Multilayer polymer biomimetic membrane for dura repair and method of making same

Abstract

The application provides a multilayer polymer biomimetic membrane for dura mater repair and a preparation method thereof. The biomimetic membrane comprises a three-layer biomimetic nanofiber composite structure: a cell adhesion inner layer on the side facing the brain tissue, an anti-adhesion outer layer on the side facing the skull, and an intermediate reinforcing layer between the cell adhesion inner layer and the anti-adhesion outer layer. The cell adhesion inner layer is a PCL / chitosan / collagen composite fiber layer, the intermediate reinforcing layer is a PCL / hydroxyapatite composite fiber layer, and the anti-adhesion outer layer is a PCL / polylactic acid-hydroxyacetic acid copolymer (PLGA) layer. The method comprises cell adhesion inner layer preparation, intermediate reinforcing layer preparation, anti-adhesion outer layer preparation, and continuous production processing. The application obtains a multilayer artificial dura mater with strong interlayer bonding, high adhesion, and biomimetic mechanical properties.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to a multilayer polymeric biomimetic membrane for dura mater repair and its preparation method. Background Technology

[0002] The dura mater, located between the skull and brain tissue, is a relatively strong connective tissue membrane that acts as a natural barrier. The natural dura mater is mainly composed of fibroblasts and collagen fibers, effectively protecting the brain and preventing cerebrospinal fluid leakage.

[0003] Dural defects can lead to cerebrospinal fluid leakage, increased risk of infection and meningitis, thus requiring timely repair to maintain anatomical integrity and promote meningeal healing. Following a dural defect, fibroblasts are activated to participate in wound healing; however, an imbalance in extracellular matrix secretion can lead to tissue adhesions, fibrosis, and scar tissue formation, hindering nerve repair.

[0004] While existing artificial dura maters possess basic biocompatibility and airtightness, they generally suffer from insufficient mechanical properties, poor adhesion to host tissues, and a lack of biomimetic hierarchical structures, making it difficult to simultaneously achieve multiple functions such as nerve repair, anti-adhesion, and mechanical support. Among existing technologies, electrospinning technology is widely used in the construction of artificial dura maters due to its ability to prepare nanofiber scaffolds; however, its weak interlayer bonding and poor structural uniformity caused by a single spinning process are significant problems. For example, multilayer structures prepared by traditional electrospinning rely on physical stacking, resulting in obvious interlayer interfaces that are prone to stress concentration leading to a decline in overall mechanical properties. Furthermore, single polycaprolactone (PCL) scaffolds lack surface functional groups, limiting their adhesion to meningeal fibroblasts and affecting wound healing efficiency. In addition, while introducing bioactive molecules (such as insulin-like growth factor IGF) or photocurable hydrogels through blend spinning can enhance functional properties, it does not fully combine innovative spinning processes (such as the synergistic effect of air-jet spinning and electrospinning), making it difficult to achieve precise construction of biomimetic hierarchical structures.

[0005] Therefore, developing a multilayer artificial dura mater that combines strong interlayer bonding, high adhesion, and biomimetic mechanical properties remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a multilayer polymer biomimetic membrane for dura mater repair and its preparation method, so as to solve the problems of weak interlayer bonding, poor cell adhesion, insufficient mechanical properties and poor anti-adhesion effect of existing products.

[0007] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, this application provides a multilayer polymeric biomimetic membrane for dura mater repair, comprising a three-layer biomimetic nanofiber composite structure: a cell-attachment inner layer facing the brain tissue, an anti-adhesion outer layer facing the skull, and an intermediate reinforcing layer located between the cell-attachment inner layer and the anti-adhesion outer layer; the cell-attachment inner layer is a PCL / chitosan / collagen composite fiber layer, the intermediate reinforcing layer is a PCL / hydroxyapatite composite fiber layer, and the anti-adhesion outer layer is a PCL / polylactic acid-glycolic acid copolymer (PLGA) layer; the total thickness of the multilayer polymeric biomimetic membrane is 250-500 μm.

[0008] Specifically, the multilayer polymer biomimetic membrane (i.e., artificial dura mater) provided in this application is composed of a three-layer biomimetic nanofiber composite structure. The cell attachment inner layer is a PCL / chitosan / collagen composite fiber layer. PCL (polycaprolactone) can effectively prevent cerebrospinal fluid leakage after brain injury due to its hydrophobic properties and good mechanical strength, maintain the stable environment of brain tissue, and provide basic structural support for nerve repair. At the same time, through chitosan (containing amino groups) and collagen (containing carboxyl and amino groups), not only are bioactive sites provided for cell adhesion, but the hydrophilicity of the material can also be improved, significantly enhancing the ability to guide the adhesion and proliferation of meningeal fibroblasts.

