and are at an angle of 90°±15°.

[0025] The present invention provides a method for preparing an artificial dura mater, the method comprising:

[0026] The anti-seepage protective layer was prepared by a tape casting method, and then a degradable repair membrane layer was prepared on the anti-seepage protective layer by an electrospinning method, and then post-processed to obtain an artificial dura mater.

[0027] Furthermore, the preparation method of the degradable repair film layer includes:

[0028] S1: dissolving the degradable polyurethane in a solvent to obtain a spinning solution;

[0029] S2: electrospinning the spinning solution to obtain a fiber orientation sheet A, then adjusting the electrospinning direction, and electrospinning on the fiber orientation sheet A to obtain a fiber orientation sheet B intersecting the orientation direction of the fiber orientation sheet A, thereby obtaining a degradable repair film layer composed of two layers of fiber orientation sheets;

[0030] S3: Repeat step S2 to obtain a degradable repair film layer composed of multiple fiber oriented sheets.

[0031] Furthermore, in step S1, the concentration of the degradable polyurethane in the spinning solution is 10-30% (w / v);

[0032] Preferably, the solvent in the spinning solution is a single solvent of N,N-dimethylformamide, a single solvent of N,N-dimethylacetamide, a mixed solvent of N,N-dimethylformamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether, or a mixed solvent of N,N-dimethylacetamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether;

[0033] More preferably, the solvent in the spinning solution is a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, wherein the volume ratio of N,N-dimethylformamide to tetrahydrofuran is 10:1-3:7, wherein the preferred volume ratio is 10:1-1:1.

[0034] Furthermore, the electrospinning conditions in step S2 include:

[0035] Negative voltage: -1KV~-4KV, positive voltage: 7KV~10KV, injection speed of the nozzle: 0.05-0.3ml / min; rotation speed of the receiving device: (800, 2500) rpm / min, distance between the nozzle and the receiving device: 0~50mm; translation speed of the nozzle is (0, 300) mm / min.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The artificial dura mater provided by the present invention is mainly composed of a degradable repair film layer and an anti-seepage protective layer compounded on the degradable repair film layer, wherein: the degradable repair film layer is obtained by compounding multiple layers of fiber oriented sheets, and the orientation direction of the oriented fibers in the fiber oriented sheets is consistent. The oriented fibers with the same orientation direction are conducive to the attachment of cells, and the cells have better mobility and growth rate on the oriented fibers, high biocompatibility, and good repair and regeneration effect; at the same time, the patch obtained by compounding fiber oriented sheets with different orientation directions in the present application has better mechanical strength. The anti-seepage protective layer is a degradable polyurethane film layer without a porous structure, which can not only play a good anti-seepage and anti-adhesion role, but also prevent the growth of fibroblasts, muscles and vascular tissue cells, etc., and play an isolation role.

[0038] Therefore, the artificial dura mater of the present application has the advantages of high biocompatibility, good mechanical strength and excellent anti-penetration and adhesion performance compared with the existing artificial dura mater through the synergistic cooperation of the degradable repair membrane layer and the anti-permeability protective layer.

[0039] The invention provides a method for preparing an artificial dura mater, wherein the method comprises: preparing an anti-seepage protective layer by a casting method, and then preparing a degradable repair film layer on the anti-seepage protective layer by an electrostatic spinning method, and performing post-processing to obtain an artificial dura mater. The above-mentioned preparation method has the technical advantages of simple processing technology and easy operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1a This is a scanning electron microscope image of the fiber orientation of the fiber orientation sheet formed by the first electrospinning in the preparation process of the degradable repair film layer in Example 1 of the present invention;

[0042] Figure 1bThis is a scanning electron microscope image of the fiber orientation of the fiber orientation sheet formed by the second electrospinning in the process of preparing the degradable repair film layer in Example 1 of the present invention;

[0043] Figure 2a This is a diagram of an artificial dura mater repair experiment provided in Experimental Example 1 of the present invention;

[0044] Figure 2b These are pictures of the repair effects of the artificial dura mater repair experiment provided in Experimental Example 1 of the present invention at 4 weeks, 8 weeks, and 12 weeks after surgery. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] According to one aspect of the present invention, an artificial dura mater comprises a degradable repair membrane layer and an anti-seepage protective layer composited on the degradable repair membrane layer;

[0047] The degradable repair film layer is prepared by compounding multiple fiber orientation sheets; the fiber orientation sheet is formed into oriented fibers by electrostatic spinning, and the orientation directions of the oriented fibers in the fiber orientation sheet are consistent;

[0048] The orientation directions of the fibers in each fiber orientation sheet in the degradable repair film layer are different.