[0009] The intermediate reinforcing layer is a PCL / hydroxyapatite composite fiber layer, mimicking the microstructure of the natural dura mater. It provides a three-dimensional scaffold for cell growth and acts as a physical barrier to prevent cerebrospinal fluid leakage and external infection. The flexibility of PCL combined with the rigidity of hydroxyapatite (HA) significantly enhances overall mechanical strength (such as tensile and tear resistance), matching the intracranial pressure environment, conforming to the dynamic movement of brain tissue, and reducing postoperative adhesions. Simultaneously, hydroxyapatite, a natural bone mineral component, possesses excellent bioactivity and osteoconductivity, stimulating fibroblast proliferation and collagen deposition at the dura mater margin, accelerating defect repair.

[0010] The composite design of PCL and HA can achieve synergistic degradation, matching the degradation rate with the tissue regeneration rate, avoiding complications caused by premature or delayed degradation. Furthermore, both PCL and HA are biocompatible materials, reducing the risk of foreign body reactions and chronic inflammation. This composite fiber layer can more effectively reduce the risk of meningeal adhesions and scar formation. The hydroxyapatite particles have a particle size of 50-200 nm.

[0011] The outer anti-adhesion layer is a PCL / polylactic acid-glycolic acid copolymer (PLGA) layer. The blended structure of PCL and PLGA combines good biocompatibility and controllable degradation. Simultaneously, this outer anti-adhesion layer is a dense fibrous membrane that isolates brain tissue from the skull or subcutaneous tissue, preventing adhesions caused by excessive proliferation of fibrous connective tissue postoperatively (such as meningeal-brain adhesions or dura mater-skull adhesions). It also forms a gradient degradation with the intermediate reinforcing layer, gradually exposing the inner HA layer and guiding cell migration for orderly repair. During PLGA degradation, a microporous structure is formed, gradually exposing the intermediate reinforcing layer HA and guiding orderly tissue regeneration, avoiding dense scarring. Furthermore, heparin is covalently grafted onto the fiber surface, utilizing its anti-adhesion properties to prevent postoperative adhesion between the meninges and skull.

[0012] Preferably, the thickness of the inner cell adhesion layer is 90-100 μm, the thickness of the intermediate reinforcing layer is 100-200 μm, and the thickness of the outer anti-adhesion layer is 60-200 μm.

[0013] Preferably, the pore size distribution of the inner cell attachment layer is 50-200 nm, the pore size distribution of the middle reinforcement layer is 200-700 nm, and the pore size distribution of the anti-adhesion outer layer is 50-200 nm.

[0014] Preferably, the porosity of the inner cell attachment layer is 70-85%, the porosity of the intermediate reinforcing layer is 40-60%, and the porosity of the outer anti-adhesion layer is 20-40%.

[0015] Specifically, the inner cell attachment layer has a porosity of 70-85%, providing ample space for fibroblast infiltration, proliferation, and extracellular matrix secretion, thus accelerating the regeneration of the host dura mater. The middle reinforcing layer has a porosity of 40-60%, ensuring mechanical support without hindering nutrient delivery and cell migration. The dense structure of the anti-adhesion outer layer effectively prevents excessive fibroblast proliferation, reducing the risk of tissue adhesion. Furthermore, a porosity gradient transition is formed between the three layers, with the porosity gradually decreasing from the inside to the outside. This reduces stress concentration at the interlayer interfaces and guides fibroblasts to migrate orderly from the inner to the middle layers, promoting tissue integration.

[0016] Secondly, this application provides a method for preparing the above-mentioned multilayer polymer biomimetic membrane, comprising the following steps: (1) Equipment layout and spinning solution introduction: Three sets of spinning devices are arranged circumferentially above the receiving device, with a spacing of 5-15cm, to ensure that the next layer is deposited immediately before the previous layer dries. The surface of the receiving device is covered with polytetrafluoroethylene material, which not only ensures uniform deposition but also facilitates subsequent peeling. The first set of spinning devices is introduced with the cell adhesion inner layer spinning solution, the second set of spinning devices is introduced with the intermediate reinforcement layer spinning solution, and the third set of spinning devices is introduced with the anti-adhesion outer layer spinning solution. (2) Preparation of multilayer composite membrane: The first spinning device uses an alternating air-jet spinning-electro-spinning method to deposit an inner cell attachment layer in situ on the receiving device. Before the inner cell attachment layer dries, the second spinning device uses an alternating air-jet spinning-electro-spinning method to continuously deposit an intermediate reinforcing layer on the inner cell attachment layer. Before the intermediate reinforcing layer dries, the third spinning device uses an electro-spinning method to continuously deposit an anti-adhesion outer layer on the intermediate reinforcing layer. After the third spinning device is turned off, a multilayer composite membrane is obtained on the receiving device. Specifically, in the preparation of the cell attachment inner layer, PCL / chitosan / collagen are used as spinning solution raw materials. A "porous substrate-oriented fiber" composite structure is formed through alternating air-spinning and electrospinning deposition, mimicking the heterogeneity of the fiber orientation in the natural dura mater and guiding cells to grow and align along the fiber axis. In the preparation of the intermediate reinforcing layer, an air-spinning process is first used to form a three-dimensional interwoven reinforcing framework. Then, PCL nanofibers are deposited via electrospinning to fill the mesh pores, forming a "framework-fiber" interlocking structure. This significantly improves the overall mechanical strength of the biomimetic membrane, with its tensile strength being significantly higher than that of a single electrospun layer.