[0049] The artificial dura mater provided by the present invention is mainly composed of a degradable repair film layer and an anti-seepage protective layer compounded on the degradable repair film layer, wherein: the degradable repair film layer is obtained by compounding multiple layers of fiber oriented sheets, and the orientation direction of the oriented fibers in the fiber oriented sheets is consistent. The oriented fibers with the same orientation direction are conducive to the attachment of cells, and the cells have better mobility and growth rate on the oriented fibers, high biocompatibility, and good repair and regeneration effect; at the same time, the patch obtained by compounding fiber oriented sheets with different orientation directions in the present application has better mechanical strength. The anti-seepage protective layer is a degradable polyurethane film layer without a porous structure, which can play a good anti-seepage role, and can also prevent fiber cells, muscles and vascular tissue cells from growing into the artificial dura mater and causing adhesion.

[0050] Therefore, the artificial dura mater of the present application has the advantages of high biocompatibility, good mechanical strength and excellent anti-permeability and adhesion performance compared with the existing artificial dura mater through the mutual cooperation of the degradable repair membrane layer and the anti-permeability protective layer. It has been verified that the degradable period of the artificial dura mater of the present application is 6 to 12 months, the tensile strength is 9 to 45N / cm; the elongation at break is 90 to 750%; the suture strength is 4 to 25N, and the softness is 130.55mN to 376.09mN; at the same time, the anti-permeability is good, and no leakage is found in the test sample under a pressure of 165.0mmHg for 60s.

[0051] In a preferred embodiment of the present invention, the oriented fibers are obtained by electrospinning degradable polyurethane or polylactic acid;

[0052] In the above preferred embodiment, the weight average molecular weight Mw of the degradable polyurethane is 18W to 22W, and the weight average molecular weight Mw of the polylactic acid is 18W to 28W;

[0053] The weight average molecular weight of the degradable polyurethane may be, but not limited to, 18W, 19W, 20W, 21W or 22W, or any value between 18W and 22W. The weight average molecular weight of the polylactic acid may be, but not limited to, 18W, 19W, 20W, 21W, 22W, 23W, 24W, 25W, 26W, 27W or 28W, or any value between 18W and 28W.

[0054] Preferably, the diameter of the oriented fibers in the fiber oriented sheet is 1 nm to 20 μm, and the porosity is 10 to 40%.

[0055] It should be noted that multiple cells are involved in artificial dura mater repair, such as fibroblasts: During the repair process, fibroblasts usually proliferate and have a diameter of about 10 to 30 microns. These cells play an important role in wound healing and tissue repair. Macrophages: These immune cells are involved in the removal of damaged tissue and infection, with a diameter of about 10 to 30 microns, or even larger. Glial cells: Such as astrocytes and oligodendrocytes, the size is usually between 10 and 30 microns, depending on the specific type and function. Endothelial cells: During the repair process, endothelial cells (such as vascular endothelial cells) are usually between 10 and 25 microns in diameter and are involved in vascular regeneration and healing.

[0056] At the same time, it has been found through research that the artificial dura mater repair cells have better adhesion properties and better bioaffinity on fibers with a diameter smaller than their own. When the transverse diameter of the electrospinning is controlled to be smaller than the diameter of the artificial dura mater repair cells, it is beneficial for the cells to attach and grow on the electrospinning. Therefore, the diameter of the oriented fibers in the fiber orientation sheet is controlled to be 1nm to 20μm, and the porosity is 10 to 40%.

[0057] In a preferred embodiment of the present invention, the anti-seepage protective layer is prepared from degradable polyurethane by a casting method;

[0058] The anti-seepage protective layer is a degradable polyurethane film layer without a pore structure.

[0059] In a preferred embodiment of the present invention, the total thickness of the artificial dura mater is 0.1-0.3 mm, wherein the thickness of the degradable repair membrane layer is 0.1-0.29 mm, and the thickness of the anti-seepage protective layer is 0.02-0.1 mm.

[0060] The total thickness of the artificial dura mater may be, but is not limited to, 0.1 mm, 0.15 mm, 0.2 mm, 0.25 mm or 0.3 mm, or any value between 0.1 and 0.3 mm. The thickness of the degradable repair film layer may be, but is not limited to, 0.1 mm, 0.15 mm, 0.18 mm, 0.2 mm, 0.23 mm, 0.28 mm or 0.29 mm, or any value between 0.1 and 0.29 mm. The thickness of the anti-seepage protective layer may be, but is not limited to, 0.02 mm, 0.05 mm, 0.08 mm, m or 0.1 mm, or any value between 0.02 and 0.1 mm.

[0061] In a preferred embodiment of the present invention, the degradable repair film layer is prepared by compounding two layers of fiber oriented sheets;

[0062] Preferably, the two-layer fiber-oriented sheet comprises a first fiber-oriented sheet and a second fiber-oriented sheet, and the first fiber-oriented sheet and the second fiber-oriented sheet have different orientation directions and are at an angle of 90°±15°.

[0063] As a preferred embodiment, the present application has found through research that a degradable repair membrane layer prepared by a composite of two layers of fiber-oriented sheets "wherein the orientation directions of the first fiber-oriented sheet and the second fiber-oriented sheet are different and are at an angle of 90°±15°" has better mechanical strength than a degradable repair patch with two layers having the same orientation direction.

[0064] Mainly manifested in:

[0065] 1. Compared with the multi-layer structure, the porosity of the degradable repair membrane layer prepared by the above two layers of fiber oriented sheets is easier to repair the combination of cells and patches, and the biocompatibility is better; at the same time, the cell migration rate is good and the cell growth rate is high.

[0066] 2. The degradable repair film prepared by two layers of fiber oriented sheets with vertical or nearly vertical orientation has good mechanical strength in both the transverse and longitudinal directions and is not easy to be torn. Compared with the patch with multiple fiber structures arranged at other angles, it has better strength.

[0067] 3. The thickness is thinner and has a wider applicability; the degradation is better controllable and can be effectively degraded after forming tissue engineering support, avoiding the problem of long degradation time of multi-layer structure patches affecting healing.

[0068] In a preferred embodiment of the present invention, the tensile strength of the artificial dura mater is 9-45 N / cm; the elongation at break is 90-750%; the suture strength is 4-25 N, and the softness is 130.55 mN-376.09 mN.

[0069] According to one aspect of the present invention, a method for preparing an artificial dura mater comprises:

[0070] The anti-seepage protective layer was prepared by a tape casting method, and then a degradable repair membrane layer was prepared on the anti-seepage protective layer by an electrospinning method, and then post-processed to obtain an artificial dura mater.

[0071] The invention provides a method for preparing an artificial dura mater, which comprises: preparing an anti-seepage protective layer by a casting method, then preparing a degradable repair membrane layer on the anti-seepage protective layer by an electrostatic spinning method, and performing post-treatment to obtain an artificial dura mater. The above-mentioned preparation method has the technical advantages of simple processing technology and easy operation.

[0072] In a preferred embodiment of the present invention, the method for preparing the degradable repair film layer comprises:

[0073] S1: dissolving the degradable polyurethane in a solvent to obtain a spinning solution;

[0074] S2: electrospinning the spinning solution to obtain a fiber orientation sheet A, then adjusting the electrospinning direction, and electrospinning on the fiber orientation sheet A to obtain a fiber orientation sheet B intersecting the orientation direction of the fiber orientation sheet A, thereby obtaining a degradable repair film layer composed of two layers of fiber orientation sheets;

[0075] S3: Repeat step S2 to obtain a degradable repair film layer composed of multiple fiber oriented sheets.

[0076] In a preferred embodiment of the present invention, the concentration of the degradable polyurethane in the spinning solution in step S1 is 10-30% (w / v);

[0077] Preferably, the solvent in the spinning solution is a single solvent of N,N-dimethylformamide, a single solvent of N,N-dimethylacetamide, a mixed solvent of N,N-dimethylformamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether, or a mixed solvent of N,N-dimethylacetamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether;

[0078] More preferably, the solvent in the spinning solution is a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, wherein the volume ratio of N,N-dimethylacetamide to tetrahydrofuran can be but not limited to 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 3:1 or 3:7, and can also be any ratio between 10:1 and 3:7, preferably 10:1 to 1:1.

[0079] In a preferred embodiment of the present invention, the conditions for electrospinning in step S2 include:

[0080] Negative voltage: -1KV~-4KV, positive voltage: 7KV~10KV, injection speed of the nozzle: 0.05-0.3ml / min; rotation speed of the receiving device: (800, 2500) rpm / min, distance between the nozzle and the receiving device: 0~50mm; translation speed of the nozzle is (0, 300) mm / min.

[0081] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0082] Example 1

[0083] A method for preparing an artificial dura mater, the method comprising:

[0084] (1) Anti-seepage protective layer:

[0085] Polyurethane particles with a molecular weight (Mw) of 80,000 were added to tetrahydrofuran (THF) solvent at a solid-liquid ratio (w / v) of 25%, and stirred to completely dissolve.

[0086] Pour the solution onto a glass plate, place a coater with the height adjusted, push it with an automatic coater, and air-dry under a laminar flow hood.

[0087] (2) Preparation of degradable repair film layer:

[0088] 1. Preparation of degradable polyurethane (PU) solution:

[0089] A 20% polyurethane solution was prepared by using degradable polyurethane as solute and a mixed solution of tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) as solvent.

[0090] The ratio of solvents: tetrahydrofuran (THF): N,N-dimethylformamide (DMF) = 1:1.

[0091] The specific preparation operation is as follows:

[0092] 1) Take 150 mL of tetrahydrofuran and 150 mL of N,N-dimethylformamide respectively and add them into a three-necked flask. Turn on the stirrer and adjust the speed to (200±100) r / min and stir evenly.