[0017] Spinning parameters for the inner cell attachment layer and intermediate reinforcing layer: airflow spinning pressure 0.5-1.0 MPa, flow rate 5-10 mL / h; electrospinning voltage 10-15 kV, flow rate 1-2 mL / h; receiving distance 15-25 cm. The alternation frequency between airflow spinning and electrospinning is every 30-60 seconds to ensure uniform fiber distribution and form a biomimetic structure of alternating "loose-oriented" fibers. Spinning parameters for the anti-adhesion outer layer: voltage 10-15 kV, flow rate 1-2 mL / h, receiving distance 15-25 cm.

[0018] Controlling the conditions during the spinning process ensures that the thickness of each film layer meets the requirements, and that the electrostatic field voltage remains consistent during deposition, thus preventing gaps between layers.

[0019] (3) Cross-linking functionalization: The multilayer composite membrane is immersed in the heparin cross-linking solution along with the receiving device and stirred at room temperature in the dark for 2-10 hours to covalently bond heparin sodium to the surface of the PCL / PLGA fiber membrane.

[0020] The outer layer of PCL / PLGA acts as a dense fibrous membrane, isolating brain tissue from the skull or subcutaneous tissue, preventing adhesions caused by excessive proliferation of fibrous connective tissue postoperatively, and reducing scar formation. The degradation products of PLGA, lactic acid and glycolic acid, can regulate local inflammatory responses, inhibit excessive fibroblast activation, and reduce the risk of scar tissue formation. The multilayer composite membrane is immersed in heparin sodium solution for an EDC / NHS cross-linking reaction, allowing heparin to be covalently grafted onto the fiber surface. The anti-adhesion properties of heparin are utilized to prevent postoperative adhesion between the meninges and skull.

[0021] (4) Post-processing: The cross-linked multilayer composite membrane is removed from the receiving device, rinsed with deionized water 3-5 times to remove unreacted reagents, and dried in a vacuum drying oven at 30-50℃ for 24 hours to obtain a multilayer polymer biomimetic membrane, i.e., artificial dura mater.

[0022] This application achieves the preparation of a multilayer polymer biomimetic membrane, namely an artificial dura mater, through the above-described method. Using PCL / chitosan / collagen as the spinning solution raw material, a cell-attached inner layer with a "porous substrate-oriented fiber" composite structure is formed through alternating air-spinning and electrospinning, guiding cells to grow along the fiber axis. Then, using PCL / hydroxyapatite spinning solution as the raw material, a three-dimensional interwoven reinforcing framework is first formed by air-spinning, followed by electrospinning to deposit PCL nanofibers to fill the mesh pores, forming a "framework-fiber" interlocking structure, significantly improving the overall mechanical strength of the biomimetic membrane; its tensile strength is significantly higher than that of a single electrospun layer. Finally, a PCL / polylactic acid-glycolic acid fiber membrane is formed using PCL / polylactic acid-glycolic acid spinning solution and electrospinning. The multilayer composite membrane is then immersed in a heparin crosslinking solution and dried to obtain a multilayer artificial dura mater with strong interlayer bonding, high adhesion, and biomimetic mechanical properties.

[0023] Preferably, the cell attachment inner layer spinning solution comprises: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL, chitosan and type I collagen were then added and stirred at 25°C for 4-6 hours until completely dissolved. The mixture was then filtered through a 0.45μm filter membrane to remove impurities and allowed to stand for 30 minutes to degas.

[0024] The mass-volume ratio of chitosan to the mixed solvent is 0.5-2% (w / v), the mass-volume ratio of type I collagen to the mixed solvent is 1-3% (w / v), and the balance is PCL.

[0025] Preferably, the intermediate reinforcing layer spinning solution comprises: A mixed solvent was prepared by mixing dichloromethane and anhydrous ethanol at a volume ratio of 8:2. PCL and hydroxyapatite were added, and the mixture was ultrasonically dispersed for 30 min (300 W power). The mixture was then stirred for 2-3 h until the PCL was completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 min to remove bubbles before use. The mass ratio of PCL to hydroxyapatite was 8-10:1, and the mass-volume ratio of PCL to the mixed solvent was 1-5% (w / v).

[0026] Preferably, the anti-adhesion outer spinning solution comprises: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL and PLGA were then added and stirred for 3-4 hours until completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 minutes to remove bubbles before use.

[0027] The mass ratio of PCL to PLGA is 1:1, and the total mass of both is 1-5% (w / v) of the mixed solvent.

[0028] Specifically, the mixed solvent of dichloromethane / anhydrous ethanol has both hydrophobicity and a certain degree of hydrophilicity. It can fully dissolve hydrophobic polymers such as PCL and PLGA, and can also uniformly disperse biomacromolecules such as chitosan and collagen to avoid agglomeration. At the same time, the dispersion stability of hydroxyapatite is improved in the mixed solvent, ensuring the uniformity of the spinning solution.