[0093] 2) Weigh 60g of polyurethane material and add it into a three-necked flask. Different volumes of solution can be prepared according to the amount of solution used.

[0094] 3) Stir at room temperature for more than 4 hours to completely dissolve the PU. Let it stand for more than 4 hours to remove bubbles and set aside.

[0095] 2. Electrospinning:

[0096] 1) Wipe the electrospinning machine clean to ensure that the equipment is in a clean state.

[0097] 2) Fix the anti-seepage protective layer on the release paper, with the other side of the release paper facing the receiving device, and fix it on the receiving device to ensure that the release paper is not loose.

[0098] 3) Adjust the distance between the spinneret and the receiver to 20±1cm, determine the position and tighten it.

[0099] 4) The receiver receiving speed is set to (1000±100)rpm / min; the push injection speed is set to 0.1±0.05mm / min.

[0100] 5) The translation speed is set to 450±50mm / min and the translation distance is set to 300±50mm.

[0101] 6) Use the prepared (20±1%) PU solution and slowly draw the PU solution with a disposable syringe to avoid bubbles. If bubbles are generated, they must be removed before subsequent operations. Install a 22G needle (inner diameter) and fix the syringe in the buckle of the electrospinning machine push pump.

[0102] The nozzle diameter range can be: 0.15-4.2mm, preferably 18G (inner diameter 0.9mm), 19G (inner diameter 0.84mm), 20G (inner diameter 0.62mm), 21G (inner diameter 0.51mm), 22G (inner diameter 0.41mm), 23G (inner diameter 0.33mm), 24G (inner diameter 0.3mm) needles. In this embodiment, a 22G needle is selected.

[0103] 7) Set the negative voltage to (-2±1)KV and the positive voltage to (7±0.5)KV. Start electrospinning and observe the spinning effect until continuous and uniform yarn is ejected. Set the electrospinning time to 30min~45min. After the first electrospinning, rotate the release paper (with a spinning layer on the surface) 90 degrees and then fix it. Set the electrospinning time to 30min~45min again and repeat the operation for the second electrospinning.

[0104] After the second electrospinning is completed, the electrospun membrane is removed from the release paper to obtain a degradable repair membrane layer.

[0105] Figure 1a This is a scanning electron microscope image of the fiber orientation of the fiber orientation sheet formed by the first electrospinning in this example.

[0106] Figure 1b This is a scanning electron microscope image of the fiber orientation of the fiber oriented sheet formed by the second electrospinning in this example.

[0107] Depend on Figure 1a and Figure 1b It can be seen that the orientation directions of the two fiber orientation sheets in the degradable repair film layer of this embodiment are different and are at an angle of 90°±15°.

[0108] (3) Post-processing:

[0109] 1. Soak the artificial dura mater in a water tank for at least 7 days, changing the water twice a day and cleaning it every two days.

[0110] The ratio of the artificial dura mater patch surface area to the water volume in each water tank should be ≥900cm2 / L.

[0111] 2. Rinse the artificial dura mater with injection water cooled to 30°C, and lay them flat on the shelf of the oven to dry. The oven temperature is set at 35°C and the drying time is (5±1)h.

[0112] After the artificial dura mater is dried, it is inspected for appearance and then cut into patches of the required size.

[0113] It should be noted that the above-mentioned method for drying the artificial dura mater can also be carried out by freeze-drying.

[0114] Example 2

[0115] A method for preparing an artificial dura mater, the method comprising:

[0116] (1) Surface composite anti-seepage protective layer: same as in Example 1.

[0117] (2) Preparation of degradable repair film layer:

[0118] 1. Preparation of degradable polyurethane (PU) solution: same as Example 1.

[0119] 2. Electrospinning:

[0120] 1)~6) are the same as in Example 1.

[0121] 7) Set the negative voltage to (-2±1)KV and the positive voltage to (7±0.5)KV. Start electrospinning and observe the spinning effect until continuous and uniform yarn is ejected. Set the electrospinning time to 30min~45min. After the first electrospinning, rotate the release paper (with a spinning layer on the surface) 90 degrees and then fix it. Set the electrospinning time to 30min~45min again and repeat the operation for the second electrospinning.

[0122] After the second electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the third electrospinning.

[0123] After the third electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the fourth electrospinning.

[0124] After the fourth electrospinning, the electrospun membrane was removed from the release paper to obtain a degradable repair membrane layer.

[0125] (3) Post-treatment: Same as Example 1.

[0126] Example 3

[0127] A method for preparing an artificial dura mater, the method comprising:

[0128] (1) Surface composite anti-seepage protective layer: same as in Example 1.

[0129] (2) Preparation of degradable repair film layer:

[0130] 1. Preparation of degradable polyurethane (PU) solution: same as Example 1.