[0029] The purity of the dichloromethane / anhydrous ethanol mixed solvent is ≥99.5%. The spinning solution used in each layer must be filtered through a 0.45μm filter membrane after preparation to remove impurities and undissolved particles.

[0030] Preferably, the heparin crosslinking solution is prepared as follows: Prepare an aqueous solution of sodium heparin with a concentration of 5-10 mg / mL, add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) as crosslinking agents, with EDC concentration of 5-10 mg / mL and NHS concentration of 1-2 mg / mL, and the volume ratio of EDC to NHS is 4:1. Stir until completely dissolved, and the pH value of the system will naturally stabilize at 5.0-6.0 (suitable for EDC / NHS crosslinking reaction), without the need for additional acid-base adjustment, and set aside for later use.

[0031] Preferably, in the cross-linking functionalization step, the solid-liquid ratio of the heparin cross-linking solution to the multilayer composite membrane is 5-10 mL / cm², ensuring complete wetting of the composite substrate and keeping the substrate flat during the cross-linking reaction to avoid wrinkles. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 A schematic diagram illustrating the manufacturing principle of an artificial dura mater provided in one embodiment of this application; Figure 2 This is an electron microscope image of the artificial dura mater provided in Embodiment 2 of this application; Figure 3 This is a physical image of the artificial dura mater provided in Embodiment 2 of this application. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are also within the scope of protection of this application.

[0035] like Figure 1 As shown, this application provides a method for preparing the above-mentioned multilayer polymer biomimetic membrane, comprising the following steps: A method for preparing a multilayer polymeric biomimetic membrane for dura mater repair includes the following steps: (1) Preparation of spinning solution: (11) Cell attachment inner layer spinning solution: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL, chitosan, and type I collagen were then added and stirred at 25°C for 4-6 hours until completely dissolved. The mixture was filtered through a 0.45 μm filter to remove impurities and allowed to stand for 30 minutes to remove bubbles. The mass-volume ratio of chitosan to the mixed solvent was 0.5-2% (w / v), the mass-volume ratio of type I collagen to the mixed solvent was 1-3% (w / v), and the balance was PCL.

[0036] (12) Spinning solution for intermediate reinforcing layer: A mixed solvent was prepared by mixing dichloromethane and anhydrous ethanol at a volume ratio of 8:2. PCL and hydroxyapatite were added, and the mixture was ultrasonically dispersed for 30 min (300 W power). The mixture was then stirred for 2-3 h until the PCL was completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 min to remove bubbles before use. The mass ratio of PCL to hydroxyapatite was 8-10:1, and the mass-volume ratio of PCL to the mixed solvent was 1-5% (w / v).

[0037] (13) Anti-adhesion outer spinning solution: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL and PLGA were then added and stirred for 3-4 hours until completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 minutes to remove bubbles before use. The mass ratio of PCL to PLGA was 1:1, and the total mass of PCL and PLGA to the mass-volume ratio of the mixed solvent was 1-5% (w / v).

[0038] (2) Equipment layout and spinning solution introduction: Three sets of spinning devices are arranged circumferentially above the receiving device, with a spacing of 5-15cm, to ensure that the next layer is deposited immediately before the previous layer dries. The surface of the receiving device is covered with polytetrafluoroethylene material, which not only ensures uniform deposition but also facilitates subsequent peeling. The first set of spinning devices is introduced with the cell adhesion inner layer spinning solution, the second set of spinning devices is introduced with the intermediate reinforcement layer spinning solution, and the third set of spinning devices is introduced with the anti-adhesion outer layer spinning solution. (3) Preparation of multilayer composite membranes: (31) Cell attachment inner layer: The first set of spinning devices uses air-jet spinning (air pressure 0.5-1.0MPa, flow rate 5-10mL / h) - electrospinning (voltage 10-15kV, flow rate 1-2mL / h) alternating every 30-60 seconds, with a receiving distance of 15-25cm, depositing to a thickness of 90-100μm, and forming a cell attachment inner layer in situ on the receiving device.

[0039] (32) Intermediate reinforcement layer: Before the inner cell attachment layer dries (within 1-2 minutes after deposition), the second set of spinning devices is started immediately. The second set of spinning devices uses the same airflow spinning-electrostatic spinning alternating process as the first set of spinning devices to continuously deposit an intermediate reinforcement layer with a thickness of 100-200μm on the inner cell attachment layer. With the help of the slight swelling effect of the solvent, the two layers of fibers permeate each other and form a preliminary bond.

[0040] (33) Anti-adhesion outer layer: Within 1-2 minutes after the deposition of the intermediate reinforcing layer, the third spinning device is turned on to use electrospinning (voltage 10-15kV, flow rate 1-2mL / h, receiving distance 15-25cm) to continuously deposit an anti-adhesion outer layer with a thickness of 60-200μm on the intermediate reinforcing layer. After the third spinning device is turned off, a multilayer composite film is obtained on the receiving device. The liquid supply is uninterrupted during the three-layer deposition process, and the electrostatic field voltage is kept consistent to ensure that there are no gaps between the layers.