[0131] 2. Electrospinning:

[0132] 1)~6) are the same as in Example 1.

[0133] 7) Set the negative voltage to (-2±1)KV and the positive voltage to (7±0.5)KV. Start electrospinning and observe the spinning effect until continuous and uniform yarn is ejected. Set the electrospinning time to 30min~45min. After the first electrospinning, rotate the release paper (with a spinning layer on the surface) 90 degrees and then fix it. Set the electrospinning time to 30min~45min again and repeat the operation for the second electrospinning.

[0134] After the second electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the third electrospinning.

[0135] After the third electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the fourth electrospinning.

[0136] After the fourth electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the fifth electrospinning.

[0137] After the fifth electrospinning, the release paper (with a spinning layer on the surface) was rotated 90 degrees and then fixed to set the electrospinning time to 30 min to 45 min, and the operation was repeated for the sixth electrospinning.

[0138] After the sixth electrospinning, the electrospun membrane was removed from the release paper to obtain a degradable repair membrane layer.

[0139] (3) Post-treatment: Same as Example 1.

[0140] Example 4

[0141] A method for preparing an artificial dura mater, the method comprising:

[0142] (1) Surface composite anti-seepage protective layer: same as in Example 1.

[0143] (2) Preparation of degradable repair film layer:

[0144] 1. Preparation of degradable polyurethane (PU) solution: same as Example 1.

[0145] 2. Electrospinning:

[0146] 1)~6) are the same as in Example 1.

[0147] 7) Set the negative voltage to (-2±1)KV and the positive voltage to (7±0.5)KV. Start electrospinning and observe the spinning effect until continuous and uniform yarn is ejected. Set the electrospinning time to 30min~45min. After the first electrospinning, rotate the release paper (with a spinning layer on the surface) 45 degrees and then fix it. Set the electrospinning time to 30min~45min again and repeat the operation for the second electrospinning.

[0148] After the second electrospinning is completed, the electrospun membrane is removed from the release paper to obtain a degradable repair membrane layer.

[0149] (3) Post-treatment: Same as Example 1.

[0150] Comparative Example 1

[0151] A method for preparing an artificial dura mater, the method comprising:

[0152] (1) Surface composite anti-seepage protective layer: same as in Example 1.

[0153] (2) Preparation of degradable repair film layer:

[0154] 1. Preparation of degradable polyurethane (PU) solution: same as Example 1.

[0155] 2. Electrospinning:

[0156] In this comparative example, except that the rotation speed of the receiving device is (500±100) rpm / min, other process parameters are the same as those of Example 1, and the produced fiber sheet does not have an oriented structure.

[0157] (3) Post-treatment: Same as Example 1.

[0158] The difference between this embodiment and embodiment 1 is that the fibers in the degradable repair film layer do not have an oriented structure.

[0159] Comparative Example 2

[0160] A method for preparing an artificial dura mater, the method comprising:

[0161] (1) Anti-seepage protective layer: same as in Example 1.

[0162] (2) Preparation of degradable repair film layer:

[0163] 1. Preparation of degradable polyurethane (PU) solution: same as Example 1.

[0164] 2. Electrospinning:

[0165] 1)~6), Same as Example 1.

[0166] 7) Set the negative voltage to (-3±1)KV and the positive voltage to (9±0.5)KV. Start electrospinning and observe the spinning effect until continuous and uniform yarn is ejected. Set the electrospinning time to 30min~1h. After the first electrospinning, keep the release paper (with a spinning layer on the surface) fixed without rotating. Set the electrospinning time to 30min~1h again and repeat the operation for the second electrospinning.

[0167] After the second electrospinning is completed, the electrospun membrane is removed from the release paper to obtain the initial oral repair membrane.

[0168] (3) Post-treatment: Same as Example 1.

[0169] The orientation directions of the two fiber orientation sheets in the degradable repair film layer of this comparative example are in the same direction.

[0170] Test Example 1

[0171] (I) Mechanical properties test:

[0172] Experimental methods:

[0173] (1) Tensile strength: First, cut the dry sample to be tested into multiple specimens with appropriate width, flat edges, parallel sides and no visible defects, then turn on the tensile testing machine, set the parameters, perform the tensile strength test, and process the data. Tensile strength (N / cm) = tension (N) / specimen width (cm).

[0174] (2) Elongation at break: First, cut the dry sample to be tested into multiple specimens with appropriate width, flat edges, parallel sides and no visible defects. Then turn on the tensile testing machine, set the parameters, perform the elongation at break test, and process the data; elongation at break (%) = (elongation at break of specimen / original length of specimen) × 100%.