[0041] (4) Crosslinking functionalization: The multilayer composite membrane is immersed in the heparin crosslinking solution along with the receiving device. The solid-liquid ratio of the heparin crosslinking solution to the multilayer composite membrane is 5-10 mL / cm². The reaction is carried out at room temperature in the dark for 2-10 h with stirring, so that the sodium heparin is covalently bonded to the surface of the PCL / PLGA fiber membrane, thus completing the anti-adhesion function modification.

[0042] Heparin crosslinking solution: Prepare a heparin sodium aqueous solution with a concentration of 5-10 mg / mL, add EDC and NHS as crosslinking agents, with EDC concentration of 5-10 mg / mL and NHS concentration of 1-2 mg / mL, and the volume ratio of EDC to NHS is 4:1, stir well and set aside.

[0043] (5) Post-processing: Remove the cross-linked multilayer composite membrane from the receiving device, rinse it with deionized water 3-5 times to remove unreacted reagents, and dry it in a vacuum drying oven at 30-50℃ for 24h, controlling the residual solvent content to ≤0.5%, to obtain a multilayer polymer biomimetic membrane, i.e., artificial dura mater.

[0044] The technical solution of this application will be illustrated in detail below with specific embodiments. Example 1

[0045] A method for preparing a multilayer polymeric biomimetic membrane for dura mater repair includes the following steps: (1) Preparation of spinning solution: (11) Cell attachment inner layer spinning solution: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL, chitosan, and type I collagen were then added and stirred at 25°C for 4 hours until completely dissolved. The mixture was filtered through a 0.45 μm filter to remove impurities and allowed to stand for 30 minutes to remove bubbles. The mass-volume ratio of chitosan to the mixed solvent was 0.5% (w / v), the mass-volume ratio of type I collagen to the mixed solvent was 1% (w / v), and the balance was PCL.

[0046] (12) Spinning solution for intermediate reinforcing layer: A mixed solvent was prepared by mixing dichloromethane and anhydrous ethanol at a volume ratio of 8:2. PCL and hydroxyapatite were added, and the mixture was ultrasonically dispersed for 30 min (300 W). The mixture was then stirred for 2 h until the PCL was completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 min to remove bubbles before use. The mass ratio of PCL to hydroxyapatite was 8:1, and the mass-volume ratio of PCL to the mixed solvent was 1% (w / v).

[0047] (13) The anti-adhesion outer spinning solution includes: A mixed solvent was prepared by mixing dichloromethane and anhydrous ethanol at a volume ratio of 8:2. PCL and PLGA were then added and stirred for 4 hours until completely dissolved. The mixture was filtered through a 0.45 μm filter membrane to remove impurities and allowed to stand for 30 minutes to remove bubbles before use. The mass ratio of PCL to PLGA was 1:1, and the total mass of PCL and PLGA was 1% (w / v) of the volume ratio of the mixed solvent to the total mass of PCL and PLGA.

[0048] (2) Equipment layout and spinning solution introduction: Three sets of spinning devices are arranged circumferentially above the receiving device with a spacing of 5cm. The surface of the receiving device is covered with polytetrafluoroethylene material. The first set of spinning devices is introduced with the cell adhesion inner layer spinning solution, the second set of spinning devices is introduced with the intermediate reinforcement layer spinning solution, and the third set of spinning devices is introduced with the anti-adhesion outer layer spinning solution. (3) Preparation of multilayer composite membranes: (31) Cell attachment inner layer: The first set of spinning devices uses air-flow spinning (air pressure 0.5MPa, flow rate 5mL / h) - electrospinning (voltage 10kV, flow rate 1mL / h) alternating every 60 seconds, with a receiving distance of 15cm, depositing to a thickness of 90μm, and forming a cell attachment inner layer in situ on the receiving device.

[0049] (32) Intermediate reinforcement layer: 2 minutes after the deposition of the cell attachment inner layer, the second set of spinning devices is started immediately. The second set of spinning devices adopts the same airflow spinning-electrostatic spinning alternating process as the first set of spinning devices to continuously deposit an intermediate reinforcement layer with a thickness of 100 μm on the cell attachment inner layer.

[0050] (33) Anti-adhesion outer layer: Within 1 minute after the deposition of the intermediate reinforcing layer, the third spinning device is turned on to use electrospinning (voltage 10kV, flow rate 1mL / h, receiving distance 15cm) to continuously deposit an anti-adhesion outer layer with a thickness of 60μm on the intermediate reinforcing layer. After the third spinning device is turned off, a multilayer composite film is obtained on the receiving device. The liquid supply is uninterrupted during the three-layer deposition process, and the electrostatic field voltage remains consistent to ensure that there are no gaps between the layers.

[0051] (4) Crosslinking functionalization: The multilayer composite membrane is immersed in the heparin crosslinking solution along with the receiving device. The solid-liquid ratio of the heparin crosslinking solution to the multilayer composite membrane is 5 mL / cm². The reaction is carried out at room temperature in the dark for 2 hours to complete the anti-adhesion function modification.

[0052] Heparin crosslinking solution: Prepare a 5 mg / mL heparin sodium aqueous solution, add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) as crosslinking agents, with EDC concentration of 5 mg / mL and NHS concentration of 1 mg / mL, and the volume ratio of EDC to NHS is 4:1.

[0053] (5) Post-processing: The cross-linked multilayer composite membrane was removed from the receiving device, rinsed three times with deionized water to remove unreacted reagents, and dried in a vacuum drying oven at 50°C for 24 hours to obtain a multilayer polymer biomimetic membrane, i.e., an artificial dura mater. The total thickness of the multilayer polymer biomimetic membrane was 250 μm. Example 2

[0054] A method for preparing a multilayer polymeric biomimetic membrane for dura mater repair includes the following steps: The difference from Example 1 is as follows: (1) Preparation of spinning solution: (11) Cell attachment inner layer spinning solution: the mass-volume ratio of chitosan to mixed solvent is 1.5% (w / v), the mass-volume ratio of type I collagen to mixed solvent is 2% (w / v), and the balance is PCL.

[0055] (12) Intermediate reinforcing layer spinning solution: The mass ratio of PCL to hydroxyapatite is 9:1, and the mass-volume ratio of PCL to mixed solvent is 3% (w / v).

[0056] (13) Anti-adhesion outer spinning solution: The mass ratio of PCL to PLGA is 1:1, and the mass-volume ratio of the total mass of the two to the mixed solvent is 2% (w / v).

[0057] (2) Equipment layout and spinning solution introduction: Arrange the three sets of spinning devices circumferentially above the receiving device with a spacing of 10cm.

[0058] (3) Preparation of multilayer composite membranes: (31) Cell attachment inner layer: The first spinning device uses air-flow spinning (air pressure 0.7MPa, flow rate 7mL / h) - electrospinning (voltage 12kV, flow rate 1.5mL / h) alternating every 60 seconds, with a receiving distance of 15cm, depositing to a thickness of 90μm to form a cell attachment inner layer.

[0059] (32) Intermediate reinforcement layer: A 120 μm thick intermediate reinforcement layer is continuously deposited on the inner cell attachment layer.

[0060] (33) Anti-adhesion outer layer: The third spinning device is turned on and electrospinning is used (voltage 7kV, flow rate 1.5mL / h, receiving distance 15cm) to continuously deposit an anti-adhesion outer layer with a thickness of 140μm on the intermediate reinforcing layer.

[0061] (4) Cross-linking functionalization: The solid-liquid ratio of heparin cross-linking solution to multilayer composite membrane is 8 mL / cm². The reaction is carried out at room temperature in the dark for 2 hours to complete the anti-adhesion function modification.

[0062] Heparin crosslinking solution: Prepare a 7.5 mg / mL heparin sodium aqueous solution, and add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) as crosslinking agents. The concentration of EDC is 7.5 mg / mL, the concentration of NHS is 1 mg / mL, and the volume ratio of EDC to NHS is 4:1.

[0063] (5) The total thickness of the multilayer polymer biomimetic membrane is 350 μm. Example 3

[0064] A method for preparing a multilayer polymeric biomimetic membrane for dura mater repair includes the following steps: The difference from Example 1 is as follows: 1. Preparation of spinning solution: (11) Cell attachment inner layer spinning solution: the mass-volume ratio of chitosan to mixed solvent is 2% (w / v), the mass-volume ratio of type I collagen to mixed solvent is 3% (w / v), and the balance is PCL.

[0065] (12) Intermediate reinforcing layer spinning solution: The mass ratio of PCL to hydroxyapatite is 10:1, and the mass-volume ratio of PCL to mixed solvent is 5% (w / v).

[0066] (13) Anti-adhesion outer spinning solution: The mass ratio of PCL to PLGA is 1:1, and the mass-volume ratio of the total mass of the two to the mixed solvent is 5% (w / v).

[0067] (2) Equipment layout and spinning solution introduction: Arrange the three sets of spinning devices circumferentially above the receiving device with a spacing of 15cm.

[0068] (3) Preparation of multilayer composite membranes: (31) Cell attachment inner layer: The first set of spinning devices uses airflow spinning (air pressure 1MPa, flow rate 10mL / h) - electrospinning (voltage 15kV, flow rate 2mL / h) alternating every 30 seconds, with a receiving distance of 25cm, depositing to a thickness of 100μm to form a cell attachment inner layer.

[0069] (32) Intermediate reinforcement layer: A 200 μm thick intermediate reinforcement layer is continuously deposited on the inner cell attachment layer.

[0070] (33) Anti-adhesion outer layer: The third spinning device is turned on and electrospinning is used (voltage 15kV, flow rate 2mL / h, receiving distance 25cm) to continuously deposit an anti-adhesion outer layer with a thickness of 200μm on the intermediate reinforcing layer.