[0175] (3) Suture strength: First, cut the dry sample to be tested into multiple specimens with appropriate width, flat edges, parallel sides and no visible defects. Then, use a suture thread of the selected specification to sew one end of the sample to be tested with two needles and tie a knot to fix it. Suture multiple specimens in the same way, and test the thickness of each sample with a thickness gauge. Then, use a tensile testing machine to clamp the sample to be tested and test the suture strength of the suture to obtain the test data.

[0176] (4) Tear strength: First, cut the dry sample to be tested into samples with appropriate width, flat edges, parallel sides and no visible defects. Use a cutter to cut along the center of the sample to prepare a trouser-shaped sample. The cutting edge of the cutter must be sharp and must not have curled edges or gaps. The cut is located in the center of the sample width. Use a thickness gauge to measure 3 points in the tear area of ​​the sample, and the arithmetic average is the thickness. Turn on the tensile testing machine, clamp the sample to be tested, and perform a tear strength test to obtain the test data.

[0177] (5) Bursting strength: First, cut the dry sample to be tested into multiple square specimens with appropriate width, flat edges, parallel sides and no visible defects, and use a thickness gauge to test the thickness of each sample. Then, turn on the tensile testing machine, clamp the sample to be tested, and perform the bursting strength test to obtain the test data.

[0178] Experimental results:

[0179]

[0180] From the above results, it can be seen that the tensile strength of the artificial dura mater of the present invention reaches 9.05-45.27N / cm, the elongation at break reaches 90.37-752.05%, the suture strength reaches 4.06-25.66N, the tear strength reaches 9.02-55.27N, and the bursting strength reaches 90.45-456.20kPa. The artificial dura mater of the present invention has good mechanical properties and can well meet the mechanical property requirements of the artificial dura mater in clinical practice.

[0181] (II) Cell proliferation assay:

[0182] Experimental methods:

[0183] Each sample was made into a circular specimen of the same size as the bottom area of ​​a 48-well plate using a puncher. After sterilization, it was placed in a 48-well cell culture plate and immersed in MEM medium containing 10% FBS at 37°C and 60 rpm for 24 hours, with the outer layer of the specimen facing the bottom of the well.

[0184] The whole process was performed in a clean bench to ensure aseptic operation. Mouse fibroblast L-929 cells grown to the logarithmic growth phase were digested with 0.25% trypsin (containing EDTA), and the cell suspension was centrifuged (1000rpm, 5min) after digestion, the supernatant was discarded, and the cells were resuspended with MEM medium, and the cells were counted to obtain a cell suspension of 2×104 cells / mL; the soaked samples were respectively spread in 48-well plates, and 3 samples were operated in parallel in each group.

[0185] 2×10 4 300 μL of a cell suspension of 10 cells / mL was seeded into each well of a 48-well plate and cultured in a cell culture incubator (37° C., 5% CO 2 , >90% humidity).

[0186] After 24h, 3d and 6d, the original culture medium in the 48-well plate was discarded, and 30μL of MTT solution (concentration of 5mg / mL) was added to the corresponding wells of the 48-well plate, and cultured in a 37°C, 5% carbon dioxide incubator. After 4h, the supernatant was removed, and 200μL of isopropanol was added to each well to dissolve the crystals. After the crystals were completely dissolved, the sample was taken out and the liquid in the well was transferred to a 96-well plate, 100μL per well, and the absorbance value at a wavelength of 570nm was measured on an enzyme reader, and compared with the OD value of the blank group to determine whether the cells had a proliferation effect on the surface of the material and calculate its cell proliferation rate.

[0187] Experimental results:

[0188]

[0189] It can be seen from the above results that Examples 1 to 4 have very good cell proliferation rate effects, especially the cell proliferation rate effects of the two-layer fiber oriented sheets of Examples 1 and 4 are more significant.

[0190] (III) Degradation experiment:

[0191] Experimental methods:

[0192] (1) Preparation of enzyme buffer:

[0193] Prepare a solution containing potassium dihydrogen phosphate and disodium hydrogen phosphate with sterile double distilled water, containing 1.653g KH2PO4 and 7.744g NaH2PO4 per liter of buffer. The pH value of the buffer solution should be 7.4±0.2, and artificial cerebrospinal fluid is added after sterilization. The salts used in the preparation are analytically pure and dried to constant weight.

[0194] The artificial cerebrospinal fluid is artificial cerebrospinal fluid (ACSF, sterile) produced by Kingclone (Beijing) Biotechnology Co., Ltd., with the product number CB3143.

[0195] (2) Degradation test:

[0196] The sample is vacuum dried at room temperature to constant weight, and the weight of the sample is accurately measured. The sample is placed in a glass container, covered with enzyme buffer and the container is sealed. The minimum volume of the buffer should be 10 mL, and the ratio of the volume of the buffer (mL) to the mass of the sample (g) should be greater than or equal to 30:1. The sample is maintained at 37 ± 1 ° C in a constant temperature water bath or oven.