[0071] (4) Crosslinking functionalization: The solid-liquid ratio of heparin crosslinking solution to multilayer composite membrane is 10 mL / cm². The reaction is carried out at room temperature in the dark for 2 hours to complete the anti-adhesion function modification.

[0072] Heparin crosslinking solution: Prepare a 10 mg / mL heparin sodium aqueous solution, add EDC and NHS as crosslinking agents, with EDC concentration of 10 mg / mL and NHS concentration of 2 mg / mL, and the volume ratio of EDC to NHS is 4:1.

[0073] (5) The total thickness of the multilayer polymer biomimetic membrane is 350 μm.

[0074] Comparative Example 1: The difference from Example 2 is that the cell attachment inner layer was prepared using only electrospinning, without the aid of airflow spinning.

[0075] Comparative Example 2: The difference from Example 2 is that the intermediate reinforcing layer is prepared only by air-jet spinning and does not involve electrospinning.

[0076] Comparative Example 3: The difference from Example 2 is that the intermediate reinforcing layer is prepared only by electrospinning and not by airflow spinning.

[0077] Comparative Example 4: The difference from Example 2 is that the solvents in the three spinning solutions were prepared using a single dichloroethane solvent.

[0078] Comparative Example 5: The difference from Example 2 is that the preparation of the intermediate reinforcing layer and the anti-adhesion outer layer is carried out after the previous layer has dried.

[0079] Comparative Example 6: The difference from Example 2 is that the multilayer composite membrane is not immersed in the heparin crosslinking solution.

[0080] Experimental Example 1 The microstructure of the artificial dura mater obtained in Examples 1-3 was tested, and the results are as follows: Figure 2 As shown, Figure 2 This is an electron microscope image of the artificial dura mater shown in Embodiment 2 of this application. Figure 3 This is a physical image of the artificial dura mater shown in Embodiment 2 of this application.

[0081] Experimental Example 2 Mechanical property testing The mechanical properties of the artificial dura mater obtained in Examples 1-3 and Comparative Examples 1-6 were tested, such as tensile strength and interlaminar peel strength.

[0082] Tensile property testing: The test was conducted using an electronic tensile testing machine (HY3080, Shanghai Hengyi Precision Instruments Co., Ltd.) according to ISO527-3 standard. The specimens were cut into 1cm×1cm pieces in both the transverse and longitudinal directions, and then mounted on the tensile testing machine. Five samples were tested in each group at a clamp separation speed of 200mm / min. The tensile strength of the test samples was recorded. At least three parallel tests were performed for each test, and the average value was taken. The results are shown in Table 1.

[0083] The interlayer peel strength was measured according to ASTM F2256 standard. Each test was performed in at least three parallel tests, and the average value was taken. The results are shown in Table 1.

[0084] Table 1

[0085] As observed in Table 1, Comparative Examples 1-3 all used only one method of air-jet spinning / electro-spinning to prepare biomimetic membranes, but their tensile strength was significantly lower than that of Examples 1-3. This indicates that this application alternately applies air-jet spinning and electro-spinning to the preparation of artificial dura mater. Through the synergistic effect of multiple processes such as "loose substrate - directional fiber - reinforcing skeleton", a composite scaffold with a biomimetic hierarchical structure (i.e., a dense outer layer to prevent adhesion, a middle layer to enhance support, and a porous inner layer to promote adhesion) is constructed. This breaks through the structural uniformity limitation of traditional single spinning processes and improves the mechanical properties of biomimetic membranes (i.e., artificial dura mater).

[0086] Experimental Example 3 Fibroblast proliferation rate The adhesion and repair efficiency of the artificial dura maters obtained in Examples 1-3 and Comparative Examples 1-6 were tested after use, including fibroblast proliferation rate and cell adhesion rate to the inner layer. At least three parallel experiments were performed for each test, and the average value was taken. The results are shown in Table 2. The testing standard adopted was ISO 19007: Guidelines for in vitro cell proliferation assays using nanomaterials.

[0087] Table 2

[0088] As shown in Table 2, this application alternates between air-jet spinning and electrospinning in the preparation of artificial dura mater, constructing a composite scaffold with a biomimetic hierarchical structure (i.e., a dense outer layer to prevent adhesion, a middle layer to enhance support, and a porous inner layer to promote adhesion). This not only improves the mechanical properties of the biomimetic membrane (i.e., artificial dura mater) but also enhances the adhesion and repair efficiency of the artificial dura mater after use.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A multilayer polymeric biomimetic membrane for dura mater repair, characterized in that, The structure comprises a three-layer biomimetic nanofiber composite: a cell-attachment inner layer facing the brain tissue, an anti-adhesion outer layer facing the skull, and an intermediate reinforcing layer located between the cell-attachment inner layer and the anti-adhesion outer layer; the cell-attachment inner layer is a PCL / chitosan / collagen composite fiber layer, the intermediate reinforcing layer is a PCL / hydroxyapatite composite fiber layer, and the anti-adhesion outer layer is a PCL / polylactic acid-glycolic acid copolymer (PLGA) layer; the total thickness of the multilayer polymer biomimetic membrane is 250-500 μm.