[0197] (3) Determination of degradation cycle:

[0198] For sample degradation testing, samples were taken at 24 weeks, 26 weeks, 36 weeks, 38 weeks, 40 weeks, 52 weeks, 52 weeks and 53 weeks for mechanical property testing.

[0199] After wiping the liquid on the sample surface with a paper towel, load the sample into the tensile testing machine fixture and test it directly. The test rate is set to 50mm / min. The test is terminated when the sample cannot be tested for mechanical properties, and the time point for stopping the test is the degradation cycle of the sample.

[0200] Experimental results:

[0201]

[0202]

[0203] It should be noted that tissue repair is a complex process that requires a certain amount of time to ensure that the damaged part can fully recover and regain sufficient strength and stability. If the patch degrades prematurely, it may lead to repair failure or require reoperation. If the patch is left in the body for too long, it may increase the risk of infection. Excessive degradation time may also affect the normal growth and repair process of surrounding tissues, and may also cause long-term compression of surrounding tissues or other adverse reactions.

[0204] From the above results, it can be seen that the artificial dura mater prepared by the present invention can be completely degraded within 26 weeks to 52 weeks, which can provide sufficient strength support for the repair of meningeal tissue to meet clinical needs, and the existence time in the body is not too long.

[0205] (IV) Flexibility test:

[0206] Experimental methods:

[0207] The samples of Examples 1 to 4 and Comparative Examples 1 to 3 were cut into specimens of 100 mm×100 mm in size and tested according to the softness determination method in GB / T8942-2016.

[0208] Experimental results:

[0209]

[0210]

[0211] From the above results, it can be seen that the artificial dura mater prepared by the present invention is soft in texture, with a softness of 130.55 mN-376.09 mN. The smaller the softness value, the softer the artificial dura mater and the better the adhesion.

[0212] (V) Anti-penetration performance test experiment:

[0213] Experimental methods:

[0214] The samples of Examples 1-4 were cut into specimens of appropriate sizes and tested according to the method for determining the anti-permeability performance in YY / T 0500-2021.

[0215] Experimental results: The artificial dura mater of the present invention has good anti-permeability, and no leakage was found in any of the tested samples under a pressure of 165.0 mmHg for 60 seconds.

[0216] (VI) Animal experiments

[0217] Experimental animals: rabbits, 4 months old.

[0218] Experimental materials: The artificial dura mater used in the experiment is the artificial dura mater of the present invention.

[0219] Surgical procedures:

[0220] 1. Preoperative preparation:

[0221] Rabbits were given general anesthesia intravenously, the surgical site was prepared, respiratory anesthesia was maintained continuously, pain relief was given intramuscularly, and antibiotics were injected 30 minutes before the operation. If the operation lasted more than 4 hours, additional antibiotics were given.

[0222] 2. Intraoperative operation:

[0223] The animal was placed prone on the animal operating table, the head was fixed with a head frame, and the skin was disinfected with iodine. After the sterile surgical towel was spread, the hair on the top of the dog's head was shaved, and the surgical operation area was routinely disinfected and covered with towels on the animal operating table.

[0224] A longitudinal incision was made at the midline of the top of the head to fully expose the skull. A high-speed electric drill was used to grind the skull on both sides 1 cm away from the midline to form a 1.5*1.5 cm skull defect to fully expose the dura mater. Two 1*1 cm dura maters were carefully cut out on both sides with meningeal scissors (the experimental samples were soaked in sterile saline for 1 min before animal surgery) to artificially create a dura mater defect on the right top. Note that a small amount of dura mater is retained at the edge of the bone window to facilitate dura mater repair.

[0225] Place a patch of appropriate size into the defective area of ​​the right dura mater, usually about 2 mm beyond the edge of the dura mater cavity, and apply it directly on the dura mater (the smooth side of the patch faces the brain tissue, and the rough side faces upward), cover the defective area of ​​the dura mater, and use non-absorbable 6-0 sutures to perform fixed-point interrupted sutures at the four corners to prevent movement. Use non-absorbable 4-0 sutures to suture the muscle layer, fascia, and scalp in sequence.

[0226] Note: The artificial dura mater is divided into a smooth side (the side with an anti-seepage protective layer) and a rough side (the side with a degradable repair membrane layer). The smooth side faces the brain tissue, and the rough side faces the scalp. Facing the light source, the smooth side has more reflective and brighter surface.

[0227] 3. Postoperative care:

[0228] Antibiotics are injected continuously for 7 days after the operation, and additional days of antibiotics are given if necessary to prevent infection.

[0229] 4. Test results see Figure 2a , Figure 2b .

[0230] Figure 2a This is a diagram of the artificial dura mater repair experiment provided in Experimental Example 1 of the present invention.