2. The multilayer polymeric biomimetic membrane for dura mater repair according to claim 1, characterized in that, The thickness of the inner cell attachment layer is 90-100 μm, the thickness of the intermediate reinforcement layer is 100-200 μm, and the thickness of the anti-adhesion outer layer is 60-200 μm.

3. The multilayer polymeric biomimetic membrane for dura mater repair according to claim 1, characterized in that, The pore size distribution of the inner cell attachment layer is 50-200 nm, the pore size distribution of the intermediate reinforcement layer is 200-700 nm, and the pore size distribution of the anti-adhesion outer layer is 50-200 nm.

4. The multilayer polymeric biomimetic membrane for dura mater repair according to claim 1, characterized in that, The porosity of the inner cell attachment layer is 70-85%, the porosity of the intermediate reinforcing layer is 40-60%, and the porosity of the outer anti-adhesion layer is 20-40%.

5. A method for preparing a multilayer polymeric biomimetic membrane for dura mater repair, characterized in that, This method is used to prepare the multilayer polymer biomimetic membrane according to any one of claims 1-4, and the preparation method includes the following steps: (1) Equipment layout and spinning solution introduction: Three sets of spinning devices are arranged circumferentially above the receiving device with a spacing of 5-15cm. The surface of the receiving device is covered with polytetrafluoroethylene material. The first set of spinning devices is introduced with cell adhesion inner layer spinning solution, the second set of spinning devices is introduced with intermediate reinforcement layer spinning solution, and the third set of spinning devices is introduced with anti-adhesion outer layer spinning solution. (2) Preparation of multilayer composite membrane: The first spinning device uses an alternating air-jet spinning-electro-spinning method to deposit the cell attachment inner layer in situ on the receiving device. Before the cell attachment inner layer dries, the second spinning device uses an alternating air-jet spinning-electro-spinning method to continuously deposit an intermediate reinforcing layer on the cell attachment inner layer. Before the intermediate reinforcing layer dries, the third spinning device uses an electro-spinning method to continuously deposit an anti-adhesion outer layer on the intermediate reinforcing layer. After the third spinning device is turned off, a multilayer composite membrane is obtained on the receiving device. (3) Cross-linking functionalization: The multilayer composite membrane is immersed in heparin cross-linking solution along with the receiving device and stirred at room temperature in the dark for 2-10 hours; (4) Post-processing: The cross-linked multilayer composite membrane is removed from the receiving device, rinsed with deionized water 3-5 times, and dried in a vacuum drying oven to obtain the multilayer polymer biomimetic membrane, i.e., artificial dura mater.

6. The method for preparing a multilayer polymeric biomimetic membrane for dura mater repair according to claim 5, characterized in that, The cell attachment inner layer spinning solution includes: Dichloromethane and anhydrous ethanol were mixed in a volume ratio of 8:2 to obtain a mixed solvent. PCL, chitosan and type I collagen were then added and stirred until completely dissolved. The mass-volume ratio of chitosan to the mixed solvent is 0.5-2% (w / v), the mass-volume ratio of type I collagen to the mixed solvent is 1-3% (w / v), and the balance is PCL.

7. The method for preparing a multilayer polymeric biomimetic membrane for dura mater repair according to claim 5, characterized in that, The intermediate reinforcing layer spinning solution comprises: A mixed solvent was obtained by mixing dichloromethane and anhydrous ethanol at a volume ratio of 8:

2. PCL and hydroxyapatite were then added and ultrasonically dispersed for 30 min. The mass ratio of PCL to hydroxyapatite was 8-10:1, and the mass-volume ratio of PCL to the mixed solvent was 1-5% (w / v).

8. The method for preparing a multilayer polymeric biomimetic membrane for dura mater repair according to claim 5, characterized in that, The anti-adhesion outer spinning solution includes: A mixed solvent was obtained by mixing dichloromethane and anhydrous ethanol in a volume ratio of 8:

2. PCL and PLGA were then added and stirred until completely dissolved. The mass ratio of PCL to PLGA is 1:1, and the total mass of both is 1-5% (w / v) of the mixed solvent.

9. The method for preparing a multilayer polymeric biomimetic membrane for dura mater repair according to claim 5, characterized in that, The heparin crosslinking solution: Prepare a heparin sodium aqueous solution with a concentration of 5-10 mg / mL, add EDC and NHS as crosslinking agents, with EDC concentration of 5-10 mg / mL and NHS concentration of 1-2 mg / mL, and the volume ratio of EDC to NHS is 4:1, and stir evenly for later use.

10. The method for preparing a multilayer polymeric biomimetic membrane for dura mater repair according to claim 5, characterized in that, In the cross-linking functionalization step, the solid-liquid ratio of the heparin cross-linking solution to the multilayer composite membrane is 5-10 mL / cm².

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