[0231] Figure 2b These are repair effect diagrams of the artificial dura mater repair experiment provided in Experimental Example 1 of the present invention at 4 weeks, 8 weeks, and 12 weeks.

[0232] As can be seen from the above, the artificial dura mater of the present invention has the functions of promoting meningeal repair, preventing infection, preventing leakage, and preventing adhesion. Figure 2b It can be clearly seen that there was no infection, leakage or adhesion 4 weeks after surgery, and the meninges began to repair. 8 weeks after surgery, the artificial dura mater was covered with capillaries. 12 weeks after surgery, the meninges had been basically repaired completely, and there was no infection, leakage or adhesion at this time.

[0233] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An artificial dura mater, characterized in that: The artificial dura mater comprises a degradable repair membrane layer and an anti-seepage protective layer composited on the degradable repair membrane layer; The degradable repair film layer is prepared by compounding multiple fiber oriented sheets; The fiber orientation sheet is formed into oriented fibers by an electrostatic spinning method, and the orientation directions of the oriented fibers in the fiber orientation sheet are consistent; The orientation directions of the fibers in each fiber orientation sheet in the degradable repair film layer are different.

2. The artificial dura mater according to claim 1, characterized in that: The oriented fibers are obtained by electrostatic spinning of degradable polyurethane or polylactic acid; Preferably, the weight average molecular weight Mw of the degradable polyurethane is 18W to 22W; Preferably, the weight average molecular weight Mw of the polylactic acid is 18W to 28W; Preferably, the diameter of the oriented fibers in the fiber oriented sheet is 1 nm to 20 μm, and the porosity is 10 to 40%.

3. The artificial dura mater according to claim 1, characterized in that: The anti-seepage protective layer is prepared from degradable polyurethane by a casting method; The anti-seepage protective layer is a degradable polyurethane film layer without a pore structure.

4. The artificial dura mater according to claim 1, characterized in that: The total thickness of the artificial dura mater is 0.1 to 0.3 mm, wherein: The thickness of the degradable repair film layer is 0.1-0.29 mm, and the thickness of the anti-seepage protection layer is 0.02-0.1 mm.

5. The artificial dura mater according to claim 1, characterized in that: The artificial dura mater has a tensile strength of 9-45 N / cm, a breaking elongation of 90-750%, a suture strength of 4-25 N, and a softness of 130.55 mN-376.09 mN.

6. The artificial dura mater according to claim 1, characterized in that: The degradable repair film layer is prepared by compounding two layers of fiber oriented sheets; Preferably, the two-layer fiber-oriented sheet comprises a first fiber-oriented sheet and a second fiber-oriented sheet, and the first fiber-oriented sheet and the second fiber-oriented sheet have different orientation directions and are at an angle of 90°±15°.

7. A method for preparing an artificial dura mater according to any one of claims 1 to 6, characterized in that: The preparation method comprises: The anti-seepage protective layer was prepared by a tape casting method, and then a degradable repair membrane layer was prepared on the anti-seepage protective layer by an electrospinning method, and then post-processed to obtain an artificial dura mater.

8. The method for preparing the artificial dura mater according to claim 7, characterized in that: The preparation method of the degradable repair film layer comprises: S1: dissolving the degradable polyurethane in a solvent to obtain a spinning solution; S2: electrospinning the spinning solution to obtain a fiber orientation sheet A, then adjusting the electrospinning direction, and electrospinning on the fiber orientation sheet A to obtain a fiber orientation sheet B intersecting the orientation direction of the fiber orientation sheet A, thereby obtaining a degradable repair film layer composed of two layers of fiber orientation sheets; S3: Repeat step S2 to obtain a degradable repair film layer composed of multiple fiber oriented sheets.

9. The method for preparing the artificial dura mater according to claim 8, characterized in that: In the step S1, the concentration of the degradable polyurethane in the spinning solution is 10-30% (w / v); Preferably, the solvent in the spinning solution is a single solvent of N,N-dimethylformamide, a single solvent of N,N-dimethylacetamide, a mixed solvent of N,N-dimethylformamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether, or a mixed solvent of N,N-dimethylacetamide and any one or more of tetrahydrofuran, dichloroethane or dimethyl ether; More preferably, the solvent in the spinning solution is a mixed solvent of N,N-dimethylformamide and tetrahydrofuran, wherein the volume ratio of N,N-dimethylformamide to tetrahydrofuran is 10:1-3:7, wherein the preferred volume ratio is 10:1-1:

1.

10. The method for preparing the artificial dura mater according to claim 8, characterized in that: The conditions for electrospinning in step S2 include: Negative voltage: -1KV~-4KV, positive voltage: 7KV~10KV, injection speed of the nozzle: 0.05-0.3ml / min; rotation speed of the receiving device: (800, 2500) rpm / min, distance between the nozzle and the receiving device: 0~50mm; translation speed of the nozzle is (0, 300) mm / min.

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